CN102600485B - Ultrasonic contrast medium dosage formula - Google Patents

Abstract

According to the invention, a concrete dosage formula is implemented and developed in clinical researches. The dosage formula employs perfluorocarbon gas which can provide a substantially enhanced image and a long duration, and combines the gas into polymer particles, such that an ultrasonic contrast medium is prepared. The dosage formula comprises particles formed by biocompatible polymers and perfluorocarbon. Preferably, lipid is mixed in the polymer, and perfluorocarbon is in a form of gas under a body temperature. A patient is administrated with the particles with a dosage effective to enhanced ultrasonography. The duration in the ventricle is longer than 5min and/or the duration in the cardiac muscle is longer than 1min, and the dosage range is 0.025-8.0mg/kg (particles/body weight), and preferably 0.05-4.0mg/kg (particles/body weight). The dosage formula is generally provided in small bottles. In a typical formula, a dosage formula is formed by dry powder which needs to be re-prepared by using sterile water before use. Specifically, water is added to a small bottle or a syringe; the bottle or the syringe is shaken, such that isotonic or isoosmotic particle suspension fluid is formed.

Description

Ultrasonic contrast agent dosage formulation

The application is to be that June 4, application number in 2004 are dividing an application of 200480043713.0 (PCT/US2004/017813), the denomination of invention application for a patent for invention that is " ultrasonic contrast agent dosage formulation " the applying date.

Technical field

The invention belongs to general diagnosing developing agent field, particularly a kind of ultrasonic contrast medium (ultrasound contrast agent) dosage formulation that the image of enhancing and the image of persistent period length can be provided.

Background technology

When utilizing the image of the ultrasonic human or animal of acquisition internal and structure, when ultrasound wave, acoustic energy wave can be reflected during through human body with certain frequency (when surpassing this frequency, can by the perception of people's ear institute).Dissimilar bodily tissue is different to hyperacoustic reflection, and the reflection being produced by the ultrasound wave that reflects different internal structure can be detected and be converted to visual image by electronics method.

For some medical conditions, the useful image that obtains the interested organ of people or structure is especially difficult, this is because in the ultrasonography producing by lacking hyperacoustic reflection of contrast-enhancing agent, and the details of said structure can not be offered an explanation out fully from tissue around.By give ultrasonic contrast medium in interested organ or other structures, improve the contrast in ultrasonography, thereby improve in essence the detection of some physiology and pathological condition and observation.In some cases, to the detection of ultrasonic contrast medium's displacement, be very important.For example, the unique blood flow patterns being caused by specific Cardiovascular abnormality only has by blood flow being given to ultrasonic contrast medium and observing blood flow or blood volume just can be identified.

When ultrasound wave passes health and be reflected to produce the image that can make accordingly internal medicine diagnosis, the material that is used as ultrasonic contrast medium plays a role by affecting ultrasound wave.

Dissimilar material affects ultrasound wave with different degree in a different manner.In addition, by contrast-enhancing agent, caused that some effect is than the more easily measured and observation of other reagent.When selecting desirable ultrasonic contrast medium's compositions, people conventionally prefer those and ultrasound wave are had the material of remarkable effect when ultrasound wave passes health.In addition, to hyperacoustic effect, should be easy to measure.Gas is the good medium as ultrasonic contrast medium.Gas must immobilization before using, and it can be used as surfactant immobilization bubble or by wrapping in liposome or microgranule with capsule.In ultrasonography, can see three kinds of main contrast reinforced effects, that is, and back reflection, beam attenuation and velocity of sound difference.

Existing multiplely be naturally used to seal such as ultrasonic contrast mediums such as air with synthetic polymer, these polymer contribute to manufacture long-term ultrasonic contrast medium after administration.Schneider etc. exist invest.Radiol., three kinds of micron-sized, that fill air, synthetic, polymer beads have been described in Vol 27, pp.134-139 (1992).It is stable in blood plasma and under working pressure to it is reported these granules.Yet their echogenicity is low when 2.5MHz.The albumose Ruzhong from supersound process is obtained for another kind of microvesicle suspended matter.Feinstein etc., j.Am.Coll.Cardiol., Vol.11, pp.59-65 (1988).Feinstein described there is good vitro stability, size is suitable for lung and lung inner chamber between the preparation of microvesicle of passage (transpulmonary passage).Yet because these microvesicles are unstable under pressure, they are short-lived in vivo, the half-life is approximately several seconds (with a cyclic process approximately equal).Gottlieb, S. etc., j.Am.Soc.Echo., Vol.3, pp.328 (1990), summary; And Shapiro, J.R. etc., j.Am.Coll.Cardiol.vol.16, pp.1603-1607 (1990).

The microvesicle that Rasor Associates company also seals gelatin in WO 80/02365 is described.The microvesicle that described gelatin is sealed forms by " coalescent " gelatin.Molecular biosciences system house (Molecular Biosystems, Inc.) is described being encapsulated in the gas microbubbles of the enclosure interior of fluorine material in WO96/04018.

Fritzch etc. have reported with the immobilized microvesicle of galactose crystallite (SHU454 and SHU508).Fritzsch, T. etc., invest.Radiol.vol.23 (Suppl 1), pp.302-305 (1988); With Fritzseh T. etc., invest.Radiol., Vol.25 (Suppl1) 160-161 (1990).This microvesicle continues 15 minutes in vitro but continues less than 20 seconds in vivo.Rovai, D. etc., j.Am.Coll.Cardiol., Vol.10, pp.125-134 (1987); And Smith, M. etc., j.Am.Coll.Cardiol., Vol.13, pp.1622-1628 (1989).Schering Aktiengesellschaft discloses preparation and the purposes for microcapsule sealing gas or the volatile liquid of ultrasound wave video picture in European patent EP 398935, and wherein, microcapsule is formed by synthetic polymer or polysaccharide.Sintetica discloses a kind of being used for the treatment of or air or the gas micro of diagnostic purpose in European patent 458745, this microsphere can be dispersed in interface deposited polymer thin film in aqueous carrier and wrap and tie up, for being expelled in host animal body or for oral cavity, rectum or urethra administration.

A kind of preparation method of the microgranule for imaging has been described in No. 92/18164 patent of WO by Delta biological engineering company limited, and the method has by dry formation that protein aqueous solution is sprayed the hollow ball that includes gas.No. 93/25242 patent of WO has been described the synthetic method for the microgranule of ultrasonic imaging, and the structure of described microgranule is for containing gas in the enclosure of polybutylcyanoacrylate or polyester.WO 92/21382 discloses a kind of manufacture of microgranule contrast medium, and described microgranule contrast medium comprises the substrate of the covalent bonds that contains gas, and wherein, described substrate is carbohydrate.Unger is United States Patent (USP) the 5th, 334, No. 381 and 5,123, No. 414 and 5,352, in No. 435, described the liposome as ultrasonic contrast medium, it comprises gas, gaseous precursors for example pH activation or photoactivation gaseous precursors, and other liquid or solid contrast-enhancing agent.

Other people have also proposed to compare with air the application of the fluorinated gas that strengthens video picture in view of the effect of entrapped gas.Quay is in U.S. Patent No. 5,393, discloses in 524 and comprised that the plurality of reagents of perfluocarbon strengthens the application of contrast in ultrasound wave video picture.Mentioned reagent is comprised of selected gas minute bubbles or microvesicle, and it demonstrates the longer life-span in solution, and enough little to such an extent as to can pass lung, and this can be applied it in the ultrasonography of cardiovascular system and other vitals.Bracco discloses the effect of fluorinated hydrocarbons gas disintegrate when being exposed to blood stream pressure to prevention microvesicle at European patent 554213.Nycomed discloses a kind of microcapsule for video picture in WO95/23615 patent, and this microcapsule is condensed and formed by the solution that comprises perfluocarbon such as protein solution etc.The Massachusetts Institute of Technology (Massachusetts Institute of Technology) discloses a kind of microgranule being formed by polyethylene glycol-(lactide-co-glycolide) bulk polymer in patent WO95/03357, in this microgranule, be encapsulated with developer, air inclusion is air and perfluocarbon for example.As Sonus pharmaceutical companies described in the WO94/16739, when solid and reflection liquid sound wave reach a similar degree, well-known, gas is the more effective and more welcome medium as ultrasonic contrast medium.In fact, as shown in the embodiment 12 of WO 94/16739, when to miniature pig administration, protein microcapsule is not considered owing to producing safety problem (and efficacy problems).United States Patent (USP) 6,132,699 and 5,611,344 have all described the method for carrying out enhancing contrast ratio in synthetic polymer shell with perfluocarbon.United States Patent (USP) 5,837,221 describe a kind of method of producing porous polymer particle, and the method is integrated with hydrophobic agents in polymer, to increase echogenicity.

There are several ultrasonic contrast mediums all granted at US and European, for very limited cardiac tonic medication. (Amersham, Mallinkrodt) comprises the thermal denaturation human albumin microcapsule that contains octafluoropropane gas.Every milliliter of microsphere suspended matter comprises 5-8 * 10 ⁸individual average diameter is the octafluoropropane of 2-4.5 micron granularity scope and 220 μ g.These microspheres are not also approved for myocardial blood flow assessment, are only approved for ventricle potentiation.Under higher pill dosage (5mL suspended matter or 1100 μ g octafluoropropanes), ventricle potentiation continues to reach 5 minutes.

(Bristol Myers medical imaging) comprises the octafluoropropane that contains lipid microsphere, and wherein lipid shell is comprised of phospholipid DPPA, DPPC and mPEG-DPPE.Every milliliter of suspended matter comprises 1.2 * 10 ¹⁰individual microgranule, the average diameter of this microgranule is in the particle size range of 1.1-3.3 micron and have 1100 μ g octafluoropropanes.This reagent is only approved for ventricle potentiation, and is not approved for myocardial blood flow assessment.Under the gas of or 5133 μ g lower at the pill dosage (for the people of 70kg) of 700 μ L, reagent persistent period of potentiation in ventricle is approximately 3.4 minutes.

(Photogen company) comprises the lipid microsphere that contains perflexane, and wherein, lipid shell is comprised of phospholipid DMPC.Every milliliter of suspended matter comprises 1.4 * 10 ⁹the perflexane of individual microgranule and 92 μ g, wherein, this microgranule has the average diameter that is less than 3 microns.This reagent is only approved for ventricle potentiation, and is not approved for myocardial blood flow assessment.Under the gas of or 40 μ g lower at the pill dosage of 0.43 milliliter (for the people of 70kg), reagent is approximately 2.6 minutes in the average duration of intraventricular potentiation.

Under any circumstance, these commercially available reagents only have limited application, are not approved for other application except ventricle potentiation, and it is 5 minutes or still less that these reagent strengthen the persistent period in the average video picture of ventricle.Still lack and can strengthen cardiovascular system particularly cardiac muscle and the video picture of ventricle and there is the business ultrasonic contrast medium compared with long duration.Reagent described in the prior, when as pill or short preserved material administration, the persistent period of the myocardium image of generation is significantly less than processes a needed time of complete cardiac tests.Usually, its persistent period during for cardiac muscle of the image that the medicament of prior art provides was less than one minute.People wish to obtain such reagent, the enhancing video picture persistent period that it can provide in cardiac muscle for super after one minute and/or in ventricle for being greater than 5 minutes.

Therefore, the object of the present invention is to provide a kind of dosage formulation that is particularly useful for cardiac tonic, it comprises microgranule, and wherein, microgranule can provide image and the long image of persistent period of enhancing.

Another object of the present invention is to provide a kind of test kit, for the dosage formulation administration that comprises microgranule at ultrasound wave developing method.

Summary of the invention

People have developed concrete dosage formulation and have used it for clinical research, and this dosage formulation has been used polymer particles, can provide pfc gas remarkable and the enhancing video picture that the persistent period is long to integrate with wherein.This dosage formulation typically comprises one, two or until five dosage, is preferably the microgranule of one or two dosage, and described microgranule is by the biocompatible polymer formation that preferably contains lipid, and it contains when body temperature is the perfluocarbon of gas.Described microgranule to be to offer medicine to patient to strengthening the effective dosage of ultrasound wave video picture, the intraventricular persistent period for be greater than five minutes and/or in cardiac muscle for being greater than one minute, dosage range is from 0.025 to 8.0mg microgranule/kg body weight.Preferably giving the dosage range of patient's administration is from 0.05 to 4.0mg microgranule/kg body weight.In a preferred version, in ventricle, the potentiation of ultrasound wave video picture continues to be greater than 9 minutes, and/or in cardiac muscle for being greater than 2 minutes.

Dosage formulation typically provides with bottle or syringe.In a typical formula, dosage formulation is dry powder form, needs again to prepare with sterilized water, particularly: to being equipped with in the bottle of dry powder or syringe, add water, then shake to produce to wait and ooze or the microparticle suspending liquid of equipressure before using.In the preferred version of these dosage formulations, suspension comprises 1.0-3.5 * 10 ⁹microgranule/mL, or 25-50mg microgranule/mL, most preferred concentration is that suspension comprises 1.5-2.8 * 10 ⁹microgranule/mL or 30-45mg microgranule/mL.In a preferred version, the particle mean size of microgranule is less than 8 microns, preferably particle mean size is 1.8-3.0 micron.

In preferred plan, gas is CF ₄, C ₂f ₄, C ₂f ₆, C ₃f ₆, C ₃f ₈, C ₄f ₈, C ₄f ₁₀, or SF ₆.In preferred version, gas is Sonazoid (n-perfluorobutane) (C ₄f ₁₀), with the dosage of the microgranule suspended matter of 75-500 μ g/mL, provide; Preferably Sonazoid provides with the dosage of the microgranule suspended matter of 100-400 μ g/mL; Preferably Sonazoid provides with the dosage of the microgranule suspended matter of 150-350 μ g/mL; Or gas is octafluoro n-propane, with the dosage of the microgranule suspended matter of 75-375 μ g/mL, provide, the dosage being preferably with the microgranule suspended matter of 120-300u g/mL provides.

In preferred plan, microgranule is formed by synthetic polymer, described synthetic polymer for example has polyalcohols acid, comprise polylactic acid, polyglycolic acid, with poly-(lactic acid-altogether-hydroxyacetic acid), PGA (polyglycolides), polyactide and poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), poe (polyorthoesters), polyamide, Merlon, polyolefin (polyalkylenes), for example polyethylene and polypropylene, ployalkylene glycol, Polyethylene Glycol for example, polyoxygenated alkene is poly(ethylene oxide) for example, polyvinyl alcohol, poly-valeric acid, with poly-(lactide is caprolactone altogether), derivant, copolymer and composition thereof, and the polymer that contains a hydrophobic compound with the ratio of 0.01-30wt% with respect to polymer weight, be preferably the polymer that the ratio with 0.01-30wt% (lipid weight/polymer weight) contains lipid.At one particularly preferably in scheme, lipid be dioleyl phosphatidyl choline (dioleoylphosphatidylcholine) (DOPC), L-Dimyristoylphosphatidylcholine (dimyristoylphosphatidylcholin, DMPC), two pentadecanoyl phosphatidylcholine (dipentadecanoylphosphatidylcholine, DPDPC), DLPC (dilauroylphosphatidylcholine, DLPC), Dioctonoyl pnosphotidyl choline (dipalmitoylphosphatidylcholine, DPPC), DSPC (distearoylphosphatidylcholine, DSPC), two Semen arachidis hypogaeae acyl phospholipids phatidylcholine (diarachidoylphosphatidylcholine, DAPC), Er behenolyl base phosphatidylcholine (dibehenoylphosphatidylcholine, DBPC), two tricosane acyl phospholipids phatidylcholines (ditricosanoylphosphatidylcholine DTPC), two wooden fatty acyl group phosphatidylcholines (dilignoceroylphatidylcholine DLGPC), or PHOSPHATIDYL ETHANOLAMINE.

Preferably, synthetic polymer in microgranule is poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), lactide is 50: 50 (1: 1) to the ratio of Acetic acid, hydroxy-, bimol. cyclic ester, weight average molecular weight is 20,000-40, in 000 daltonian scope, the hydrophobic compound in microgranule is DAPC, and ratio is 5: 6.6% (DAPC weight/polymer weight).

Dosage formulation can provide with bottle or the syringe form that comprises microgranule dry powder, or provides with the kit form that comprises resuspension microgranule solution.Typically, in the bottle of dry powder or syringe, also comprise excipient for example sugar or Sal, solution etc. is oozed, or again ooze after preparation etc.Then, using this dosage formulation as pill or injection by being administered to patient's administration, carry out video picture in during whole 30 minutes.

Microgranule all of great use, comprises ultrasound wave video picture, nuclear magnetic resonance, fluoroscopy, X ray and computerised tomography in multiple diagnostic imaging program.For cardiology application examples, microgranule is being checked as the clinical trial of myocardium blood flow assessment and ventricle potentiation.

The specific embodiment

Here, to the method after improving, microgranule, test kit with for the dosage formulation of ultrasound wave video picture, be described.Microgranule is very useful in the application of various diagnostic ultrasound video pictures, very useful in for example for example myocardial blood flow assessment of angiography and echocardiography, the assessment of myocardium blood volume and ventricle potentiation in ultrasound procedures especially.

I. definition

Unless otherwise mentioned, with common used the same, term " microgranule " comprises " microsphere " and " microcapsule ", and other microgranule.Microgranule can be spherical or aspheric in shape.

Here " microcapsule " is generally defined as having a microgranule that surrounds the outer polymer shell of gas core." microsphere " is defined as can be solid spherical, or by the porous spherical of the formed honeycombed structure of the pore that spreads all over gassiness polymer or spongy architecture.Some microspheres can comprise by the formed outer polymer shell with honeycombed structure or spongy architecture of the gassiness pore that spreads all over polymer shell.For this class microsphere, this outer polymer shell surrounds inner gas core.

With common used the same, term " dosage (dosage) " and " dose (dose) " are synonyms, represent the amount that once gives, or produce expection diagnosis or the required amount of contrast effect.

Here the term that used " dosage formulation ", refers to for example syringe of bottle or other containers, and the requirement that it comprises one or more dosage produces the material of expection diagnosis or contrast effect.

The same with routine, " patient's position " refers to patient's specific region or part with it here.In some cases, " patient's position " refers to the position that spreads all over patient's whole body.The example at this position has other positions, tissue, organ of pulmonary, gastrointestinal portion, cardiovascular position (comprising cardiac muscular tissue or cardiac muscle (for example cardiac muscle), the chamber of ventricle, atrium, valvular function), kidney position and health etc., comprise vascular tissue and blood circulation and ill tissue, comprise cancerous tissue." patient's position " for example comprises will be by the position of diagnostic image video picture.Although patient's position " also " may be external, preferably in vivo.

The same with custom, " vascular tissue " represents blood vessel (comprising tremulous pulse, vein, blood capillary etc.) here.

The same with custom, " gastrointestinal position " comprises esophagus, stomach, small intestinal and large intestine and the defined position of rectum here.

The same with custom, " kidney position " refers to kidney and the vascular tissue that is directly connected kidney here, comprises ventral aorta.

The same with custom, " target site " and " target area " cross-reference, all represent the patient position of wishing that medicament arrives here.

The same with custom, " want video picture position " here and " video picture position " cross-reference, all represent patient's expection video picture position.

The same with custom, " ventricle blood flow or ventricle potentiation " refers to that blood flow passes the ventricle of heart with one or more cardiac cycle here.

With custom the same, here " atrium blood flow " refer to blood flow with one or more circulations the atrium through heart.

The same with custom, " myocardial blood flow " refers to that blood is with one or more periods (being included in the blood vessel of heart) mobile in the vascular tissue of cardiac muscle or cardiac muscle here.

The same with custom, " myocardium blood volume " refers to the amount of the blood in the vascular tissue of cardiac muscle or cardiac muscle here.

The same with custom, " cardiac cycle " refers to a complete contraction cycle of heart here, comprises relaxation cycle and the contraction cycle of heart.

The same with custom, here " increase brightness " refer to that the image obtaining with there is no ultrasonic contrast medium compares, the increase of image in brightness.

With custom the same, here " enhancing video picture " refer to that obtaining image with there is no ultrasonic contrast medium compares, the image that brightness has increased relatively.

With custom the same, here " persistent period " refer to the total time that the increase brightness of video picture can be detected.

The same with custom, " coronary artery expander " refers to biological active substances for example dipyridamole (dipyridamole) or adenosine here, when these medicines are taken by patient, can cause the expansion of cardiovascular position vascular tissue.

II. microgranule

In preferred version, microgranule comprises polymer, lipid and pfc gas.Microgranule can both comprise microsphere and also comprise microcapsule, or only comprised microsphere or microcapsule.

Polymer

In preferred version, microgranule is formed by synthetic polymer.The microgranule of synthesized polymer deposits yields bio-compatible and inanimate object material contamination.

In addition, preferably synthetic polymer, is because synthetic polymer renewable synthetic and degraded in vitro and in vivo.According to the needed selection of time polymer of body internal stability, this for example the time be to be distributed to the needed time of position of wanting video picture, and needed time of video picture.Synthetic polymer can be modified to produce the microgranule (for example, changing molecular weight and/or functional group) of different in kind.

Typical synthetic polymer has: polyalcohols acid is polylactic acid, polyglycolic acid and PLGA, PGA (polyglycolides), polyactide, poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) and composition thereof, polyanhydride, poe (polyorthoesters), polyamide, Merlon, polyolefin for example, for example polyethylene and polypropylene, polyglycols for example poly(ethylene oxide) polyvinyl alcohol, poly-valeric acid and poly-(lactide is caprolactone altogether) and derivant, copolymer and mixture of Polyethylene Glycol, oxidized polyolefin for example.Here " derivant " comprises that chemical group is substituted, the polymer of addition, for example the normally used alkyl of those skilled in the art, alkylene, hydroxylating, oxidation and other modifications.

The object lesson of preferred biodegradable polymer comprises: alkyd is polymer, polyactide, PGA (polyorthoesters), poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), the copolymer with PEG, polyanhydride, poly-(neighbour) ester, polyurethane, poly-(butanoic acid), poly-(valeric acid), poly-(lactide is caprolactone altogether) and the copolymer thereof of lactic acid and hydroxyacetic acid for example.Best preferred polymers is poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), and wherein lactide is that the weight average molecular weight of 50: 50 (1: 1) and polymer is at 20,000-40, within the scope of 000 dalton to the ratio of Acetic acid, hydroxy-, bimol. cyclic ester.Weight average molecular weight (Mw) is the mean molecule quantity calculating based on having the molecular mass of given molecular weight in single polymer chain distribution.Mw can measure with gel permeation chromatography (GPC).

Hydrophobic compound

In a preferred version, as U.S. Patent No. 5,837,221 is described, and polymer comprises a kind of hydrophobic compound.Conventionally, lipid for example, it is that amount hydrophobic and in polymer inside is an effective dose, in conjunction with such compound, can limit by microgranule infiltration and/or the absorption of water, has also therefore limited the loss of gas in microgranule.

This can effectively increase by comprising lipid, synthetic polymer and being encapsulated with gas persistent period of the fluorinated gas enhancing video picture that for example microgranule of perfluocarbon provides especially.

Can be used for fixing the lipid of polymer particles internal gas including, but not limited to the lipid of following kind: fatty acid and derivant thereof, monoglyceride, diglyceride and triglyceride, phospholipid, sphingolipid, cholesterol and steroid derivatives, terpenes and vitamin.

Fatty acid, cis-trans-isomer and derivative of fatty acid that fatty acid and derivant thereof can include but is not limited to saturated and unsaturated fatty acid, odd and even number comprise ethanol, ester, anhydride, hydroxy fatty acid and prostaglandin.Operable saturated and unsaturated fatty acid is including, but not limited to the straight or branched molecule with 12-22 carbon atom.The example of operable satisfied fatty acid includes, but are not limited to: lauric acid, myristic acid, Palmic acid and stearic acid.The example of operable unsaturated fatty acid includes, but are not limited to: lauric acid, physeteric acid, myristoleic acid, palmitoleic acid, petroselic acid and oleic acid.The example of operable branched chain fatty acid includes, but are not limited to: different lauric acid, different myristic acid, different Palmic acid, isostearic acid and isoprenoid.Derivative of fatty acid comprises 12-(((7 '-lignocaine coumarin-3-) carbonyl) methylamino)-stearic acid; N-[12-(((7 ' lignocaine coumarin-3-) carbonyl) methylamino) octadecanoyl] the amino Palmic acid of-2-, N-succinyl-dioleoyl phospholipid acyl ethanol amine and palmityl-homocysteine; And/or their compositions.Operable monoglyceride, diglyceride, triglyceride and derivant thereof include, but are not limited to: the carbon number of molecule is fatty acid between 6-24 or the mixture of fatty acid, digalactosyl two glyceride, DAG; 1,2-, bis-palmityls-sn-3-succinyl glycerol; With 1,3-, bis-palmityls-2-succinyl glycerol.

The phospholipid that can use includes, but are not limited to: phosphatidic acid, with saturated and lecithin, PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol, Phosphatidylserine, phosphatidylinositols, lysophosphatide acyl derivative, cuorin and β-acyl group alkyl phospholipid unsaturated lipid.The example of phospholipid includes, but are not limited to: dioleyl phosphatidyl choline (dioleoylphosphatidylcholine) (DOPC), L-Dimyristoylphosphatidylcholine (dimyristoylphosphatidylcholine, DMPC), two pentadecanoyl phosphatidylcholine (dipentadecanoylphosphatidylcholine, DPDPC), DLPC (dilauroylphosphatidylcholine, DLPC), Dioctonoyl pnosphotidyl choline (dipalmitoylphosphatidylcholine, DPPC), DSPC (distearoylphosphatidylcholine, DSPC), two Semen arachidis hypogaeae acyl phospholipids phatidylcholine (diarachidoylphosphatidylcholine, DAPC), Er behenolyl base phosphatidylcholine (dibehenoylphosphatidylcholine, DBPC), two tricosane acyl phospholipids phatidylcholines (ditricosanoylphosphatidylcholine DTPC), two wooden fatty acyl group phosphatidylcholines (dilignoceroylphatidylcholine DLPC), and PHOSPHATIDYL ETHANOLAMINE, for example dioleoyl phosphatidyl ethanolamine (dioleoylphosphatidylethanolamine) or 1-cetyl-2-palmityl phosphoglycerol ethanolamine.Also can use the synthetic phospholipid with asymmetric acyl chain (for example, with the acyl chain of 6 carbon atoms and the acyl chain of another twelve carbon atom).

Operable sphingolipid comprises ceramide, sphingomyelins, cerebroside, ganglioside, sulfolipide and haemolysis sulfolipide.The example of aphingolipid includes, but are not limited to: ganglioside GM and GM2.

Operable steroid includes, but are not limited to: cholesterol, cholesterol sulfate, Cholesteryl hemisuccinate, 6-(5-cholesterol-3 β-oxygen) hexyl-6-amino-6-deoxidation-1-sulfo--α D-lactose pyranoside, the amino 6-deoxidation-1-sulfo--α D-mannopyranose glycosides of 6-(5-cholestene-3 β-oxygen) hexyl 6-and (cholesteryl-4 '-trimethyl-35-amido) butyrate.

Operable other lipoid substance comprises tocopherol and derivant thereof, and oil and derived oils, for example stearmide.

Much cation lipid can be used, DOTMA for example, N-[1-(2,3-titanium dioxide oleoyl) propyl group-N, N, N-trimethylammonium chloride; DOTAP, 1,2-titanium dioxide oleoyl-3-(three methylaminos) propane; With DOTB, 1,2-dioleoyl-3-(4 '-trimethyl) bytyry-sn-glycerol.

Most preferred lipid is phospholipid, is preferably DPPC, DAPC, DSPC, DTPC, DBPC, DLPC, is preferably DPPC, DSPC, DAPC and DBPC.

The content range of lipid is 0.01-30% (lipid weight/polymer weight); Be preferably 0.1-20% (lipid weight/polymer weight), most preferably be 1-12% (lipid weight/polymer weight).

When microgranule is formed by method of the present invention, its size can obtain good reproduction.Here, unless otherwise mentioned, term granule " size " or " diameter " is the equal particle size of index.Enumerate an example that can be used for defining the equation of the equal particle size of number (Xn) below.

$X_n=\frac{\underset{i=1}{\overset{\∞}{\mathrm{\Σ}}}{n}_i{d}_i}{\underset{i=1}{\overset{\∞}{\mathrm{\Σ}}}{n}_i}$

Here, the powder number of a given diameter of ni=(di)

Here, term " volume mean diameter " refers to that volume weight diameter is average.Enumerate an example that can be used for defining the equation of volume mean diameter (Xv) below:

$X_v={\left[\frac{\underset{i=1}{\overset{\∞}{\mathrm{\Σ}}}{n}_i{d_i}^3}{\underset{i=1}{\overset{\∞}{\mathrm{\Σ}}}{n}_i}\right]}^{1/3}$

Here, the numbers of particles of a given diameter of ni=(di).

Grain size analysis can be used Ku Erte suspended matter determination instrument, by optical microscopy, scanning electron microscopy, transmittance electron microscopy, laser diffractometry, for example uses Malvern Mastersizer, light scattering method or propagation time interval method to carry out.Here " Ku Er special formula method " refers to: powder is dispersed in electrolyte, then with being equipped with the Coulter Multisizer II of the pipe of 50 μ m bores to analyze the suspension obtaining.The method can provide size to measure and granule density.

For the preparation of in can the preferred version through the injectable microgranule of pulmonary capillary bed, mean particle dia be less than 8 microns.Large microgranule may block lung bed, and little microgranule may not provide sufficient contrast effect.For the ultrasonic contrast medium of intravenously administrable, the size of its microgranule preferably, between 0.75～5 micron, is preferably between 1.8～3.0 microns.

In preferred version, microgranule has honeycombed structure or spongy architecture, formed, or microgranule has the polymer shell with Nidus Vespae or cavernous transformation loose structure by the pore that spreads all over polymer.In both cases, pore is all gassy.These microgranules are by comprising for example dry formation of polymer solution spraying of ammonium carbonate as described below of pore creating material.

Ultrasonic contrast medium

The example of fluorinated gas comprises CF ₄, C ₂f ₄, C ₂f ₆, C ₃f ₆, C ₃f ₈, C ₄f ₈, C ₄f ₁₀and SF ₆.Preferred Sonazoid (C ₄f ₁₀), because it can provide not agglomerative soluble gas under serviceability temperature, and be pharmaceutically acceptable.

The kind that depends on gas containing the amount of fine-grained gas, but general dosage is 75-500 μ g/mL microparticle suspending liquid.For Sonazoid, preferred gas content is that dosage is 100-400 μ g/mL microparticle suspending liquid, and preferably dosage is 150-350 μ g/mL microparticle suspending liquid.For octafluoro n-propane, preferred gas content is that dosage is 75-375 μ g/mL microparticle suspending liquid, and preferably dosage is 120-300 μ g/mL microparticle suspending liquid.

III manufactures the method for microgranule

Microgranule can be manufactured by many methods, preferably with spraying is dry, manufactures.Main standard is that, before forming microgranule, polymer must dissolve or melt in hydrophobic compound or lipid.

Solvent

During forming, polymer is dissolved in solvent conventionally.Here, polymer solvent refers to volatile organic solvent or has relatively low boiling point or can under vacuum, remove, and is can be acceptable when people is carried out to trace administration, for example dichloromethane.Also can use other solvent, for example ethyl acetate, Ethyl formate, ethanol, methanol, dimethyl formamide (DMF), acetone, acetonitrile, oxolane (THF), Methanamide, acetic acid, dimethyl sulfoxide (DMSO) and chloroform, or its mixture.Conventionally, polymer is dissolved in solvent take and form the polymer solution that concentration is 0.1～60% (weight is to volume (w/v)), is more preferably between 0.25～30% (w/v), is preferably between 0.5-10% (w/v).

Spraying is dry

The dry production of the most handy spray dried of microgranule, is dissolved in biocompatible polymer and lipid in a suitable solvent, using pore creating material as solid or Solution Dispersion in polymer solution, then that the spraying of polymer solution and pore creating material is dry, form microgranule.Here, the process of " spraying is dry " of polymer solution and pore creating material refers to polymer solution and pore creating material carries out atomization so that it forms mist, thereby and directly contacts with hot carrier gas its dry process that makes.Utilize the conventional spray dryer in this area, polymer solution and pore creating material are atomized at the air inlet of spray dryer, by least one hothouse, then with Powdered, are collected.Temperature is different and can change according to used gas or polymer.Can produce desirable product by controlling the temperature of entrance and exit.

In spray-drying process, the size of formed microgranule and form are affected by following factor: for nozzle function that polymer solution and pore creating material are sprayed, nozzle pressure, with the molecular weight of flow velocity, the polymer using, the concentration of solution polymer, the type of the kind of polymer solvent, pore creating material and amount, vapo(u)rizing temperature (entrance and exit temperature) and the polymer of the polymer solution of pore creating material.Conventionally, suppose that polymer solution concentration is the same, polymer molecular weight is higher so, and particle size is larger.

Spray-dired general technology parameter is as follows: inlet temperature=30-200 ℃, outlet temperature=5-100 ℃, polymer flow velocity=10-5,000ml/min.

Can before spraying is dry, make polymer solution and pore creating material emulsifying together with gas, thereby gaseous state diagnostic agent is sealed.Or, can produce inflation microgranule at spraying drying steps, then by microgranule is applied to pfc gas, air is replaced with to desirable gas, or microgranule evacuation is removed to the air of sealing, be then full of desirable pfc gas.If adopt evacuation step to carry out exchanging gas, can use freeze dryer or vacuum tank.

Promote the additive that micronize forms

In the forming process of microgranule, can add various surfactants.The example of operable emulsifying agent or surfactant (0.1-15%w/w polymer) comprises biologically the most acceptable emulsifying agent.Its example comprises natural and the synthetic bile salts combining with aminoacid or bile acid, and uncombined deoxidation cholyltaurine and cholic acid.

With respect to the volume of polymer solution, pore creating material is included in polymer solution with the amount of 0.01%～90wt%, to increase the formation in hole.For example, in spraying is dry, can with solid form or be dissolved in solvent for example the solution form in water use pore creating material, described pore creating material can be enumerated volatile salts for example ammonium bicarbonate, ammonium acetate, ammonium carbonate, ammonium chloride or ammonium benzoate or other volatile salts.Then, by solid pore creating material or solution emulsifying together with polymer solution of comprising pore creating material, to produce dispersion or the microdroplet of pore creating material in polymer.Then these dispersions or emulsion spray drying are removed to polymer solvent and pore creating material.After polymer precipitation, can be by the microgranule of sclerosis freezing and lyophilizing to remove all pore creating materials that are not removed in polymer settling step.

Preferably utilize polymer formation microgranule; for example lactide is that 50: 50 and weight average molecular weight are 20 to the ratio of Acetic acid, hydroxy-, bimol. cyclic ester; 000-40; poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) within the scope of 000 dalton; and phospholipid; the DAPC (diarachidoylphosphatidylcholine) ((1 of 5-6.6% (DAPC weight/polymer weight); 2-bis-Semen arachidis hypogaeae acyl group-sn-glyceryl-3-phosphocholines (DAPC)) ((1,2-diarachidoyl-sn-glycero-3-phosphocholine).Microgranule is at mannitol and tween in further formulated to be created in the microgranule dry powder of inflating again with Sonazoid on freeze dryer.Dry powder is prepared again with 5ml sterilized water before use, to add in the bottle of dry powder water then jolting the microparticle suspending liquid in the mannitol oozing such as to be created in.Preferably the character of suspension is: each dosage microparticle suspending liquid contains the Sonazoid that gas content is 150-350 μ g/ml, and the dosage of microparticle suspending liquid is 1.5-2.8 * 10 ⁹individual microgranule/milliliter, the dosage of microparticle suspending liquid is 30-45mg microgranule/milliliter, particle mean size is in the scope of 1.8-3.0 micron.

The application of IV microgranule

1. give the formula of patient's administration

Microgranule can further process to produce dry powder with excipient.While again preparing with pharmaceutically acceptable carrier before giving patient's administration, excipient provides tension force or permeability or alleviates the suspension of microgranule.Be suitable for providing the excipient of permeability or tension force to have sugar and salt, sugar includes, without being limited to mannitol, dextrose (dextrose) or glucose, and salt includes, without being limited to sodium chloride or sodium phosphate.Be suitable for providing the excipient that alleviates microsphere suspension to comprise the acceptable wetting agent of any pharmacy or surfactant, include, without being limited to polysorbate80 (tween ), TWEEN-20 (tween ), Pluronic (Pluronic) or Polyethylene Glycol.As a reference, here describing some is suitable for providing permeability or tension force maybe can be used as the excipient of wetting agent, for example < < handbook of pharmaceutical excipients (Handbook of Pharmaceutical Excipients) > > is (the 4th edition, medicine association of Great Britain imperial family, science with put into practice publishing house) or < < Lei Mingdun: pharmaceutical science with put into practice > > (the 19 edition, Mack publishing company).The dry powder of microgranule and excipient produces by suspended particulates in the solution of excipient.If necessary, further use size fractionated step.Microgranule in excipient solution is loaded in bottle or syringe, freezing, lyophilizing, generation dry powder formulation.When step of freeze drying finishes, by recharge freeze dryer with pfc gas, make microgranule be full of pfc gas.Then bottle or syringe being clogged or cover lid with stopper, is in the situation of bottle, turns down stopper edge is curling.This causes being full of perfluocarbon at the headroom of bottle or syringe.

Or, can use drug excipient and microgranule dry mixed, then pack bottle or syringe into.Little bottled syringe is placed on to evacuation on freeze dryer or in vacuum tank, then allows microgranule be full of pfc gas.Then bottle or syringe being clogged or cover lid with stopper, is in the situation of bottle, turns down stopper edge is curling.This causes being full of perfluocarbon at the headroom of bottle or syringe.

2. dosage unit

Can use the microgranule of different size dosage unit.For example small dosage units can comprise the microgranule of 25-75mg.A middle dosage unit can comprise 75-150mg.A large dosage unit can comprise the microgranule of 150-250mg.An especially big dosage unit can comprise the microgranule of 150-1000mg.

When forming microparticle suspending liquid after again preparing, in suspension, the mass concentration of microsphere generally changes within the scope of 20～60mg/mL.The preferred 25-50mg/mL of the mass concentration of microsphere in suspension, in suspension, the mass concentration of microsphere most preferably is 30-45mg/mL.Preferred 1.0-3.5 * 10 of the concentration of microgranule in suspension ⁹microgranule/mL, in suspension, the concentration of microgranule most preferably is 1.5-2.8 * 10 ⁹microgranule/milliliter.Preferably microgranule has the particle mean size that is less than 8 microns, is preferably in the scope of 1.8-3.0 micron.

Pharmaceutically acceptable carrier comprises water for injection, sterilized water, saline, the saline that contains glycerol, contains tween saline, contain tween saline, etc. the dextrose (2.5%) that oozes of the glucose (5%), 1/2 etc. that oozes, etc. the mannitol (2.5%) that oozes of the mannitol (5%), 1/2 etc. that oozes, contain tween grade ooze mannitol, contain tween grade ooze mannitol.

3. test kit

The test kit of the microgranule parenteral that can be provided for comprising pfc gas.This test kit comprises at least two components.A dry powder contrast medium that composition comprises a dosage unit in bottle or syringe, another composition comprises the pharmaceutically acceptable carrier in bottle or syringe.Before to patient's administration, pharmaceutically acceptable carrier is added in the dosage unit of dry powder contrast medium, forms the microparticle suspending liquid that is filled with gas, and described microparticle suspending liquid can be used as ultrasound wave video picture contrast medium in the diagnostic imaging of any route of administration.

4. for bottle or the container of microgranule

For test kit, do not need special bottle or syringe or adapter, common bottle, syringe and adapter can be used for microgranule.To unique requirement of bottle, be exactly between stopper and container, to have good sealing.Therefore, the quality of sealing just becomes the problem of major concern, and the decline of any sealing integrity all will cause undesirable material to enter bottle or gas leak.Except guarantee aseptic, for the product under reduced pressure clogging, for guaranteeing the correct preparation again of safety, the maintenance of vacuum is absolutely necessary.About stopper, can be to take elastomer to be individualized compound or the multi-component formula on basis, for example poly-(isobutene .) or " butyl rubber ", and must be able to not allow used gas permeation.According to the accumulated dose of dry powder in bottle, select the size of bottle.Gravel size decision 5mL, 10mL, 20mL and the 30mL of bottle.According to the accumulated dose of dry powder in syringe, select the size of syringe.Gravel size decision 5mL, 10mL, 20mL and the 50mL of syringe.

5. diagnostic application

Microparticle compositions can be used for, in many different diagnostic application, comprising ultrasound wave video picture, nuclear magnetic resonance, fluoroscopy, X ray and computerised tomography.

In preferred version, microgranule is used to ultrasound procedures, and for example angiography and echocardiography, include, without being limited to diagnosis and the ejection fraction assessment of ventricle video picture, myocardial blood flow assessment, the assessment of myocardium blood volume, coronary artery disease.

Microgranule can be used for angiography, for detection of liver and kidney disease, for detection of with characterize tumor mass and tissue and measure peripheral blood speed.Microgranule also can link together with ligand, and described ligand minimizes or make the concrete position in granule targeting body by the adhesion of tissue.

Obtain the conventional method of image

Dry powder microgranule is prepared again with pharmaceutically acceptable carrier before use, then adopts suitable approach for example, as being injected into blood vessel (intravenous (i.v) or intra-arterial (i.a)) or oral, one of patient's administration is effectively measured, for detection of.Microparticle compositions can with inject or the mode of short transfusion (being less than 30 minutes) to patient's intravenous administration.Preferably inject in the time that is controlled at 15 seconds to 20 minutes, be preferably 30 seconds to 15 minutes.Usually, for patient, conventionally each intravenous injection is controlled in the dosage range of 0.025 to 8mg/kg body weight, is preferably 0.05 to 4mg/kg dosage range.

For diagnostic ultrasound application, at least a part for patient body is applied to energy so that destination organization video picture.Then obtain the visual picture at position in patient body, can determine thus and have or do not exist ill tissue.The ultrasonic imaging technique that comprises Second Harmonic Imaging and gate imaging (gated imaging) is all well known in this area, and also has some to describe, Uhlendorf for example, iEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, 14 (1): 70-79 (1994); With Sutherland etc., journal ofthe American Society ofEchocardiography, 7 (5): 441-458 (1994), at this, is all incorporated to it herein and is incorporated by reference.

Can apply ultrasound wave with sensor.If desired, ultrasonic can be pulse or can be continuous.Therefore, diagnostic ultrasound generally includes the application of echo, and subsequently, in a listening period, sonac receives reflected signal.Can use harmonic wave, higher harmonics or subharmonic.Can effectively use the second harmonic mode of accepting 2x frequency, x is non-staple frequency here.This can be used for reducing the signal from background material, utilizes developer to strengthen the position of wishing from this developer targeting of signal of sensor, for example clot.Other harmonic signal, for example odd harmonic signal, for example 3x or 5x, also can similarly receive in this way.Rd harmonic signal is x/2 and x/3 for example, also can be received simultaneously and process to form image.

In addition, can apply power Doppler (Power Doppler) or color Doppler (Color Doppler).The in the situation that of power Doppler, power Doppler's relatively high energy can allow pore resonate.This can be created in the acoustic irradiation in the scopes such as subharmonic or higher hamonic wave, identical with the supersonic frequency applying.

Concrete video picture application

Microgranule as described herein can be used to cardiology and radiology purposes.For the application of cardiology, to patient's delivery of particulate compositions, by Ultrasound Instrument scan patients, obtain the visual image at cardiovascular position.As selection, microparticle compositions can with the stresser of materia medica or the stresser administering drug combinations of physics.Suitable materia medica stresser comprises the vasodilations coronarius such as dipyridamole or adenosine, dobutamine etc. affect the medicament (for example strengthening cardiac contractile force) of contractility, or the myocardium speed change agent (for example strengthening contraction frequency) such as dobutamine.Suitable mechanical stress thing comprises physical training, for example, use treadmill or stationary bicycle.

For radiologic application, to patient's delivery of particulate compositions, by Ultrasound Instrument scan patients, obtain the visual image of patient's wish check point.

Microgranule can be used for evaluating function and evaluation myocardial blood flow or myocardium blood volume or the diagnosis of coronary heart disease (coronary artery disease) of cardiovascular system.For example, microgranule can strengthen ventricle video picture, therefore can help to carry out local cardiac function by blood vessel wall motion analysis and analyze, and by ejection fraction measurement, contributes to comprehensive cardiac function to analyze.This microgranule can also be used to evaluate myocardial blood flow so that functional heart tissue and ischemia (blood flow is not enough) heart tissue or the difference of infraction (dead) heart tissue are come.The contrasting signal detecting in cardiac muscle can be as the estimation of myocardium blood volume, because ultrasonic contrast medium resides in blood vessel after intravenously administrable.In a period of time, disappearance or the minimizing of a specific myocardial sites, setting off by contrast intensity or brightness of image are the signs of Oligemia (namely not enough).

General, unless patient has serious coronary heart disease, as for example evaluated by the method for ultrasound contrast, the blood flow at each position of heart should show as normally.In order to detect the blood flow anomalies that there is no serious cardiopathic patient, or it is not enough to detect less myocardial blood flow, must increase the demand of blood flow to heart by bringing out stress.Can by allow patient take exercise or administration medicine compound for example stress is brought out in vasodilation, the medicament that affects contractility or myocardium speed change agent.During exercise or medicine stress, blood flow deficiency can more easily be detected, this is because at the position of the coronary artery blood supply by with stenosis, the ability that increases blood flow has reduced.Myocardium ultrasonography contrast after ultrasonic contrast medium's administration can be before pre-stress state be made under (being rest state) and stress state.During stress video picture, finding does not need the myocardial sites highlighting and during quiet video picture, not to find above-mentioned position, the sign of Here it is ischemia.In the myocardial sites of all finding not need to highlight during stress video picture and during quiet video picture, it is the sign of blocking.

In one embodiment, myocardial blood flow can be measured by following steps: inject first to patient (1), delivery of particulate compositions, (2) by Ultrasound Instrument scan patients, carry out imaging to obtain the visual image at cardiovascular position, (3) with medicine stresser or exercise, bring out patient's stress state, (4) drug administration by injection microparticle compositions continuation scanning for the second time, and (5) are by with the naked eye directly observing or with quantitative image analysis, the image difference obtaining in step (2) and (4) being evaluated.

For radiology, apply, microgranule can be used for improving the ability for the ultrasound wave video picture of radiology indication, comprise the video picture of kidney, liver and peripheral vascular disease, increase the visibility of blood flow and blood flow patterns, and improve the trickle pathological changes of body interior depths or the detection of structure.Microgranule can be used to the indication of trunk (macrovascular) and capillary vessel (microvascular).In the indication of trunk (morbid state of health aorta and vein and the diagnosis of condition), by range estimation intracranial vessel, microgranule can contribute to detect the situation of apoplexy and pre-apoplexy, by evaluating the not closed and peripheral vessel thrombosis of degree, the vascular grafts of carotid artery stenosis, microgranule can contribute to detect the atherosclerosis of in large blood vessel (for example carotid artery).For capillary vessel indication (morbid state and by analyzing the diagnosis of capillary vessel blood flow patterns), microgranule can contribute to identify pathological changes, tumor or other the disease (for example adenoma or hemangioma) in liver, for example, with pathological changes, tumor or other the disease (aneurysm of splenic artery) in kidney, spleen, and the pathological changes of chest, ovary, other tissue and organ, tumor or other disease.

Can, by then carrying out video picture with ultrasonic scanning patient to patient's delivery of particulate compositions, to obtain patient's visual image of any pathological tissues with it, thereby patient's pathological tissues be diagnosed.Pathological tissues can show as the position of certain brightness enhancing or not demonstrate the position that brightness strengthens.

The enhancing image obtaining with microparticle compositions

Microgranule produces and strengthens image after administration.Strengthening image can be by comparing the increase of brightness of image when not taking ultrasonic contrast medium or the basic elimination by artificial image in image is showed.Therefore, the ultrasound wave video picture that comprises heart tissue and the vascular tissue being connected with cardiovascular position is associated, and the basic elimination that strengthens the artificial image occurring in increase that image can be by for example cardiovascular station diagram image brightness and/or cardiovascular station diagram picture is showed.After single medicament administration, figure image persistence is 10 seconds to 60 minutes.Image preferably continues 20 seconds to 30 minutes, preferably continues 30 seconds to 20 minutes.In a preferred version, in ventricle, ultrasound wave video picture strengthens and to continue to be greater than 5 minutes, and/or in cardiac muscle for being greater than 1 minute.

Can be naked depending on observing or with quantitative Imaging Analysis, the increase of brightness of image being evaluated by naked eyes.By specific gray scale (approximately from 0 to about 255VDUs or colour code), as above differentiating with reference to doing, preferably brightness increase level has about 10VDUs (gray scale) at least.More preferably brightness increase level is greater than about 10VDUs, and for example about 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100VDUs.In some embodiments, brightness increase is greater than about 100VDUs, for example, and about 105,110,115,120,125,130,135,140,145 or 150VDUs.In other embodiment, brightness increase is greater than about 150VDUs, for example, and about 155,160,165,170,175,180,185,190,195 or 200VDUs.For example, or brightness is greater than about 200VDUs,, about 205,210,215,220,225,230,235,240,245,250 or 255VDUs.

By can further understanding said method and compositions in conjunction with following nonrestrictive embodiment.

Embodiment

Material

Acetic acid, ammonium bicarbonate, mannitol USP and polysorbate80 (not being derived from the composition of animal) are purchased from Spectrum Chemicals company (Gardena, CA).Polymer (poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) (PLGA) (50: 50)) and DAPC (1,2-bis-Semen arachidis hypogaeae acyl-sn-glyceryl-3-phosphocholines (DAPC)) respectively purchased from the (Ingelheim of Boehringer Ingelheim company, German) and Avanti company (Alabaster, AL).Dichloromethane is purchased from EM Science company (EMD Chemicals, Gibbstown, NJ).Bottle (30 milliliters of tubulose bottles) and stopper (20 millimeters, Lycoperdon polymorphum Vitt, single QI KOU, Fluro-Tec company) are from West Pharmaceutical Services company (Lionville, PA).Sonazoid (DFB) gas is purchased from F2 Chemical Products Ltd. (Lancashire, Britain).

Analytical method

The quantitative analysis of particle mass concentration

In bottle, the mass concentration of microgranule adopts ICP-MS method (feeling even plasma mass spectrography) to carry out quantitatively.In microgranule, the quantity of polymer is by measuring stannum analysis by ICP-MS method.The quantity that is present in the polymer in microgranule is to compare and measure with being present in for manufacturing in the polymer of certain share of microgranule the quantity of stannum according to the quantity that is present in the stannum in microgranule.In microgranule, the quantity of phospholipid is by measuring phosphorus analysis with ICP-MS.The quantity that is present in the phosphorus in microgranule is to compare and measure with the quantity that is present in the phosphorus of phospholipid itself according to the quantity that is present in the phosphorus in microgranule.In every milliliter of suspension, the quality of microgranule is to calculate like this: the quantity of polymer and phospholipid in every bottle is added up, then divided by the volume (5mL) of again preparing.

Grain size analysis

The particulate samples of preparation is again added in electrolyte solution, with the Coulter Multisizer II that 50 μ m mouth pipes are housed, the granular size of the suspension obtaining and particle concentration are analyzed.

The gas content of microgranule

Dry powder bottle is prepared again with 5mL water, and jolting produces microparticle suspending liquid.With pin and syringe, through stopper, take out one group of 0.3 ml aliquots sample, the DFB content in the suspension obtaining is analyzed.These aliquots are injected to the headspace vial of seal.Headspace vial is balance at least 10 hours at room temperature.Then sample is heated to 45 ℃, in headspace sampler calorstat, keeps 20 minutes.Adopt gas chromatography, with import and the flame ionic detector of the parcel of blowing, the head space gas above suspension is analyzed.Adopt area single-point calibration method to carry out quantitatively.

Gas chromatography systems parameter and temperature program(me) are listed in table 1 and table 2.

Table 1:GC systematic parameter

Sample:	Head space, 1mL injection annulus
		Detector	FID
Chromatographic column	Supelco 60/80Carbopack B 5％Fluorocol
		Injector temperature	150℃

Detector temperature	325℃
		Carrier gas	Helium (25mL/min)
FID gas	Hydrogen (60mL/min)
			Air (350mL/min)
	Nitrogen (5mL/min)

Table 2:GC temperature program(me)

	Initial temperature	Speed	Final temperature	Retention time
					Initial condition	40℃	N/A	N/A	2.0min
The first gradient	40℃	5℃/min	65℃	0.0min
					The second gradient	65℃	10℃/min	130℃	0.0min
The 3rd gradient	130℃	50℃/min	200℃	0.0min
					Final condition	200℃	N/A	N/A	3.1min

Embodiment 1: as ultrasonic contrast medium's microgranule manufacture

At 25 ℃ by the acetate dissolution of the DAPC of the PLGA of 176g, 10.6g (1,2-, bis-Semen arachidis hypogaeae acyl-sn-glyceryl-3-phosphocholines (DAPC)) and 2.26g in 5.88L dichloromethane, be mixed with organic solution.Ammonium bicarbonate by 68.5g is dissolved in to the aqueous solution forming in 338ml water for injection and is added in above-mentioned organic solution, with rotor stator emulsifying mixer in 10L homogenate groove with the rotating speed homogenate of 4310RPM 10 minutes.

With nitrogen, as atomization gas and dry gas, the emulsion producing is sprayed dry.Emulsion is sprayed dry on workbench top, spray dryer adopts purchased from the Spraying Systems (Wheaton of company, IL) air-atomizing nozzle and purchased from the glass hothouse/cyclone separator of Buchi company (Brinkmann, Westbury, NY).Spraying drying condition is as follows: Emulsion flow velocity is 40ml/min, and atomization gas flow velocity is 30L/min, and dry gas flow velocity is 46kg/hr, and outlet temperature is 12 ℃.

By disperseing, freezing and step of freeze drying further processes the dried product of spraying.In 5.0L water, dissolve 140g mannitol and 4.10g polysorbate80, preparation water-bearing media.Concentration by the dried microgranule of spraying with 25mg/ml is dispersed in above-mentioned medium.Use rustless steel purchased from Misonix company (Farmingdale, New York), 800 grades, flow cell ultrasonoscope dispersion is carried out to decondensation and make its diameter 10 that sieves " vibration sieve (RBF-10) (purchased from Vorti-Siv company (Salem, OH)).Ultrasonoscope is protected with the chuck of 4 ℃, prevents dispersion heating.Dispersion is with sieve the successively sieve of 25 μ m and 20 μ m of the speed of 150mL/min.Pack the dispersion after sieving into bottle (10ml dispersion packs the bottle of 30ml into), stopper in partial plugs, immerses in liquid nitrogen freezing.

After freezing, by bottle lyophilizing.After lyophilizing completes, spacing container, is inflated to Sonazoid (DFB) in bottle again, so that the pressure before jumping a queue reaches 5 kPas.

Dry powder is prepared again with 5ml sterilized water before use, in the bottle of dry powder, adds then jolting of water, to obtain microgranule, the suspension in the mannitol oozing such as is suspended from.Suspension comprises 2.2 * 10 ⁹microgranule/mL, 37mg microgranule/mL, the particle mean size of microgranule is 2.2 microns.

Embodiment 2: the Leakage Gas speed of microgranule

With gas chromatography (GC), two batches of microgranules producing according to the method for embodiment 1 (batch 1 and batch 2) Leakage Gas speed is separately evaluated, referred to above-mentioned analytical method one joint.The 3rd batch of microsphere (criticizing 3) produced with the method for similar embodiment 1, just in producing microgranule process, saves phospholipid, two Semen arachidis hypogaeae acyl phosphoric acid fat choline (1,2-, bis-Semen arachidis hypogaeae acyl-sn-glyceryl-3-phosphocholines (DAPC)).

Table 3: the gas content of microgranule and gas leak rate

The microgranule that comprises DAPC has been lost about 10% initial gas content after 70 minutes, and containing the microgranule of DAPC, has not lost 87% initial gas content.In addition, the microgranule that comprises DAPC has a higher initial gas content with respect to the microgranule that there is no DAPC.This shows to comprise DAPC is very important for the formation of the inner loose structure of microgranule in spray-drying process and the gas of maintenance microgranule inside.

After patient's administration, wish that the ultrasonic contrast medium's total duration using is different and different according to the kind of applied cardiology or radiology ultrasonic examination, be approximately 30 seconds to 60 minutes conventionally.Therefore, if surpass the cycle of ultrasonic examination, just nonsensical to the mensuration of the microgranule gas loss that comprises lipid DAPC so.

Embodiment 3: the cardiac image as microgranule dosage function strengthens

In normal adults, with it the microgranule of producing according to method described in embodiment 1 is studied.Dry powder is preparation again before use, to adding 5mL sterilized water jolting bottle in bottle 10 times.The ultimate density of the microsphere in the suspension producing is approximately 37mg/mL.Experimenter receives the single dosage of 0.5mg/kg, 2.0mg/kg or 4.0mg/kg body weight.Experimenter carries out transthoracic ultrasonic imaging with consecutive second harmonic imaging (frame frequency 15Hz, sensor frequency 2.1/4.2MHz).Image is carried out to the visual evaluation of potentiation intensity and persistent period.

Under the dosage of 2mg/kg and 4mg/kg, the ventricle potentiation persistent period all surpasses 9 minutes.Under these two dosage, when experimenter during by re-imaging, had 13 people still can see contrast effect after 30 minutes in 15 people, this shows that microgranule provides long potentiation.

The persistent period of ventricle potentiation is summarized in table 4.

Table 4: the persistent period of left ventriculography image intensifying

Dosage (mg/kg body weight)	The average duration of ventricle potentiation (minute)
		0.5	2.6
2.0	＞9.6
		4.0	＞9.6

Embodiment 4: the comparison in assess cardiac image of microgranule and commodity

The adult that two individual weights and cardiac function are matched carries out the comparative study of heart ultrasonic video picture.To first experimenter, give the microgranule unitary agent that the method for embodiment 1 is produced.Dry powder is preparation again before use, to adding then jolting bottle 10 times of 5ml sterilized water in bottle.In the suspension producing, the ultimate density of microsphere is approximately 37mg/mL, and the gas content of suspension is approximately 250g/mL.First experimenter accepts the dosage of 4mg microgranule/kg, and this dosage is equivalent to the gas dosage of 27g/kg body weight.Second experimenter accepts the commercially available ultrasonic contrast medium of single dose (Amersham Health), it contains the perfluoropropane that comprises albumen endosperm microsphere.What two experimenters accepted is the gas (27 μ g/kg body weight) of uniform amt, and described gas is sound wave active component.Two experimenters carry out transthoracic ultrasonic imaging with consecutive second harmonic imaging (frame frequency 15Hz, sensor frequency 2.1/4.2MHz).Image is carried out to the visual evaluation of potentiation intensity and persistent period.

The persistent period of ventricle potentiation and myocardium potentiation is summarized in table 5.

Table 5: the persistent period of different ultrasonic contrast medium figure image intensifying

With the microgranule that the method described in embodiment 1 is produced, all can provide the image of enhancing in ventricle and cardiac muscle, the persistent period of the image of described enhancing is significantly longer than for thering is the suitable persistent period by complete cardiac tests of ultrasonic enforcement.

Embodiment 5: by microparticle formulations, myocardial blood flow is assessed to evaluate ischemia

The microgranule that the method for experimenter's administrable embodiment 1 that is diagnosed as coronary heart disease is produced.Experimenter has accepted two amicron injections in 60 minutes.(" quiet injection ", 1.7mg/kg) is used for evaluating the cardiac muscle under rest state in microgranule injection first.Carry out, before microgranule injection for the second time, with vasodilation dipyridamole (0.56mg/kg), making experimenter produce pharmacological stress.After stress induction, experimenter accepts microgranule injection for the second time, and (" stress injection ", 1.3mg/kg), evaluates the cardiac muscle under stress state.

Peace and quiet after experimenter's delivery of particulate and stress state image are compared, demonstrate the myocardial sites of the increase minimum of figure image intensifying, and this position long-pending change greatly after stress-induced.This existing infraction in region that shows cardiac muscular tissue also has ischemic part.Ischemic detection is confirmed with another diagnostic techniques-core video picture.The core that carries out quiet and stress after administration 99Tc (MIBI) pours into (nulear perfusion), with commercially available gamma counter to experimenter's imaging.The damage of recording on the ultrasonoscopy of quiet and stress is confirmed in the core perfusion image of quiet and stress.

Claims (33)

1. a test kit, it comprises a kind of dosage particles and for again preparing the solution of this dosage particles,

Wherein this dosage particles comprises microsphere, and described microsphere contains polyalcohols acid copolymer and hydrophobic compound, and described microsphere to be encapsulated with under body temperature be the pfc gas of gas,

Wherein, described polyalcohols acid copolymer is poly-(lactide altogether Acetic acid, hydroxy-, bimol. cyclic ester), and described hydrophobic compound is two Semen arachidis hypogaeae acyl phospholipids phatidylcholines, and described pfc gas is Sonazoid, and

Wherein said microsphere is the porous spherical with honeycombed structure or spongy architecture, and

Wherein said microsphere is dry powder, and

Wherein said microsphere has the particle mean size of 1.8～3.0 microns, and

Described microsphere is again prepared to form to wait with solution and is oozed suspension, and the microsphere concentration in this suspension is 1.0 * 10 ⁹to 3.5 * 10 ⁹microsphere/mL suspension or microsphere mass concentration are 25～50mg microsphere/mL suspension,

The amount of wherein said Sonazoid gas is 75～500 μ g/mL suspensions, and

Wherein the dosage range of microsphere described in dosage particles is 0.5～4.0mg microsphere/kg body weight, thereby be suitable for providing in cardiac muscle, compares the enhancing ultrasonography that is greater than 1 minute when not taking contrast medium.

2. test kit as claimed in claim 1, is characterized in that, the microsphere concentration range in described suspension is 1.5 * 10 ⁹to 2.8 * 10 ⁹the suspension of microsphere/mL or microsphere mass concentration are 30 to 45mg microsphere/mL suspension.

3. test kit as claimed in claim 1, is characterized in that, described microsphere has the particle mean size of 1.9～2.6 microns.

4. test kit as claimed in claim 1, is characterized in that, described dosage is selected from 0.5mg microsphere/kg body weight, 2.0mg microsphere/kg body weight and 4.0mg microsphere/kg body weight.

5. test kit as claimed in claim 1, is characterized in that, with the microsphere suspension of 100～400 μ g/mL dosages, provides Sonazoid.

6. test kit as claimed in claim 5, is characterized in that, with the microsphere suspension of 150～350 μ g/mL dosages, provides Sonazoid.

7. test kit as claimed in claim 1, is characterized in that, described hydrophobic compound mixes with the ratio of 0.01～30% (hydrophobic compound weight/polyalcohols acid copolymer weight) with described polyalcohols acid copolymer.

8. test kit as claimed in claim 1, it is characterized in that, the lactide of described poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) and the ratio of Acetic acid, hydroxy-, bimol. cyclic ester are that 1:1, weight average molecular weight are 20～40kDa, and described DAPC is to mix with described polyalcohols acid copolymer with the ratio of 5～6.6% (DAPC weight/polyalcohols acid copolymer weight).

9. test kit as claimed in claim 1, is characterized in that, described suspension further contains one or more excipient that is selected from sugar, salt and surfactant.

10. manufacture, for a method for the compositions of ultrasound contrast video picture, comprising:

The microsphere that suspends and contain polyalcohols acid copolymer and hydrophobic compound in solution;

Described suspension is put into bottle or syringe;

Freezing described suspension;

Make described bottle lyophilizing to produce dry powder in bottle or syringe; With

Be used under body temperature as the pfc gas of gas recharges freeze dryer to obtain compositions,

Wherein said compositions comprises microsphere, and described microsphere is encapsulated with pfc gas, and

Wherein said microsphere is the porous spherical with honeycombed structure or spongy architecture, and

Wherein said microsphere is dry powder, and

Wherein said microsphere has the particle mean size of 1.8～3.0 microns,

Wherein, described polyalcohols acid copolymer is poly-(lactide altogether Acetic acid, hydroxy-, bimol. cyclic ester), and described hydrophobic compound is two Semen arachidis hypogaeae acyl phospholipids phatidylcholines, and described pfc gas is Sonazoid, and

While again preparing with pharmaceutically acceptable carrier before microsphere is being used, they can form the second suspension, and this second suspension is to wait to ooze suspension, and microsphere concentration is wherein 1.0 * 10 ⁹to 3.5 * 10 ⁹microsphere/mL the second suspension or microsphere mass concentration are 25～50mg microsphere/mL the second suspension,

The amount of wherein said Sonazoid gas is 75～500 μ g/mL suspensions, and

Wherein the dosage range of microsphere described in dosage particles is 0.5～4.0mg microsphere/kg body weight, thereby be suitable for providing in cardiac muscle, compares the enhancing ultrasonography that is greater than 1 minute when not taking contrast medium.

11. methods as claimed in claim 10, is characterized in that, described suspension further contains excipient.

12. 1 kinds of manufactures are used for the method for the compositions of ultrasound contrast video picture, comprising:

By microsphere dry mixed in the solution that contains polyalcohols acid copolymer and hydrophobic compound;

Mixture is put into bottle or syringe;

After evacuation, be used under body temperature as the pfc gas of gas is full of bottle or syringe to obtain compositions,

Wherein said compositions comprises microsphere, and described microsphere is encapsulated with pfc gas, and

Wherein said microsphere is the porous spherical with honeycombed structure or spongy architecture, and

Wherein said microsphere is dry powder, and

Wherein said microsphere has the particle mean size of 1.8～3.0 microns,

Wherein, described polyalcohols acid copolymer is poly-(lactide altogether Acetic acid, hydroxy-, bimol. cyclic ester), and described hydrophobic compound is two Semen arachidis hypogaeae acyl phospholipids phatidylcholines, and described pfc gas is Sonazoid, and

While again preparing with pharmaceutically acceptable carrier before microsphere is being used, they can form etc. oozes suspension, and the microsphere concentration in this suspension is 1.0 * 10 ⁹to 3.5 * 10 ⁹microsphere/mL suspension or microsphere mass concentration are 25～50mg microsphere/mL suspension,

The amount of wherein said Sonazoid gas is 75～500 μ g/mL suspensions, and

Wherein the dosage range of microsphere described in dosage particles is 0.5～4.0mg microsphere/kg body weight, thereby be suitable for providing in cardiac muscle, compares the enhancing ultrasonography that is greater than 1 minute when not taking contrast medium.

13. methods as claimed in claim 12, is characterized in that, described mixture further contains excipient.

14. 1 kinds of dosage particles that are dry powder form, it is used as injectable enhancing ultrasound contrast developer after again preparing with pharmaceutically acceptable carrier, and described dosage particles comprises

(a) for placing the container of dry powder,

(b) the dry powder microgranule that dry powder comprises the amount that is selected from the group consisting of 150-250mg and 250-1000mg, described dry powder microgranule comprises polyalcohols acid copolymer and hydrophobic compound, wherein microgranule is the porous sphere with honeycomb texture or sponge structure, and the particle mean size of microgranule is 1.8-3.0 micron, and

(c) Sonazoid (C ₄f ₁₀) gas,

Described polyalcohols acid copolymer is poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), and described hydrophobic compound is two Semen arachidis hypogaeae acyl phospholipids phatidylcholines,

Wherein, by adding the pharmaceutically acceptable carrier that is applicable to injection of ormal weight again to prepare the dry powder in container, thereby form particle concentration, be 1.5 * 10 ⁹-2.8 * 10 ⁹microgranule/mL or particle mass concentration are the suspension of 25-50mg microgranule/mL,

The amount of wherein said Sonazoid gas is 75～500 μ g/mL suspensions, and

Wherein microparticle suspending liquid provides the myocardium enhancing ultrasound wave video picture that is greater than 1 minute.

15. dosage particles as claimed in claim 14, it provides the enhancing ultrasound wave video picture of the ventricle of at least 30 minutes.

16. dosage particles as claimed in claim 14, it comprises sterilized water as the pharmaceutically acceptable carrier that is applicable to injection.

17. dosage particles as claimed in claim 16, it forms particle mass concentration is the suspension of 30-45mg microgranule/mL.

18. dosage particles as claimed in claim 14, wherein the particle mean size of microgranule is 1.9-2.6 micron.

19. dosage particles as claimed in claim 14, the dosage that it comprises 0.5-4.0mg microgranule/kg body weight.

20. dosage particles as claimed in claim 19, wherein said dosage is selected from the group consisting of 0.5mg microgranule/kg body weight, 2.0mg microgranule/kg body weight and 4.0mg microgranule/kg body weight.

21. dosage particles as claimed in claim 14, wherein Sonazoid (C ₄f ₁₀) with the dosage of the microparticle suspending liquid of preparation again of 100-400 μ g/mL, provide.

22. dosage particles as claimed in claim 21, wherein Sonazoid (C ₄f ₁₀) with the dosage of the microparticle suspending liquid of preparation again of 150-350 μ g/mL, provide.

23. dosage particles as claimed in claim 14, wherein contain hydrophobic compound with respect to polyalcohols acid or its copolymer or its mixture with the ratio of 1-12% (hydrophobic compound weight/polymer weight).

24. dosage particles as claimed in claim 14; the lactide of wherein said poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) is that the weight average molecular weight of 1:1 and polymer is 20 to the ratio of Acetic acid, hydroxy-, bimol. cyclic ester; 000-40; within the scope of 000 dalton, the ratio of described two Semen arachidis hypogaeae acyl phospholipids phatidylcholines is 5-6.6% (two Semen arachidis hypogaeae acyl phospholipids phatidylcholine weight/polymer weight).

25. dosage particles as claimed in claim 14, wherein said container is bottle or syringe.

26. dosage particles as claimed in claim 25, wherein said bottle or syringe further comprise one or more and are selected from the excipient in the group consisting of sugar, salt and surfactant.

27. dosage particles as claimed in claim 14, it mainly consists of 1 or 2 dosage.

28. dosage particles as claimed in claim 14, it mainly consists of maximum 5 dosage.

The ultrasound contrast video picture test kit of 29. 1 kinds of enhancings, comprises

(i) dry powder dose preparation, it comprises

(a) container,

(b) be selected from the dry powder microgranule of the amount of the group being formed by 150-250mg and 250-1000mg; described dry powder microgranule comprises polyalcohols acid copolymer and hydrophobic compound; described polyalcohols acid copolymer is poly-(lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether); described hydrophobic compound is two Semen arachidis hypogaeae acyl phospholipids phatidylcholines

Wherein microgranule is the porous sphere with honeycomb texture or sponge structure, and the particle mean size of microgranule is 1.8-3.0 micron, and

(c) Sonazoid (C ₄f ₁₀) gas, and

(ii) be applicable to the pharmaceutically acceptable carrier of injection, for preparaton volume preparation again,

Wherein, using pharmaceutically acceptable carrier again to prepare the dry powder dose preparation in container, is 1.5 * 10 thereby form particle concentration ⁹-2.8 * 10 ⁹microgranule/mL or particle mass concentration are the microparticle suspending liquid of 25-50mg microgranule/mL,

The amount of wherein said Sonazoid gas is 75～500 μ g/mL suspensions,

Wherein, when microgranule is during with intravenously administrable, microgranule provides the myocardium enhancing ultrasound wave video picture that is greater than 1 minute.

30. test kits as claimed in claim 29, wherein said container is bottle or syringe, and test kit further comprises second bottle or syringe, this second bottle or syringe have the use of ormal weight so that the pharmaceutical acceptable carrier of particle suspension.

31. test kits as claimed in claim 29, wherein pharmaceutically acceptable carrier is selected from lower group of material: water for injection, sterilized water, saline, the saline that contains glycerol, contain tween saline, contain tween saline, etc. the dextrose (2.5%) that oozes of the glucose (5%), 1/2 etc. that oozes, etc. the mannitol (2.5%) that oozes of the mannitol (5%), 1/2 etc. that oozes, contain tween grade ooze mannitol, contain tween grade ooze mannitol.

32. test kits as claimed in claim 31, wherein pharmaceutically acceptable carrier is water for injection.

33. dosage particles as claimed in claim 14, the concentration of the outstanding suspension of the microgranule of wherein again preparing through carrier is suitable for the intravenously administrable of bolus infusion.