CN1747721A - Composition for chemoembolotherapy of solid tumors - Google Patents

Abstract

A composition for chemoembolotherapy of solid tumours comprises particles of a water-insoluble water-swellable synthetic anionic polymer and, absorbed therein an anthracycline. Suitably the polymer is a poly(vinyl alcohol) based polymer and the drug is doxorubicin.

Description

The compositions that is used for the chemoembolization treatment of solid tumor

The present invention relates to contain polymerization embolization material and a kind of compositions that is attached to the therapeutic agent in this polymeric matrix.This compositions is used for occlusion of bone tumors and discharges cytotoxic agent to tumor.

Embolotherapy is field of developing in the interventional medicine, and still it will rely on conduit and arrive assigned address through tremulous pulse usually, thereby discharges a kind of reagent to block specific blood vessel.This treatment has been used to block the blood supply such as some hypervascular tumor of liver cell tumor, and is just becoming a general choice of fibroma uteri treatment recently.

The embolization material of clinical practice has certain scope, needs to be discharged into the thromboembolism position through conduit, thereby is released in the blood flow and with its blocking-up.This can realize from physically blocking blood vessel by using little particle or bead, perhaps under the liquid embolizing agent situation, need certain phase transformation or reaction that fluidity substance is solidified and formation casting mold thing (cast) in blood vessel.

The most general suppository based on microgranule is polyvinyl alcohol (PVA) foam particle (as ivalon) of having used many decades.Recently, the microgranule rather than the sheet form of this material occurred, therefore need before release, not make graininess by surgeon.

In WO-A-0168720, narrated embolotherapy compositions based on PVA.Earlier the PVA derivatization is formed the big monomer with acrylic acid side-chain radical.Choose wantonly then and in the presence of comonomer, make these acrylic acid groups polymerizations, form to meet the inflatable but water-insoluble polymer substrate of water.Polyreaction can take place in position, and PVA has had water-insoluble after being released into blood vessel arrival thromboembolism position by this.In addition, polymerization also can be carried out before release, forms microsphere usually and is discharging with the form of suspension in aqueous media.

In WO-A-0168720, suggestion can add bioactivator in embolizing compositions, and activating agent can be discharged from formed hydrogel.One class activating agent is a chemotherapeutant.The example of chemotherapeutant has cisplatin, amycin and mitomycin.The document has provided about activating agent being attached to some general guidances of the method in the embolizing compositions.If compositions is a kind of liquid of in-situ solidifying, activating agent can be at an easy rate and liquid mixing.If these goods are preformed, then suggestion can come up in conjunction with activating agent by " sealing " or by being coated onto the surface.Also therapeutic agent is not attached to example successful in the compositions of any kind.

With aldehyde cross-linking agent such as glutaraldehyde make poly hydroxy ethyl acrylate, the microsphere of the hydrogel materials that is cross-linked to form through the polymethyl methacrylate and the PVA of hydrolysis is also as suppository.Hydroxyethyl methylacrylate can with comonomer as containing the comonomer copolymerization of acidic-group.For example, the cross-linked copolymer that hydroxyethyl methylacrylate and about 1-2 mole % acrylic acid form by the crosslinked back of 0.3-1.0 mole % ethylene glycol dimethacrylate, its equilibrium moisture content scope and has been used for many years with the contact lens form between 55-60% (weight).

A kind of thromboembolism product on the market is sold by Biosphere company, and this product contains three acryloyl gelatin (trisacrylgelatin) microspheres that have collagen coating.Collagen protein has positive charge generally under physiological pH.At Ball, D.S.et al., J.Vasc.Interv.Radiol. (2003), 14, among the 83-88, Biosphere company proves, when a series of medicament mixed that microsphere and normal and embolizing compositions give simultaneously, the mechanical property of microsphere is not adversely affected.Amycin, cisplatin and mitoxantrone have been carried out special test.

Amycin and other anthracene nucleus classes have been attached in many delivery systems based on polymeric matrix, for example polylactide class or poly-Acetic acid, hydroxy-, bimol. cyclic ester class microsphere and crosslinked fibrin is former and albumin microsphere.Juni, and people such as K. (Chem.Pharm.Bull. (1985), 33 (1), 313-318) described and be attached to amycin in the polylactic acid microsphere and make the said composition intra-arterial be released into the liver of Canis familiaris L..The said composition thromboembolism peripheral arterial of liver.The microsphere of these types is harder, is difficult for storage and release.That amycin is covalently bound to cross-linking polyvinyl alcohol surface and detected its cell toxicant characteristic (Wingard, L B et al.CancerResearch (1985) 45 (8) 3529-3536).Because medicine is covalently bound on the polymer, before discharging, must downcut from the surface, therefore under physiological condition, possibly can't discharge.

Jones, people such as C. (Brit.J.Cancer (1989) 59 (5)) have described and amycin is attached on the ion exchange microsphere and with said composition the rat tumor model has been carried out the chemoembolization treatment.

A kind of new compositions that is applicable to thromboembolism of the present invention comprises and has a kind of water inflatable but water-insoluble polymer substrate and a kind of particle that is absorbed in the water-soluble therapeutic agents in this substrate of meeting, it is characterized in that: this polymer totally has negative charge in the pH6-8 scope, when particle expands in water when reaching balance, its particle size is between 40-1500 μ m, and therapeutic agent is to have at least one amino anthracycline compound.

Polymer among the present invention must be that chance water is inflatable but water-fast.Therefore, in the presence of waterborne liquid, polymer can form hydrogel.Generally speaking this polymer is a covalent cross-linking, but this polymer part ion is crosslinked at least also is suitable.This polymer can be by the ethylene linkage unsaturated monomer in difunctionality or more carry out polymerization in the presence of the cross-linking monomer of high functionality and make, and the ethylene linkage unsaturated monomer comprises anionic monomer.Can use the copolymer of hydroxyethyl methylacrylate, acrylic acid and cross-linking monomer such as ethylene glycol dimethacrylate or methylene-bisacrylamide, as be used for copolymer based on the contact lens of etafilcon A.

Another kind ofly can be used to form that to meet water inflatable but polymer water-fast substrate is the polyvinyl alcohol with aldehydes cross-linking agent such as glutaraldehyde cross-linking.For this product, polyvinyl alcohol must be an anionic, for example by making the monomer and the hydroxyl reaction that contain acidic functionality that the anionic side chains group is provided.The example of suitable reagent has diacid, as dicarboxylic acids.

If polymeric matrix all contains the polyvinyl alcohol macromonomer of a more than ethylene linkage unsaturated terminal chain group by each molecule, by making with the ethylene linkage unsaturated monomer combined polymerization that comprises a kind of acid monomer, then the present invention has special value.The PVA macromonomer can have suitable molecular weight such as 1000-500 by for example providing, between the 000D, and preferably 10,000-100, between the 000D, the PVA polymer that has ethylene or acrylic acid side-chain radical is made.The acrylic acid side-chain radical can be reacted by some hydroxyl by the acid of acrylic or methacrylic for example and PVA and generate ester bond and form.Can make vinyl group be aggregated to method on the polyvinyl alcohol, narration to some extent among the preferred US 5,508,317 and 5,583,163 at for example US 4,978,713.For example, preferred macromonomer comprises the skeleton of a polyvinyl alcohol, and this skeleton is connected with (alkyl) acrylamide moieties by a cyclic acetal key.Embodiment 1 has described the synthetic of a kind of like this macromonomer.Preferred each molecule has the PVA macromonomer of about 2-20 (for example 5-10) thiazolinyl side-chain radical.

When the PVA macromonomer when comprising a kind of ethylene linkage unsaturated monomer combined polymerization of acid monomer, described acid monomer preferably has general formula I

Y ¹BQ

Y wherein ¹Be selected from:

CH ₂＝C(R)-CH ₂-O-，CH ₂＝C(R)-CH ₂OC(O)-，CH ₂＝C(R)OC(O)-，CH ₂＝C(R)CH ₂＝C(R)CH ₂OC(O)N(R ¹)-，R ²OOCCR＝CRC(O)-O-，RCH＝CHC(O)O-，RCH＝C(COOR ²)CH ₂-C(O)-O-，

With

Wherein:

R is hydrogen or C ₁-C ₄Alkyl;

R ¹Be hydrogen or C ₁-C ₄Alkyl;

R ²Be hydrogen or C _1-4Alkyl or BQ, wherein B and Q are defined as follows:

A is-O-or-NR ¹-;

K ¹Be group-(CH ₂) _rOC (O)-,-(CH ₂) _rC (O) O-,-(CH ₂) _rOC (O) O-,-(CH ₂) _rNR ³-,-(CH ₂) _rNR ³C (O)-,-(CH ₂) _rC (O) NR ³-,-(CH ₂) _rNR ³C (O) O-,-(CH ₂) _rOC (O) NR ³-,-(CH ₂) _rNR ³C (O) NR ³-(radicals R wherein ³Identical or different) ,-(CH ₂) _rO-,-(CH ₂) _rSO ₃-or optional and B ¹In conjunction with and form a covalent bond, r is 1 to 12, R ³Be hydrogen or C ₁-C ₄Alkyl;

B is straight or branched alkane two bases, oxyalkylene, alkane two basic oxygen alkane two bases or alkane two base oligomeric (oxygen alkane two bases) chains, and optional one or more fluorine atom that comprises of this chain replaces chain up to forming perfluor; Perhaps, if Q or Y ¹Comprising a terminal carbon with the B keyed jointing, is a covalent bond;

Q is an anionic group.

Described anionic group can be such as carboxylate, carbonate, sulfonate, sulfate, nitrate, phosphonate or phosphate group, preferred sulfonate groups.Monomer can be with the form polymerization of free acid or salt.The pKa value of preferred conjugate acid is less than 5.

In the monomer of general formula I, Y ¹Be preferably CH ₂=CRCOA-group, wherein R is hydrogen or methyl, preferable methyl, A is preferably NH.B is preferably 1-12 carbon atom, alkane two bases of preferred 2-6 carbon atom.

A kind of particularly preferred monomer type is (alkyl) acrylamide alkyl sulfonic acids, as 2-acrylamido-2-methyl isophthalic acid-propane sulfonic acid (AMPS).

In the ethylene linkage unsaturated monomer, can comprise diluting monomer, for example non-ionic monomer.This monomer has the pKa that is beneficial to the control acidic-group, and the hydrophilic or the hydrophobicity of control product provide hydrophobic region in polymer, perhaps only play inert diluent.The example of nonionic diluting monomer has (alkyl) alkyl acrylate and (alkyl) acrylamide, especially contains this compounds of the alkyl of 1-12 carbon atom; (alkyl) alkyl acrylate that hydroxyl and dihydroxy replace and (alkyl) acrylamide; Vinyl lactam; Styrene and other fragrant monomers.

The ethylene linkage unsaturated monomer can also comprise zwitterionic monomer, for example hydrophilic, lubricity, biocompatibility and/or the blood compatibility in order to increase particle.Suitable zwitterionic monomer is narration to some extent in our previous disclosed WO-A-9207885, WO-A-9416748, WO-A-9416749 and WO-A-9520407.Zwitterionic monomer is preferably 2-methacryloxy-2 '-trimethylamine ethyl phosphonic acid inner salt (MPC).

In polymeric matrix, the anion level is preferably at 0.1-10meq g ^-1Scope, preferably be at least 1.0meq g ^-1

When PVA macromonomer and other ethylene linkage unsaturated monomer combined polymerizations, PVA macromonomer and other monomeric weight ratios are preferably in 50: 1 to 1: 5 scopes, more preferably in 20: 1 to 1: 2 scopes.In the ethylene linkage unsaturated monomer, the content of anionic monomer is preferably at 10-100 mole % scope, preferably at least 25 moles of %.

By weight analysis determining, chance water is inflatable but water-insoluble polymer preferably has the equilibrium water content of 40-99% (weight), preferred 75-95%.

Polymer can form particle by several method.For example, crosslinked polymer can be made whole block material such as thin slice or bulk, is ground into required size then.In addition, cross linked polymer also can itself just be made particulate forms, for example carries out polymerization with monomeric droplet in the decentralized photo of the successive carrier that do not dissolve each other.Known example such as suitable Water-In-Oil polymerization can produce the particle that has the expection size when expanding.For example, US 4,224, and 427 have described and exist under the situation of suspending agent, and by water-soluble monomer being distributed in the successive solvent phase, forming maximum gauge is the method for the spherical beads of the same size of 5mm.Can add stabilizing agent and surfactant size with the control dispersed phase particles.After the polymerization, crosslinked microsphere reclaims with known method, cleans and optional sterilization.Particle for example microsphere preferably expands in waterborne liquid and carries out classification according to their size.

Therapeutic active substance used in the present invention is a kind of anthracycline compound, and it comprises the anthraquinone radicals that is connected with an amino sugar.It is believed that the amino on the sugar combines with anionic group in the polymeric matrix, thereby realize the controlled release after high-caliber drug loading and the administration.

The example of suitable anthracycline compound has general formula I I

We have found that the amycin that the effectiveness of various tumors has been obtained full test has interesting especially medicine carrying and release characteristics.As if this medicine has special affinity to polyvinyl alcohol-grafting-acrylamido propane sulfonic acid copolymer, and therefore high-caliber amycin can be attached in this polymer and how in a few days discharge.

In the present invention, non-covalent being attached on the polymeric matrix of medicine is very important.

Therapeutic active substance can be attached in the polymeric matrix by several different methods.In one approach, polymerization or crosslinked before therapeutic active substance can mix the mixture of for example a kind of monomer or macromonomer mixture or a kind of crosslinkable polymer and cross-linking agent with a kind of polymer precursor.In addition, active substance also can reload wherein behind crosslinked polymer.For example, microgranular dry polymer can expand in the solution (preferred aqueous solutions) of therapeutic active substance, optional subsequently remove unabsorbed medicine also/or evaporating solvent.Active substance is dissolved in the solution such as organic solvents such as ethanol (or more preferably water), can be ejected on the moving bed of particle, and medicine is being absorbed in the particle body when desolvating removing by this.Be the most easily, we have found that can be only with the expansion particle and the drug solution Long contact time that are suspended in continuous liquid medium such as the water, and medicine is absorbed in the particle body thus.It is believed that this is similar to cation exchange type process.Inflating medium can be removed subsequently, perhaps easily inflating medium and particle be remained as the part of product together, be used as suppository later on.

In an especially preferred embodiment, the expansion particle by simple gel/liquid isolation technics with do not separated by the inflating medium of matrix absorption, for example the filter that via hole diameter is suitable (being glass filter easily) filters.Have seldom or do not have the expansion particle slurry of the outer liquid of particle can be pumped in the suitable hold-up vessel and preserve with sterilization with former state.Find that this particle slurry is enough stable,, also do not have drug loss because in the storage process of this form, seldom there is liquid to ooze out.

In addition, the suspension of particle also can be removed any residual medicine carrying solution by filtering, and particle can come dry by any classical way that is used for the dry drug product.This air drying under room temperature or high temperature or decompression or the vacuum; Classical lyophilization; Atmospheric freeze drying; The solution of supercritical fluid is strengthened dispersion (SEDS).In addition, the microsphere of medicine carrying can replace water with organic solvent and dewater by series of steps, then makes more volatile organic solvent evaporation.Organic solvent should select can not dissolved substance solvent.

In brief, a typical classical freezing dry process can followingly carry out: a duplicate samples packing in some vials that partly clogged, is placed on bottle in the freeze dryer on the refrigerative temperature control frame.Reduce the temperature of temperature control frame, sample is freezed to determine temperature uniformly.After freezing the finishing, the pressure that the pressure in the reduction exsiccator is extremely determined is to start elementary drying.In elementary dry run, steam is progressively removed from freeze piece by distillation, and the temperature of temperature control frame is controlled in constant low temperature simultaneously.Secondary drying starts by improving temperature control frame temperature and further reducing constant pressure, and therefore the water that is absorbed in the partial desiccation piece can be removed the level that reduces to hope up to the moisture of remnants.Bottle can seal in position, if desired can be at the protective atmosphere lower seal.

The atmospheric freeze drying method is to finish by the Rapid Cycle of carrying out very exsiccant air on frozen product.Compare with the freeze-drying method of classics, antivacuum lyophilization has many advantages.The circulation dry gas has improved the heat and the mass transfer of freezing sample, and this does to such an extent that be identical sooner with washings in the weather of wind is arranged.Most of work in this field are all relevant with food production, and the reservation amount of having observed the volatile aromatic chemical compound increases to some extent, and it is still uncertain to the exsiccant potential benefit of biological product.Attractive especially is that what use normal pressure spray drying means obtained is free-pouring fine powder rather than block.Can obtain to have the particle of submicron order diameter, this is than usually by grinding little ten times on the particle that obtains.The essence of microgranule and its high surface area cause producing and are easy to rehydrated product, at present also can not be to can sucking and use required particle size through skin and carry out refined control, but still there are potentiality in this field.

For suffering from solid tumor such as liver cell tumor and need carrying out the compositions that the patient of embolotherapy gives is the water slurry that contains the expansion particle that absorbs the drug.Usually wish that suspension mixed as the conventional contrast agent that is used for the gel-type embolizing compositions with preparation before discharging.For example, the water slurry that contains the expansion particle that absorbs the drug can mix with liquid contrast agent such as 2: 1 to 1: 2 by volume amount of iodized oil (preferred about 1: 1) that suppository uses with common before facing administration.Absorb the drug but seldom or not contain the embodiment of the expansion particle slurry of the outer liquid of particle for containing among the present invention, particle slurry and contrast agent can mix before facing release similarly, for example, preferably mix in the amount of 1: 2 to 1: 1 scope by volume 1: 5 to 2: 1 scope.When the compositions that contains medicine is supplied with use with exsiccant form, exsiccant particle can be added in the contrast agent, perhaps preferred elder generation expands in aqueous medium such as normal saline with formation serosity or suspension, mixes with contrast agent before discharging then.In addition, except that anthracycline compound, particle can also load contrast agent in advance.The compositions of administration also can be mixed mutually with the other treatment agent, perhaps also can separate administering drug combinations with the other treatment agent.Usually, conventional releasing device such as the administration from injector syringe of intra-arterial conduit of compositions.

Need the patient's of embolotherapy embolizing compositions to discharge with disposable single dose.The thromboembolism process adopts routine techniques to monitor by following the tracks of contrast agent.May find, be preferably in after for the first time dosage gives a period of time, as after usefulness contains the combination treatment 4-10 week of amycin for the first time, discharge the embolizing compositions of second-dose, with the new blood vessel that forms of thromboembolism to tumor feeding, the embolizing compositions of preferred second-dose preferably can be used for chemoembolization compositions of the present invention.Compositions is with each treatment 25-100mg/m ²The drug dose administration of scope, but also can behind the sufficient safety evaluation of process, use higher dosage.The preferred dose of the each treatment of amycin can be greater than 50mg/m ², for example reach 100mg/m ²Or it is higher.It has been generally acknowledged that the dosage that the each treatment of each patient is higher than 150mg is worthless.

As a second aspect of the present invention, provide a kind of anthracycline compound to be used for solid tumor is carried out the purposes of the compositions of embolotherapy in preparation, in this treatment, anthracycline compound discharges from a kind of polymeric matrix, and described polymeric matrix is to be formed by polyvinyl alcohol macromonomer and the unsaturated anionic monomer combined polymerization of ethylene linkage that each molecule has at least two ethylene linkage unsaturated terminal chain groups.

In this aspect of the invention, polymeric matrix can form in position.Like this, the fluid composition that contains macromonomer, anionic monomer and anthracycline compound can be released in patient's the blood circulation and be placed in and can form the thromboembolism gel thus under the condition of target site initiated polymerization.In addition, polymeric matrix also can be pre-formed before administration, as described in a first aspect of the present invention.

PVA macromonomer and anionic monomer are preferably as described in the above first aspect.Other monomers also can carry out combined polymerization as described in first aspect present invention.

The present invention is specified by following examples, and its result is as shown below:

Fig. 1 shows the result of embodiment 2;

Fig. 2 shows the result of embodiment 3;

Fig. 3 shows the result of embodiment 4;

Fig. 4 shows the result of embodiment 5;

Fig. 5 shows the result of embodiment 6;

Fig. 6 a and b show the result of embodiment 7.

Embodiment 1: the method for preparing microsphere general introduction

Nelfilcon B macromonomer is synthetic:

Synthetic first step in stage of microsphere comprises preparation Nelfilcon B, and it is a kind of polymerizability macromonomer from widely used water-soluble polymer PVA.(88% hydrolysis contains 12% acetate, and mean molecule quantity is about 67, and 000D) (150g) (Clariant, Charlotte, NC USA) joins in one 2 liters the glass reaction container with Mowiol 8-88 polyvinyl alcohol (PVA) powder.Along with light and slow gently stirring the down adds 1000ml water and will make the mixing speed increase bring up to 400rpm.Dissolve fully in order to ensure PVA, the temperature raising is increased to 99 ± 9 ℃ kept 2-3 hour.Cold really but after room temperature, with the time N-acrylamide ethylhexanal (NAAADA) (Ciba Vision, Germany) (PVA of 2.49g or 0.104mmol/g) is mixed in the PVA solution, adds concentrated hydrochloric acid (100ml) subsequently, with the addition of catalyzing N AAADA by ester interchange and PVA.Be reflected at room temperature and carried out 6-7 hour, use the sodium hydroxide solution of 2.5M to be neutralized to pH7.4 then and come cessation reaction.The sodium chloride that is generated and all unreacted NAAADA remove (step 2) with diafiltration.

The diafiltration of macromonomer:

The principle of diafiltration (tangential flow filtration) is to make the feedstock solution (being Nelfilcon B solution in this example) that needs purification stride across the circulation continuously of filter membrane surface, filter membrane can make unwanted material (NaCl, NAAADA) see through becomes garbage, has enough little aperture simultaneously and can stop reservation liquid to be stayed in the circulation by making it.

Nelfilcon B diafiltration uses rustless steel Pellicon 2 pony supports to carry out, and the molecular cut off that is stacked with some its apertures on this support is 3000 0.1m ²Cellulose filter membrane (Millipore Corporation, Bedford, the MA U.S.).The weight average molecular weight of Mowiol 8-88 is 67000, therefore sees through the limited in one's ability of filter membrane.

In the flask that macromonomer is housed, put into magnetic stirring bar and flask is placed on the agitating plate.Use LS24 VI type conduit, the MasterflexLS peristaltic pump through Easy Load II pump head is installed joins solution in the diafiltration assembly.Nelfilcon with about 50 pounds/square inch in the filter membrane cocycle to quicken infiltration.When solution is concentrated to about 1000ml, add entry to keep constant volume, up to extra adding 6000ml water with the speed identical with depleted filter liquor gathering speed.After finishing immediately under 25 ℃ with solution concentration to the 20-23% solid, viscosity is the 1700-3400 centipoise.Nelfilcon comes qualitative evaluation by GFC, NMR and viscosity.

Microsphere is synthetic:

With the synthetic microsphere of suspension polymerization, wherein water (Nelfilcon B) is joined in the organic facies immiscible with it (butyl acetate).Adopt rapid mixing can make water disperse to form drop, the size of drop and stability be subjected to such as the ratio of mixing speed, viscosity, water and organic facies and influence biphase between the control of factors such as use of the stabilizing agent of interface energy and surfactant.Made the microsphere of two series, shown in promptly low AMPS series and the higher AMPS series, they composed as follows.

The high AMPS of A:

Water: about 21%w/w Nelfilcon B solution (about 400 ± 50g)

About 50%w/w 2-acrylamido-2 methyl propane sulfonic acid sodium salt (140 ± 10g)

Purified water (137 ± 30g)

Potassium peroxydisulfate (5.22 ± 0.1g)

Tetramethylethylenediamine TMEDA (6.4 ± 0.1ml)

Organic facies: n-butyl acetate (2.7 ± 0.3L)

10%w/w cellulose acetate-butyrate in the ethyl acetate (46 ± 0.5g) (stabilizing agents)

Purified water (19.0 ± 0.5ml)

B hangs down AMPS:

Water: about 21%w/w Nelfilcon B solution (about 900 ± 100g)

About 50%w/w 2-acrylamido-2-methyl propane sulfonic acid sodium salt (30.6 ± 6g)

Purified water (426 ± 80g)

Potassium peroxydisulfate (20.88 ± 0.2g)

TMEDA(25.6±0.5ml)

Organic facies: n-butyl acetate (2.2 ± 0.3L)

10%w/w cellulose acetate-butyrate (CAB) (92 ± 1.0g) in the ethyl acetate

Purified water (16.7 ± 0.5ml)

The 4000ml reactor of the heating bath of the feedback transducer that computerizeds control (Julabo PN 9-300-650) heating tape interlayer with continuous detecting reaction temperature.

Under 25 ℃ butyl acetate is added in the reactor, add CAB solution and water then.System uses nitrogen purging 15 minutes, adds the PVA macromonomer then.Add TMEDA and under nitrogen, temperature is risen to 55 ℃ of maintenances 3 hours, cause the crosslinked of dispersive PVA solution.Cross-linking reaction is undertaken by the polyreaction of redox initiation, that is, the amino of TMEDA and the peroxidating radical reaction in the potassium peroxydisulfate generate free radical.The polymerization of the two keys on these free radicals initiation PVA and the AMPS and crosslinked then, thus dispersive PVA-AMPS droplet is changed into insoluble polymer microballoon.After being cooled to 25 ℃, product is transferred to and carries out purification on the filtration reactor, wherein removes by filter butyl acetate, carries out following steps then:

Clean to remove butyl acetate and CAB with 2 * 300ml ethyl acetate

Balance is 30 minutes in ethyl acetate, filters then

Clean under vacuum filtration with 2 * 300ml ethyl acetate

Balance 30 minutes and remove by filter ethyl acetate, CAB and water in acetone

Clean under vacuum filtration with 2 * 300ml acetone

Equilibrate overnight in acetone

Clean under vacuum with 2 * 300ml acetone

55 ℃ of following vacuum dryings 2 hours are to remove residual solvent

Dyeing:

This step is chosen wantonly, but usually when medicine loads with coloured active matter this step be unnecessary (just can provide color) because of this coloured active matter itself.When hydration, microsphere contains the water of have an appointment 90% (w/w), is difficult to observe.For the ease of observing in clinical setting, microsphere is dyed blueness with reactive blue 4 dyestuff (RB4).RB4 is a kind of soluble chloride triasine dyes, and it can generate the covalency ehter bond with the hydroxyl side-chain radical reaction on the PVA skeleton under alkali condition.Be reflected under the pH12 (NaOH) and carry out, the HCl that is therefore generated can be neutralized and generate NaCl.

Before dyeing, microsphere is that portion is divided into many parts (handling respectively) by hydration fully again and with 35g.Dyeing liquor is prepared as follows: 0.8g RB4 is dissolved in 2.5M NaOH solution (25ml) and the water (15ml), it is joined in 2 liters of microspheres in the 80g/l saline solution then.Mix after 20 minutes, remove unreacted dyestuff piece with 32 μ m sieve collection product and rinsing.

Extract:

Adopt a kind of thick extracting method to remove all RB4 unconjugated or non-specific adsorption.Used scheme is as follows:

Balance is 5 minutes in 2 premium on currency.On sieve, collect and rinsing.Repeat 5 times.

Balance in the solution of 2 liters of 80mM sodium hydrogen phosphates in 0.29% (w/w) saline solution.Be heated to boiling 30 minutes.Cooling is collected on sieve and is washed with 1 liter of saline solution.Repeat again 2 times.

Collect, washing on sieve, and in 2 premium on currency balance 10 minutes.

Collection was also dewatered 30 minutes in 1 liter of acetone.

Merge all each part microspheres and equilibrate overnight in 2 liters of acetone.

Screening:

Prepared microspheres product magnitude range makes its classification at the 100-1200 micron thereby must use the certain limit slot size to sieve, and obtains following specified distribution.

1.100-300μm

2.300-500μm

3.500-700μm

4.700-900μm

5.900-1200μm

The screening before, with the microsphere vacuum drying to eliminate solvent, then in 60 ℃ in water balance with hydration fully again.Use has 15 inches rustless steel screening dishes, slot size sieves microsphere at 316 liters of rustless steel vortex screen devices (vortisieve unit) (MMIndustries, Salem Ohio) of 32-1000 mu m range.Make saline solution after the filtration by this device recirculation with the help classification.Discard the microsphere of collecting in 32 tm screen.

Embodiment 2: the loading of amycin

The low AMPS microsphere as embodiment 1 preparation is used in this experiment.To employed each big or small microsphere, get 0.5ml and be transferred in 2 1ml syringes, one is used to absorb the drug, and another is with comparing.Testing selected size is 106-300 μ m, 300-500 μ m, 500-710 μ m and 850-1000 μ m.For the reliability of verification operation, prepare the syringe of 3 dress 500-710 μ m microspheres more in addition.11 10ml vials are covered with paper tinsel, to prevent that amycin is by light degradation in experimentation.The drawing standard curve.With 80ml concentration is that the drug solution of 20mg/ml prepares following concentration and measures its light absorption (483nm): 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml and 3.125 μ g/ml.Resulting light absorption is depicted as curve chart, with the drug level that is absorbed by microsphere in the curvilinear equation experiment with computing.When adding microsphere, in 4 vials, pack 5ml distilled water (ROMIL) into comparing.The drug solution that adds the 5ml desired concn in 7 remaining vials.Initial light absorption and corresponding solution concentration thereof are just learnt when the production standard curve.(, it must be diluted 200 times and use the concentration of 100 μ g/ml in order to measure the light absorption of 20mg/ml solution.This 1: 200 diluent all adopts in to the whole process of the absorption of solution measuring microsphere always.) when joining first vial that medicine is housed, first microsphere just begins to use the chronometer timing immediately, and microsphere adds in each bottle of all the other 6 vials by the order from the minimum to the maximum.Vial with sealing of lid after, put it on the impeller immediately.Control sample repeats same operation.With the time interval determination light absorption of the order identical in 0.167 hour (10 minutes), 0.5 hour, 1 hour, 2 hours, 24 hours and 96 hours with setting up vial.Can calculate the medication amount (mg number) of every 1ml microsphere and the drug absorption percentage rate of every 1ml microsphere from these data.The result as shown in Figure 1.

Embodiment 3: the influence of drug level to loading

Method according to embodiment 2 is summarized can be loaded into the amycin of certain limit variable concentrations on the high AMPS microsphere goods.According to observations, most of medicine was loaded into microsphere (500-710 μ m magnitude range) and goes up (see figure 2) in several hours.Can observe heap(ed) capacity by weight far above low AMPS goods.

Embodiment 4: the influence of microsphere size to loading

The loading of carrying out amycin on the microsphere of several different magnitude range is so that the comparative drug absorbtivity.To load medicine very fast although observe less microsphere, and the result who loads continuously in 24 hours shows, the drug loading that waits heavy microsphere to reach after the balance is also roughly the same.Absorption faster is owing to the surface area than minimicrosphere increases (see figure 3).

Embodiment 5: the repeatability of loading

In order to measure the repeatability that amycin loads, the loading experiment of summarizing among the embodiment 2 is repeated repeatedly.The high AMPS microsphere of 500-710 μ m magnitude range loads medicine with the 20mg/ml pharmaceutical aqueous solution, and the absorption (Fig. 4) in time of monitoring medicine.

Embodiment 6: the eluting of amycin from microsphere

High AMPS microsphere is eluted to (Fig. 5) in the 250ml distilled water with the amycin loading of variable concentrations and with microsphere.

The medicine of eluting still was lower than detectability in the time of 3 hours on 133.2 μ g/ml and the 2mg/ml medicine carrying microballoons.For higher drug loading, tangible burst effect (burst effect) was arranged in initial a few minutes, be the release of delaying time more slowly subsequently.By inference, the prominent free drug that has reflected that eluting goes out the water that carries in microsphere of releasing, and the time-delay eluting is by drug induced to the microsphere of the ionic interaction " combination " between charged group by main.With regard to high drug load (loading solution from 20mg/ml), burst effect has been represented the about 45% of the total drug loading of microsphere, remainder from the carrier fully eluting need a couple of days.Studies show that final 100% medicine can both be from eluting on the microsphere.

Embodiment 7: high AMPS microsphere is to the observation of amycin Absorption

Solution (66.6 μ g/ml) and the 3mlPBS adding of 1ml amycin in phosphate buffer PBS is equipped with about 0.5g size in the bottle of the high AMPS microsphere of 850-1000 mu m range (hand sieving).Microsphere is placed under the CCD photographing unit, took pictures once, continue 2.5 hours every 2 minutes.Stirring does not appear in the interior sample of section at this moment, but observes because the small amount of motion due to the local heat of light source.Therefore initial with last microsphere is identical, all is comparable in the whole time period.By enhancing red in the microsphere and on every side solution minimizing and observe the absorption (Fig. 6) of medicine.

Embodiment 8: the preparation of exsiccant medicine carrying microballoons

Microsphere can load amycin according to embodiment 2 described methods.Microsphere makes dehydration with the following method: microsphere to be drained off is placed in the plastic containers, and is used in 10% acetone (ROMIL) the solution covering of preparation among the PBS (InverclydeBiologicals).Microsphere was placed in solution 10 minutes, stirred several times each 30 seconds in this process.Then solution is toppled over out, this process is repeated twice again.Increase acetone concentration to 25%, 50%, 75% and final 100%, repeat this step.After final 100% dehydration, pour out acetone and microsphere is placed in 50 ℃ of baking boxs and be dried to constant weight.

The desciccate that obtains can suspend before thromboembolism operation again/again hydration in saline/contrast agent.Hydration rate is very fast, be expanded to＞volume during 80% complete hydration only needs a few minutes.

Embodiment 9: the preparation of microsphere pulp

The high AMPS microsphere of producing by top embodiment 1 was expanded 30 minutes in 20mg/ml amycin aqueous solution.Observe the outer liquid of particle after this and decolour substantially, color (redness) is limited in the microsphere substantially.Suspension removes by filter supernatant through agglomerating glass funnel.Microsphere is used the distilled water wash of two volumes on filter under slight negative pressure.Then microsphere is transferred in the wide mouthed bottle, and it is pumped into the glass syringe from wide mouthed bottle with peristaltic pump.Sterilize with the syringe sealing and with gamma ray with the syringe plug after removing pump.

Embodiment 10: loading-target medicine carrying dosage and actual medicine carrying dosage

Preparation concentration is a series of amycin solution of 22-80mg/ml in water.Get these solution of 1ml and add in the high AMPS microsphere of 1ml the ultraviolet detection drug absorption.Sample stirs on the rolling vortex mixer.Getting 10,20,30,60 minutes time points, is thereafter 2 hours, is 24 hours after again.By remaining amycin calculated uptake in the solution.Microsphere can load with the various dose that is up to every ml hydration microsphere 80mg, and in less than 30 minutes, 99% drug solution all is in the microsphere.

Embodiment 11: the high dose amycin

The amycin solution that in embodiment 10, has prepared 80mg/ml.This is a kind of heavy-gravity colloidal mixture, is unsuitable for daily use.Repeat this high dose, but use the amycin solution of 4ml 20mg/ml.The ultraviolet detection drug absorption has obtained to be loaded with in the high AMPS microsphere of 1ml hydration the final drug loading of 80mg medicine again.

Embodiment 12: loading-medicament sources

The amycin in three kinds of sources of use prepares the microsphere of 25mg/ml drug loading among the present invention.

Adriamycin ^TMPFS is that the concentration of a kind of commercially available (Pharmacia and Upjohn) is the solution of 2mg/ml.

Adriamycin ^TMRDF be a kind of commercially available (Pharmacia and Upjohn) adding the powder of lactose with hydrotropy.

Amycin EP.

To Adriamycin RDF and amycin EP solution, get 2ml and add in the 2ml microsphere ultraviolet detection drug absorption.To Adriamycin PFS solution, the solution of getting 50ml 2mg/ml adds in the 2ml microsphere, and detection of drugs absorbs.The solution of 30 minutes latter two 25mg/ml all loads, and the solution of 2mg/ml tracked just showed absorption (Fig. 8) fully in 24 hours.

Embodiment 13: the loading of other anthracene nucleus classes

The sample of high AMPS microsphere (900-1200 μ m) the 8ml glass container of packing into of 4 1ml hydrations of preparation.Measure microsphere in the 1ml phosphate buffer (PBS) with the 10ml glass cylinder, and it is transferred in the glass container.Then, shift out all PBS in each sample with the Pasteur glass pipette.Loading solution is prepared as follows: the 20mg daunorubicin in 1 bottle (Beacon Pharmaceuticals) is restored with 1ml water (ROMIL), reach final concentration 20mg/ml.The instant liquid of 50mg epirubicin (Pharmacia) in 1 bottle is restored with 2.5ml water, reach final concentration 20mg/ml.Preparation 20mg/ml amycin (DaburOncology) solution is to compare with the sample of front.Measure the uv absorption of solution after the preparation immediately, and the preparation diluent is to make the standard curve of every kind of drug solution at the 483nm place.

Get every kind of 1ml and load solution and 1ml water (in contrast) and add in each bottle that the microsphere that 1ml prepares as mentioned above is housed, and pick up counting.Bottle all is placed on the rolling vortex mixer in the whole experiment.Take out 50 μ l at preset time point (0,10,20,30,45 and 60 minute), dilute on demand and at 483nm place reading.By these readings, calculate the solution concentration of each time point according to the respective standard curve under every kind of situation.The minimizing that is loaded into the medication amount solution Chinese medicine when extracting with this device in the microsphere is measured.By the mg number of the every 1ml hydration of these data computation microsphere medicine carrying and be figure (Fig. 9).This shows that tested anthracene nucleus class all loads in an identical manner.

Embodiment 14: the eluting of other anthracene nucleus classes

Microsphere with medicine carrying as described in embodiment 13 is determined the drug release form.Every type medicine carrying microballoons is got 1ml and is transferred in the brown glass container that 100ml PBS is housed and pick up counting.Container places 37 ℃ of water-baths in whole experiment.Take out 1ml solution at preset time point (0,0.16,0.5,1,2 and 72 hour), put back to behind the reading in the container, make volume keep constant like this.Sample is at 483nm place reading, and by the equation calculating concentration of the standard curve of embodiment 12 determined each anthracene nucleus classes.The mg number of the medicine that is gone out by eluting in the every 1ml microsphere of these data computation also is figure (Figure 10+11).This shows that the anthracene nucleus class studied has identical eluting form during 0 eluting from microsphere.

Embodiment 15: the anthracene nucleus class is to the influence of microsphere size

Use embodiment 13 described microspheres, determine size distribution, distinguish its diameter with Image Pro Plus 4.05 then with CCD photographing unit and the captured microsphere image of microscope.The microsphere that has loaded different anthracene nucleus classes is transferred in the minicell culture bottle every image taking 50-1500 microsphere.Image Pro Plus 4.05 distinguishes 100-1500 diameter of micro ball according to magnitude range.With diameter tabulation and convert the bar diagram of magnitude range to, be figure (Figure 12) with its standardization and with Excel to the frequency of occurrences.This shows that the anthracene nucleus class has identical influence to the microsphere size.

Embodiment 16: the medicine carrying of other commercially available microspheres

According to steps outlined among the embodiment of front, amycin is loaded in microsphere of the present invention and the another kind of commercially available embolism microball (Embosphere is coated with three acryloyl microspheres of collagen coating), and it is compared.Obviously, as seen from Figure 15, microsphere of the present invention has the ability that absorbs the drug, and the commercially available prod of standard does not then have.

Embodiment 17: medicine carrying-physical effect

By the size behind loading of mensuration medicine and the eluting and the releasability of compression and amycin medicine carrying microballoons, the loading of assessment amycin and eluting are to the influence of high AMPS microsphere.When medicine effectively when the hydration microsphere replaces water, the loading of medicine makes a little decline of whole magnitude range (Figure 14), compressible a little decline of this simultaneous.Observe size and compressibility behind the eluting and all be not subjected to persistent influence (as determined) by measuring Young's modulus with Instron tension test instrument.Microsphere remains unchanged in the medicine carrying process by the ability that standard catheter discharges.

Embodiment 18: the influence of eluting-microsphere size

Load the microsphere of all size of the present invention with 70mg/ml amycin solution.Then microsphere is put into 500ml phosphate buffer, ultraviolet determination burst size.External elution profile shows high to the form release initial 2 hour to dash forward released of 40% amycin that loads at eluting, remaining then medicine eluting at least 12 days time.The release of all magnitude range all has similar feature, differs ± 5% with interior (Figure 15).

Embodiment 19: the influence of eluting-medium

The microsphere of the 25mg/ml of being loaded with amycin of the present invention is positioned in the different media and in 60 minutes monitors eluting.In blood plasma and PBS, be presented in initial 60 minutes and discharge slowly.Release in the water is lower than the ultraviolet detection limit.Ionic existence is depended in the release of this explanation medicine, because could displace the bonded medicine of ionic bond (Figure 16) from the polymeric matrix that has anionic charge like this.

Embodiment 20: the stability of amycin

Measure the influence of loading and dispose procedure to medicine stability.Measured the stability when amycin solution is preserved under different condition.By loading and release that HPLC follows the tracks of amycin in the microsphere of the present invention, whether be subjected to the influence of this process with the USP method to determine amycin.The chromatogram that produces all occurs unimodal in similar retention time, show that microsphere loads and the process that discharges to amycin without any adverse influence.

Embodiment 21: microsphere-material compatibility that amycin loads

Microsphere of the present invention loads with the 25mg/ml amycin.Then microsphere is suspended in contrast agent and the saline, then at release catheter (Progreat ^TM, Terumo) and in the syringe (Merit) placed 24 hours.At different time points, measure the stability (amycin ultraviolet/HPLC method, component detects by an unaided eye/the SEM method) of amycin and each component.Do not observe the degraded of component or medicine at room temperature 24 hours.

Embodiment 22: preload product-loading dosage

In the magnitude range of microsphere of the present invention the serial dosage of preparation be 5,10,20, the sample of 45mg/ml.The drug loading of each sample is measured by uv detection method.The data of 25 independent trialss are listed in the table below:

Table 1: load the actual dose that the solution ultraviolet detection records

Target dose mg/ml	45.0	45.0	45.0	45.0	45.0	45.0	45.0	45.0	45.0
											Mean dose mg/ml	44.79	45.25	46.60	46.56	45.66	46.56	45.74	46.57	44.025
SD	0.42	0.45	0.02	0.06	0.06	0.02	0.02	0.02	0.0397
											％CV	0.93	1.00	0.05	0.12	0.14	0.04	0.04	0.04	0.0901
Microsphere size μ m	100-300	100- 300	300- 500	500- 700	500- 700	700-900	700- 900	700- 900	900- 1200
											Measure number of times	30	28	9	15	23	24	13	15	5
											Target dose mg/ml	20.0	20.0	20.0	20.0	20.0	20.0
Mean dose mg/ml	21.31	21.12	21.05	21.04	21.02	19.65
											SD	0.01	0.01	0.03	0.02	0.03	0.0099
％CV	0.04	0.05	0.15	0.09	0.16	0.0504
											Microsphere size μ m	100-300	100- 300	300- 500	500- 700	700- 900	900- 1200
Measure number of times	33	28	9	9	9	5
Target dose mg/ml	5.0	5.0	5.0	5.0	5.0	10.0	10.0	10.0	10.0	10.0
											Mean dose mg/ml	4.99	5.14	4.98	4.98	5.13	9.98	9.98	9.96	9.93	9.93
SD	0.01	0.006	0.00	0.01	0.009	0.00	0.00	0.01	0.009	0.007
											％CV	0.12	0.12	0.08	0.21	0.18	0.01	0.04	0.11	0.09	0.07
Microsphere size μ m	100-300	300- 500	500- 700	700- 900	900- 1200	100-300	300- 500	500- 700	700-900	900- 1200
											Measure number of times	5	5	5	5	5	5	5	5	5	5

The dosage range that records is:

(45mg/ml:44.37-45.77mg/ml 3.11% scope)

(20mg/ml:21.11-21.32mg/ml 1.05% scope)

(10mg/ml:9.93-9.98mg/ml 0.5% scope)

(5mg/ml:4.98-5.14mg/ml 3.2% scope)

These data show almost there is not difference between different tests, can reach accurate and precise drug loading.

Embodiment 23: preload product-lyophilizing weightlessness

The microsphere that amycin among the present invention is loaded carries out lyophilizing with a kind of special-purpose circulation.The microsphere of all magnitude range with 25 independent trialss measure that dosage are 5,10,20, percent weight loss (percent with medicine carrying microballoons is represented table 2) during 45mg/ml.Obtained consistent weight loss, the product of any weight change that shows medicine carrying microballoons before the lyophilizing after to lyophilizing all not have to influence.During the data show lyophilizing of Figure 17 because dehydration always causes the weightlessness greater than 82%.

Table 2: the weightlessness of amycin medicine carrying microballoons when lyophilizing

Dosage mg/ml	45	45	45	45	45	45	45	45	45
											Microsphere size μ m	100-300	100- 300	300- 500	500- 700	500-700	700-900	700- 900	700- 900	900- 1200
Average weightless %	85.7	84.4	85.76	84.10	81.57	84.51	82.1	86.82	83.98
											SD	1.4	3.5	3.89	1.69	1.53	3.88	1.9	1.90	3.99
％CV	1.7	4.2	4.53	2.01	1.87	4.59	2.3	2.18	4.75
											Measure number of times	30	28	9	15	23	24	13	15	5
											Dosage mg/ml	20.0	20.0	20.0	20.0	20.0	20.0
Microsphere size μ m	100-300	100- 300	300- 500	500- 700	700- 900	900- 1200
											Average weightless %	90.6	92.7	91.92	92.48	91.38	90.65
SD	3.3	1.3	5.03	1.35	2.34	0.85
											％CV	3.6	1.4	5.48	1.46	2.56	0.94
Measure number of times	33	28	9	9	9	5
Dosage mg/ml	5.0	5.0	5.0	5.0	5.0	10.0	10.0	10.0	10.0	10.0
											Microsphere size μ m	100-300	300- 500	500- 700	700- 900	900- 1200	100- 300	300- 500	500- 700	700- 900	900- 1200
Average weightless %	94.8	94.26	93.2	93.1	94.90	93.7	93.30	90.3	94.5	93.04
											SD	0.2	1.11	0.5	0.5	0.78	0.2	0.26	8.3	0.2	0.29
％CV	0.2	1.18	0.6	0.6	0.82	0.2	0.28	9.2	0.2	0.31
											Measure number of times	5	5	5	5	5	5	5	5	5	5

Embodiment 24: preload product-residual moisture

Preparation with 5,10,20, the microsphere of the present invention of all magnitude range of the amycin loading of 45mg/ml, lyophilizing is sterilized with gamma ray then.Use moisture residual in the gravimetric detemination sample then, be included in 70 ℃ of heating microspheres to reaching constant weight.Record moisture residual in all samples and be less than 5%.

Table 3

	Drug dose mg/ml
						Drug dose mg/ml	0 5 10 20 45
Magnitude range	Residual moisture %
						100-300μm	2.65	2.10	2.46	3.70	3.52
300-500μm	2.74	2.13	2.17	1.60	1.86
						500-700μm	2.68	2.34	2.58	0.90	4.50
700-900μm	2.83	2.42	2.51	1.07	1.40
						900-1200μm	2.71	1.64	2.71	2.88	2.85

Embodiment 25: preload product-discharge after the hydration again

Preparation with 5,20, the microsphere of the present invention of all magnitude range of the amycin loading of 45mg/ml, lyophilizing is handled with gamma ray then.Make sample hydration again in water then, ultraviolet is followed the tracks of the medicine release in PBS 100 hours (Figure 18).

Embodiment 26: the size of the amycin medicine carrying microballoons of hydration again

The microsphere of the present invention (300-500 μ m) that preparation loads with the 20mg/ml amycin, lyophilizing is carried out gamma ray to sample segment then and is handled.Sample is hydration again in water, and (Image Pro-Plus Figure 19) measures size with the image analyzer of calibrating then.

Size is not handled and measured to the sample of medicine carrying too.Size slightly changed after the data show of Figure 20 was carried out different processing, but did not all make product exceed the acceptability limit of 250-500 μ m.

Embodiment 27: amycin behind the transcatheter arterial chemoembolization in the Hepar Leporis seu Oryctolagi cancer model (Vx-2)

From the amycin medicine carrying microballoons, continue to discharge

The purpose of this research is in order to be evaluated at the performance of the amycin medicine carrying microballoons of the present invention that discharges by transcatheter arterial chemoembolization (TACE) in the Hepar Leporis seu Oryctolagi cancer model.This research is carried out in the John Hopkins Hospital (The John Hopkins Hospital) of U.S. Baltimore.

27.1 materials and methods

Animal is divided into 6 groups (the 1st, 2,3,4,5,6 groups), 5 every group (4 of laboratory animals, 1 of control animal).The control animal of all groups is intra-arterial injection amycin (identical with laboratory animal concentration) all, and laboratory animal is handled with the medicament elution microsphere that contains amycin according to modified chemoembolization scheme.Animal is not on the same group put to death at following time point:

The 1st group: chemoembolization was operated back 1 hour

The 2nd group: chemoembolization was operated back 12 hours

The 3rd group: chemoembolization was operated back 24 hours

The 4th group: chemoembolization was operated back 3 days

The 5th group: chemoembolization was operated back 7 days

The 6th group: chemoembolization was operated back 14 days

27.2 the preparation of animal

The VX2 tumor cell line was expelled to the back leg of carrier rabbit (New Zealand white rabbit) and grown cultures 14 days.Collect the tumor that produces in each carrier rabbit upper body, and by every rabbit is dissected vivo tumor tissue,, the aseptic cutting chopping and the stainless steel filtering net sieve that stimulated the menstrual flow prepare tumor from every rabbit and starch.The rabbit intramuscular mixture that gives acepromazine (1mg/kg) and ketalar (20mg/kg) is in advance anaesthetized rabbit in advance.After about 15 minutes, do venous inlet, and vein gives animal penthiobarbital (40mg/kg), in operation process, to keep narcotism through auricular vein.Scrape off the hair of abdominal part, with the benzidine preserved skin and do a midline incision.Cutting open the belly by centre center exposes the liver of every rabbit, uses a tumor serosity of 21G vein blood vessel puncture needle (angiocath) direct injection (0.2ml) to the lobus sinister of the liver that exposes with around forming enough hepatic parenchymal unique isolated damage to be arranged then.Per two experimental rabbits are starched with a tumor.Tumor was grown in Hepar Leporis seu Oryctolagi 14 days, its size is reached according to the size of the desired diameter of former experiment in the 2.5-3.5cm scope.Electricity consumption calcination control over bleeding.Then abdominal part is sewed up and closed with running seam running suture, skin stitches with suture and gauze and merging bandages.All adopt suitable aseptic technique in the whole operation.After the operation, animal is put into cage, warming and carry out end-tidal CO with woollen blanket ₂Concentration monitor is revived from anesthesia until animal.If the misery on pain or the health obviously appears in animal, the subcutaneous 0.02-0.05mg/kg analgesic that gives is like general sieve coffee, and per 12 hours once, for three days on end.

27.3 the preparation of amycin eluting microsphere:

Press embodiment 22 preparations magnitude range of the present invention at the microsphere that the 100-300 micron also loads with the 45mg/ml amycin, sterilize by embodiment 23 lyophilizing and with gamma ray.Face use before with microsphere with the hydration of 1ml sterilized water, and adding 2ml Omnipaque and 1ml saline.Solution was ready to before will injecting at least in 10 minutes, and every rabbit arterial is injected the 1ml (as described below) in total solution.

27.4 chemoembolization operation:

Tumour transplatation is gone into rabbit liver after two weeks, animal is fetched done " chemoembolization ".By above-mentionedly anaesthetizing, do venous inlet in advance, anaesthetizing with penthiobarbital.Do the inlet that enters femoral artery,common, then a conduit is inserted common hepatic artery.Injection of contrast medium inserts a 2F JB1 conduit and makes it as far as possible near tumor after confirming the position at tumor place.If desired, all Transsend seal wires with catheter guidance to target artery.In case conduit is suitably located, as mentioned above amycin eluting microsphere is expelled in the tumor bed immediately.Matched group is only injected the amycin of same concentrations, and does not carry out the thromboembolism operation.After finishing " chemoembolization ", shift out conduit, tremulous pulse is with being stopped blooding by re-absorbed suture material ligation.All adopt suitable aseptic technique in whole operation and the subsequent operation.To the greatest extent institute might reduce not accommodate misery as far as possible, comprises the operative incision that limit tumour transplatation, subcutaneous injection seemingly general sieve coffee easing the pain as far as possible, clinical at any time calling when the health of needs assessment animal and pain and uncomfortable level.Then animal is put back to cage.All animals are put to death according to above-mentioned time point.

Put to death animal according to the veterinary administration rule.All animals in each group are being put to death under by the deep anaesthesia state due to the intravenous injection 100mg/kg thiopental IV according to its time point after the processing.

27.5 pathology and Histological evaluation

Cut the liver of rabbit, carefully extract and be placed in the container that 5% formaldehyde is housed.Liver is cut into slices to be used for macroscopy with the spacing of 5mm.Each section is cut into 4 μ slabs then and is closed Yihong dyeing processing with hematoxylin all with the complete embedding of paraffin.Estimate the tumor survival rate by perusal, and represent with the percentage rate of the tumor area of living in every section.Measure in the tumor and the concentration of amycin in the no tumor hepatic tissue with HPLC.

27.6 gather blood to carry out the amycin analysis

By in ear, inserting ductus arteriosus, during from injectable drug eluting microsphere, at 20 minutes, 40 minutes, 60 minutes, 120 minutes and 180 acquisition time 3ml whole bloods, transfer to then in the vacuum tube that is added with heparin respectively, and centrifugal 10 minutes of 2000g at room temperature.Isolate blood plasma and it is moved on in the polypropylene pipe with cover of labelling.Make its quick freezing in methanol/ice, in-20 ℃ of preservations for analyzing.

27.7 tissue is handled to determine in the tumor and the concentration of amycin in the no tumor hepatic tissue

Downcut tumor and no tumor hepatic tissue (about 100mg) and remove top epidermis and dead cell.Use the weight and the record of the accurate weighing tissue of test tube weigh in advance, put it to immediately then on the dry ice, then in-80 ℃ of preservations for analyzing.

27.8 result:

Drug distribution is summed up:

1. the concentration of amycin in tumor reached the highest in back 7 days in treatment, secondly was 3 days groups (Figure 20).

2. the concentration of amycin in tumor even still very high in 14 days groups shows amycin continuous eluting (Figure 20) from microsphere.

3. all time point amycin and the concentration of catabolite adriamycinol (doxorubicinol) in blood plasma thereof are very low.This after administration 20 minutes remarkable especially, and the concentration of amycin in blood plasma almost continue to descend with linearity at 20-180 minute.When intra-arterial injection does not have the amycin of microsphere, in the blood plasma the high 10-17 of the concentration of amycin times (Figure 21).

Histological observation:

1. 7-14 days neoplasm necrosis maximums (Figure 22).

2. notice that 1 hour group and 12 hours groups can be considered matched group, because the chemoembolization agent does not also begin to kill cell.

3. last, 50% and 37% tumor cell was arranged neither complete death neither be survived fully in the group respectively at 3 days and 7 days.These cells possibilities " impaired ", even may apoptosis.

4. about the distribution of microsphere, the same with expection all do not found any microsphere in the control animal.In laboratory animal, find that all microspheres all are retained in the small artery that the blood vessel diameter with expection is the 100-300 micron.Flee from space in the blood vessel without any microsphere.

5. in 14 days organized, the tumor implantation position was as if major part is downright bad in the hepatic tissue left side.Downright bad tumor and Normocellular difference are difficult to determine.Therefore, we can infer that the effectiveness of medicament elution microsphere has reached maximum after 14 days.These results are consistent.

6. histologic analysis has confirmed that also the medicament elution microsphere kills the effectiveness of tumor cell.We recognize that from many groups of experiments of front intra-arterial injection amycin or carboplatin (concentration is identical) only cause 30% neoplasm necrosis, and animal about equally with not treatment (i.e. contrast) for this.On the contrary, rabbit is treated the necrosis that causes 50-100% with the medicament elution microsphere, downright bad the most complete at 14 days, be significantly higher than independent intra-arterial injection (Figure 22).

Embodiment 28: clinical trial

With carrying out clinical trial, to estimate it in the safety, pharmacokinetics form and the effectiveness that are used for unresectable liver cell tumor (HCC) patient is treated according to the microsphere pulp of embodiment 9 preparations.The patient age that is fit to this research is in 18-75 year, suffers from the HCC that should not carry out eradication therapy such as excision, liver transplantation, transdermal therapeutic, has good liver function (Child-PughA type), loses compensatory without any liver.Get rid of following patient:

A) once treat the patient of HCC before with any anti-cancer therapies;

B) suffers from the patient of another kind of primary tumor;

C) suffers from the patient of severe hepatic disease: Child-Pugh B-C type or activeness gastrointestinal hemorrhage, encephalopathy or ascites.Bilirubin level＞3mg/dL;

D) suffer from the patient of Cancerous disease: (involvement of blood vessel comprises the segmental Portal Venous Obstruction to C type BCLC; Liver external diffusion or cancer related symptoms=PST are 1-4) or D type BCLC (WHO physical ability situation 3 or 4, Okuda III phase);

E) suffers from the patient of any liver thromboembolism surgical contraindication: door body shunting, from liver blood stream, the unusual (platelet count＜50.000/mm of blood coagulation experiment ³Or prothrombin activity＜50%), renal failure, serious atherosclerosis;

F) suffer from patient (serum bilirubin 〉=5mg/dL, the numeration of leukocyte≤3.000 cells/mm of amycin administration contraindication ³, cardiac ejection fraction＜50%).

Chemoembolization was at 0 o'clock and carry out February.Treatment will be ended owing to the formation of any exclusion standard or by patient's decision.Carry out thromboembolism with the blended amycin medicine carrying microballoons of contrast agent by being injected at before facing administration, until reaching flow stagnation.The PVA diameter of micro ball is selected by surgeon, and its size may be for about 500 μ m.Do not use antibiotic prophylaxis.

In order to analyze the pharmacokinetics form of amycin microsphere, to the patient of bilirubin level＜1.5mg/dL, the dosage that begins to scale up with following dosage: at every turn treat 25mg/m ²(2 patients), 50mg/m ²(2 patients), 75mg/m ²(2 patients), 100mg/m ²(2 patients).Patient at 1.5-3mg/dL begins with following dosage to bilirubin level: 25mg/m ²(2 patients), 50mg/m ²(2 patients), 75mg/m ²(2 patients), 100mg/m ²(2 patients).If do not observe any dose-limiting toxicity at certain dosage level, then increase dosage in proportion in next group, the maximum accumulated dose of amycin is 150mg in the single therapy.

Pharmacokinetics is estimated: after operation 1 hour, 6 hours, 24 hours, 48 hours and 7 days, in patient's while in hospital or in out-patient department, the collection sample carries out the amycin horizontal analysis from peripheral blood.

Safety: record and the analysis complication relevant in 6 months whole research process with treatment.Hospital stay (4 days) record untoward reaction incident the 0th month and February.At the 7th day, the 14th day and the 1st, 3 and June, in out-patient department the patient is paid a return visit, it is carried out clinical examination and determination experiment chamber parameter.All all can find the untoward reaction incident in paying a return visit behind the begin treatment, comprising bone marrow depression and other toxic appearance relevant with amycin, liver failure, renal failure, infection (cholecystitis, hepatomegaly, spontaneous bacterial peritonitis, bacteremia, Ischaemia hepatitis or biliary tract stenosis), gastrointestinal hemorrhage.

Curative effect: the 3rd and the spiral-fault photography strengthened by contrast agent June estimate objective reaction.Its amplitude defines according to EASL's unified standard: complete reaction: no Cancerous disease sign; Partial reaction: the tumor total amount descends＞50%; No change: decline＜50% or increase＜25%; PD: increase＞25%.Therefore, objective reaction has reflected complete reaction and partial reaction.

Claims (26)

1. compositions, said composition comprises and has a kind of water inflatable but water-insoluble polymer substrate and a kind of particle that is absorbed in the water-soluble therapeutic agents in this substrate of meeting, it is characterized in that: described polymer totally has negative charge in the pH6-8 scope, when described particle expands in water when reaching balance, its particle size is between 40-1500 μ m, and described therapeutic agent is for having the anthracycline compound of an amino at least.

2. according to the compositions of claim 1, wherein said polymer is a covalent cross-linking.

3. according to the compositions of claim 1 or 2, wherein said polymer comprises crosslinked polyvinyl alcohol.

4. according to the compositions of claim 3, said composition contains the polyvinyl alcohol macromonomer of at least two ethylene linkage unsaturated terminal chain groups by each molecule and comprises that the ethylene linkage unsaturated monomer combined polymerization of anionic monomer forms.

5. according to the compositions of claim 4, wherein said anionic monomer has general formula I

Y ¹BQ

Y wherein ¹Be selected from:

CH ₂＝C(R)-CH ₂-O-，CH ₂＝C(R)-CH ₂OC(O)-，CH ₂＝C(R)OC(O)-，CH ₂＝C(R)CH ₂＝C(R)CH ₂OC(O)N(R ¹)-，R ²OOCCR＝CRC(O)-O-，RCH＝CHC(O)O-，RCH＝C(COOR ²)CH ₂-C(O)-O-，

With

Wherein:

R is hydrogen or C ₁-C ₄Alkyl;

R ¹Be hydrogen or C ₁-C ₄Alkyl;

R ²Be hydrogen or C _1-4Alkyl or BQ, wherein B and Q are defined as follows;

A is-O-or-NR ¹-;

K ¹Be group-(CH ₂) _rOC (O)-,-(CH ₂) _rC (O) O-,-(CH ₂) _rOC (O) O-,-(CH ₂) _rNR ³-,-(CH ₂) _rNR ₃C (O)-,-(CH ₂) _rC (O) NR ³-,-(CH ₂) _rNR ³C (O) O-,-(CH ₂) _rOC (O) NR ³-,-(CH ₂) _rNR ₃C (O) NR ³-(wherein, radicals R ³Identical or different) ,-(CH ₂) _rO-,-(CH ₂) _rSO ₃-or optional and B ¹In conjunction with and form a covalent bond, r is 1 to 12, R ³Be hydrogen or C ₁-C ₄Alkyl;

B is straight or branched alkane two bases, oxyalkylene, alkane two basic oxygen alkane two bases or alkane two base oligomeric (oxygen alkane two bases) chains, and optional one or more fluorine atom that comprises of this chain replaces chain up to forming perfluor; Perhaps, if Q or Y ¹Comprising a terminal carbon with the B keyed jointing, is a covalent bond;

Q is an anionic group.

6. according to the compositions of claim 5, Y wherein ¹Be CH ₂=CRCOA, wherein R is H or methyl, and A is NH, and wherein B is C _1-12Alkane two bases.

7. according to the compositions of claim 5, wherein Q is carboxylate, carbonate, sulfonate, sulfate, nitrate, phosphonate or phosphate group, preferred sulfonate groups.

8. according to each compositions among the claim 3-7, wherein the PVA macromonomer has 1,000-500, and the mean molecule quantity of 000D scope, preferably 10,000-100,000D scope.

9. according to each compositions among the claim 3-8, wherein the thiazolinyl side-chain radical is connected with the oxygen atom of adjacent hydroxyl by the cyclic acetal key, the reaction that described cyclic acetal key preferably passes through the aldehyde of N-(alkyl) acrylamido replacement forms, be generally the dialkyl acetal form, preferred N-acrylamide ethylhexanal dimethylacetal.

10. according to the compositions of the arbitrary claim in front, wherein said anthracycline compound is the chemical compound with general formula I I:

Preferred amycin.

11. according to the compositions of the arbitrary claim in front, wherein said particle is a microsphere.

12. according to the compositions of the arbitrary claim in front, wherein said particle expands and is suspended in the waterborne liquid.

13. according to the compositions of claim 12, said composition further comprises a kind of preparation, preferred contrast agent.

14. according to each compositions among the claim 1-11, wherein said particle is pumpable serosity form, described slurry package is contained in expansible particle in the waterborne liquid, and is substantially free of the outer liquid of particle.

15. according to the compositions of claim 14, said composition is contained in a sterilization storage capsule, in the preferred syringe.

16. according to each compositions among the claim 1-11, said composition is a substantially dry.

17. an anthracycline compound is used for solid tumor is carried out the purposes of compositions of any aforementioned claim of basis of embolotherapy in preparation.

18. one kind according to each composition manufacturing method among the claim 1-16, wherein in the presence of water, a kind of inflatable but microgranule water-insoluble polymer of water of meeting is contacted with a kind of solution of anthracycline compound, by this anthracycline compound is absorbed in the polymeric matrix.

19. according to the method for claim 18, wherein said contact is by carrying out in the aqueous solution that polymer particle is suspended in anthracycline compound.

20. according to the method for claim 19, the polymer particle that is absorbed with anthracycline compound in its mesostroma reclaims from suspension and drying, preferably by the freeze-drying drying.

21., wherein the particle of expanded polymer is separated with supernatant and transfer in the storage capsule that is preferably syringe when liquid expands, and sterilization and preserving in this container in still being inflated according to the method for claim 19.

22. an anthracycline compound is used for solid tumor is carried out the purposes of the compositions of embolotherapy in preparation, discharge from a kind of polymeric matrix at anthracycline compound described in this treatment, described polymeric matrix is to be formed by polyvinyl alcohol macromonomer and the unsaturated anionic monomer combined polymerization of a kind of ethylene linkage that each molecule has at least two ethylene linkage unsaturated terminal chain groups.

23. according to the application of claim 22, wherein contain macromonomer and monomeric fluid composition and initiated polymerization by introducing in patient's blood circulation, original position forms polymeric matrix in patient's vascular system.

24. Therapeutic Method wherein makes according to each compositions among claim 1-12 and the 14-16 and mixes with a kind of contrast agent, gives this mixture then with the thromboembolism solid tumor in patient's blood vessel.

25. Therapeutic Method wherein gives compositions according to claim 13 with the thromboembolism solid tumor in patient's blood vessel.

26. according to the method for claim 24 or 25, wherein tumor is a hepatocarcinoma.

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