CN102099016A - Drug loaded polymeric nanoparticles and methods of making and using same - Google Patents

Abstract

The present disclosure generally relates to nanoparticles having about 0.2 to about 35 weight percent of a therapeutic agent; and about 10 to about 99 weight percent of biocompatible polymer such as a diblock poly(lactic) acid-poly(ethylene)glycol. Other aspects of the invention include methods of making such nanoparticles.

Description

Carry the polymer nano particle and its preparation and application of medicine

Related application

The U.S.S.N.61/061760 submitted this application claims on June 16th, 2008；The U.S.S.N.61/105916 that on October 16th, 2008 submits；The U.S.S.N.61/106777 that on October 20th, 2008 submits；The U.S.S.N.61/169514 that on April 15th, 2009 submits；The U.S.S.N.61/175209 that on May 4th, 2009 submits；The U.S.S.N61/061704 that on June 16th, 2008 submits；The U.S.S.N.61/169519 that on April 15th, 2009 submits；The U.S.S.N.61/175219 that on May 4th, 2009 submits；The U.S.S.N.61/061697 that on June 16th, 2008 submits；The U.S.S.N.61/088159 that August in 2008 is submitted on the 12nd；The U.S.S.N.61/169541 that on April 15th, 2009 submits；The U.S.S.N.61/175226 that on May 4th, 2009 submits；The U.S.S.N.61/173784 that on April 29th, 2009 submits；The priority for the U.S.S.N.61/173790 that 29, U.S.S.N.61/182300 and 2009 on April that on May 29th, 2009 submits submit；Each of which is incorporated herein by reference in their entirety.

Background technology

For a long time, it has been recognized that it is beneficial that some drugses are delivered into the system of patient's (for example, targetting specific tissue or cell type or the specific diseased tissue of targeting and non-normal tissue) or the system of Drug controlled release.

For example, comprising active medicine and for example, targetting the therapeutic agent of those of the specifically diseased tissue of tissue or cell type or targeting specific and non-normal tissue, it is possible to reduce amount of the medicine in the non-targeted tissue of body.When treatment such as cancer (wherein it is desirable to the medicine delivery of cell toxic amount to cancer cell without kill around cancerous tissue) illness when, this is especially important.Effective drug targeting effect can reduce common undesirable and life-threatening sometimes side effect in anticancer therapy.In addition, this therapeutic agent can allow medicine to reach some tissues that they can not otherwise be reached.

The therapeutic agent of offer control release and/or targeted therapies also allows for delivering the medicine of effective dose, and the medicine is known limitation in other nanoparticle delivery systems.For example, it can solve following problems：Nanoparticle systems (the related each nanoparticle of the medicine containing appropriate amount) are prepared, while keeping the size of the nanoparticle sufficiently small, so that with favourable delivery characteristics.Wish to load nanoparticle with the therapeutic agent of a large amount, and use the Nanoparticulate formulations of too high drug load (drug load) excessive nanoparticle for actual therapeutic application will to be produced.

It is accordingly, there are to nanoparticle therapeutic agent and the demand for preparing the nanoparticle method, it can deliver the medicine for the treatment of level to treat diseases such as cancer, while also reducing the side effect of patient.

The content of the invention

On the one hand, the present invention provides therapeutic nanoparticle, and the therapeutic nanoparticle includes activating agent or therapeutic agent, for example taxane and one, two or three kind of bio-compatible polymer.For example, therapeutic nanoparticle is disclosed herein, the therapeutic nanoparticle includes the therapeutic agent of the percentage by weights of about 0.2- about 35；Polylactic acid-block-the ethylene glycol copolymer or PLA of the percentage by weights of about 10- about 99-co-poly glycolic-block-ethylene glycol copolymer；The PLA or PLA-co-poly glycolic of the percentage by weights of about 0- about 50.Exemplary treatment agent includes anti-tumor agents, such as such as taxane, docetaxel and the therapeutic agent that can include the percentage by weights of about 10- about 30, for example, taxane medicament.

The Hydrodynamic diameter of disclosed nanoparticle can be, for example, about 60- about 120nm or about 70- about 120nm.

Exemplary treatment nanoparticle can include the PLA-PEG copolymer of the percentage by weights of about 40- about 90 or the PLA-PEG copolymer of the percentage by weights of about 40- about 80.This polylactic acid-block-ethylene glycol copolymer can include having about 15-20kDa (or e.g., from about 15- about 100kDa, e.g., from about 15- about 80kDa) number-average molecular weight PLA, with with about 2- about 10kDa, for example, the polyethylene glycol of about 4- about 6kDa number-average molecular weight.For example, disclosed therapeutic nanoparticle can comprising the percentage by weights of about 70- about 90 the percentage by weight of PLA-PEG and about 15- about 25 docetaxel or PLA-PEG, the PLA or PLGA of the percentage by weights of about 30- about 50 of the percentage by weights of about 30- about 50, and the percentage by weights of about 15- about 25 Docetaxel (doxetaxel).This PLA (PLA) can have about 5- about 10kDa number-average molecular weight.This PLGA (polylactic-co-glycolic acid (poly (lacticacid-co-glycolic acid))) can have about 8- about 12kDa number-average molecular weight.

Disclosed therapeutic nanoparticle can be stable (for example substantially retaining most of activating agents), keep at least 5 days, for example, can be kept in vitro (such as in sucrose solution) stable more than 5 days at 25 DEG C.In another embodiment, when disclosed particulate is placed in room temperature or 37 DEG C of phosphate buffer solution, it substantially can discharge immediately below about 2% below about 5% or even below about 10% therapeutic agent.In one embodiment, disclosed nanoparticle can retain size and/or molecular weight more than one week or one month or longer.

In some embodiments, disclosed nanoparticle further can include the percentage by weights of about 0.2- about 10 PLA-PEG (it is targeted ligand functionalized) and/or PLA-co-poly glycolic-block-PEG of the percentage by weights of about 0.2- about 10 can be included (it is targeted ligand functionalized).In some embodiments, this targeting ligand can be covalently bond to PEG, for example, being bound to PEG, such as PLA-PEG- alkylidenes-GL2 via alkylidene connexon (alkylene linker).For example, disclosed nanoparticle can include the PLA-PEG-GL2 or PLA-co-poly glycolic-PEG-GL2 of the molar percentages of about 0.2- about 10.It should be understood that being related to following part with reference to PLA-PEG-GL2 or PLGA-PEG-GL2：Alkylidene connexon (such as C that PEG is connected to GL2 can be included₁-C₂₀, for example, (CH₂)₅).For example, disclosed nanoparticle can be selected from following polymer compounds：

Wherein R₁Selected from group H, and the C being optionally optionally substituted by halogen₁-C₂₀Alkyl group；

R₂It is key, ester linker (linkage) or acid amides linker；

R₃It is C₁-C₁₀Alkylidene or key；

X is 50- about 1500, e.g., from about 170- about 260；

Y is 0- about 50, and such as y is 0；With

Z is about 30- about 456 or about 30- about 200, for example, z is about 80- about 130.

In one embodiment, therapeutic nanoparticle can include the therapeutic agent of the percentage by weights of about 0.2- about 35；The PLA-PEG copolymer or PLGA-PEG of the percentage by weights of about 30- about 99；The PLA or PLA-co-poly glycolic of the percentage by weights of about 0- about 50；The PLA-PEG-GL2 or PLA of the percentage by weights of about 0.2- about 10 or the percentage by weights of about 0.2- about 30-co-poly glycolic-PEG-GL2.For example, PLA-PEG-GL2 can include the PLA of the number-average molecular weight with about 10,000Da- about 20,000Da and with about 4,000- about 8, the polyethylene glycol of 000 number-average molecular weight.

Composition, the composition such as comprising multiple disclosed nanoparticles and pharmaceutically acceptable excipient are provided.In some embodiments, this composition can contain the palladium below about 10ppm.

Exemplary composition can include multiple polymer nano particles, the PLA-PEG copolymer or PLGA-PEG of taxane medicament and the percentage by weights of about 10- about 99 respectively comprising the percentage by weights of about 0.2- about 35；With pharmaceutically acceptable excipient, such as sucrose.Nanoparticle preparaton is also provided herein, the nanoparticle preparaton is included：Multiple disclosed nanoparticle, sucrose and water；Wherein for example, the weight rate of the nanoparticle/sucrose/water is about 5-10%/10-35%/60-90% (w/w/w) or about 4-10%/10-30%/60-90% (w/w/w).

Treating cancer, the method for such as prostate cancer is also provided herein, including gives the therapeutic nanoparticle of patient in need's effective dose, the therapeutic nanoparticle includes the antineoplastic such as Docetaxel of the percentage by weights of about 0.2- about 35；The PLA-PEG copolymer or PLGA-PEG of the percentage by weights of about 30- about 90；Optionally, the PLA or PLA-co-poly glycolic of the percentage by weights of about 5- about 20；With, optionally, the PLA-PEG-GL2 or PLA-co-poly glycolic-PEG-GL2 of the percentage by weights of about 0.2- about 30 (for example, the percentage by weights of about 0.2- about 20 or the percentage by weights of about 0.2- about 10).

Brief description of the drawings

The diagram of one embodiment of the disclosed nanoparticle of Fig. 1 descriptions.

The scheme of the exemplary synthesis of the disclosed nanoparticle of Fig. 2 descriptions.

Fig. 3 is the flow chart for the emulsion process to form disclosed nanoparticle.

Fig. 4 is the flow chart of disclosed emulsion process.

Fig. 5 describes influence of thick emulsion (coarse emulsion) preparation to the particle size of quenching.Using standard aqueous phase (1% sodium taurocholate, 2% phenmethylol, 4% ethyl acetate) W: O be 5: 1 emulsification, use the placebo organic matter (placebo organic) of 30% solid.

Fig. 6 describes influence of the feed pressure (feed pressure) to the particle size of gained.

Fig. 7 describes particle size depending on scale (scale).

Fig. 8 describes influence of the solid concentration to particle size.

Fig. 9 descriptions carry the influence of the solid concentration of medicine.

Figure 10 describes the influence that homopolymer PLA and PLGA-PEG or PLA-PEG loads (loading) to DTXL (docetaxel).

Influence of Figure 11 descriptions as a part of homopolymer PLA of nanoparticle to the drug release rate of nanoparticle.

Figure 12 describes influence of the cetanol to the initial rate of the insoluble drug release of nanoparticle.

Figure 13 descriptions are relative to conventional docetaxel, and docetaxel is from the release in vitro in disclosed nanoparticle.

Figure 14 describes the influence of solid concentration and PLA homopolymer to the loading percentage of sirolimus (rapamycin).

Figure 15 describes the sirolimus of disclosed nanoparticle with the release in vitro of time.

Figure 16 describes influence of the PLA homopolymer to the loading percentage of CCI-779.

Figure 17 describes influence of the solid concentration to the particle size of the particulate containing CCI-779.

Figure 18 describes the CCI-779 of disclosed nanoparticle with the release in vitro of time.

Figure 19 describes the extracorporeal releasing characteristic of the nanoparticle (including vinorelbine) of illustrative disclosure.

The extracorporeal releasing characteristic of the disclosed nanoparticle of Figure 20 descriptions (including vincristine or docetaxel).

Figure 21 describes the pharmacokinetics of vincristine and vincristine PTNP in rats.

Figure 22 describes the mean tumour volume after giving disclosed nanoparticle (including docetaxel) in the MX-1 transplanting mouse models of breast cancer.

Figure 23 descriptions are after vein gives disclosed nanoparticle (including docetaxel) 24 hours, the docetaxel concentration in the mouse tumor of the MX-1 xenograft mouse models of breast cancer.

Figure 24 is after giving and being inoculated with the mouse of mankind's LNCaP prostate gland cancer cells, the tumor of prostate distribution of the disclosed nanoparticle with docetaxel.

Figure 25 is shown in give the disclosed nanoparticle containing docetaxel after, the Tumor growth inhibition in the mouse for being inoculated with mankind's LNCaP prostate gland cancer cells.

Detailed description of the invention

This invention relates generally to polymer nano particle, comprising activating agent or therapeutic agent or medicine, and its method for preparing and using this therapeutic nanoparticle.In general, " nanoparticle " refers to less than 1000nm, any particulate of e.g., from about 10nm- about 200nm diameter.Disclosed therapeutic nanoparticle can include the nanoparticle of the diameter with about 60- about 120nm or about 70- about 130nm or about 60- about 140nm.

Disclosed nanoparticle can include the activating agent of the percentage by weights of about 0.2- about 35, the percentage by weights of about 3- about 40, the percentage by weights of about 5- about 30, the percentage by weights of 10- about 30,15-25 percentage by weights or the percentage by weights of even about 4- about 25, such as antineoplastic, such as taxane medicament (such as docetaxel).

Nanoparticle disclosed herein include one, two, three or more plant bio-compatibles and/or Biodegradable polymeric.For example, it is contemplated that nanoparticle can be comprising the percentage by weights of about 10- about 99 one or more block copolymerization-polymer (comprising Biodegradable polymeric and polyethylene glycol), and the percentage by weights of about 0- about 50 biodegradable homopolymer.

In one embodiment, disclosed therapeutic nanoparticle, which may be embodied in, effectively treats disease or obstacle, the targeting ligand of such as prostate cancer, for example, low-molecular weight PSMA parts in patient in need.In some embodiments, low-the molecular weight ligands are conjugated to polymer, and part-conjugated polymer (conjugated polymer) (for example, PLA-PEG- part) of the nanoparticle comprising some ratios (ratio) and non-functionalized polymer (such as PLA-PEG or PLGA-PEG).Nanoparticle can have both polymer of Optimal Ratio so that the part of effective dose is associated with the nanoparticle for the treatment of disease or obstacle.Combined (cell combination/target intake) for example, increased ligand density can increase target so that nanoparticle " target specificity ".Alternatively, some of nanoparticle concentration non-functionalized polymer (for example, non-functionalized PLGA-PEG copolymers) can control inflammation and/or immunogenicity (i.e., cause the ability of immune response/response), with permission nanoparticle there is circulating half-life (to be applied to treatment disease or obstacle (for example, prostate cancer).In addition, in some embodiments, non-functionalized polymer can be via reticuloendothelial system (RES) reduction from the clearance rate in the circulatory system.So as to which non-functionalized polymer can provide the nanoparticle with the feature that particulate can be allowed to be advanced after administration by body.In some embodiments, non-functionalized polymer can balance the part of other high concentration, and the part otherwise can accelerate to remove by subject, and then cause the lower delivering to target cell.

For example, the nanoparticle of the conjugated functionalized polymeric to part can be included by being disclosed herein, the part constitutes about 0.1-50, for example, 0.1-30, for example, 0.1-20, for example, the nanoparticle (that is, functionalization+non-functionalized polymer) of the whole polymer composition of 0.1-10 molar percentages.In further embodiment, also disclosed herein is comprising with one or more it is low-molecular weight ligands are conjugated (for example, the covalently nanoparticle of the polymer (i.e. by connexon (such as alkylidene connexon) or key), wherein low-molecular weight ligands are for total polymer, its percentage by weight is about 0.001-5, for example, about 0.001-2, for example, about 0.001-1.

The polymer nano particle of the activating agent comprising about 2 about 20 percentage by weights is also provided herein.For example, the composition comprising this nanoparticle can deliver effective dose to the target drone body region (body area) of such as patient.

For example, disclosed nanoparticle be able to can be effectively bonded to biology body (biological entity) or otherwise with biology body association (associate), the biology body is for example, specific membrane component or cell surface receptor.The targeting (for example, targeted to specific tissue or cell type, targeted to specific diseased tissue but and non-normal tissue etc.) of therapeutic agent is wished, for treating tissue specific disease such as solid tumor cancer (such as prostate cancer).For example, compared with the systemic delivery of cell toxicant anticancer agent, nanoparticle disclosed herein can substantially prevent medicament to kill healthy cell.Extraly; disclosed nanoparticle can be in order to the administration (compared with not containing the effective dose medicament given of disclosed nanoparticle or preparaton) of the medicament of relatively low-dose, and the disclosed nanoparticle can reduce undesirable side effect typically relevant with traditional chemotherapy.

Polymer

In some embodiments, the matrix of nanoparticle of the invention comprising polymer and therapeutic agent.In some embodiments, therapeutic agent and/or targeting moiety (that is, low-molecular weight PSMA parts) can associate with least part polymer substrate.For example, in some embodiments, the surface association that targeting moiety (such as part) can covalently with polymer substrate.In some embodiments, non-covalent association (covalent association) is mediated by connexon.Therapeutic agent can with the surface association of polymer substrate, encapsulate in the polymer matrix, surrounded and/or scattered throughout polymer substrate by polymer substrate.

Multiple polymers and be known for forming the method for particulate therefrom in drug delivery field.In some embodiments, disclosure is related to at least two high molecular nanoparticles, wherein the first described macromolecule includes the first polymer for being bound to low-molecular weight ligands (such as targeting moiety)；The second polymer for not being bound to targeting moiety is included with second of macromolecule.Nanoparticle can be optionally comprising one or more extra non-functionalized polymer.

Any polymer can be used according to the present invention.Polymer can be natural or non-natural (synthesis) polymer.Polymer can be homopolymer or copolymer, and it includes two or more monomers.For sequence, copolymer can be random sequence, block sequence or comprising random and block sequence combination.Usually, it is organic polymer according to Inventive polymers.

Terms used herein " polymer ", is provided, i.e. include the molecular structure of the one or more repeat units (monomer) connected by covalent bond with the usual implication that this area is used.The repeat unit can all be equal or can exist in the polymer more than a type of repeat unit in some cases.In some cases, polymer can be biologically derivative, i.e. biopolymer.Non- limitation example includes peptide or protein matter.In some cases, extra part can also be present in polymer, and for example biological part retinal diseases are such as following in those.If it exceeds a type of repeat unit is present in polymer, then the polymer is " copolymer ".It should be appreciated that in any embodiment using polymer, the polymer used in some cases can be copolymer.The repeat unit for forming copolymer can be with any pattern.For example, repeat unit with random order, using any alternate sequential arrangement or can be used as block copolymer, i.e., comprising one or more regions (respectively comprising the first repeat unit (for example, first block), with one or more regions (respectively comprising the second repeat unit (for example, second block)) etc..Block copolymer can have the different blocks of two (diblock copolymers), three (triblock copolymers) or more quantity.

Disclosed particulate can include copolymer, in some embodiments, describe generally by two or more polymer together covalent bond two or more polymer (all as described herein those) so that having associated each other.So as to, copolymer can include first polymer and second polymer, they are conjugated to form block copolymer together, wherein it can be the second block of the block copolymer that the first polymer, which can be the first block and the second polymer of the block copolymer,.Certainly, those skilled in the art will be understood that, in some cases, block copolymer can contain the polymer of a variety of blocks, and " block copolymer " used herein is not limited to unique block copolymer only with single first block and single second block.For example, block copolymer can include the first block containing first polymer, the second block containing second polymer and three block containing third polymer or first polymer etc..In some cases, block copolymer can the first block containing any number of first polymer and second polymer the second block (and in some cases, three block, the 4th block etc.).Further it is to be noted that ground is, block copolymer can also be formed from other block copolymers in some cases.For example, first block copolymer can (it can be homopolymer, biopolymer with conjugated to other polymer, other block copolymers etc.), to form the new block copolymer containing polytype block, and/or it is conjugated to other parts (for example, conjugated to non-polymer part).

In some embodiments, polymer (for example, copolymer, for example, block copolymer) can be amphiphilic, i.e. with hydrophilic segment and hydrophobic part or relative hydropathic part and relative hydrophobic part.Hydrophilic polymer can be that the polymer and hydrophobic polymer of usually attraction water can be the polymer for usually repelling water.Hydrophilic or hydrophobic polymer can be identified, for example, sample and measurement its contact angle with water by preparing polymer (usually, polymer is by the contact angle with less than 60 °, and hydrophobic polymer is by with greater than about 60 ° of contact angle).In some cases, the hydrophily of two or more polymer can be measured relative to each other, i.e. first polymer can be more more hydrophilic than second polymer.For example, first polymer can have the contact angle smaller than second polymer.

In one group of embodiment, the polymer (for example, copolymer, for example, block copolymer) considered herein includes the polymer of bio-compatible, i.e. this polymer：When it is inserted into or injected into subject living, do not induce unfavorable response typically, for example, do not cause significant inflammation and/or polymer by the acute rejection of immune system (for example, via T- cell responses).Correspondingly, the therapeutic particulate considered herein can be non-immunogenicity.The finger Endogenous Growth Factors of terms used herein non-immunogenicity are in its inmature (naive) state, it does not produce or only produced the circulating antibody of floor level, T- cells or reaction immunocyte generally, and is generally produced not in individual for the immune response of its own.

Biocompatibility usually refers to material by the acute rejection of at least a portion immune system, i.e., the immune response in the subject caused by the material implantation subject of biocompatible, the immune response can seriously cause material to a certain degree the material must to be removed from the subject from fully being controlled, and often up to by the rejection of immune system enough.The simple experiment of one measure biocompatibility can make to be exposed to cell outside polymeric acceptor；The polymer of bio-compatible is this polymer：It is in intermediate concentration, for example, in 50 microgram/10⁶The concentration of cell usually will not cause notable cell death.For example, when the polymer of bio-compatible is exposed to cell such as fibroblast or epithelial cell, or even if when being swallowed or absorbed in another manner by the cell, it can cause the cell death below about 20%.Can the non-limitation example of the polymer of useful bio-compatible in the various embodiments of the present invention include poly- twoAlkanone (PDO), polyhydroxyalkanoate, poly butyric ester, poly- (decanedioic acid glyceride) (poly (glycerol sebacate)), polyglycolide, polylactide (polylactide), PLGA, polycaprolactone or copolymer or derivative comprising these and/or other polymer.

In some embodiments, it is considered to the polymer of bio-compatible can be biodegradable, i.e. the polymer can degrade in chemistry and/or biologically in physiological environment (such as internal)." biodegradable " polymer used herein is those, when being introduced into cell, by molecular machinery (biologically degradable) and/or pass through chemical process, such as the component that cell can be recycled or removed is resolved into hydrolysis (chemically degradable), and does not have notable toxic action to cell.In one embodiment, Biodegradable polymeric and their degradation by-products can be with bio-compatibles.

For example, it is contemplated that polymer can be when exposed to water (for example, in subject) while the polymer of hydrolysis, the polymer can degrade when exposed to hot (for example, in about 37 DEG C temperature).The degraded of polymer can occur with the speed of change, depending on the polymer or copolymer used.For example, the half-life period (wherein 50% polymer can be degraded into the time of monomer and/or other non-polymeric portions) of polymer may be about a couple of days, several weeks, several months or several years, depending on polymer.Polymer can be biologically degradable, for example, by enzymatic activity or molecular machinery, in some cases, for example, by exposed to lysozyme (for example, with relatively low pH).In some cases, polymer can resolve into other non-polymers that monomer and/or cell can be recycled or removed, and there is no notable toxic action to cell (for example, polylactide can hydrolyze to form lactic acid, polyglycolide can hydrolyze to form glycolic etc.).

In some embodiments, polymer can be polyesters, including the copolymer containing lactic acid and glycolic acid units, such as polylactic-co-glycolic acid and poly- third hands over fat-co-glycolide (poly (lactide-co-glycolide)), and " PLGA " is referred to as jointly herein；With the homopolymer comprising glycolic acid units, at referred to herein as " PGA ", and lactic acid units, such as Poly-L-lactide, poly- D-ALPHA-Hydroxypropionic acid, poly- D, Pfansteihl, PLLA (lactide), poly- D- lactides, with poly- D, L- lactides are referred to as " PLA " jointly herein.In some embodiments, Exemplary polyesters class includes, for example, polyhydroxy acids；The polymer and lactide of PEGylation and the copolymer of glycolide are (for example, the PLGA and its derivative of the PGA of the PLA of PEGylation, PEGylation, PEGylation.In some embodiments, polyesters include, for example, polyanhydrides, poly- (ortho esters) of poly- (ortho esters) PEGylation, poly- (caprolactone), poly- (caprolactone) of PEGylation, polylysine, the polylysine of PEGylation, poly- (aziridine), poly- (aziridine) of PEGylation, poly- (the poly- 1B of L- lactide-cos) (poly (L-lactide-co-L-lysine)), poly- (serine ester), poly- (CHP ester), poly- [α-(4- aminobutyls)-L- glycolics], and its derivative.

In some embodiments, polymer can be PLGA.PLGA is the co-polymer of bio-compatible and biodegradable lactic acid and glycolic, and various forms of PLGA feature can be lactic acid: the ratio of glycolic.Lactic acid can be Pfansteihl, D-ALPHA-Hydroxypropionic acid or D, Pfansteihl.PLGA degradation rate can be adjusted by changing lactic acid-ethanol ratio.In some embodiments, lactic acid can be according to the present invention PLGA to be used feature: the ratio of glycolic is about 85: 15, about 75: 25, about 60: 40, about 50: 50, about 40: 60, about 25: 75 or about 15: 85.In some embodiments, the ratio of lactic acid and glycolic acid monomers in the polymer (for example, PLGA block copolymers or PLGA-PEG block copolymers) of particulate can be selected to various parameters optimization (such as release of water intake, healing potion and/or depolymerization dynamics can be optimized).

In some embodiments, polymer can be one or more acrylate copolymers.In some embodiments, acrylate copolymer includes, for example, acrylic acid and methacrylic acid copolymer, methylmethacrylate copolymer, ethoxyethyl methacrylates, cynaoethyl methacrylate (cyanoethyl methacrylate), amino alkyl methacrylate copolymer, poly- (acrylic acid), poly- (methacrylic acid), methacrylic acid alkylamide copolymer, poly- (methyl methacrylate), poly- (methacrylic acid polyacrylamide, amino alkyl methacrylate copolymer, glycidyl methacrylate copolymer, polycyanoacrylate and the combination for including one or more aforementioned polymers.Acrylate copolymer can include the copolymer of complete-polymerization of acrylic acid and methacrylate (quaternary ammonium group containing low content).

In some embodiments, polymer can be cationic polymer.In general, cationic polymer can be condensed and/or protect nucleic acid (such as DNA, RNA or derivatives thereof) electronegative chain.In some embodiments, consider in disclosed particulate using the polymer containing amine such as poly- (lysine), polyethyleneimine (PEI) and poly- (acyl ammonia amine) dendrimers (dendrimer).

In some embodiments, polymer can be the degradable polyesters for carrying cationic side chain.The example of these polyesters includes poly- (the poly- 1B of L- lactide-cos), poly- (serine ester), poly- (CHP ester).

Particulate disclosed herein can contain or can not contain PEG.In addition, some embodiments can be related to the copolymer containing poly- (ester-ether), for example, having by ester bond (for example, R-C (O)-O-R ' keys) and ehter bond (for example, R-O-R ' keys) connection repeat unit polymer.In certain embodiments of the present invention, Biodegradable polymeric, such as hydrolyzable polymer (containing hydroxy-acid group) can be conjugated to be formed poly- (ester-ether) with PEG repeat unit.Polymer (for example, copolymer, for example, block copolymer) containing PEG repeat unit can also be referred to as " PEGylation " polymer.

It can be end-blocking (terminated) and comprising end group to consider PEG, for example, when PEG do not have it is conjugated to part when.For example, PEG can terminate hydroxyl, methoxyl group or other alkoxy bases, methyl or other alkyl groups, aromatic yl group, carboxylic acid, amine, acid amides, Acetyl Groups, guanidino group or imidazoles.The end group of other considerations includes azide, alkynes, maleimide, aldehyde, hydrazides, azanol, alkoxyamine or thiol moiety.

Those skilled in the art will know methods and techniques for PEGylation polymer, for example, by using EDC (1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochloride) and NHS (n-hydroxysuccinimide) so that the PEG group of polymer to termination amine is reacted, pass through ring-opening polymerisation technology (ROMP) etc..

In one embodiment, can optimize the molecular weight of polymer is used for effective treatment disclosed herein.For example, the molecular weight of polymer can influence microparticle degradation speed (such as when the molecular weight of Biodegradable polymeric can be conditioned), solubility, water intake and drug release kinetics.For example, the molecular weight of polymer can be adjusted to, particulate is biodegradable within the rational period (week, 3-4 weeks, 5-6 weeks, 7-8 weeks etc. from a few houres to 1-2) in treated subject.Disclosed particulate can be with the diblock copolymer for example comprising PEG and PL (G) A, wherein for example, peg moiety can have about 1,000-20,000, for example, about 2,000-20,000, for example, about 2- about 10,000 number-average molecular weight, and PL (G) part A can have about 5,000- about 20,000 or about 5,000-100,000, for example, about 20,000-70,000, for example, about 15,000-50,000 number-average molecular weight.

For example, it disclosed herein is exemplary treatment nanoparticle, the therapeutic nanoparticle includes the PLA-PEG copolymer or PLGA-PEG of the percentage by weights of about 10- about 99, or the percentage by weights of about 20- about 80, the percentage by weights of about 40- about 80 or the percentage by weights of about 30- about 50 or the percentage by weights of about 70- about 90 PLA-PEG copolymer or PLGA-PEG.Exemplary PLA-PEG copolymer can include the polyethylene glycol of the PLA of about 15- about 20kDa or about 10- about 25kDa number-average molecular weight and about 4- about 6 or about 2kDa- about 10kDa number-average molecular weight.

Disclosed nanoparticle can the optionally PLA comprising the percentage by weights of about 1- about 50 or PLA-co-poly glycolic (not including PEG), or can optionally include the PLA or PLA-co-poly glycolic of the percentage by weights of about 1- about 50 or the percentage by weights of about 10- about 50 or the percentage by weights of about 30- about 50.For example, PLA or PLA-co-poly glycolic can have about 5- about 15kDa or about 5- about 12kDa number-average molecular weight.Exemplary PLA can have about 5- about 10kDa number-average molecular weight.Exemplary PLGA can have about 8- about 12kDa number-average molecular weight.

In some embodiments, the polymer of nanoparticle can be with conjugated to lipid (lipid).For example, polymer can be the PEG of lipid-end-blocking.Described below, the lipid moieties of polymer can be used for and other polymer self-assembly so that be easy to form nanoparticle.For example, hydrophilic polymer can be conjugated to lipid, the lipid will be with hydrophobic polymer self-assembly.

In some embodiments, lipid is oil.In general, any oil known in the art can be with conjugated to the of the invention polymer used.In some embodiments, oil can include one or more fatty acid groups or its salt.In some embodiments, fatty acid group can be comprising digestible, long-chain (for example, C₈-C₅₀), substitution or the hydrocarbon that is unsubstituted.In some embodiments, fatty acid group can be C₁₀-C₂₀Aliphatic acid or its salt.In some embodiments, fatty acid group can be C₁₅-C₂₀Aliphatic acid or its salt.In some embodiments, aliphatic acid can be undersaturated.In some embodiments, fatty acid group can be monounsaturated.In some embodiments, fatty acid group can be how unsaturated.In some embodiments, the double bond of unsaturated fat acid groups can be cis-configuration.In some embodiments, the double bond of unsaturated fat acid groups can be anti-configuration.

In some embodiments, fatty acid group can be the one or more in butyric acid, caproic acid, octanoic acid, capric acid, laurate, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acids or lignoceric acid.In some embodiments, fatty acid group can be the one or more in palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linolenic acid, gamma-linoleic acid, arachidonic acid, gadoleic acid, argininosuccinic acid, EPA, docosahexaenoic acid or erucic acid.

In a specific embodiment, lipid is Formula V：

And its salt, wherein each R is independently C_1-30Alkyl.In an embodiment of Formula V, lipid is 1,2 distearyl-sn- glycerol-3-phosphates monoethanolamines (DSPE), and its salt, for example, sodium salt.

In one embodiment, optional small molecule targeting moiety bonding, for example, covalently bonded to the lipidic component of nanoparticle.For example, it provided herein is nanoparticle, the nanoparticle includes therapeutic agent, polymer substrate (including functionalization and non-functionalized polymer) and lipid and low-molecular weight PSMA targeting ligands, wherein described targeting ligand bonding, for example, covalently bonded to the lipidic component of nanoparticle.In one embodiment, the lipidic component for being bonded to low-molecular weight targeting moiety is Formula V.In another embodiment, the present invention provides targeting-specific nanoparticle, and the nanoparticle includes therapeutic agent, polymer substrate, DSPE and low-molecular weight PSMA targeting ligands, wherein the part is bonding, for example, covalently bonded to DSPE.For example, the nanoparticle of the present invention can include the polymer substrate containing PLGA-DSPE-PEG- parts.

The nanoparticle of consideration can include the part-conjugated polymer and non-functionalized polymer of effective treatment prostate cancer of certain ratio, wherein hydrophilic, part-conjugated polymer is conjugated extremely by the lipid with hydrophobic polymer self-assembly so that hydrophobic and hydrophilic polymer constitutes the nanoparticle of Non-covalent binding." self-assembly " refers to higher order structure (higher order structure) self-assembly process, and this naturally attracts each other dependent on the component (for example, molecule) of higher order structure.Typically, it is based on size, shape, composition or chemical characteristic, occurs by the random motion of molecule and the formation of key.For example, this method includes providing first polymer, the first polymer is reacted with lipid, to form polymer/lipid conjugates.Then polymer/the lipid conjugates react to prepare polymer/lipid conjugates of part-combination with low-molecular weight ligands；Mixed with by polymer/lipid conjugates of the part-combination and second, non-functionalized polymer, and therapeutic agent；So as to form nanoparticle.In some embodiments, the first polymer is PEG, so as to form the PEG of lipid-end-blocking.In one embodiment, the lipid of Formula V is, for example, 2 distearyl-sn- glycerol-3-phosphates monoethanolamines (DSPE) and its salt, for example, sodium salt.Then, the PEG of the lipid-end-blocking can be with for example, mix to form nanoparticle with PLGA.

Targeting moiety

It provided herein is nanoparticle, the nanoparticle can include arbitrary targeting moiety, i.e., biology body or the part otherwise associated with biology body can be bound to, the biology body is for example, membrane component, cell surface receptor, PSMA or the like.The targeting moiety existed in microparticle surfaces can allow particulate to be adapted to be confined to specific target site, for example, tumour, affected part, tissue, organ, type of cell etc..So, then the nanoparticle can be " target specificity ".In some cases, then medicine or other Payloads (payload) can discharge from particulate and be allowed to and specific target site local interaction.

In one embodiment, disclosed nanoparticle includes targeting moiety, and the targeting moiety is low-molecular weight ligands, for example, low-molecular weight PSMA parts.Terms used herein " with reference to " refers to the interaction between a pair corresponding molecules or part thereof, mutual affinity or binding ability is shown between described molecule or part thereof, usually due to the combination of specific or non-specific or interaction, including but not limited to biochemistry, physiology and/or chemical interaction." biology combination " defines the type of interaction, and the interaction occurs in molecule (including protein, nucleic acid, glycoprotein, carbohydrate, hormone or the like) between.Term " binding partners (binding partner) ", which refers to, can undergo the molecule combined with specific molecular." specific binding " refers to be bound to or recognized the molecule (such as polynucleotide) of binding partners (or binding partners of limited quantity), and it is to combine or recognize relative to other, similar biological body substantially higher degree.In one group of embodiment, the targeting moiety, which has, is below about 1 micromole, at least about 10 micromoles or at least about 100 micromolar affinity (being measured via dissociation constant).

For example, targeting moiety can cause the tumour (such as solid tumor) that particulate is adapted to be confined in subject's body, affected part, tissue, organ, cell type, this depends on the targeting moiety used.For example, low-molecular weight PSMA parts can be adapted to be confined to solid tumor, such as mammary gland or tumor of prostate or cancer cell.The subject can be the mankind or non-human animal.The example of subject includes but is not limited to mammal such as dog, cat, horse, donkey, rabbit, cow, pig, sheep, goat, rat, mouse, cavy, hamster, primate, the mankind, or the like.

Consider that targeting moiety includes small molecule.In some embodiments, term " small molecule " refers to regardless of whether being that naturally occurring or artificially generated (for example, via chemical synthesis) has relative low molecule amount and be not the organic compound of protein, polypeptide or nucleic acid.Small molecule generally has carbon-to-carbon multikey.In some embodiments, the size of small molecule is less than about 2000g/Mol.In some embodiments, small molecule is below about 1500g/mol or below about 1000g/mol.

In some embodiments, small molecule is below about 800g/mol or below about 500g/mol, e.g., from about 100g/mol- about 600g/mol or about 200g/mol- about 500g/Mol.

For example, targeting moiety can be with small targeting (small target) prostate cancer, such as targeting moiety can be PSMA peptidase inhibitorses.These parts are also referred to herein as " low-molecular weight PSMA parts ".When compared with the expression in normal structure, PSMA (PSMA) is overexpressed at least 10- times in the expression of malignant prostate relative to expression in the normal tissue, (Silver et al. 1997 is dialled further up with the level that transfer phase PSMA expression is developed to disease, Clin.cancer Res., 3：81).

In some embodiments, the low-molecular weight PSMA parts are Formulas I, II, III or IV：

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification；

Wherein m and n are 0,1,2 or 3 independently of one another；P is 0 or 1；

R¹、R²、R⁴And R⁵Substituted or unsubstituted alkyl is each independently selected from (for example, C_1-10- alkyl, C_1-6- alkyl or C_1-4- alkyl), substituted or unsubstituted aryl (for example, phenyl or pyridine radicals (pyrdinyl)), and its arbitrary combination；And R³It is H or C_1-6- alkyl is (for example, CH₃)。

For Formulas I, II, III and IV compound, R¹、R²、R⁴Or R⁵Comprising the point of nanoparticle is connected to, for example, being connected to the point of polymer (part of disclosed nanoparticle being formed, for example, PEG).The point of connection can be formed by following keys：Covalent bond, ionic bond, hydrogen bond, by adsorbing the key of (including chemisorbed and physical absorption) formation, by Van der Waals key or the key of dispersion force formation.If for example, R¹、R²、R⁴Or R⁵It is defined as aniline or C_1-6- alkyl-NH₂Group, any hydrogen (for example, amino hydrogen) of these functional groups can be removed so that low-molecular weight PSMA parts are covalently bond to the polymer substrate (for example, PEG- blocks of polymer substrate) of nanoparticle.Terms used herein " covalent bond " refers to the key formed between two atoms by least one pair of shared electron institute.

In Formulas I, II, III or IV specific embodiment, R¹、R²、R⁴And R⁵It is C independently of one another_1-6- alkyl or phenyl or C_1-6Any combination of-alkyl or phenyl, it is independently by OH, SH, NH₂Or CO₂H replaces one or more times, and wherein described alkyl group can be separated by N (H), S or O.In another embodiment, R¹、R²、R⁴And R⁵It is CH independently of one another₂-Ph、(CH₂)₂-SH、CH₂-SH、(CH₂)₂C(H)(NH₂)CO₂H、CH₂C(H)(NH₂)CO₂H、CH(NH₂)CH₂CO₂H、(CH₂)₂C(H)(SH)CO₂H、CH₂-N(H)-Ph、O-CH₂- Ph or O- (CH₂)₂- Ph, wherein each Ph can be independently by OH, NH₂、CO₂H or SH substitutions are one or more times.For these formulas, the NH₂, OH or SH groups serve as the point for being covalently attached to nanoparticle (for example ,-N (H)-PEG ,-O-PEG or-S-PEG).

In yet another embodiment, the low-molecular weight PSMA parts are selected from

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification, and wherein described NH₂, OH or SH groups serve as the point for being covalently attached to nanoparticle (for example ,-N (H)-PEG ,-O-PEG or-S-PEG).

In further embodiment, the low-molecular weight PSMA parts are selected from

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification, wherein R is independently selected from NH₂、SH、OH、CO₂H, by NH₂, SH, OH or CO₂The C of H substitutions_1-6- alkyl, and by NH₂, SH, OH or CO₂The phenyl of H substitutions, and wherein R serves as and is covalently attached to nanoparticle (for example ,-N (H)-PEG ,-S-PEG ,-O-PEG or CO₂- PEG) point.

In further embodiment, the low-molecular weight PSMA parts are selected from

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification, wherein the NH₂Or CO₂H groups, which are served as, is covalently attached to nanoparticle (for example ,-N (H)-PEG or CO₂- PEG) point.These compounds further can be replaced by following radicals：NH₂、SH、OH、CO₂H, by NH₂, SH, OH or CO₂The C of H substitutions_1-6- alkyl or by NH₂, SH, OH or CO₂The phenyl of H substitutions, wherein these functional groups can also act as being covalently attached to the point of nanoparticle.

In further embodiment, the low-molecular weight PSMA parts are

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification, wherein n is 1,2,3,4,5 or 6.For the part, NH₂Group serves as the point for being covalently attached to nanoparticle (for example ,-N (H)-PEG).

In yet another embodiment, the low-molecular weight PSMA parts are

And its enantiomter, stereoisomer, rotational isomer, dynamic isomer, diastereoisomer or racemic modification.Especially, butylamine compound has the advantage being readily synthesized, especially because it lacks phenyl ring.In addition, it is undesirable to be bound by theory, the butylamine compound will likely resolve into naturally occurring molecule (that is, lysine and glutamic acid), thus make xicity related minimum.

In some embodiments, it can be used for targeting and solid tumor (such as, prostate or breast cancer tumour) the small molecule targeting moiety of relevant cell includes PSMA peptidase inhibitorses, such as 2-PMPA, GPI5232, VA-033, phenylalkyl phosphonic amide (phenylalkylphosphonamidate) and/or its analogs and derivatives.In some embodiments, can be used for targetting the small molecule targeting moiety of the cell relevant with prostate cancer includes mercaptan and benzazolylthiol derivative, such as 2-MPPA and 3- (2- mercaptoethyls) -1H- indole-2-carboxylic acid derivatives.In some embodiments, can be used for targetting the small molecule targeting moiety of the cell relevant with prostate cancer includes hydroxamate derivative.In some embodiments, can be used for targetting the small molecule targeting moiety of the cell relevant with prostate cancer comprising based on PBDA- and urea inhibitor (such as ZJ 43, ZJ 11, ZJ 17, ZJ 38 and/or and and the like and derivative), androgen receptor target medicament (ARTAs), polyamine (such as putrescine, spermine and spermidine), also referred to as enzyme glutamic acid carboxylase II (enzyme glutamate carboxylase II) (GCPII) inhibitor, NAAG peptases or NAALAD enzymes.

In the further embodiment of the present invention, the targeting moiety can be the part for targetting Her2, EGFR or toll acceptor.

For example, it is contemplated that the targeting moiety can include nucleic acid, polypeptide, glycoprotein, carbohydrate or lipid.For example, targeting moiety can be the nucleic acid targeting moiety (such as fit, for example, A10 is fit) for being bound to cell-type-specific markers' thing (marker).In general, fit is the oligonucleotides (for example, DNA, RNA or its analog or derivative) for being bound to specific target (such as polypeptide).In some embodiments, targeting moiety can be the naturally occurring or synthesized part of cell surface receptor, for example, growth factor, hormone, LDL, transferrins etc..Targeting moiety can be antibody, and the term intention includes antibody fragment, the part of the feature of antibody, and single-stranded targeting moiety can be with for example, be identified using operation such as phage display.

Targeting moiety can be targeting peptides or targeting peptidomimetic (peptidomimetic), and it has the length of up to about 50 residues.For example, targeting moiety can include amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acid or conservative variant or its peptidomimetic.In specific embodiment, the targeting moiety is to include amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN peptide, and wherein X and Z are variable amino acids, and with less than 20, the length of 50 or 100 residues.The CREKA (Cys Arg Glu Lys Ala) peptides or its peptidomimetic or pungent peptide AXYLZZLN are further contemplated as targeting moiety, and peptide or conservative variant or its peptidomimetic are (incorporating collagen IV or target tissue basement membrane (for example, the target tissue basement membrane of blood vessel) or formed compound), it may be used as targeting moiety.Exemplary targeting moiety includes targeting ICAM (ICAIU, such as ICAM-1) peptide.

Targeting moiety disclosed herein usually conjugated to disclosed polymer or copolymer (such as PLA-PEG), and this polymer conjugate can form the disclosed nanoparticle in part.For example, disclosed therapeutic nanoparticle can optionally include the PLA-PEG or PLGA-PEG of the percentage by weights of about 0.2- about 10, wherein the PEG is targeted part (such as PLA-PEG- parts) functionalization.Consider that therapeutic nanoparticle can include, for example, the PLA-PEG-GL2 or PLA of the molar percentages of about 0.2- about 10-co-poly glycolic-PEG-GL2.For example, PLA-PEG-GL2 can include about 10kDa- about 20kDa number-average molecular weight and about 4,000- about 8,000 number-average molecular weight.

In some embodiments, this targeting ligand can be covalently bond to PEG (for example, being bound to PEG via alkylidene connexon), such as PLA-PEG- alkylidenes-GL2.For example, disclosed nanoparticle can include the PLA-PEG-GL2 or PLA-co-poly glycolic-PEG-GL2 of the molar percentages of about 0.2- about 10.It should be appreciated that referring to alkylidene connexon (such as C that can include that PLA-PEG or PLGA-PEG are connected to GL2 with reference to PLA-PEG-GL2 or PLGA-PEG-GL2₁-C₂₀, for example, (CH₂)₅) part.

Exemplary polymer conjugates includes：

Wherein R₁Selected from H, and by one, two, three or more optionally substituted C of halogen₁-C₂₀Alkyl group；

R₂It is key, ester linker or acid amides linker；

R₃It is C₁-C₁₀Alkylidene or key；

X is 50- about 1500 or about 60- about 1000；

Y is 0- about 50；With

Z is about 30- about 200 or about 50- about 180.

In various embodiments, x represents the molar fractions of 0- about 1；The molar fractions of about 0- about 0.5 can be represented with y.In an exemplary embodiment, x+y can be about 20- about 1720, and/or z can be about 25- about 455.

For example, disclosed nanoparticle can include the polymer target part shown in Formula IV：

Wherein n is about 200- about 300, for example, about 222, and m is about 80- about 130, e.g., from about 114.In some embodiments, disclosed nanoparticle can comprising about 0.1- about 4% (by weight) such as Formula IV polymer conjugate, or about 0.1- about 2% or about 0.1- about 1% or about 0.2%- about 0.8% (by weight) such as Formula IV polymer conjugate.

In an exemplary embodiment, disclosed nanoparticle includes the nanoparticle containing PLA-PEG- alkylidene-GL2 conjugates, wherein, for example, PLA has about 16,000Da number-average molecular weight, PEG has about 5000Da molecular weight, and for example, alkylidene connexon is C₁-C₂₀Alkylidene, such as (CH₂)₅。

For example, disclosed nanoparticle can include the conjugates being expressed from the next：

Wherein y is that about 222 and z is about 114.

Disclosed polymer conjugate can use the conjugated technology of any suitable to be formed.For example, use following two kinds of compound (polymer of such as targeting moiety and bio-compatible of technology, polymer and PEG of bio-compatible etc.) can be conjugated together, such as EDC-NHS chemistry (1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and n-hydroxysuccinimide) involves the reaction of maleimide or carboxylic acid, and it can be with conjugated to mercaptan, amine or similarly one end of the polyethers of functionalization.This polymer it is conjugated (for example, poly- (ester) and poly- (ether) it is conjugated to be formed poly- (ester-ether)) can be carried out in following organic solvents, such as, but not limited to dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, acetone or the like.Conventional experiment is used no more than, those ordinarily skilled in the art can determine specific reaction condition.

In another group of embodiment, conjugated reaction can react to carry out by other parts of the polymer with polymer or comprising amine (such as targeting moiety) comprising carboxylic acid functional (for example, poly- (ester-ether) compound).For example, targeting moiety, such as low-molecular weight PSMA parts can be with amine reaction to form the part containing amine, and then the part containing amine can be with the conjugated carboxylic acid to polymer.This reaction can be used as the reaction appearance of list-step, i.e. described conjugated to be carried out without using intermediate such as n-hydroxysuccinimide or maleimide.In one group of embodiment, by the way that the part containing amine is added in the solution of the polymer containing carboxylic acid-end-blocking, the conjugated reaction between the polymer (such as poly- (ester-ether) compound) of the part containing amine and carboxylic acid-end-blocking can be realized, it is described to be partially dissolved in organic solvent such as (but not limited to) dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridine, two containing amine

Alkane or dimethyl sulfoxide (DMSO).The polymer of the carboxylic acid-end-blocking can be contained in organic solvent such as, but not limited to dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran or acetone.In some cases, the reaction between the polymer of the part containing amine and carboxylic acid-end-blocking can spontaneously occur.Not conjugated reactant can be washed after this reaction, and the polymer can be in solvent such as, for example, being precipitated in diethyl ether, hexane, methanol or ethanol.

As specific example, low-molecular weight PSMA parts can be prepared as targeting moiety in the particle as follows.Carboxyl acid modified poly- third hands over fat-co-glycolide (PLGA-COOH) can be with the conjugated isodigeranyl function PEG (NH to amine-modification₂- PEG-COOH) to form PLGA-PEG-COOH copolymer.Use low-molecular weight PSMA parts (NH of amine-modification₂- Lig), PLGA-PEG-Lig triblock polymer can be formed by the amine functional group on the carboxylic acid end part by PEG.Then multi-block polymer can be used, such as discussed below for example, for therapeutic application.

Terms used herein " alkyl " includes the aliphatic group of saturation, including straight-alkyl group (for example, methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl etc.), branch-alkyl group (isopropyl, tert-butyl, isobutyl group etc.), cycloalkyl (alicyclic ring) group (cyclopropyl, cyclopenta, cyclohexyl, suberyl, cyclooctyl), alkyl-substituted group of naphthene base and the alkyl group of cycloalkyl substitution.

Term " aryl " includes such group, it include can containing 0-4 heteroatomic 5- and 6- member list-cyclic aromatic groups, for example, phenyl, pyrroles, furans, thiophene, thiazole, isothiazole, imidazoles, triazole, tetrazolium, pyrazoles,

It is azoles, different

Azoles, pyridine, pyrazine, pyridazine and pyrimidine, and it is such.In addition, term " aryl " includes polyaromatic group, for example, it is three rings, bicyclic, for example, naphthalene, benzo

Azoles, benzo two

Azoles, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl base, quinoline, isoquinolin, anthryl, phenanthryl, naphthyridines (napthridine), indoles, benzofuran, purine, benzofuran, deazapurine (deazapurine) or indolizine.Those can also be referred to as " aryl-heterocyclic ", " heterocycle ", " heteroaryl " or " heteroaromatics " in ring structure containing heteroatomic aromatic yl group.The aromatic ring can be replaced in one or more ring positions by this substituent as described above, for example, alkyl, halogen, hydroxyl, alkoxy, alkyl carbonyl epoxide, aryl carbonyl epoxide, alkoxy-carbonyl oxy, aryloxycarbonyl epoxide, carboxylate radical (carboxylate), alkyl-carbonyl, alkyl amino-carbonyl (alkylaminoacarbonyl), Aralkylaminocarbonyl, alkenyl amino carbonyl, alkyl-carbonyl, aryl carbonyl, aromatic alkyl carbonyl, alkenyl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl thiocarbonyl, phosphate radical (phosphate), phosphonate radical (phosphonato), phosphinic acids root (phosphinato), cyano group, amino (including alkyl amino, dialkyl amido, arylamino, ammonia diaryl base, and alkyl aryl amino), acyl amino (including alkyl-carbonyl-amino, aryl-amino-carbonyl, carbamoyl and urea groups), amidino groups, imido grpup, sulfydryl, alkylthio group, arylthio, thiocarboxylic acid root, sulfate radical, Alkylsulfinyl, sulfonate radical (sulfonato), sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano group, azido, heterocyclic radical, alkylaryl, or aromatics or heteroaromatic moiety.Aromatic yl group can also be condensed or bridged to form polycyclic (for example, naphthane) with alicyclic ring or heterocycle (non-aromatic ring).

Targeting moiety can be with for example, further by functional group (it can react with the polymer (for example, PEG) of the present invention) substitution, so as to produce the conjugated polymer to targeting moiety.The functional group includes the arbitrary portion that can be used for producing covalent bond with polymer (for example, PEG), such as amino, hydroxyl and thio.In a specific embodiment, small molecule can be by NH₂, SH or OH substitution, they are bonded directly to small molecule or via extra group, for example, alkyl or phenyl is bound to small molecule.In a non-limiting examples, the small molecule disclosed in herein cited patent, patent application and non-patent references can be bound to aniline, alkyl-NH₂(for example, (CH₂)_1-6NH₂) or alkyl-SH (for example, (CH₂)_1-6NH₂), wherein the NH₂It can be reacted with SH groups with polymer (for example, PEG), to form covalent bond with polymer, i.e. form polymer conjugate.

For example, disclosed herein is nanoparticle, the nano particle includes therapeutic agent；With the first macromolecule, first macromolecule includes conjugated PLGA-PEG copolymers or PLA-PEG copolymers to part, the part have about 100g/mol-500g/mol molecular weight, wherein the conjugated PLGA-PEG copolymers or PLA-PEG copolymers to part be the molar percentages of about 0.1- about 30 total polymer content or the molar percentages of about 0.1- about 20 or the molar percentages of about 0.1- about 10 or the molar percentages of about 1- about 5 nanoparticle total polymer content.This nanoparticle can further include the second macromolecule, and second macromolecule includes PLGA-PEG copolymers or PLA-PEG copolymers, wherein the copolymer is not bound to targeting moiety；With pharmaceutically acceptable excipient.For example, relative to total polymer content, first copolymer can the part containing about 0.001-5 percentage by weight.

Exemplary nano particulate can include therapeutic agent；And polymer composition, wherein the polymer composition is included：The first macromolecule containing the first polymer for being bound to part；With containing being not associated with to the second macromolecule of the second polymer of targeting moiety；Wherein described polymer composition includes the part of the percentage by weights of about 0.001- about 5.0.This part can have about 100g/mol- about 6000g/mol or below about 1000g/mol, e.g., from about 100g/mole- about 500g/mol molecular weight.In another embodiment, it provided herein is pharmaceutical composition, comprising a variety of targets-specific polymer nano particle, respectively comprising therapeutic agent；And polymer composition, wherein first macromolecule containing the first polymer that is bound to part of the polymer composition comprising the molar percentages of about 0.1- about 30 or the molar percentages of about 0.1- about 20 or the molar percentages of about 0.1- about 10；With containing being not associated with to the second macromolecule of the second polymer of targeting moiety；With pharmaceutically acceptable excipient.

Nanoparticle

Disclosed nanoparticle can have made of substantially spherical (that is, described particulate is usually seemingly spherical) or the spherical structure of non-.For example, the particulate, can be using the spherical structure of non-after expansion or shrinkage.In some cases, the particulate can include polymer blend.For example, polymer blend can be formed, it includes the first polymer of the polymer containing targeting moiety (that is, low-molecular weight PSMA parts) and bio-compatible, and does not contain containing the polymer of bio-compatible the second polymer of targeting moiety.By controlling ratio of first and second polymer in final polymer, the concentration of the targeting moiety in the final polymer and position control easily can be arrived into any suitable degree.

Disclosed nanoparticle can have the characteristic dimension below about 1 micron, and the characteristic dimension of wherein particulate is with the just spherical diameter with the particulate same volume.For example, the particulate can have the characteristic dimension of following particulates：In some cases can be below about 300nm, below about 200nm, below about 150nm, below about 100nm, below about 50nm, below about 30nm, below about 10nm, below about 3nm or below about 1nm.In specific embodiment, nanoparticle of the invention has about 80nm-200nm, about 60nm- about 150nm or about 70nm- about 200nm diameter.

In one group of embodiment, the particulate can have internal and surface, wherein described surface has different from internal composition, i.e., internally can at least there is a kind of compound (or vice versa as the same) being not present on surface, and/or at least one compound with different concentration is present on internal and surface.For example, in one embodiment, compound, such as targeting moiety of polymer conjugate of the invention are (i.e., low-molecular weight ligands), the inside and surface of the particulate are may reside in, but relative to the inside of the particulate, it is present in the surface of the particulate with higher concentration, although in some cases, the concentration of the particulate internally can be substantially non-zero, i.e. there is the compound of detectable amount in the inside of the particulate.

In some cases, the inside of the particulate is more hydrophobic than the surface of the particulate.For example, relative to the surface of the particulate, the inside of the particulate can be relative hydrophobic, and medicine or other Payloads can be hydrophobic, and easily the center with the particulate of relative hydrophobic is associated.So as to which the medicine or other Payloads can be contained in the inside of the particulate, this can make its cover and from contacting the external environment condition around the particulate (or vice versa as the same).For example, giving would be protected from comprising medicine or other Payloads in the particle for subject contacts subject's body, and body also will be with the medical separation.The yet still another aspect of the present invention is related to polymer particles, and the polymer particles contain the polymer or macromolecule for having more than a kind of presence, and involve this polymer or high molecular storehouse (libraries).For example, in one group of embodiment, particulate, which can contain, has more than a kind of recognizable polymer (for example, copolymer, for example, block copolymer), and the ratio of two kinds of (or more plant) polymer can be independently controlled, and this is easy to the control of the characteristic of the particulate.For example, first polymer can be the polymer conjugate of the part containing targeting moiety and bio-compatible, and targeting moiety can not be contained containing the part of bio-compatible for second polymer or second polymer can be containing the part with the recognizable bio-compatible of first polymer.Therefore, the control of the amount of these polymer in polymer particles can be used for a variety of physics for controlling the particulate, biology or chemical characteristic, for example, the size of the particulate is (for example, by the molecular weight for changing one or both of polymer), surface charge is (for example, by the ratio for controlling the polymer, if the polymer has different electric charges or end group), surface hydrophilicity is (for example, if the polymer has different molecular weight and/or hydrophily), the superficial density of the targeting moiety is (for example, by the ratio for controlling two or more polymer) etc..

It is used as specific example, particulate can include the first diblock polymer of the targeting moiety containing PEG and the PEG, with containing PEG without targeting moiety or second polymer containing PEG and targeting moiety, wherein the PEG of the second polymer has the length (or number of repeat unit) different relative to the PEG of the first polymer.It is used as yet another embodiment, particulate can include the first polymer of the part containing the first bio-compatible and targeting moiety, with the second polymer of the part of the second bio-compatible containing the part different from the first bio-compatible (for example, with it is different constitute, the substantially different number of repeat unit etc.) and targeting moiety.As also yet another embodiment, first polymer can include part and the first targeting moiety of bio-compatible, and second polymer can the part comprising bio-compatible and the second targeting moiety different from the first targeting moiety.

For example, disclosed herein is can be bound to the therapeutic polymer nano particle of target, the first non-functionalized polymer is included；The second optional non-functionalized polymer；Functionalized polymeric containing targeting moiety；And therapeutic agent；The functionalized polymeric of functionalized polymeric of the wherein described nanoparticle comprising the molecules of about 15- about 300 or the molecules of about 20- about 200 or the molecules of about 3- about 100.

In a specific embodiment, the first or second high molecular polymer of nanoparticle of the present invention is PLA, PLGA or PEG or its copolymer.In a particular embodiment, the first high molecular polymer is PLGA-PEG copolymers, and the second macromolecule is PLGA-PEG copolymers or PLA-PEG copolymers.For example, exemplary nano particulate can have PEG hats (corona), its density is about 0.065g/cm³Or about 0.01- about 0.10g/cm³。

Disclosed nanoparticle can be stable (for example retaining substantially all activating agent), for example, keep stable at least about 3 days, about 4 days or at least about 5 days in the solution containing sugar in room temperature or at 25 DEG C.

In some embodiments, disclosed nanoparticle can also include fatty alcohol, and it can increase the speed of insoluble drug release.For example, disclosed nanoparticle can include C₈-C₃₀Alcohol such as cetanol, octanol, stearyl alcohol, arachidic alcohol, docosyl alcohol (docosonal) or octasonal.

Nanoparticle can have control release characteristic, for example, in the period of extension, such as 1 day, in the time of 1 week or longer, it may be possible to a certain amount of bioactive agent delivery be delivered into patient, for example, the specific site of patient.In some embodiments; the substantially release immediately of disclosed nanoparticle (such as at about 1 minute-about 30 minutes) below about 2%, activating agent (such as taxane) medicament below about 5% or below about 10%, such as when being placed on room temperature and/or phosphate buffer solution at 37 DEG C.

For example, the disclosed nanoparticle containing therapeutic agent can be with, in some embodiments, when being placed on for example, in the 25C aqueous solution therapeutic agents can be discharged to substantially correspond to following speed：A) about 0.01- about 20% total therapeutic agent is discharged after about 1 hour；B) about 10- about 60% therapeutic agent was discharged after about 8 hours；C) about 30- about 80% total therapeutic agent is discharged after about 12 hours；And d) release is not less than about 75% total therapeutic agent after about 24 hours.

In some embodiments, after disclosed nanoparticle or composition containing disclosed nanoparticle to be given to subject or patient, if relative to the C for the therapeutic agent (for example, not as part of nanoparticle) individually given_maxFor, peak serum concentration (C of the therapeutic agent in the patient_max) substantially higher.

In further embodiment, relative to the t for the therapeutic agent individually given_maxFor, when giving subject by the disclosed nanoparticle containing therapeutic agent, the t for the therapeutic agent that the therapeutic agent can have_maxIt is substantially longer.

This particulate storehouse can also be formed.For example, by changing two kinds of (or more the plant) ratios of polymer in the particulate, these storehouses can be used for filler test, height-flux determine, or the like.By those characteristics as described above, the entity (Entities) in storehouse can change, and in some cases, more than one characteristics of the particulate can change in storehouse.Correspondingly, an embodiment of the invention is related to the storehouse of the nanoparticle of the polymer (having different qualities) with different ratios.The storehouse can include the polymer of any suitable ratio.

Fig. 1 explanations can produce storehouse using polymer those polymer as described above.For example, in Fig. 1, polymer particles include the hydrophobic polymer containing bio-compatible, the hydrophilic polymer of bio-compatible, with the first macromolecule of low-molecular weight PSMA parts, with the hydrophobic polymer containing bio-compatible and the second macromolecule of the hydrophilic polymer of bio-compatible, the polymer particles can be used for the storehouse for producing the first and second high molecular particulates with different ratios.

This storehouse can be used for realizing the particulate with any number of desired characteristic, for example characteristic such as surface functionality, surface charge, size, zeta (ζ) current potential, hydrophobicity, the ability for controlling immunogenicity, or the like.

As specific example, in certain embodiments of the present invention, the storehouse includes the particulate of the polymer conjugate of the polymer containing bio-compatible and low-molecular weight ligands, as discussed herein.With reference to Fig. 1, this particulate of display is non-limitation example.In the figure, disclosed polymer conjugate is used to form particulate 10.The polymer for forming particulate 10 includes and is present in low-molecular weight ligands 15 of the microparticle surfaces, and bio-compatible part 17.In some cases, such as shown here, targeting moiety 15 can be the conjugated part 17 to bio-compatible.However, not the compatible part 17 of all biological be shown it is conjugated to targeting moiety 15.For example, in some cases, particulate, such as particulate 10 can use the first polymer of the part 17 containing bio-compatible and low-molecular weight ligands 15, and the part 17 containing bio-compatible and the second polymer without targeting moiety 15 are formed.By controlling the ratio of the first and second polymer, the particulate with different qualities can be formed, and in some cases, the storehouse of this particulate can be formed.In addition, medicine 12 is included in the center of particulate 10.In some cases, due to hydrophobic effect, medicine 12 may be embodied in the particulate.For example, relative to the surface of the particulate, the inside of the particulate can be relative hydrophobic, and medicine can be the dewatering medicament with the relative hydrophobic center association of the particulate.In one embodiment, therapeutic agent and the surface association of nanoparticle, be encapsulated in nanoparticle, by nanoparticle surround or be dispersed in whole nanoparticle.In another embodiment, the therapeutic agent is encapsulated by the hydrophobic core of nanoparticle.

It is used as specific example, particulate 10 can include the polymer of the polymer of the bio-compatible containing relative hydrophobic and the targeting moiety 15 of relative hydropathic, so that during particulate formation, the polymer of the hydrophobic bio-compatible of the hydrophilic targeting moiety of higher concentration exposure on the surface with higher concentration is present in the inside of the particulate.

In some embodiments, the polymer of bio-compatible is hydrophobic polymer.The non-limitation example of the polymer of bio-compatible includes polylactide, polyglycolide, and/or poly- third friendship fat-co-glycolide.

In various embodiments, the disclosure provides nanoparticle and includes 1) polymer substrate；2) optionally, the compound or layer of amphiphilic, it surrounds the polymer substrate or the scattered continuous or discrete shell for forming the particulate in the polymer matrix；3) non-functionalized polymer, it can be with forming portion segregation polymer matrix, and 4) is covalently attached to the low molecule amount PSMA parts of polymer, and it can be with forming portion segregation polymer matrix.For example, the layer of amphiphilic, which can reduce penetration by water, enters nanoparticle, thus strengthen drug encapsulation efficiency and slow down insoluble drug release.

Terms used herein " amphiphilic ", which refers to wherein molecule, has the characteristic of polar portion and non-polar part.Usually, the compound of amphiphilic has the polar head for being connected to long hydrophobic tail.In some embodiments, polar portion is water-soluble, and non-polar portion is water insoluble.In addition, the polar portion can have effective positive charge or effective negative electrical charge.Alternatively, the polar portion can have effective positive charge and effective negative electrical charge, and be amphion or inner salt.For the intent of the present invention, the compound of the amphiphilic can be, but not limited to following one or more：Natural derivative lipid, surfactant or with hydrophilic and hydrophobic partially synthetic compound.

The instantiation of the compound of amphiphilic includes but is not limited to, phospholipid, such as 1, 2 distearyl-sn- glycerol-3-phosphates monoethanolamines (DSPE), DPPC (DPPC), DSPC (DSPC), palmitoylphosphatidylcholine (DAPC), two behenyl phosphatidyl cholines (DBPC), two or two tridecanoyl phosphatidyl cholines (DTPC), with dilignoceroylphatidylcholine (DLPC), with 0.01-60 (weight lipid/w polymer), most preferably 0.1-30 (weight lipid/w polymer) ratio incorporation.The phospholipid that can be used includes but is not limited to; phosphatidic acid, the phosphatidyl choline containing saturation and undersaturated lipid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, lysophosphatidyl derivatives, cuorin and β-acyl group-y- alkyl phospholipids.The example of phospholipid includes but is not limited to, phosphatidyl choline, such as DOPC, dimyristoyl phosphatidyl choline, two pentadecanoyl phosphatidyl cholines, DLPC, DPPC (DPPC), DSPC (DSPC), palmitoylphosphatidylcholine (DAPC), two behenyl phosphatidyl cholines (DBPC), two or two tridecanoyl phosphatidyl cholines (DTPC), dilignoceroylphatidylcholine (DLPC)；And cytoskeletal protein, such as DOPE or 1- cetyl -2- palmitoyl glycerols phosphoric acid (glycerophos)-phosphoethanolamine (phoethanolamine).The synthetic phospholipid containing asymmetric acyl chain (for example, acyl chain of acyl chain and another 12 carbon containing 6 carbon) can be used.

In a specific embodiment, the component of the amphiphilic for the layer that can be used for forming amphiphilic is lecithin, and, especially, phosphatidyl choline.Lecithin be amphiphilic lipid and, so, formed with towards hydrophilic (polarity) head (it is often aqueous) around them, and hydrophobic tail relative to each other phospholipid bilayer.Lecithin has the advantage of the natural lipid (for example, soybean) of available form, and has obtained FDA approval for other delivery apparatus.In addition, the mixture of lipid such as lecithin is relative to single pure lipid advantageously.

In some embodiments, disclosed nanoparticle has the individual layer of amphiphilic, it is intended that the layer is not phospholipid bilayer, but as single continuous or discrete layer is around nanoparticle or exists wherein.The layer of the amphiphilic is the nanoparticle " association " with the present invention, it is intended that it is located near polymer substrate, such as surrounds the outside of polymer shell or is dispersed in the polymer of composition nanoparticle.

The preparation of nanoparticle

The system and method for relating in one aspect to prepare disclosed nanoparticle again of the disclosure.In some embodiments, in different ratios using two or more different polymer (for example, copolymer, for example, block copolymer) and from the polymer (for example, copolymer, for example, block copolymer) particulate is produced, control the characteristic of the particulate.For example, a kind of polymer is (for example, copolymer, for example, block copolymer) low-molecular weight PSMA parts can be included, and in order to which the ability of the immunogenicity of the particle obtained by its biocompatibility and/or control can select another polymer (for example, copolymer, for example, block copolymer).

In one group of embodiment, the solution of one or more polymer is included by providing, and the solution is contacted with polymer nonsolvent to produce the particulate, to form the particulate.The solution can be miscible or immiscible with the polymer nonsolvent.For example, water-miscible liquid such as acetonitrile can contain the polymer, and particulate is formed as acetonitrile contacts (for example, by the way that acetonitrile is dumped into water with speed control) with water, polymer nonsolvent.Comprising polymer in the solution, when polymer nonsolvent is contacted, it can then precipitate to form particulate such as nanoparticle.In environment temperature and pressure, when a kind of liquid is at least 10% (by weight) insoluble in alternative degree, two kinds of liquid are referred to as " immiscible " or unmixing each other.Usually, organic solution is (for example, dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridine, two

Alkane, dimethyl sulfoxide (DMSO) etc.) and liquid, aqueous (for example, water or salt or water, cell or the biological media of other species, ethanol etc. containing dissolving) immiscible each other.For example, the first solution can be poured into the second solution (with suitable speed or speed).In some cases, as the first solution contacts immiscible second liquid, particulate such as nanoparticle can be formed, for example, polymer precipitation causes polymer formation nanoparticle, and the entrance second liquid that the first solution is toppled in contact, in some cases, for example, when the speed of introducing is to be carefully controlled and keep relatively slow speed, nanoparticle can be formed.By one of ordinary skill in the art be used only routine experimentation can this particulate of easily optimal control formed.

, can height control characteristic such as surface functionality, surface charge, size, zeta (ζ) current potential, hydrophobicity, the ability for controlling immunogenicity, and such using disclosed method.For example, the storehouse of particulate can be synthesized, and screening, to differentiate the particulate of the polymer with specific ratio, the polymer makes the particulate there is the part (for example, low-molecular weight PSMA parts) with specific density on the surface of the particulate.This allows the particulate with one or more specific characteristics to be produced, for example, the part of specific size and specific superficial density, without excessive work.Correspondingly, some embodiments of the invention are directed to use with the triage techniques in this storehouse, and any particulate differentiated using this storehouse.In addition, can be differentiated by the method for any suitable.For example, discriminating can be direct or indirect or quantitative progress or qualitative progress.

In some embodiments, operated using the operation identical described with those for producing ligand-functionalised polymer conjugate, the nanoparticle functionalization of-formation is made with targeting moiety.For example, the first copolymer (PLGA-PEG, poly- third friendship fat-co-glycolide and PEG) mixes to form particulate with therapeutic agent.Then, the particulate is with low-molecular weight ligands association to form nanoparticle, and the nanoparticle can be used for treating cancer.Thus the particulate can change the treatment feature of the nanoparticle with different amounts of low-molecular weight ligands association so as to control the ligand surface density of the nanoparticle.In addition, for example, by the molecular weight of control parameter such as molecular weight, PEG, and nano-particle surface electric charge, point-device control particulate can be obtained.

There is provided nanometer emulsified process in further embodiment, the process represented by such as Fig. 3 and 4.For example, therapeutic agent, first polymer are (for example, di-block copolymer-polymer such as PLA-PEG or PLGA-PEG, any of which one can be optionally in combination with to part, for example, GL2) and arbitrary second polymer (such as (PL (G) A-PEG or PLA) and organic solution the first organic phase of formation.This first phase can include the solid of the weight of about 5- about 50%, the solids of e.g., from about 5- about 40% or the solids of about 10- about 30%.First organic phase can combine to form the second phase with first aqueous solution.The organic solution can include, for example, toluene, MEK, acetonitrile, tetrahydrofuran, ethyl acetate, isopropanol, isopropyl acetate, dimethylformamide, dichloromethane, dichloromethane, chloroform, acetone, phenmethylol, Tween 80, sorbester p17 etc., and combinations thereof.In one embodiment, the organic phase can include phenmethylol, ethyl acetate and combinations thereof.Second phase can be about 1-50 weight %, for example, about 5-40 weight % solids.The aqueous solution can be water, optionally be combined with one or more sodium taurocholates, ethyl acetate, polyvinyl acetate and phenmethylol.

For example, oil or organic phase can use the only part solvent miscible with non-solvent (water).Therefore, when with sufficiently low ratio mixing and/or when using water with organic solvent presaturation, the oil phase keeps liquid.Use, for example, high energy disperse liquid system, such as homogenizer or sonicator, the oil phase can be emulsified into the aqueous solution and as liquid droplet, shearing enters nanoparticle.The surfactant solution that (otherwise referred to as " aqueous phase ") containing water section of emulsion can be made up of sodium taurocholate, and by ethyl acetate and phenmethylol presaturation.

Emulsifying second can be carried out with forming newborn phase in one or two emulsifying steps.For example, colostric fluid (primary emulsion) can be prepared to form miniemulsion.For example, using being simply mixed, high pressure homogenizer (high pressure homogenizer), Probe Ultrasonic Searching ripple crush instrument, stirrer (stir bar) or rotor-stator homogenizer can form the colostric fluid.Instrument or high pressure homogenizer are crushed by using such as Probe Ultrasonic Searching ripple, for example, pass through (passes) using by 1,2, the 3 or more secondary of refiner, the colostric fluid can turn into miniemulsion.For example, when using high pressure homogenizer, the pressure used can be about 1000- about 8000psi, about 2000- about 4000psi, 4000- about 8000psi or about 4000- about 5000psi, for example, about 2000,2500,4000 or 5000psi.

Solvent can be needed to evaporate or dilute the extraction to complete solvent and solidify the particulate.For dynamic (dynamical) more preferably control and the more telescopic process to extraction, the solvent dilution via aqueous quench fluid (quench) can be used.For example, emulsion can be diluted being enough to dissolve whole organic solvents to form the concentration of quenching (quench) phase to reach into cold water.Quenching can be carried out at least partially in about 5 DEG C or lower temperature.For example, the water used in quenching may be at the temperature (about 5 DEG C of about 10 DEG C of e.g., from about 0- or about 0-) less than room temperature.

In some embodiments, the particulate is all encapsulated in the stage and not all therapeutic agent (such as docetaxel), and solubilizing agents for drugs is added to quenching to form solubilized phase (solubilized phase).The solubilizing agents for drugs can be for example, Tween 80, polysorbas20, kollidon, cyclodextrin, lauryl sodium sulfate or sodium taurocholate.For example, Tween-80 can be added to quenching nanoparticle suspension to dissolve the formation of free drug and prophylactic agent crystal.In some embodiments, the ratio of solubilizing agents for drugs and therapeutic agent (such as docetaxel) is about 100: 1- about 10: 1.

Solubilising can be filtered to reclaim nanoparticle.For example, milipore filter can be used for concentrating the nanoparticle suspension and substantially eliminate organic solvent, free drug, and other processing aids (surfactant).Exemplary filtering can use tangential flow filtration system to carry out.For example, allow the film in the aperture that solute, micelle and organic solvent pass through by using with suitably nanoparticle is retained, can be with Selective Separation nanoparticle.The exemplary film of the molecular weight of the section with about 300-500kDa (~5-25nm) can be used.

Diafiltration (diafiltration) can use constant volume method to carry out, percolate (about 5 DEG C of cold deionized water, e.g., from about 0- or about 10 DEG C of 0-) can be added to feed suspension by intention with the speed identical speed for removing filtrate from suspension.In some embodiments, filtering can be including the use of about 10 DEG C of about 5 DEG C of about 0- or 0- the first temperature, and about 35 DEG C of about 30 DEG C of about 20- or 15- second temperature first filtering.For example, filtering can be included in about 5 DEG C of about 0- processing about 6 filtration volumes (diavolume) of about 1-, and in about 20- about 30 DEG C handle at least one filtration volume (e.g., from about 1- about 3 or about 1-2 filtration volume).

Nanoparticle suspension is being purified and is concentrating, the particulate can be by one, two or more sterilizings and/or depth filter, for example, with~0.2 μ m thick prefilter.

In the further embodiment for preparing nanoparticle, organic phase is made up of and formed the mixture of therapeutic agent (for example, docetaxel) and polymer (homopolymer, co-polymer and co-polymer) with part.The organic phase is mixed with water with about 1: 5 ratio (oil phase: aqueous phase), wherein the aqueous phase is made up of the solvent of surfactant and some dissolvings.By being simply mixed or by using rotor-stator homogenizer by two combined formation colostric fluids.Then, the colostric fluid is made to turn into miniemulsion by using high pressure homogenizer.Then, it is quenched miniemulsion by adding deionized water under mixing.Quench fluid: emulsion ratio is about 8.5: 1.Then the solution of tween (for example, Tween 80) is added into quench fluid to realize about 2% tween altogether.This is to dissolve free, non-encapsulated medicine.Then nanoparticle is separated by centrifugation or ultrafiltration/diafiltration.

Will be expected, can be different from the amount of final preparaton available for the polymer and therapeutic agent or the amount of activating agent for preparing preparaton.For example, some activating agents can be not suitable for mixing nanoparticle completely, and this free therapeutic agent can be filtered out for example.For example, in one embodiment, can prepare preparaton using the activating agent (such as docetaxel) of about 20 percentage by weights and about 80 Weight Percent Polymers (such as polymer can include about 2.5 molar percentages PLA-PEG-GL2 and the PLA-PEG of about 97.5 molar percentages), the preparaton produces the final nanoparticle for example comprising about 10 Weight Percent Active agent (such as docetaxel) and about 90 Weight Percent Polymers (wherein described polymer can include the PLA-PEG-GL2 of about 1.25 molar percentages and the PLA-PEG of about 98.75 molar percentages).This method can provide the final nanoparticle that suitably patient is administered, and the nanoparticle includes the therapeutic agent of the percentage by weights of about 2- about 20, the therapeutic agent of e.g., from about 5, about 8, about 10, about 15 percentage by weights.

Therapeutic agent

According to the present invention, including, for example, therapeutic agent (such as anticarcinogen), diagnostic medicament (such as contrast agent；Radionuclide；With fluorescence, luminous and magnetic part), any medicament of preventative medicament (such as vaccine) and/or trophism medicament (nutraceutical agent) (such as vitamin, mineral matter) can be delivered by the disclosed nanoparticle.Include but is not limited to small molecule (such as cytotoxic agent), nucleic acid (for example according to the Exemplary Agents of the invention to be delivered, siRNA, RNAi and mircoRNA medicament), protein (such as antibody), peptide, lipid, carbohydrate, hormone, metal, radioactive element and compound, medicine, vaccine, immuning agent, and/or its combination.In some embodiments, the medicament to be delivered is useful medicament in treating cancer (for example, prostate cancer).

For example, if used, targeting moiety can target or the particulate is adapted to the specific part for being confined to subject, and Payload can be delivered to those parts.In a specific embodiment, the medicine or other Payloads can be discharged from the particulate in the way of control release and allow it partly to be interacted with specific target site (for example, tumour).Terms used herein " control release " (and variant of the term) is (for example, " control-release system " of context) usually intention cover in the site of selection or be otherwise in speed, be spaced and/or measure controllable h substance (for example, medicine).Control release covers, but be not necessarily limited to, substantially continuous delivering, pattern delivery (patterned delivery) are (for example, it is interrupted delivering within the period by what regular or irregular time interval was interrupted), delivering with the pill of selection material is (for example, if predetermined, separation the amount of material release within the relatively short period (for example, several seconds or a few minutes)).

Activating agent or medicine can be therapeutic agents, such as antineoplastic, such as mTor inhibitor is (for example, sirolimus, CCI-779 or everolimus), vinca alkaloids, such as vincristine, forskolin or taxane, such as taxol (or its derivative, such as DHA- taxols or PG- taxols (paxlitaxel)) or docetaxel.

In one group of embodiment, the Payload is medicine or more than a kind of combination of medicine.This particulate can be useful in following embodiments：For example, wherein targeting moiety can be used for the specific local location that the particulate containing medicine is directed to subject, such as, it is allowed to the appearance of the local delivery of medicine.Exemplary treatment agent includes chemotherapeutic agents, such as Doxorubicin (adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, Etoposide, methotrexate (MTX), vinorelbine (venorelbine), 5 FU 5 fluorouracil (5-FU), vinca alkaloids such as vincaleukoblastinum or vincristine；Bleomycin,Taxol (PTX),Docetaxel (taxotere),Aldesleukin,Asparaginase,Busulfan,Carboplatin,Cladribine,Camptothecine,CPT-11,SN-38 (SN38),Dacarbazine,S-I capecitabines,Tegafur,5 ' doxifluridines (5 ' deoxyflurouridine),UFT,Eniluracil,Deoxycytidylic acid,5- azepine cytimidines,5-aza-2'-deoxycytidine (5-azadeoxycytosine),Allopurinol,2- chlorine adenosines,Trimetrexate,Aminopterin,Methylene -10- goes azepine aminopterin (MDAM),oxaplatin,picoplatin,Four platinum,Satraplatin,Platinum-DACH,Ormaplatin,CI-973,JM-216 and the like,Epirubicin,Etoposide phosphate,9-aminocamptothecin,10,11- methylenedioxy camptothecines,karenitecin,9-nitrocamptothecin,TAS 103,Eldisine,Phenylalanin-Lost,ifosphamidemefosphamide,Perfosfamide,Trofosfamide BCNU,Semustine,epothilones A-E,Tomudex,6-MP,6- thioguanines,Amsacrine,Etoposide phosphate,karenitecin,ACV,Valaciclovir,GCV,Amantadine,Rimantadine,Lamivudine,Zidovudine,Avastin,Herceptin,Rituximab,5 FU 5 fluorouracil,And combinations thereof.

The non-limitation example of potential suitable medicine includes anticarcinogen, including for example, docetaxel, mitoxantrone and mitoxantrone hydrochloride.In another embodiment,The Payload can be anticarcinogen,Such as 20-epi-1,25 dihydroxy vitamin d3s,4- ipomeanols,5-ethinyluracil,9- dihydro taxols,Abiraterone,Acivicin,Aclarubicin,Hydrochloric acid acodzole,Acronine,acylfiilvene,Gland cyclopentanol,Adozelesin,Aldesleukin,Whole-tk antagonists,Hemel,Ambamustine,Ambomycin,Ametantrone acetate,amidox,Amifostine,Aminoglutethimide,Aminol evulinic acid,Amrubicin,Amsacrine,Anagrelide,Anastrozole,Andrographolide,Angiogenesis inhibitor,Antagonist D,Antagonist G,Antarelix,Anthramycin,Anti- dorsalization morphogenetic proteins -1 (anti-dorsalizdng morphogenetic protein-1),Antiestrogen,Antineoplaston,ASON,Aphidicolin glycinate,Apoptosis gene conditioning agent,Apoptosis regulators,Apurinic acid,ARA-CDP-DL-PTBA,Arginin deaminase,Asparaginase,Asperline,asulacrine,Atamestane,Atrimustine,Ocean Lidar 1 (axinastatin 1),Ocean Lidar 2,Ocean Lidar 3,Azacitidine,Azasetron,Azalomvcin,Azatyrosine,Azetepa,Azotomycin,Baccatin III derivative,balanol,Batimastat,Benzo chlorin,Benzodepa,benzoylstaurosporine,Beta-lactam derivative,β-alethine,betaclamycin B,Birch olic acid,BFGF inhibitor,Bicalutamide,Bisantrene,Bisantrene hydrochloride,bisazuidinylspermine,Bisnafide,Two methanesulfonic acid bisnafides,bistratene A,Bizelesin,Bleomycin,Bleomycin Sulphate,BRC/ABL antagonists,breflate,Brequinar sodium,Bropirimine,Budotitane,Busulfan,Fourth methyllanthionine-sulphoxide imine,Act-C,Calcipotriol,calphostin C,Calusterone,Camptothecin derivative,Canary pox IL-2,Capecitabine,caraceraide,Carbetimer,Carboplatin,Carboxylic acid amides-amino-triazole,Carboxamide triazole,carest M3,BCNU,earn 700,Inhibitor derived from cartilage (cartilage),Carubicin hydrochloride,Carzelesin,Casein kinase 2 enzyme inhibitor,Australia castanospermine (castanosperrnine),Cecropin B,Cedefingol,Cetrorelix,Chlorambucil,Chlorin,Sulfonamides Lv quinoxalines,Cicaprost,Cirolemycin,Cis-platinum,Cis porphyrin (cis-porphyrin),Cladribine,Clomifene analog,Clotrimazole,collismycin A,collismycin B,Combretastatin A4,Combretastatin analogs,conagenin,crambescidin 816,Crisnatol,Crisnatol methanesulfonates,cryptophycin  8,Cryptophycin A derivatives,curacin  A,cyclopentanthraquinones,Endoxan,cycloplatam,cypemycin,Cytarabine,cytarabine ocfosfate,Cytolytic factor,Hexestryl diphosphate,Dacarbazine,Dacliximab,Actinomycin D,Daunorubicin hydrochloride,Decitabine,APL,Deslorelin,Right ifosfamide,Dexormaplatin,Dexrazoxane,Dexverapamil,Dezaguanine,Dezaguanine mesilate,Diaziquone,Didemnin B,didox,diethyhiorspermine,Dihydro-U-18496,dioxamycin,Diphenyl spiromustine,Docetaxel,Tadenan,Dolasetron,Doxifluridine,Doxorubicin,Doxorubicin hydrochloride,Droloxifene,Citric acid Droloxifene,Masterone,Dronabinol,Duazomycin,duocannycin SA,Ebselen,Ecomustine,Edatrexate,Edelfosine,Edrecolomab,eflomithine,Eflomithine hydrochlorides,Olive alkene,elsarnitrucin,Emitefur,Enloplatin,Enpromate,Epipropidine,Epirubicin,Epirubicin hydrochloride,Epristeride,Erbulozole,Red blood cell gene therapy medium (vector) system,Esorubicin hydrochloride,Estramustine,Estramustine analog,Estramustine phosphate sodium,Estrogen agonist,Estrogen antagonist,Etanidazole,Etoposide,Etoposide phosphate,Etoprine,Exemestane,Fadrozole,Fadrozole hydrochloride,Fazarabine,Suwei A amine,Filgrastim,Finasteride,flavopiridol,Fluorine

STING,Floxuridine,fluasterone,Fludarabine,Fludarabine phosphate,Fluorodaunorunicin hydrochlorides,Fluorouracil,Flurocitabine,Forfenimex,Formestane,Fosquidone,Fostriecin,Fostriecin sodium,Fotemustine,gadoliniumtexaphyrin,Gallium nitrate,Galocitabine,Ganirelix,Gelatinase inhibitor,Gemcitabine,Gemcitabine hydrochloride,Glutathione inhibitor,hepsulfam,heregulin,Hexamethylene bisacetamide,Hydroxycarbamide,Hypericin,Ibandronic acid,Idarubicin,Idarubicin hydrochloride,Idoxifene,Idramantone,Ifosfamide,ihnofosine,Ilomastat,Imidazo acridone,Imiquimod,Immunostimulatory peptides,The acceptor inhibitor of insulin-like growth factor -1,Interferon activator,Interferon α-2 A,Interferon α-2 B,Interferon-' alpha '-N1,Interferon-' alpha '-N3,Interferon beta-IA,Interferon gamma-IB,Interferon,Interleukins,MIBG,Iododoxorubicin,iproplatm,Irinotecan,Irinotecan hydrochloride,Iroplact,Irsogladine,isobengazole,isohomohalicondrin B,Itasetron,Promote microfilament polymerizer (jasplakinolide),kahalalide F,Piece spiral shell element-N triacetates,Lanreotide,Lanreotide acetate,leinamycin,Lenograstim,Lentinan sulfate,leptolstatin,Letrozole,Aleukemic leukemia inhibiting factor,Leucocyte alpha interferon,Leuprolide acetate,Leuproside/estrogen/progesterone,Leuprorelin,Levamisol,Liarozole,Liarozole hydrochloride,Straight-chain polyamine analog,The glycopeptide of lipophilic two,Lipophilic platinum compounds,lissoclinamide,Lobaplatin,Lombricine,Lometrexol,Lometrexol sodium,Lomustine,Lonidamine,Losoxantrone,Losoxantrone hydrochloride,Lovastatin,Loxoribine,Lurtotecan,lutetiumtexaphyrin lysofylline,Bacteriolyze peptide,Maitansine,mannostatin A,Marimastat,Masoprocol,maspin,Matrilysin inhibitor,NMPI,Maytansine,Mustargen hydrochloride,Megestrol acetate,Melengestrol acetate,Melphalan,Menogaril,merbarone,Mercaptopurine,Meterelin,Methioninase,Methotrexate (MTX),Methotrexate sodium,Metoclopramide,Metoprine,Meturedepa,Microalgae inhibitors of protein kinase C,MIF inhibitor,Mifepristone,Miltefosine,Mirimostim,Misplace double-stranded RNA,Mitindomide,mitocarcin,Mitocromin,Mitogillin,Mitoguazone,Mitolactol,Mitomalcin,Mitomycin,Mitomycin analogs,Mitonafide,Mitosper,Mitotane,Mitotoxin fibroblast growth factors-saporin,Mitoxantrone,Mitoxantrone hydrochloride,Mofarotene,Molgramostim,Monoclonal antibody,Mankind's chorionic gonadotrophin,Monophosphoryl lipid a/ Mycobacterial cell walls SK,Mopidamol,MDRG inhibitor,Therapy (multiple tumor suppressor 1-based therapy) based on multiple tumor suppressor 1,Mustargen anticancer,Indian Ocean sponge B (mycaperoxide B),Mycobacterial cell wall extract,Mycophenolic Acid,myriaporone,N- acetyl group dinaline,Nafarelin,nagrestip,Naloxone/pentazocine,napavin,Naphthalene terpinum,Nartograstim,Nedaplatin,Nemorubicin,Neridronic Acid,Neutral endopeptidase,Nilutamide,nisamycin,Nitric oxide modulator,Nitric oxide antioxidant,nitrullyn,Nocodazole,Nogalamycin,The benzamide of n- substitutions,O6-BG,Octreotide,okicenone,Oligonucleotides,Onapristone,Ondansetron,oracin,Stomatocyte factor derivant,Ormaplatin,Osaterone,Oxaliplatin,oxaunomycin,Oxisuran,Taxol,Paclitaxel analogs,Paclitaxel derivatives,palauamine,Palmityl rhizomycin,Pamidronic acid,Panaxytiol,Panomifene,Secondary coccus element,Pazelliptine,Pegaspargase,Peldesine,Peliomycin,Neptamustine,Pentosan Polysulfate Sodium,Pentostatin,pentrozole,Peplomycin sulfate,Perflubron,Perfosfamide,Perilla alcohol,Benzene azine mycin,Phenylacetate,Inhibitors of phosphatases,Molten chain bacterium,Pilocarpinum hydrochloride,Pipobroman,Piposulfan,THP,Piritrexim,Piroxantrone hydrochloride,placetin A,placetin B,Plasminogen Activator inhibitor,Platinum complex,Platinum compounds,The amine complex of platinum-three,Plicamycin,Plomestane,Porfimer Sodium,Porfiromycin,Prednimustine,Procarbazine hydrochloride,Propyl group pair-acridone,Prostaglandin J2,Prostate cancer antiandrogen,Proteasome inhibitor,Immunomodulator based on a-protein,Inhibitors of protein kinase C,Protein-tyrosine-phosphatase inhibitor,Purine nucleoside phosphorylase inhibitor,Puromycin,Puromycin hydrochloride,Alizarinopurpurin,pyrazorurin,Pyrazoloacridine,Pyridoxal Hemoglobin Polyoxyethylene compound (pyridoxylated hemoglobin polyoxyethylene conjugate),RAF antagonists,Raltitrexed,Ramosetron,RAS farnesyl protein transferase inhibitors,RAS inhibitor,RAS-GAP inhibitor,Demethylation retelliptine,The Etidronic Acids of rhenium RE 186,Rhizomycin,Riboprine,Ribozyme,RH retinarnide,RNAi,Rogletimide,Rohitukine,Romurtide,Roquinimex,rubiginone B1,ruboxyl,Safingol,Safingol hydrochloride,saintopin,sarcnu,sarcophytol A,Sargramostim,SDI1 analogies,Semustine,Inhibitor 1 derived from aging,Sense oligonucleotides,Signal transduction inhibitor,Signal transduction modulators,Simtrazene,Single chain antigen binding protein,Sizofiran,Sobuzoxane,Sodium Borocaptate,Sodium phenylacetate,solverol,Somatomedin associated proteins,Sonermin,sparfosafe sodium,Sparfosic acid,Sparsomycin,spicamycin D,Spirogermanium hydrochloride,Spiromustine,Spiroplatin,splenopentin,spongistatin 1,Squalamine,Stem cell inhibitors,Dry-cell division inhibitor,stipiamide,Broneomycin,Streptozotocin,The plain inhibitor of mesenchyma dissolving,sulfinosine,Sulofenur,Potent vasoactive intestines peptide antagonists,suradista,Suramin,Sphaerophysine,The glycosaminoglycan of synthesis,Talisomycin,Tallimustine,TAM methiodide,Tauromustine,Tazarotene,Tecogalan sodium,Tegafur,tellurapyrylium,Telomerase inhibitor,Teloxantrone hydrochloride,Temoporfin,Temozolomide,Teniposide,Teroxirone,Testolactone,tetrachlorodecaoxide,Red tetrazolium (tetrazomine),thaliblastine,Thalidomide,Thiapurine,Thiocoraline,Thioguanine,Phosphinothioylidynetrisaziridine,TPO,Thrombopoietin mimetics,Thymalfasin,Thymopoietin receptor stimulating agent,Thymotrinan,Thyroid-stimulating hormone (TSH),tiazofurin,The first purpurine of ethyl tin,Tirapazamine,Titanocene dichloride,Hycamtin hydrochloride,topsentin,Toremifene,Toremifene Citrate,The myeloid-lymphoid stem cell factor,Translation inhibitor,Trestolone acetate,Tretinoin,Triacetyluridine,Triciribine,Phosphoric acid triciribine,Trimetrexate,Trimetrexate glucuronate,Triptorelin,Tropisetron,Tubulozole hydrochloride,Turosteride,Tyrosine kinase inhibitor,Tyrphostin,UBC inhibitor,Ubenimex,Uracil mustard,Uredepa,Urogenital sinus-derivative growth inhibitory factor,Urokinase receptor antagonist,Vapreotide,variolin B,Velaresol,Black false hellebore is obviously,verdins,Verteporfin,Vinblastine sulfate,Vincristine sulphate,Eldisine,Vindesine sulfate,Sulfuric acid vinepidine,Sulfuric acid vinglycinate,Sulfuric acid leurosine,Vinorelbine or vinorelbine tartrate,Sulfuric acid vinrosidine,vinxaltine,Sulfuric acid vinzolidine,vitaxin,Vorozole,Zanoterone,Zeniplatin,Zilascorb,Zinostatin,Zinostatin stimalamer or zorubicin hydrochloride.

Pharmaceutical formulation

According to another aspect of the invention, nanoparticle disclosed herein can form pharmaceutical composition with pharmaceutically acceptable carrier combinations.It will be appreciated by those skilled in the art that the carrier can be selected based on method of administration as described below, the position of targeting problem, the medicine being delivered, time course of delivering medicine etc..

The pharmaceutical composition of the present invention can give patient by mode well known in the art (including oral and parenteral outer approach).Terms used herein " patient ", refers to the mankind and non-human class, including, for example, mammal, bird, reptile, amphibian and fish.For example, non-human class can be mammal (for example, rodent, mouse, rat, rabbit, monkey, dog, cat, primate or pig).Parental routes are wished in some embodiments, and its reason is that they avoid contacting with being found to be present in gastral digestive ferment.According to this embodiment, the present composition can be given by following manner：Inject (for example, intravenous, subcutaneous or intramuscular, intraperitoneal injection), per rectum, Via vagina, part (by powder, creme, ointment or drops) or by suck (by spray).

In a specific embodiment, subject in need is given by the nanoparticle whole body of the present invention, for example, being transfused or injecting by IV.

Ejection preparation, for example, the aqueous or oleaginous suspension of sterile injectable, can be prepared according to known technology using suitable dispersant or wetting agent and suspending agent.Aseptic injection preparation can also be aseptic injectable solution, suspension or the emulsion in nontoxic parenteral acceptable diluent or solvent, for example, the solution in 1,3-BDO.It is water, Ringer's solution, U.S.P, and isotonic sodium chlorrde solution in the acceptable medium and solvent that can be used.In addition, conventional be used as solvent or suspension media using sterile, fixed oil.Therefore, the glycerine one or diester of synthesis can be included using any gentle fixed oil.In addition, using aliphatic acid, such as oleic acid prepares injection.In one embodiment, conjugates of the present invention is suspended in the tween of carrier fluid, sodium carboxymethylcellulose of the carrier fluid comprising 1% (w/v) and 0.1% (v/v)^TM 80.The injection preparaton can be with, for example, retain the filter of bacterium by being filtered through or sterilized by mixing the bactericide of aseptic solid composite form, the aseptic solid composite can be using being preceding dissolved or dispersed in sterilized water or other sterile injectable mediums.

For the solid dosage forms of oral administration, including capsule, tablet, pill, powder and fine granule.In this solid dosage forms, encapsulating or non-encapsulated conjugates is mixed with following at least one：Inertia, pharmaceutically acceptable excipient or carrier, such as sodium citrate or Dicalcium Phosphate and/or (a) filler or swelling agent, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) adhesive, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum, (c) NMF, such as glycerine, (d) disintegrant, such as aga agar, calcium carbonate, potato or tapioca, alginic acid, some silicate, and sodium carbonate, (e) solution retarding agents (solution retarding agent), such as paraffin, (f) absorbsion accelerator, such as quaternary ammonium compound, (g) wetting agent, such as, for example, cetanol and glycerol monostearate, (h) absorbent, such as white bole and bentonite, lubricant (i), such as talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, NaLS, and its mixture.In the case of capsule, tablet and pill, the formulation can also include buffer solution.

It will be recognized that each doctor considers the patient to be treated to select the exact dose of PSMA- Target Particles, it is however generally that, regulating dosage and administration are to provide the PSMA- Target Particles of effective dose to treated patient.The PSMA- Target Particles of " effective dose " used herein refer to, and cause the required amount of desired biologically/response.As one of ordinary skill in the art it will be recognized that the PSMA- Target Particles of effective dose can change, this depends on all factors described as follows：Desired biologic endpoint, the medicine to be delivered, target tissue, method of administration etc..For example, the effective dose of the PSMA- Target Particles containing cancer therapy drug is probably such amount, it causes tumor size to reduce by the desired amount wished in the period.Admissible extra factor includes the seriousness of morbid state；Age, weight and the sex of patient under consideration；Diet, the time of administration and frequency；Drug regimen；The sensitiveness of reaction；With/response is resistant to therapy.

The nanoparticle of the present invention can be prepared in dosage device (dosage unit) form of easy administration and dose uniformity.Statement " dosage unit form " used herein refers to the unit of the nanoparticle being physically isolated for the patient for being applicable to treatment.However, it is to be understood that the summation of the daily composition using the present invention will be determined by the doctor in charge in the range of its rational medicine judges.For arbitrary nanoparticle, initially the effective dosage therapeutic can be determined or usually estimated in the animal model of mouse, rabbit, purchase or pig in cell culture.The animal model is also used for realizing desired concentration range and method of administration.Then, this information may be used to determine whether useful dosage and method of administration in the mankind.The treatment effect and toxicity of nanoparticle can be determined by the standard pharmaceutical procedures in cell culture or experimental animal, for example, ED₅₀(the effective dosage in the treatment in 50% colony) and LD₅₀(making dosage dead in 50% colony).The dose ratio of toxicity and therapeutic effect is therapeutic index, and the therapeutic index can be expressed as, LD₅₀/ED₅₀Ratio.The pharmaceutical composition for showing big therapeutic index is useful in some embodiments.The data obtained from cell culture measure and zoopery can be used for a series of dosage for preparing people.

In one embodiment, compositions disclosed herein can include the palladium below about 10ppm or the palladium below about 8ppm or below about 6ppm.For example, such composition provided herein, it includes the nanoparticle with polymer conjugate PLA-PEG-GL2, wherein the composition has the palladium below about 10ppm.

In an exemplary embodiment, disclosed pharmaceutical composition includes multiple nanoparticles, respectively comprising therapeutic agent；The first macromolecule containing PLGA-PEG copolymers or PLA-PEG copolymers of the total polymer content of the total polymer content or the molar percentages of about 0.1- about 20 or the molar percentages of about 0.1- about 10 of the nanoparticle of the molar percentages of about 0.1- about 30 or the nanoparticle of the molar percentages of about 1- about 5, the copolymer is conjugated to the part with about 100g/mol-500g/mol molecular weight；With the second macromolecule containing PLGA-PEG copolymers or PLA-PEG copolymers, wherein the copolymer is not associated with to targeting moiety；With pharmaceutically acceptable excipient.For example, first copolymer can have the part of the about 0.001-5 percentage by weights relative to total polymer content.

In some embodiments, it is contemplated that suitable for the composition of freezing, including nanoparticle disclosed herein and the solution suitable for freezing, for example, sucrose solution is added into nanoparticle suspension.The sucrose can be with for example, prevent the cryoprotector that the particulate is assembled in freezing.For example, it provided herein is nanoparticle preparaton, the nanoparticle preparaton includes multiple disclosed nanoparticles, sucrose and water；Wherein described nanoparticle/sucrose/water is about 3-30%/10-30%/50-90% (w/w/w) or about 5-10%/10-15%/80-90% (w/w/w).

Treatment method

In some embodiments, it can be used for the breaking-out for treating, alleviating, improve, relaxing, delay one or more symptoms or disease, obstacle and/or illness according to the Target Particles of the present invention, suppress the progress of one or more symptoms or disease, obstacle and/or illness, mitigate the seriousness of one or more symptoms or disease, obstacle and/or illness, and/or reduce the incidence or the feature of disease, obstacle and/or illness of one or more symptoms.In some embodiments, Target Particles of the invention can be used for treating solid tumor, for example, cancer and/or cancer cell.In some embodiments, the Target Particles of the present invention can be used for treating any cancer, and wherein PSMA is expressed on the surface of the cancer cell of patient in need or in Tumor neovasculature (neovasculature for including prostate or non-prostate solid tumor).The example of indication relevant PSMA- includes but is not limited to, prostate cancer, breast cancer, non-ED-SCLC, the carcinoma of the rectum and glioblastoma.

Term " cancer " include it is pernicious before (pre-malignant) and malignant cancer.Cancer includes but is not limited to, prostate, stomach cancer, colorectal cancer, cutaneum carcinoma, for example, melanoma or basal-cell carcinoma, lung cancer, breast cancer, cancer, bronchiolar carcinoma, cancer of pancreas, carcinoma of urinary bladder, brain or the central nervous system cancer of head and neck, peripheral nervous system cancer, the cancer of the esophagus, mouth cancers or pharynx cancer, liver cancer, kidney, carcinoma of testis, cancer of bile ducts, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal, osteosarcoma, chondrosarcoma, the cancer of blood tissues, and it is such." cancer cell " can be the form of tumour, separately exist in subject (for example, leukaemia) or the cell line derived from cancer.

Cancer can be relevant with a variety of physical symptoms.The symptom of cancer is generally dependent on type and the position of tumour.For example, lung cancer can cause to cough, be short of breath and pectoralgia, and colon cancer frequently results in diarrhoea, constipation and had blood in stool.But there is provided but only several examples, following symptoms are usually usually related to many cancers：Fever, shiver with cold, night sweat, cough, expiratory dyspnea, weight saving, poor appetite, apocleisis, nausea,vomiting,diarrhea, anaemia, jaundice, hepatomegaly, hemoptysis, fatigue, dispirited, cognition dysfunction, depression, hormone disturbance, neutrophils reduction, pain, not-healing ulcer, enlargement of lymph nodes, peripheral neuropathy and sex dysfunction.

In invention, on the one hand there is provided the method for treating cancer (such as prostate or breast cancer).In some embodiments, treating cancer includes giving the Target Particles of the invention of subject in need's therapeutically effective amount with this amount necessary to realizing desired result and time.In some embodiments of the present invention, " therapeutically effective amount " of the Target Particles of the present invention is such amount, the breaking-out of one or more symptoms or cancer is effectively treated, alleviated, improving, relaxing, being delayed to the amount, suppress the progress of one or more symptoms or cancer, mitigate the seriousness of one or more symptoms or cancer, and/or reduce the incidence or the feature of cancer of one or more symptoms.

There is provided the method for giving the subject's present composition for suffering from cancer (such as prostate cancer) in one aspect of the invention.In some embodiments, subject is given particulate with this amount necessary to realizing desired result (that is, treating cancer) and time.In some embodiments of the present invention, " therapeutically effective amount " of the Target Particles of the present invention is such amount, the breaking-out of one or more symptoms or cancer is effectively treated, alleviated, improving, relaxing, being delayed to the amount, suppress the progress of one or more symptoms or cancer, mitigate the seriousness of one or more symptoms or cancer, and/or reduce the incidence or the feature of cancer of one or more symptoms.

Therapeutic scheme of the present invention, which involves, gives the Target Particles of the invention that healthy individuals (that is, any symptom of cancer are not shown and/or the subject with cancer is not diagnosed) therapeutically effective amount.For example, before the progress of cancer and/or the paresthesia epilepsy of cancer, healthy individuals " immune " can be made with the Target Particles of the present invention；Individual among risk is (for example, the patient with cancer family history；Carry the patient of one or more gene mutations relevant with cancer progression；Patient with the genetic polymorphism relevant with cancer progression；Pass through the patient of the virus infection relevant with cancer progression；With patient of habit and/or life style relevant with cancer progression etc.) breaking-out (for example, at 48 hours, at 24 hours or at 12 hours) that can be substantially with cancer symptoms treats simultaneously.Certainly, it is known that the individual with cancer can receive the treatment of the present invention at any time.

In other embodiments, nanoparticle of the invention can be used for suppressing cancer cell, for example, the growth of prostate gland cancer cell.Terms used herein " suppression cancer cell growth " refers to the prevention or the killing of cancer cell of the slowing down of the speed of any cancer cell proliferation and/or migration, cancer cell proliferation and/or migration so that rate reduction of the speed of cancer cell growth relative to observation or the prediction of untreated control cancer cell.Term " suppress growth " can also refer to cancer cell or the size of tumour is reduced or disappeared, and its metastatic potential reduction.Preferably, it is this to suppress that in molecular level the cancer size of patient, the growth of cancers for preventing patient, the cancer infringement for reducing patient or prevention or the cancer metastasis stove for suppressing patient are reduced.By arbitrary a variety of suitable marks, those skilled in the art can readily determine that whether growth of cancer cells suppresses.

The suppression of cancer cell growth can be confirmed by following manner：For example, cancer cell is prevented by the moment in the cell cycle, for example, being prevented in the G2/M phases in cancer cell cycle.The suppression of cancer cell growth can also be confirmed by following manner：By directly or indirectly measuring cancer cell or tumor size.In human patients with cancer, it is used generally following methods and carries out this measurement：Use known imaging method, such as magnetic resonance imaging, computed axial computerized tomography and X-ray.Cancer cell growth can also indirect determination, such as by determine circulate carcinomebryonic antigen level, PSA or other cancer-specific antigens, they are related to cancer cell growth.The general existence also to the extension of subject of suppression of growth of cancers and/or increased health and Ankang are related.

It is also provided herein and gives patient the method for nanoparticle disclosed herein; the nanoparticle includes activating agent; wherein; when patient is administered; independent (i.e. and private nanoparticle) relative to medicament is administered, and this nanoparticle substantially reduces the volume of distribution and/or substantially reduces free Cmax.

Embodiment

The present invention typically now is described, be will readily understand that by reference to following examples (including being intended only to certain aspect and embodiment example to the present invention), and be not intended to limit the present invention in any way.

Embodiment 1：The synthesis of low-molecular weight PSMA parts (GL2)

5g (10.67mmol) initial compounds are dissolved in 150mL dry DMF.Allyl bromide, bromoallylene (6.3mL, 72mmol) and K are added into the solution₂CO₃(1.47g, 10.67mmol).2h is stirred into reaction, solvent is removed, thick material is dissolved in AcOEt and H is used₂O is washed, until pH is neutrality.Use MgSO₄It is (anhydrous) to dry organic phase and evaporation to obtain the material of 5.15g (95%).(TLC is in CH₂Cl₂: Rf=0.9 in MeOH 20: 1, initial compounds Rf=0.1, it is disclosed with ninhydrin and uv light).

To compound (5.15g, 10.13mmol) in CH₃Et is added in solution in CN (50mL)₂NH (20mL, 0.19mol).Reaction is stirred at room temperature and reaches 40min.Remove solvent and by column chromatography (hexane: AcOEt 3: 2) purifying compound to obtain 2.6g (90%).(TLC is in CH₂Cl₂: Rf=0.4 in MeOH 10: 1, it is disclosed (compound has purple) with ninhydrin.¹H-NMR(CDCl₃, 300MHz) and δ 5.95-5.85 (m, 1H ,-CH₂CHCH₂), 5.36-5.24 (m, 2H ,-CH₂CHCH₂), 4.62-4.60 (m, 3H ,-CH₂CHCH₂, NHBoc), 3.46 (t, 1H, CH (Lys)), 3.11-3.07 (m, 2H, CH₂NHBoc), 1.79 (bs, 2H, NH₂), 1.79-1.43 (m, 6H, 3CH₂(Lys)), 1.43 (s, 9H, Boc).

At -78 DEG C, to diallyl glutamate (diallyl glutamate) (3.96g, 15mmol) and triphosgene (1.47g, 4.95mmol) in CH₂Cl₂Added in agitating solution in (143mL) in CH₂Cl₂Et in (28mL)₃N (6.4mL, 46mmol).So that reactant mixture is warmed to room temperature and stirred up to 1.5h.Then, added at -78 DEG C in CH₂Cl₂Lysine derivative (2.6g, 9.09mmol) in the solution of (36mL) and reaction is stirred at room temperature reaches 12h.The solution CH₂Cl₂Dilution, uses H₂O is washed twice, in MgSO₄Dried on (anhydrous) and (TLC is in CH to obtain 4g (82%) by column chromatography (hexane: 3: 1 → 2: 1 → AcOEt of AcOEt) purifying₂Cl₂: 20: 1Rf=0.3 in MeOH, it is disclosed with ninhydrin).¹H-NMR(CDCl₃, 300MHz) and δ 5.97-5.84 (m, 3H, 3-CH₂CHCH₂), 5.50 (bt, 2H, 2NH ureas), 5.36-5.20 (m, 6H, 3-CH₂CHCH₂), 4.81 (bs, 1H, NHBoc), 4.68-4.40 (m, 8H, 3-CH₂CHCH₂, CH (Lys), CH (glu)), 3.09-3.05 (m, 2H, CH₂NHBoc), 2.52-2.39 (m, 2H, CH₂(glu.)), 2.25-2.14 and 2.02-1.92 (2m, 2H, CH₂(glu.)), 1.87-1.64 (m, 4H, 2CH₂(Lys)), 1.51-1.35 (m, 2H, CH₂(Lys)), 1.44 (s, 9H, Boc).

CH is being dried to compound (4g, 7.42mmol)₂Cl₂The TFA (9mL) of 0 DEG C of addition in solution in (40mL).Reaction is stirred at room temperature and reaches 1h.Solvent is removed under vacuo until being completely dried, to obtain 4.1g (quantitative).(TLC is in CH₂Cl₂: Rf=0.1 in MeOH 20: 1, it is disclosed with ninhydrin).¹H-NMR(CDCl₃, 300MHz) and δ 6.27-6.16 (2d, 2H, 2NH urea), 5.96-5.82 (m, 3H, 3-CH₂CHCH₂), 5.35-5.20 (m, 6H, 3-CH₂CHCH₂), 4.61-4.55 (m, 6H, 3-CH₂CHCH₂), 4.46-4.41 (m, 2H, CH (Lys), CH (glu)), 2.99 (m, 2H, CH₂NHBoc), 2.46 (m, 2H, CH₂(glu.)), 2.23-2.11 and 2.01-1.88 (2m, 2H, CH₂(glu.)), 1.88-1.67 (m, 4H, 2CH₂(Lys)), 1.45 (m, 2H, CH₂(Lys))。

Under argon, at 0 DEG C, Pd (PPh are added into solution of the compound (2g, 3.6mmol) in DMF (anhydrous) (62mL)₃)₄(0.7g, 0.6mmol) and morpholine (5.4mL, 60.7mmol).Reaction is stirred at room temperature and reaches 1h.Remove solvent.Crude product CH₂Cl₂Wash twice, be then dissolved in H₂O.To the molten middle addition NaOH (0.01N) dilute solution until pH claim it is extremely alkaline.Solvent is removed under reduced pressure.Use CH₂Cl₂, AcOEt and MeOH-CH₂Cl₂The mixture of (1: 1) washs solid again, is dissolved in H₂O is neutralized and is used Amberlite IR-120H⁺Resin is neutralized.Evaporation solvent, and compound precipitates with MeOH, to obtain the GL2 of 1g (87%).¹H-NMR(D₂O, 300MHz) δ 4.07 (m, 2H, CH (Lys), CH (glu)), 2.98 (m, 2H, CH₂NH₂), 2.36 (m, 2H, CH₂(glu.)), 2.08-2.00 (m, 1H, CH₂(glu)), 1.93-1.60 (m, 5H, CH₂(glu.), 2CH₂(Lys)), 1.41 (m, 2H, CH₂(Lys)).Quality ESI (Mass ESI)：320.47[M+H⁺], 342.42 [M+Na⁺]。

Embodiment 2：The synthesis of low-molecular weight PSMA parts (GL1)

130mg (0.258mmol) initial compounds are dissolved in 3mL DMF (anhydrous).Allyl bromide, bromoallylene (150 μ L, 1.72mmol) and K are added into the solution₂CO₃(41mg, 0.3mmol).1h is stirred into reaction, solvent is removed, crude product is dissolved in AcOEt, and uses H₂O washings are neutrality until pH.Use MgSO₄It is (anhydrous) to dry organic phase and evaporation to obtain 130mg (93%).(TLC is in CH₂Cl₂: 20: 1Rf=0.9 in MeOH, initial compounds Rf=0.1 is disclosed with ninhydrin and uv light).¹H-NMR(CDCl₃, 300MHz) and δ 7.81-7.05 (12H, aromatic compounds), 6.81 (bs, 1H, NHFmoc), 5.93-5.81 (m, 1H ,-CH₂CHCH₂), 5.35-5.24 (m, 2H ,-CH₂CHCH₂), 5.00 (bd, 1H, NHboc), 4.61-4.53 (m, 5H ,-CH₂CHCH₂, CH₂(Fmoc), CH (pheala.)), 4.28 (t, 1H, CH (Fmoc)), 3.12-2.98 (m, 2H, CH₂(pheala.), 1.44 (s, 9H, Boc).

CH is being dried to compound (120mg, 0.221mmol)₂Cl₂The TFA (1mL) of 0 DEG C of addition in solution in (2mL).Reaction is stirred at room temperature and reaches 1h.Solvent is removed under vacuo, is added water and is removed again, adds CH₂Cl₂Remove until being completely dried to obtain 120mg (quantitative) again.(TLC is in CH₂Cl₂: Rf=0.1 in MeOH 20: 1, it is disclosed with ninhydrin and uv light).¹H-NMR(CDCl₃, 300MHz) and δ 7.80-7.00 (13H, aromatic compounds, NHFmoc), 5.90-5.75 (m, 1H ,-CH₂CHCH₂), 5.35-5.19 (m, 3H ,-CH₂CHCH₂, NHboc), 4.70-4.40 (2m, 5H ,-CH₂CHCH₂, CH₂(Fmoc), CH (pheala.)), 4.20 (t, 1H, CH (Fmoc)), 3.40-3.05 (m, 2H, CH₂(pheala.))。

At -78 DEG C, to diallyl glutamate (110mg, 0.42mmol) and triphosgene (43mg, 0.14mmol) in CH₂Cl₂Added in agitating solution in (4mL) in CH₂Cl₂Et in (0.8mL)₃N (180 μ L, 1.3mmol).So that reactant mixture is warmed to room temperature and stirred up to 1.5h.Then, will be in CH at -78 DEG C₂Cl₂(1mL) and Et₃Phenylalanine derivative (140mg, 0.251mmol) in N (70 μ L, 0.5mmol) solution, which adds and reaction is stirred at room temperature, reaches 12h.The solution CH₂Cl₂Dilution, uses H₂O is washed twice, in MgSO₄Dry and purified by column chromatography (hexane: AcOEt 3: 1) on (anhydrous), (TLC is in CH to obtain 100mg (57%)₂Cl₂: Rf=0.3 in MeOH 20: 1, disclosed with ninhydrin and uv light).¹H-NMR(CDCl₃, 300MHz) and δ 7.80-6.95 (13H, aromatic compounds, NHFmoc), 5.98-5.82 (m, 3H, 3-CH₂CHCH₂), 5.54 (bd, 1H, NH ureas), 5.43-5.19 (m, 7H, 3-CH₂CHCH₂, NH ureas), 4.85-4.78 (m, 1H, CH (pheala.)), 4.67-4.50 (m, 9H, 3-CH₂CHCH₂, CH₂(Fmoc), CH (glu.)), 4.28 (t, 1H, CH (Fmoc)), 3.05 (d, 2H, CH₂(pheala.)), 2.53-2.33 (m, 2H, CH₂(glu.)), 2.25-2.11 and 1.98-1.80 (2m, 2H, CH₂(glu.))。

To raw material (60mg, 0.086mmol) in CH₃Et is added in solution in CN (1mL)₂NH (1mL, 10mmol).Reaction is stirred at room temperature and reaches 40min.Remove solvent and compound is purified by column chromatography (hexane: AcOEt 2: 1), to obtain 35mg (85%).(TLC is in CH₂Cl₂: Rf=0.5 in MeOH 10: 1, initial compounds Rf=0.75, it is disclosed with ninhydrin (compound has purple) and uv light).¹H-NMR(CDCl₃, 300MHz) and δ 6.85 and 6.55 (2d, 4H, aromatic compounds), 5.98-5.82 (m, 3H, 3-CH₂CHCH₂), 5.56 (bd, 1H, NH ureas), 5.44-5.18 (m, 7H, 3-CH₂CHCH₂, NH ureas), 4.79-4.72 (m, 1H, CH (pheala.)), 4.65-4.49 (m, 7H, 3-CH₂CHCH₂, CH (glu.)), 3.64 (bs, 2H, NH₂), 3.02-2.89 (m, 2H, CH₂(pheala.)), 2.49-2.31 (m, 2H, CH₂(glu.)), 2.20-2.09 and 1.91-1.78 (2m, 2H, CH₂(glu.))。

It is (anhydrous in DMF to compound (50mg, 0.105mmol) under argon, at 0 DEG C；Pd (PPh are added in solution in 1.5mL)₃)₄(21mg, 0.018mmol) and morpholine (154 μ L, 1.77mmol).Reaction is stirred at room temperature and reaches 1h.Remove solvent.Thick material CH₂Cl₂Wash twice, and be dissolved in H₂In O.To the dilute solution of the molten middle addition NaOH (0.01N) until pH is in extremely alkalescence.Solvent is removed under reduced pressure.The solid CH₂Cl₂, AcOEt and MeOH-CH₂Cl₂The mixture of (1: 1) is washed again, is dissolved in H₂O is neutralized and is used AmberliteIR-120H⁺Resin is neutralized.Evaporation solvent and compound is precipitated with MeOH, to obtain the GL1 of 25mg (67%).¹H-NMR(D₂O, 300MHz) δ 7.08 and 6.79 (2d, 4H, aromatic compounds), 4.21 (m, 1H, CH (pheala.)), 3.90 (m, 1H, CH (glu.)), 2.99 and 2.82 (2dd, 2H, CH₂(pheala.)), 2.22-2.11 (m, 2H, CH₂(glu.)), 2.05-1.70 (2m, 2H, CH₂(glu.))。¹³C-NMR(D₂O, 75MHz) δ 176.8,174.5,173.9 (3COO), 153.3 (NHCONH), 138.8 (H₂N-C (Ph)), 124.5,122.9,110.9 (aromatic compounds), 51.3 (CH (pheala.)), 49.8 (CH (glu.)), 31.8 (CH₂(pheala.)), 28.4 and 23.6 (2CH₂-glu.)).Quality ESI：354.19[M+H⁺], 376.23 [M+Na⁺]。

Embodiment 3：PLA-PEG preparation

Synthesis passes through d, the ring-opening polymerisation of l- lactides and the Alpha-hydroxy-ω as macromolecule-initiator (macro-initiator)-methoxyl group PEG acts on completing, and use the 2 ethyl hexanoic acid tin (II) as catalyst to carry out in elevated temperature, following display (PEG Mn ≈ 5,000Da；PLA Mn≈16,000Da；PEG-PLA M_n≈21,000Da)

Polymer is purified by being dissolved in dichloromethane, and is precipitated in the mixture of hexane and ether.The polymer reclaimed from the step will be dried in drying box.

Example 4：It is prepared by PLA-PEG- parts

Reaction of the synthesis (display is in fig. 2) by FMOC, BOC lysine and allyl bromide, bromoallylene and potassium carbonate in dimethylformamide, FMOC, BOC lysine are converted into FMOC, BOC, pi-allyl lysine to start, then the diethylamine processing in acetonitrile.The BOC, pi-allyl lysine then react with triphosgene and diallyl glutamate, and then the trifluoroacetic acid in dichloromethane handles to form compound " GL2P ".

Then, by adding hydroxyl-PEG- carboxylic acids and EDC and NHS makes the pendant amine of lysine in GL2P by PEGylation.GL2P is conjugated to PEG via acid amides linker.The structure of the gained compound is labeled " HO-PEG-GL2P ".After PEGylation, the ring-opening polymerisation effect (ROP) of d, l- lactide and the oh group in HO-PEG-GL2P (being used as initiator) is used to polylactide block polymer being connected to HO-PEG-GL2P generations " PLA-PEG-GL2P " via ester bond.2 ethyl hexanoic acid tin (II) is used as being used for the catalyst that ring-opening polymerisation is acted on.

Finally, in dichloromethane morpholine and four (triphenylphosphines) closes palladium (being used as catalyst) and removes the allyl group on PLA-PEG-GL2P, to produce final product PLA-PEG- parts.The final compound is purified by being precipitated in 30/70% (v/v) ether/hexane.

Example 5：Nanoparticle preparation-nanoprecipitation (nanoprecipitation)

Nanoparticle can use GL1 or GL2 parts to prepare.According to the process shown in scheme 1, the PSMA inhibitor GL2 (it has the amino group being located in not crucial region is combined for PSMA) based on urea raw material Boc-Phe (4NHFmoc)-OH and diallyl glutamic acid commercially available from are synthesized.Nanoparticle is formed using nanoprecipitation：Polymeric ligands conjugates is dissolved in the miscible organic solvent of water and the other medicaments of medicine absorbed for spike particulate.Ligand surface density can be adjusted comprising extra non-functionalized polymer.Polymer solution is scattered in aqueous phase and gained particulate is collected by filtration.The particulate can be dry or be absorbed for cell in vitro or the test immediately of anti-tumor of prostate activity in vivo.

Scheme 1

Example 6：Nanoparticle preparation-emulsion process

Organic phase is formed by 5% solid (wt%), and fat-co-glycolide-PEG diblock copolymer (PLGA-PEG is handed over comprising 2% poly- third；45kDa-5kDa), 2% PLA (PLA；8.5kDa) with 1% docetaxel (DTXL), wherein docetaxel has structure

Organic solvent is ethyl acetate (EA) and phenmethylol (BA), and wherein BA includes 20% (wt%) organic phase.It is used to dissolve docetaxel using BA parts.The organic phase and aqueous phase are mixed with about 1: 5 ratio (oil phase: aqueous phase), wherein the aqueous phase is made up of 0.5% sodium taurocholate in water, 2%BA and 4%EA (wt%).By being simply mixed or by using rotor-stator homogenizer by two combined formation colostric fluids.Then, crushing instrument or high pressure homogenizer by using Probe Ultrasonic Searching ripple makes colostric fluid turn into miniemulsion.

Then, it is quenched miniemulsion by adding (0-5 DEG C) of the cold quench fluid of deionized water under mixing.The quench fluid: the ratio of emulsion is about 8.5: 1.Then, 25% (wt%) Tween 80 solution is added to quench fluid to reach about 2% whole Tween 80s.Then, the nanoparticle is separated by centrifugation or ultrafiltration/diafiltration.Then, nanoparticle suspension can use cryoprotector, and such as 10wt% sucrose is freezed.

It moreover has been found that except PLGA-PEG copolymers, adding PLA significantly increasing medicament loads.It is likely to, similarly plays a part of increasing encapsulation efficiency in itself using BA, encapsulation efficiency is also increased even if BA dissolvings DTXL is not needed.It was found that the temperature of quenching serves the effect of key in medicine is carried.Load medicine during relative to the quench fluid for using room temperature, load medicine is dramatically increased using cold quench fluid (typically remaining at 0-5 DEG C).

DTXL has extremely low water solubility, and it was found that non-encapsulated DTXL frequently forms crystal, and the crystal is difficult to separate from the nanoparticle of formation.Solubilizing agents for drugs (Tween 80) is added after miniemulsion is quenched.Tween 80 can effectively dissolve DTXL crystal and allow nanoparticle to be separated from non-encapsulated DTXL by following manner：By preventing the formation of DTXL crystal, and/or any DTXL crystal formed by effectively dissolving when miniemulsion is quenched.One group of nanometer emulsified condition of standard is as follows：

Control：

Add increases drug load as the homopolymer of the additive produced, and particle size is reduced, as follows：

It is quenched temperature

Here, being different from above-mentioned control for the control compared, because those are carried out in cold quenching temperature.

Exemplary parameter

The emulsion process of embodiment 7

Following processes use the solids content of increased oil phase.The general flow chart of process is described in figure 3, and the flow chart of process is described in Fig. 4.By reducing the solvent of emulsification oil phase, when nanoparticle is hardened, less drug loss is in cooling fluid.Selection solid and solvent system are excessively sticky to avoid, and this, which can be limited, is emulsified into~the ability of 100nm droplets.Preparaton is allowed to keep sufficiently low viscosity highly filled using relatively low molecular weight copolymer (~16kDa-5kDa PLA-PEG) and low molecular mass homopolymers (~7kDa PLA).There is the solvent system of suitable solvent ability to cause medicine to keep high concentration in the solution for selection.The use of co-solvent system (usually 79: 21 ethyl acetate: phenmethylol) is with 80: 20 polymer：The successive soln of up to 50% solid of docetaxel admixture creates conditions.

Organic phase is made up of and formed docetaxel (DTXL) and polymer (homopolymer, co-polymer and co-polymer) with part.The organic phase and aqueous phase are mixed with about 1: 5 ratio (oil phase: aqueous phase), wherein the aqueous phase is made up of surfactant and some dissolution solvents.In order to realize high load medicine, about 30% solid in organic phase is used.

Organic phase is made up of and formed the mixture of docetaxel (DTXL) and polymer (homopolymer, co-polymer and co-polymer) with part.Composition and organic solvent are listed in table.The organic phase and aqueous phase are mixed with about 1: 5 ratio (oil phase: aqueous phase), wherein the aqueous phase is made up of surfactant and some dissolution solvents.By being simply mixed or by using rotor-stator homogenizer by two combined formation colostric fluids.Then, colostric fluid is made to turn into miniemulsion by using high pressure homogenizer.Then, under mixing, in given temperature (being listed in the table below), be quenched the miniemulsion by adding deionized water.The quench fluid: the ratio of emulsion is about 8.5: 1.Then, 25% (wt%) Tween 80 solution is added to quench fluid to reach about 2% whole Tween 80s.This is applied to dissolve free, non-encapsulated medicine, and makes nanoparticle separation process feasible.Then, by centrifugation or ultrafiltration/diafiltration, the nanoparticle is separated.

Control

One group of nanometer emulsified condition of standard is provided below.Form containing whetheing there is-the particulate (non-targeted nano particulate) of part.

10% solid

20% solid

40% solid

30% solid with high surfaces surfactant concentration reduced for particle size；Targeted nano microparticle batches.

Embodiment 8：Nanoparticle preparation-emulsion process 2

Organic phase is made up of and formed docetaxel (DTXL) and polymer (homopolymer, co-polymer and co-polymer) with part.The organic phase and aqueous phase are mixed with about 1: 5 ratio (oil phase: aqueous phase), wherein the aqueous phase is made up of surfactant and some dissolution solvents.In order to realize high load medicine, about 30% solid in organic phase is used.

By be simply mixed or by using rotor-stator homogenizer by the beginning of two combined formation, thick emulsion.Rotor/stator produces uniform milky solution, and stirrer produces significantly greater thick emulsion.Observation stirring submethod causes the significant oil phase droplet that is attached to feed containers side, shows thick emulsion size for quality and non-key procedure parameter, and it should be made suitably to refine to prevent loss of yield or phase separation.Therefore, although in more mass rapid blender be probably suitable, but use the rotor stator as the standard method for forming thick emulsion.

Then, colostric fluid is made to turn into miniemulsion by using high pressure homogenizer.After continuously across (103) by refiner, the size of thick emulsion does not significantly affect the particle size.M-110-EH (Fig. 5).

Significantly affected it was found that refiner feed pressure has on the particle size of gained.On pneumatic (pneumatic) and electronic M-110EH refiners, it is found that reduction feed pressure also reduces the particle size (Fig. 6).Therefore, it is 4000-5000psi/ interaction chambers (interaction chamber) for the standard operating pressures that M-110EH is used, this is the minimum treat pressure on unit.The M-110EH also has the selection of one or two interaction chamber.It matches somebody with somebody restrictive Y- chambers according to standard, and 200 μm of Z- chambers of itself and lower limit are connected.It was found that when removing Y- chambers and being replaced with blank chamber, the particle size is actually reduced.In addition, removing Y- chambers dramatically increases the flow velocity of emulsion in process.

2-3 times pass through after, the particle size is not substantially reduced, and continuously across even particle size can be caused to increase.The result is summarized in the figure 7, and wherein placebo organic phase is by 25.5% polymer raw material (stock) (50: 50 16.5/5PLA/PEG：8.2PLA) constitute.Organic phase is 5: 1 emulsifications by O: W with standard aqueous phase, and carries out repeatedly (multiple) with caution by (discreet passes), each through quenching fraction emulsion later.The scale of instruction represents the total solid of preparaton.

The unexpected scale dependence of influence display of the scale to particle size.The trend is shown in the range of 2-10g batch size, and larger batch is produced compared with little particulate.Have confirmed, when considering the scale batch higher than 10g, scale dependence disappears.The amount of the solid used in oil phase is about 30%.Fig. 8 and 9 describes influence of the solid concentration to particle size and load medicine；In addition to 15-175 groups, all batches are placebos.For placebo batch, the value of % solids is represented, % solids are that medicine exists with standard 20%w/w.

Table A summarizes the parameter of emulsion process.

Table A

Then, it is quenched miniemulsion by adding deionized water in given temperature under mixing.In quenching unit operation, emulsion is added to cold aqueous quench fluid under agitation.This is suitable for the oil phase solvent for extracting signal portion, the nanoparticle effectively hardened for downstream filter.Cold quench fluid significantly improves drug encapsulation.Quench fluid: the ratio of emulsion is about 5: 1.

Then, 35% (wt%) Tween 80 solution is added to quench fluid to reach about 2% whole Tween 80s.Then, after emulsion quenching, the solution of Tween-80 is added, it acts as solubilizing agents for drugs, is easy to effectively remove non-encapsulated medicine during filtering.Table B points out the parameter of each quenching process.

Table B：Summarize the parameter of quenching process.

Temperature must keep enough cold T to be maintained in the particulate with suspension (sufficiently low solvent strength) dilute enough_gBelow.If Q: E ratio is not high enough to, then the solvent of higher concentration is plasticized the particulate and is easy to drug leakage.On the contrary, colder temperature is easy to the high drug encapsulation in low Q: E ratio (to~3: 1) so that the process may more effectively be run.

Then, by tangential flow filtration process separate nanoparticle with concentrate the nanoparticle suspension and buffer solution by solvent, free drug and solubilizing agents for drugs from quenching solution exchange into water.Using with the regenerated cellulose film that molecular weight section (MWCO) is 300.

Carry out constant volume diafiltration (DF) and remove quenching solvent, free drug and Tween-80.In order to carry out constant volume DF, buffer solution is added by retentate container with the phase same rate for removing filtrate.The procedure parameter of TFF operations is summarised in table C.Cross flow one speed refers to solution and flows through feeding-passage (channel) and the speed through film.The flowing provides removing can be with the power of polluted membrane and the molecule of limitation filtrate flows.Transmembrane pressure is the power that the permeable molecule of driving passes through film.

Table C：TFF parameters

Then, during post processing (workup), the nanoparticle slurry thermal cycle of filtering to elevated temperature.After first time is exposed to 25 DEG C, the medicine of the encapsulating of fraction (usually 5-10%) quickly discharges from nanoparticle.Because the phenomenon, the batch of cold state is kept to be easy to form free drug or medicine crystal during the non-chilled storage of delivering or arbitrary portion during whole post processing.During post-processing, by the way that nanoparticle slurry is exposed into elevated temperature, with the droplet (small drop) in medicine is carried for cost, ' should loosely encapsulate ' medicine can be removed and improve the stability of product.Table D is summarised in two embodiments of 25 DEG C of processing.Other experiments in~2-4 filtration volume it has been shown that be exposed to after 25 DEG C, product is sufficiently stable, and most of medicines encapsulated are not lost.The amount of the cold treatment before 25 DEG C are handled is used as using 5 filtration volumes.

Table D：

¹25 DEG C of post processings：After at least five filtration volume of various periods, sub-batch time (sublot) is exposed to 25 DEG C.Reporting range, its reason is have multiple sub-batch time to be exposed to 25 DEG C.

²Stability：Data are represented, are formed in the slurry before crystal, end-product can be 25 DEG C of times kept in 10-50mg/ml nanoparticle concentration (visual by microscopy).

³External cracking (burst)：Represent that medicine discharges in first time point (substantially immediately).

After the filtering process, nanoparticle suspension is by sterilizing level filter (0.2 μm, absolute (abosolute)).Using prefilter to protect sterilizing level filter, thereby using rational filter area/time for the process.Value is summarized in table E.

Table E：

Filtering group (filtration train) is E rtel Alsop Micromedia XL depth filter M953P films (0.2 μm, nominal)；Pall SUPRAcap, equipped with Seitz EKSP thickness filter medium (0.1-0.3 μm, nominal)；0.65/0.2 microns of sterilizing level PES filters of Pall Life Sciences Supor EKV.

It can use, the 0.2m for depth filter²Filter area/kg nanoparticle and 1.3m for the level filter that sterilizes²Filter area/kg nanoparticle.

Embodiment 9

The selectively targeted nanoparticle that can be prepared, the polymer comprising bio-compatible, its is conjugated to such as PEG, chemotherapeutics described herein, and optionally conjugated to GL1 or GL2.Exemplary nano particulate is displayed in Table 1：

Embodiment 10

The nanoparticle being displayed in Table 2, is prepared using the operation in embodiment 8.Prepared as shown in experiment 1 and 2 macromolecule and PLGA-PEG- comprising PLGA-PEG it is small-the high molecular nanoparticle of molecule ligand (SML).In experiment 3 and 4, the macromolecule comprising PLA-PEG, PLGA-PEG-SML macromolecule and PLA high molecular nanoparticle (DB=diblock copolymers) are prepared.

Small-molecular targeted part-functionalization macromolecule and unfunctionalized high molecular ratio can be adjusted, and use experiment 1, can prepare the nanoparticle with polymer composition, the polymer composition be about 0.94 mole of %, 4.63 moles of % and 9.01 mole of % functionalization macromolecule (with reference to " total Poly DB-GL2 mol% ").Extraly, using these methods, can prepare comprising about 0.015,0.073 and 0.143 weight % (for total polymer) small-molecule ligand nanoparticle (with reference to " Wt.%wrt GL2poly ").

The nanoparticle with functionalized polymeric can also be prepared, the polymer constitutes about 0.1-30, such as, 0.1-20, such as, the nanoparticle of the whole polymer composition of 0.1-10 molar percentages, and low-molecular weight ligands of the percentage by weight of nanoparticle are 0.001-5 for total polymer, for example, 0.001-2, for example, 0.001-1.

Embodiment 11

As described in table F and comparing, various nanoparticle preparatons are formed using embodiment 8：

Table F：

As shown in Figure 10, drug load is not sacrificed without using homopolymer PLA and significantly, it is possible to achieve optimal particle size.For the batch separately made using co-polymer, the batch release medicine with PLA homopolymer is significantly faster (Figure 11).Various polymer types and molecular weight are not added with optimizing the extra value of drug load and particle size.On the contrary, in 15% total solid with " candidate polymer " types of particles, its size is generally higher than 100-120nm target size.Typically increase in-vitro release rate (Figure 12) in the incorporation of 5wt% cetanols.

The cryoprotector of embodiment 12

Freezing nanometer, which emulsifies suspension of the nanoparticle in single deionized water, causes particles agglomerate.It is believed that this is due to crystallization and winding (Jaeghere et al. of the PEG chains on nano-particle surface；Pharmaceutical Research 16 (6), p 859-852).Excipient (sucrose, trehalose or mannitol) based on sugar can play a part of these nanoparticles of cryoprotection under the conditions of freeze/thaw, and concentration is low as 1wt% for dilution (~10mg/ml) nanoparticle suspension.A kind of preparaton includes 10wt% sucrose, and it contains required excessive sucrose and is to have identical osmolality with physiological saline.

Table G shows that 16/5PLA-PEG co-polymers are not easy to freeze-thaw aggregation.

Table G

Embodiment 13 goes palladium removing

Dosage level (ug/ days) based on human clinical trial, maximum acceptable palladium level is about 10ppm in PLA-PEG-GL2 compositions.Polymer (PLA-PEG-GL2) solution (20 or 35mg/mL) in dichloromethane (DCM) is loaded on 5g resin columns (with the advance solvations of 10mLDCM) and then uses 30mL DCM systems under gravity.Polymer removes solvent by using rotary evaporation, is then reclaimed in room temperature in vacuo drying.Polymer recovery gravimetric analysis is determined, and remaining palladium content passes through inductively coupled plasma (ICP) spectroscopic assay in Galbraith LaboratoriesInc.

Table H

Such as table H findings, it have rated in polymer charge/unit weight resin mercaptan, TMT, urea and thiourea functional group so that palladium level is less than 50ppm.However, only TMT (trithiocyanuric acid (trimecaptotriazine)) resin produces good (＞ 90%) polymer recovery.In addition, TMT resins also produce the palladium content under 10ppm acceptance thresholds.As a result seem there are some variability, this depends on the experiment condition used.Especially, when 5g TMT resin columns are loaded with 1050mg polymer, palladium is more effectively removed.This may be due to the more long residence time of type of polymer and palladium catalyst under experimental conditions.

The preparaton of embodiment 14

The preparaton of PLA-PEG- parts, PLA, PLA-PEG and docetaxel comprising nanoparticle is formed in sucrose/water composition：

Component	Nominal concentration (mg/mL)
		Docetaxel	5
PLA-PEG- parts	1.1
		PLA-PEG	21.4
PLA	22.5
		Sucrose	100
Water	In right amount

The release in vitro of embodiment 15

Release in vitro method is used to determine the initial cracking under conditions of room temperature and 37 DEG C and discharged from these nanoparticles.In order to keep sink condition (sink condition) and prevention nanoparticle to enter release sample, electrodialysis system is designed.After acquisition can granulate the ultracentrifuge of 100nm particulates, remove dialyser and centrifuge for the separation release medicine from the medicine of encapsulating.

Electrodialysis system is as follows：Pass through pipettor, 3mL docetaxel nanometers particulate (about 250 μ g/mL medicines/PLGA/PLA nanoparticles, corresponding to 2.5mg/mL solid concentrations) is placed on to the inner tube of 300kDa MWCO dialyzers (dialyzer) in the slurry of DI- water.The nanoparticle is suspension in the medium.Dialyzer is put into the vial (the 2.5% hydroxyl beta cyclodextrin in PBS) containing 130ml dissolution mediums, is stirred continuously to prevent the water layer not stirred in film/outer solution interface formation using oscillator in 150rpm.At predetermined time point, fetch the sample (1mL) of aliquot from outer solution (dialysate) and docetaxel concentration is analyzed by HPLC.

Centrifugation systems are run using condition of similarity in relatively low suspension vol (no dialysis bag).Sample centrifuges 30 minutes in 60,000g and measures the medicine of release for medicine assay supernatant.

The release in vitro of the docetaxel nanometer particulate of embodiment 16

By suspension (docetaxel of 10% weight and polymer (1.25wt%PLA-PEG-GL2 and 98.75wt%PLA-PEG, the Mn PLA=16Da of 90% weight of the docetaxel nanometer particulate such as prepared in embodiment 8；Mn PEG=5Da) it is placed in dialysis box and is incubated under agitation in 37 DEG C of PBS banks.The sample of dialysate is collected, and uses analysed by reverse phase HPLC docetaxel.In order to compare, conventional docetaxel experience identical process.Figure 13 describes release in vitro feature of the nanoparticle relative to conventional docetaxel.The docetaxel of encapsulating first in 24 hours it is linear from the gradually release of the release of polymer substrate substantially with remainder from the particulate within the period of about 96 hours.

The sirolimus nanoparticle of embodiment 17

Using the general operation of embodiment 8, with the total solid % of 80% (w/w) polymer-PEG or PLA containing homopolymer polymer-PEG (each 40% (w/w)), a collection of 5%, 15% and 30%, nanoparticle batch is prepared.Use solvent：21% phenmethylol and 79% ethyl acetate (w/w).For each 2 grams of batch size, the medicine using 400mg and the 16-5 polymer-PEG or 0.8g using 1.6g 16-5 polymer-PEG+0.8g 10kDa PLA (homopolymer).Use diblock polymer 16-5PLA-PEG or PLGA-PEG (L: G of 50: 50), and if used, the homopolymer：PLA, containing Mn=6.5kDa, Mw=10kDa and Mw/Mn=1.55.

Organic phase (medicine and polymer) is prepared in 2g batches：Medicine and polymer are added into 20mL scintillation vials.In solid concentration %, it is necessary to the quality of solvent show as follows：

I.5% solid：7.98g phenmethylol+30.02g ethyl acetate

Ii.15% solids：2.38g phenmethylol+8.95g ethyl acetate

Iii.30% solids：0.98g phenmethylol+3.69g ethyl acetate

The aqueous solution is prepared with 0.5% sodium taurocholate, 2% phenmethylol and 4% ethyl acetate in water.7.5g sodium taurocholates, 1402.5g DI water, 30g phenmethylol and 60g ethyl acetate are added in 2L bottles, and is mixed on agitating plate (stir plate) until dissolving.

In order to form emulsion, the aqueous phase and the ratio of oil phase used is 5: 1.Organic phase is poured into the aqueous solution and 10 seconds are homogenized in room temperature to form thick emulsion using IKA.Solution is supplied in 9Kpsi (45psi, on measuring instrument) by refiner (110S) for 2 times with caution to pass through to form nano-emulsion.

Emulsion is poured into the quench fluid (D.I. water) in 5 DEG C of ＜ while being stirred on agitator disk.The ratio of quench fluid and emulsion is 8: 1.35% (w/w) Tween 80 is added to the water to the ratio of the Tween 80: medicine with 25: 1 to be quenched.The nanoparticle concentrated by TFF and quench fluid on the TFF with 500kDa Pall boxes (2 film) with being concentrated into~100mL.The cold DI water of use~20 filtration volume (2 liters) is percolated to carry out, and the volume is reduced to lowest volume, then collects final slurry ,~100mL.The solid concentration of unfiltered final slurry is determined by following manner：Using the 20mL scintillation vials of taring are with the addition final slurries of 4mL and dried under vacuo on lyophilized/drying box, and determine the weight that is completely dried in the slurry of (dry down) of the nanoparticle in 4mL.The sucrose (0.666g/g) of concentration is added final slurry sample to obtain 10% sucrose.

The solid concentration of the final slurry of 0.45um filterings is determined by following manner：By filtering about 5mL final slurry sample, sucrose is then added by 0.45 μm of syringe filter；4mL filtered sample is added into the 20mL scintillation vials of taring and is dried under vacuo on lyophilized/drying box.

The remaining sample of unfiltered final slurry is freezed with sucrose.

Rapamycin (sirolimus) preparaton

The influence of solids content and the influence of the inclusion of PLA homopolymer are shown in fig. 14.

Extracorporeal releasing experiment is studied by the way that nanoparticle is dispersed in the PBS of 37 DEG C of the polysorbas20 (T20) containing 10% (w/w).Using T20 with increase solubility of the rapamycin in PBS with the detection holding level by HPLC and keep sink condition.In the dissolution medium for the 130mL that 3mL drug-carried nanometer is redispersed in tank with concentration known (about 250 μ g/ml).Select these volumes to ensure maxima solubility of Cmax of the medicine in dissolution medium always less than 10%, i.e. sink condition.The medium and nanoparticle suspension are stirred in 150rpm.At predetermined time point, centrifuge 1hr to separate nanoparticle from elution media in 50,000rpm (236,000g) 4ml aliquot.By elution media injected into HPLC to determine medicine from the release in nanoparticle.The release of rapamycin shows slow and sustained release, and such as Figure 15 shows.

Embodiment 18- CCI-779s

Nanoparticle such as is prepared in embodiment 17 and 8, except using the CCI-779 with 30% solids content before emulsification in organic phase：

The weight % and Figure 17 that Figure 16 describes CCI-779 describe nanoparticle for the different polymer nano particles with CCI-779.Such as the extracorporeal releasing experiment of embodiment 17 result show such as Figure 18 slow and sustained releases shown CCI-779 slow and sustained release.

The vinorelbine nanoparticle of embodiment 19

Using the general operation of embodiment 8, with the total solid % of 80% (w/w) polymer-PEG or PLA containing homopolymer polymer-PEG (each 40% (w/w)), a collection of 5%, 15% and 30%, nanoparticle batch is prepared.Use solvent：21% phenmethylol and 79% ethyl acetate (w/w).For each 2 grams of batch size, the vinorelbine using 400mg and the 16-5 polymer-PEG or 0.8g using 1.6g 16-5 polymer-PEG+0.8g 10kDaPLA (homopolymer).Use diblock polymer 16-5PLA-PEG or PLGA-PEG (L: G of 50: 50), and if used, the homopolymer：PLA, containing Mn=6.5kDa, Mw=10kDa and Mw/Mn=1.55.

Organic phase (medicine and polymer) is prepared in 2g batches：Medicine and polymer are added into 20mL scintillation vials.In solid concentration %, it is necessary to the quality of solvent show as follows：

I.5% solid：7.98g phenmethylol+30.02g ethyl acetate

Ii.15% solids：2.38g phenmethylol+8.95g ethyl acetate

Iii.30% solids：0.98g phenmethylol+3.69g ethyl acetate

The aqueous solution is prepared with 0.5% sodium taurocholate, 2% phenmethylol and 4% ethyl acetate in water.7.5g sodium taurocholates, 1402.5g DI water, 30g phenmethylol and 60g ethyl acetate are added into bottle, and is mixed on agitating plate (stir plate) until dissolving.

In order to form emulsion, the aqueous phase and the ratio of oil phase used is 5: 1.Organic phase is poured into the aqueous solution and 10 seconds are homogenized in room temperature to form thick emulsion using IKA.Solution is supplied in 9Kpsi (45psi, on measuring instrument) by refiner (110S) for 2 times with caution to pass through to form nano-emulsion.

Emulsion is poured into the quench fluid (D.I. water) in 5 DEG C of ＜ while being stirred on agitator disk.The ratio of quench fluid and emulsion is 8: 1.35% (w/w) Tween 80 is added to the water to the ratio of the Tween 80: medicine with 25: 1 to be quenched.The nanoparticle is concentrated by TFF and quench fluid is concentrated into~100mL in the TFF with 500kDa Pall boxes (2 film).The cold DI water of use~20 filtration volume (2 liters) is percolated to carry out, and the volume is reduced to lowest volume, then collects final slurry ,~100mL.The solid concentration of unfiltered final slurry is determined by following manner：Using the 20mL scintillation vials of taring are with the addition final slurries of 4mL and dried under vacuo on lyophilized/drying box, and determine weight of the nanoparticle in the 4mL slurry being completely dried.The sucrose (0.666g/g) of concentration is added final slurry sample to obtain 10% sucrose.

The solid concentration of the final slurry of 0.45um filterings is determined by following manner：By filtering about 5mL final slurry sample, sucrose is then added by 0.45 μm of syringe filter；4mL filtered sample is added into the 20mL scintillation vials of taring and is dried under vacuo on lyophilized/drying box.

The remaining sample of unfiltered final slurry is freezed with sucrose.

Vinorelbine preparaton：

^*=the release in vitro on sample

In three preparaton (16-5PLA-PEG of 30% total solid；16-5PLA-PEG+PLA；And 16-5PLGA-PEG+PLA) on carry out release in vitro, and use 10% urea in PBS solution to collect release in vitro data as dissolution medium in air chamber at 37 DEG C.Result is described in following table and Figure 19：

Embodiment 20- vincristine

Using the general operation of embodiment 8, the nanoparticle preparaton for including vincristine is prepared.

Vincristine preparaton

Ref. No.	Component	Composition (based on Wt. (%))
			50-103-3-5	MPEG (5k)-lPLA (16K)/vincristine	96/4
50-117-1-5	MPEG (5k)-lPLA (16K)/vincristine	95/5
			50-117-2-5	MPEG (5k)-lPLA (16K)/vincristine	96/4
50-103-4	MPEG (5k)-lPLA (16K)/lPLA (16K)/vincristine	46/46/8
			50-103-2	MPEG (5k)-lPLA (16K)/lPLA (16K)/vincristine	47/47/6

The analysis feature of vincristine preparaton

Carry out release in vitro on vincristine preparaton, and in air chamber use 10% urea in PBS solution to collect release in vitro data as dissolution medium at 37 DEG C.Figure 20 describes the release in vitro of some reference batch.

The pharmacokinetics of embodiment 21

The nanoparticle with vincristine and the pharmacokinetics (PK) of the nanoparticle with docetaxel such as prepared in embodiment 8 such as prepared in embodiment 20 is determined in Sprague-Dawley (SD) rat.In time=0, to rat (about 300g male Sprague Dawley, jugular vein is intubated) give passive target nanoparticle (the 10wt% medicines that medicine containing 5mg/kg and PTNP entrapped drug are given in dosage in the single dose intravenous of 0.5mg/kg free drugs or single dose intravenous, 90wt polymer (PLA-PEG, Mn PLA=16Da；Mn PEG=5Da, PTNP).Different time points upon administration, from jugular vein intubation by blood sample collection to the tubule containing heparin lithium, and prepare blood plasma.Then blood plasma level is determined by extracting medicine from blood plasma by lcms analysis.

Figure 21 describes vincristine and vincristine PTNP, and docetaxel and docetaxel PTNP PK features.

The analysis of the particle size of embodiment 22

Particle size passes through two kinds of technology-dynamic light scatterings (DLS) and laser diffraction analysis.Use the 660nm laser that is located in 90 ° of scatterings to carry out DLS at 25 DEG C, using Brookhaven ZetaPals equipment, in the waterborne suspension of dilution, and use Cumulants and NNLS methods (TP008) analysis.With Horiba LS950 equipment, in the waterborne suspension of dilution, using the HeNe laser and the LED positioned at 405nm positioned at 633nm in 90 ° of scatterings, laser diffraction is carried out and using Mie optical models (TP009) analysis.It is related from DLS output and the hydrodynamic radius of the particulate (including PEG ' hats '), and laser diffraction apparatus and the physical dimension of PLA particulates ' core ' are more relevant.

Embodiment 23- ligand densities

It is assumed that whole mean particle dia is equal to the Hydrodynamic diameter by Brookhaven particulate fields, nanoparticle is just spherical, and all hydrophilic PEG and part are expressed on the surface, and whole PEG is fully hydrated, the model of the microparticle surfaces can be set up, as shown in Table I：

Table I：The nano-particle surface model of the 100nm particulates of 16/5 co-polymer and 6.5kDa homopolymers

Embodiment 24- breast cancer tumours are targetted

There is the mouse kind of MX-1 xenograft in transplanting, have rated nanoparticle (10wt% docetaxels, 90wt the polymer (~1.25wt%PLA-PEG-GL2 of the intravenous administration such as prepared in example 8；With~98.75%PLA-PEG, Mn PLA=16Da；Mn PEG=5Da) (be labeled as BIND-14) for the conventional docetaxel with identical drug/polymer composition (PTNP) and non-targeting control particulate, suppress the ability of non-Prostate Tumor Growth.When tumour reaches 300mm³Average external volume, give test article (sucrose, docetaxel, PTNP, BIND-14) 3 times within 4 days every to mouse.Each treatment group is shown in Figure 22 with the mean tumour volume of time.

It has rated in the mouse for carrying mankind's MX-1 breast cancer xenografts, the ability that targeted nano particulate (BIND-14) enhancing docetaxel is delivered to tumour after intravenous administration, wherein mean tumour volume are 1700mm³.Using LC/MS/MS, after IV is administered 24 hours, in the tumor resection of the animal from the docetaxel for giving BIND-14, PTNP and routine, the concentration (ng/mg) of the docetaxel for docetaxel content is analyzed, and it is shown in fig 23.

The prostate cancer of embodiment 25 is targetted

It has rated in the Male SCID mice of allogeneic of mankind's LNCaP prostate cancers is carried, after intravenously administrable, nanoparticle (10wt% docetaxels, 90wt the polymer (~1.25wt%PLA-PEG-GL2 such as prepared in embodiment 8 is used；With~98.75%PLA-PEG, Mn PLA=16Da；Mn PEG=5Da；BIND-14), delivering from the nanoparticle of docetaxel to tumour.Male SCID mice subcutaneous vaccination has mankind's LNCaP prostate gland cancer cells.Three after inoculation are to surrounding, and single IV gives 5mg/kg docetaxel, and the docetaxel is the docetaxel of BIND-014 or routine.Mouse is killed after administration 2h or 12h.Docetaxel is determined from each group tumor resection and by LC-MS methods.

After 12 hours of the BIND-14 that 50mg/kg is given in single, receive the tumour docetaxel concentration in BIND-14 animal, for the tumour docetaxel concentration in conventional DTXL animals is received, it is high about 7 times, show that length-cycle P SMA- targeted nanos particulate as shown in figure 24 delivers more DTXL to tumor sites.

As shown in figure 25, also it have rated in LNCaP xenograft tumor models, the ability that BIND-014 suppresses tumour growth is given in repetition.Male SCID mice subcutaneous vaccination has mankind's LNCaP prostate gland cancer cells.After inoculation three to surrounding, mouse BIND-014, conventional docetaxel (DTXL), the DTXL being encapsulated in non-targeted nano particulate (PTNP) and medium (control) 4 times were given every 1 day.After 4 5mg/kg administrations, in the docetaxel for receiving routine or the animal of non-Target Particles (PTNP), reduction of the gross tumor volume in BIND-014 animal is received is bigger.The increase of docetaxel concentration causes more significant cytotoxic effect in tumour.

Equivalent

Normal experiment is used no more than, those skilled in the art will be recognized that or it was determined that a variety of equivalents of the embodiment of invention described herein.This equivalent is intended to covered in the claims below.

It is incorporated by reference into

Herein cited whole patents, the application of disclosed patent, the full content mode herein especially in full of network address and other bibliography are incorporated herein by reference.

Claims (52)

1. therapeutic nanoparticle, comprising：

The therapeutic agent of the percentage by weights of about 0.2- about 35；

The diblock PLA-PEG copolymer or diblock PLGA-PEG of the percentage by weights of about 10- about 99；With

The PLA or PLA-co-poly glycolic of the percentage by weights of about 0- about 75.

2. the therapeutic nanoparticle of claim 1, wherein the therapeutic agent is taxane.

3. the therapeutic nanoparticle of claim 1, wherein the therapeutic agent is vinca alkaloids.

4. the therapeutic nanoparticle of claim 3, wherein the therapeutic agent is vincristine or vinorelbine.

5. the therapeutic nanoparticle of claim 1, wherein the therapeutic agent is mTOR inhibitors.

6. the therapeutic nanoparticle of claim 5, wherein the therapeutic agent is sirolimus, CCI-779 or everolimus.

7. any one of claim 1-6 therapeutic nanoparticle, wherein the Hydrodynamic diameter of the therapeutic nanoparticle is about 60- about 120nm.

8. any one of claim 1-7 therapeutic nanoparticle, wherein the Hydrodynamic diameter is about 70- about 120nm.

9. any one of claim 1-8 therapeutic nanoparticle, wherein the therapeutic nanoparticle substantially retains therapeutic agent at least 5 days at 25 DEG C.

10. any one of claim 1-2 or 7-9 therapeutic nanoparticle, include the taxane medicament of the percentage by weights of about 10- about 20.

11. any one of claim 1,3,4 or 7-9 therapeutic nanoparticle, include the vinca alkaloids of the percentage by weights of about 10- about 20.

12. any one of claim 1 or 5-9 therapeutic nanoparticle, include the sirolimus of the percentage by weights of about 10- about 20.

13. any one of claim 1-12 therapeutic nanoparticle, includes the PLA-PEG copolymer of the percentage by weights of about 40- about 90.

14. the therapeutic nanoparticle of claim 13, wherein the PLA-PEG copolymer includes the polyethylene glycol of the PLA of the number-average molecular weight with about 15-100kDa and the number-average molecular weight with about 2- about 10Da.

15. the therapeutic nanoparticle of claim 13, wherein the PLA-PEG copolymer includes the polyethylene glycol of the PLA of the number-average molecular weight with about 15-20kDa and the number-average molecular weight with about 4- about 6kDa.

16. any one of claim 1-15 therapeutic nanoparticle, wherein when the particulate is placed in the phosphate buffer solution of room temperature, substantially release is below about 5% therapeutic agent immediately in 1 hour for it.

17. any one of claim 1-15 therapeutic nanoparticle, wherein when the particulate is placed in the phosphate buffer solution of room temperature, substantially release is below about 10% therapeutic agent immediately in 24 hours for it.

18. any one of claim 1-17 therapeutic nanoparticle, wherein when in the phosphate buffer solution that the particulate is placed on 37 DEG C, substantially release is below about 10% therapeutic agent immediately for it.

19. any one of claim 1-18 therapeutic nanoparticle, the PLA-PEG comprising the percentage by weights of about 30- about 50, the PLA or PLGA of the percentage by weights of about 30- about 50 and the percentage by weights of about 15- about 25 activating agent.

20. any one of claim 1-19 therapeutic nanoparticle, wherein the PLA has about 5- about 10kDa number-average molecular weight.

21. any one of claim 1-19 therapeutic nanoparticle, wherein the PLA has about 5- about 100kDa number-average molecular weight.

22. any one of claim 1-19 therapeutic nanoparticle, wherein the PLGA has about 8- about 12kDa number-average molecular weight.

23. any one of claim 1-19 therapeutic nanoparticle, wherein the PLGA has about 8- about 100kDa number-average molecular weight.

24. any one of claim 1-23 therapeutic nanoparticle, also includes the PLA-PEG for the percentage by weights of about 0.2- about 30 for being targeted ligand functionalized.

25. any one of claim 1-23 therapeutic nanoparticle, also includes the PLA-co-poly glycolic-PEG for the percentage by weights of about 0.2- about 30 for being targeted ligand functionalized.

26. the therapeutic nanoparticle of claim 24 or 25, wherein the targeting ligand is covalently bond to PEG.

27. any one of claim 1-23 therapeutic nanoparticle, also includes the PLA-PEG for the percentage by weights of about 0.2- about 10 for being targeted ligand functionalized.

28. any one of claim 1-27 therapeutic nanoparticle, PLA-PEG-GL2 or PLA-co-poly glycolic-PEG-GL2 also comprising the molar percentages of about 0.2- about 10.

29. any one of claim 1-27 therapeutic nanoparticle, also comprising selected from following polymer compounds：

Wherein R₁The C selected from H and being optionally optionally substituted by halogen₁-C₂₀Alkyl group；

R₂It is key, ester linker or acid amides linker；

R₃It is C₁-C₁₀Alkylidene or key；

X is 50- about 1500；

Y is 0- about 50；With

Z is about 30- about 200.

30. any one of claim 1-27 therapeutic nanoparticle, also comprising the polymer compound being expressed from the next：

Wherein y is that about 222 and z is about 114.

31. therapeutic nanoparticle, comprising：

The therapeutic agent of the percentage by weights of about 0.2- about 35；

The PLA-PEG copolymer or PLGA-PEG of the percentage by weights of about 30- about 99；

The PLA or PLA-co-poly glycolic of the percentage by weights of about 0- about 50；With

The PLA-PEG-GL2 or PLA of the percentage by weights of about 0.2- about 10-co-poly glycolic-PEG-GL2.

32. the therapeutic nanoparticle of claim 31, wherein the PLA-PEG-GL2 includes the PLA of the number-average molecular weight with about 10,000Da- about 20,000Da and with about 4,000- about 8, the polyethylene glycol of 000 number-average molecular weight.

33. the therapeutic nanoparticle of claim 32, comprising selected from following polymer compounds：

Wherein R₁Selected from H, and the C being optionally optionally substituted by halogen₁-C₂₀Alkyl group；

R₂It is key, ester linker or acid amides linker；

R₃It is C₁-C₁₀Alkylidene or key；

X is about 100- about 300；

Y is 0- about 50；With

Z is about 70- about 200.

34. the therapeutic nanoparticle of claim 33, wherein y is 0.

35. the therapeutic nanoparticle of claim 33 or 34, wherein x is about 170- about 260.

36. any one of claim 31-35 therapeutic nanoparticle, wherein z is about 80- about 130.

37. any one of claim 31-36 therapeutic nanoparticle, wherein PLA-PEG-GL2 is expressed from the next：

Wherein y is that about 222 and z is about 114.

38. any one of claim 1-37 therapeutic nanoparticle, also comprising fatty alcohol.

39. the therapeutic nanoparticle of claim 38, wherein the fatty alcohol is cetanol.

40. composition, multiple nanoparticles comprising any one of claim 1-39, and pharmaceutically acceptable excipient.

41. the composition of claim 39, includes the palladium below about 10ppm.

42. pharmaceutical composition, comprising：

Multiple polymer nano particles, respectively include the therapeutic agent of the percentage by weights of about 0.2- about 35, the polymer of the bio-compatible of the percentage by weights of about 70- about 99, and optionally fatty alcohol；The polymer of wherein described bio-compatible is selected from：A) PLA-PEG copolymer；B) PLGA-PEG；C) a) or b) and PLA or PLA-co-glycolic copolymer combination；With

Pharmaceutically acceptable excipient.

43. the pharmaceutical composition of claim 42, wherein the pharmaceutically acceptable excipient is sucrose.

44. the pharmaceutical composition of claim 43, wherein when a hour in the phosphate buffer solution that the nanoparticle is placed on 25 DEG C, it discharges the taxane medicament below about 10%.

45. any one of claim 42-44 pharmaceutical composition, wherein the nanoparticle substantially retains therapeutic agent at least 5 days at 25 DEG C.

46. any one of claim 42-45 pharmaceutical composition, wherein the therapeutic agent is selected from docetaxel, vincristine, vinorelbine, sirolimus and tersirolimus.

47. nanoparticle preparaton, comprising：

Any one of claim 1-39 multiple nanoparticles；

Sucrose；With

Water.

48. the nanoparticle preparaton of claim 47 the, wherein nanoparticle/sucrose/water is about 5-10%/10-35%/60-90% (w/w/w).

49. the nanoparticle acceptable in the treatment suitable for treating solid tumor, comprising：

Wherein y is that about 222 and z is about 114；The activating agent of the percentage by weights of about 5- about 25.

50. the nanoparticle acceptable in the treatment of claim 49, wherein the activating agent is selected from mTOR inhibitors, taxane or vinca alkaloids.

51. treating the method for prostate or breast cancer, including give any one of the claim 1-50 of patient in need's effective dose any therapeutic nanoparticle or composition.

52. treating the method for prostate or breast cancer, including the therapeutic nanoparticle of patient in need's effective dose is given, the therapeutic nanoparticle is included：

The Docetaxel of the percentage by weights of about 0.2- about 35；

The PLA-PEG copolymer or PLGA-PEG of the percentage by weights of about 30- about 90；

Optionally, the PLA or PLA-co-poly glycolic of the percentage by weights of about 5- about 75；With

The PLA-PEG-GL2 or PLA of the percentage by weights of about 0.2- about 10-co-poly glycolic-PEG-GL2.

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