WO2006072198A1 - Adjuvant pour un traitement par ultrasons focalises a haute intensite et son procede de depistage - Google Patents

Adjuvant pour un traitement par ultrasons focalises a haute intensite et son procede de depistage Download PDF

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Publication number
WO2006072198A1
WO2006072198A1 PCT/CN2005/001367 CN2005001367W WO2006072198A1 WO 2006072198 A1 WO2006072198 A1 WO 2006072198A1 CN 2005001367 W CN2005001367 W CN 2005001367W WO 2006072198 A1 WO2006072198 A1 WO 2006072198A1
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eef
hifu
adjuvant
tissue
treatment
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PCT/CN2005/001367
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English (en)
French (fr)
Inventor
Zhibiao Wang
Faqi Li
Yanbing Xiao
Ziwen Xiao
Liping Liu
Zhilong Wang
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Chongqing Haifu(Hifu)Technology Co., Ltd
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Priority to US11/794,928 priority Critical patent/US20090117052A1/en
Application filed by Chongqing Haifu(Hifu)Technology Co., Ltd filed Critical Chongqing Haifu(Hifu)Technology Co., Ltd
Priority to JP2007549780A priority patent/JP4773458B2/ja
Priority to EP05781912A priority patent/EP1842560A4/en
Priority to CA002593638A priority patent/CA2593638A1/en
Priority to BRPI0518499-1A priority patent/BRPI0518499A2/pt
Priority to AU2005324271A priority patent/AU2005324271A1/en
Publication of WO2006072198A1 publication Critical patent/WO2006072198A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of medicine and medical treatment. Specifically, the present invention relates to the field of ultrasonic therapy, and more particularly to a HIFU therapeutic adjuvant capable of increasing tissue energy deposition in a target area during HIFU treatment and a screening method thereof. Background technique
  • High-intensity focused ultrasound (HIFU) technology has been clinically recognized as a new method for treating tumors and other diseases. It focuses on ultrasound to form high-intensity, continuous ultrasound energy on the lesion, resulting in transient high-temperature effects (65 ⁇ 100 °C), cavitation, mechanical and sonochemical effects, selectively causing coagulative necrosis of the lesion , the ability of the tumor to lose proliferation, infiltration and metastasis.
  • HIFU High-intensity focused ultrasound
  • Another object of the present invention is to provide a screening method for a high intensity focused ultrasound therapeutic aid.
  • Still another object of the present invention is to provide a high intensity focused ultrasound therapeutic aid for enhancing the effect of HIFU in treating diseases.
  • one aspect of the present invention provides a HIFU therapeutic auxiliary agent, which is a substance capable of enhancing the absorption of ultrasonic energy in a target area to be treated by HIFU after being administered to a living body, that is, A substance that reduces the amount of ultrasound energy required to damage a target tissue (tumor and non-tumor tissue) per unit volume.
  • a HIFU therapeutic auxiliary agent which is a substance capable of enhancing the absorption of ultrasonic energy in a target area to be treated by HIFU after being administered to a living body, that is, A substance that reduces the amount of ultrasound energy required to damage a target tissue (tumor and non-tumor tissue) per unit volume.
  • the type of substance of the HIFU therapeutic auxiliary is not excessively limited as long as the substance can change the acoustic environment of the target tissue after being applied to the target tissue, and promote the absorption and deposition of the therapeutic ultrasonic energy by the target tissue, thereby causing the target tissue.
  • the energy efficiency factor (EEF) can be effectively reduced.
  • the adjuvant for HIFU treatment of the present invention may be a solid, a liquid or a gas.
  • injury refers to a substantial change in the physiological state of a tumor tissue, usually referred to as coagulative necrosis of the tumor tissue.
  • the amount of ultrasonic energy required to damage the target tissue per unit volume can be quantified by energy efficiency factor (EEF).
  • EEF , in J/mm 3 , represents the ultrasound energy required to damage a unit volume of tumor tissue.
  • II represents the HIFU transducer focus coefficient, which reflects
  • a preferred embodiment of the invention is that the HIFU therapeutic adjuvant is administered to the target group After weaving, the EEF of the target tissue can be lowered, so that the basic EEF (ie, EEF( s )) measured before the application of the HIFU adjuvant and the EEF measured by the tissue after application of the HIFU adjuvant (ie, EEF)
  • the ratio between ( f ) is greater than 1, preferably greater than 2, more preferably greater than 4. There is no limit to the upper limit of the ratio, and the larger the better.
  • the HIFU therapeutic adjuvant is a biocompatible substance having a particle size ranging from 10 n ⁇ to 8 ⁇ , which can be administered by intravenous, intraarterial or topical injection.
  • the substance can reduce the EEF of the target tissue after administration to the target tissue, so that the basic EEF (ie, EEF (the base) of the tissue measured before the application of the HIFU adjuvant and the EEF (the EEF measured after the application of the HIFU adjuvant) That is, the ratio between the EEFs is greater than 1, preferably greater than 2, and more preferably greater than 4. There is no limit to the upper limit of the ratio, and the larger the better.
  • the HIFU adjuvant of the present invention may be encapsulated by a lipid film, a protein film or a sugar film, or may be a naked form that is not wrapped.
  • a lipid film a protein film or a sugar film
  • HIFU adjuvants can be encapsulated in lipid membranes to improve the targeting of the adjuvant. If the HIFU adjuvant itself does not cause vascular embolization when administered intravenously, a nude form that is not wrapped by a lipid film, a protein film or a sugar film may be used.
  • the tumor tissue or lesion may be added to the adjuvant.
  • a substance with specific affinity such as an antibody that recognizes a tumor.
  • the HIFU therapeutic adjuvant comprises a continuous phase composed of a discontinuous phase composed of a film-forming material-wrapped core and an aqueous medium, wherein the discontinuous phase is uniformly dispersed in the continuous phase
  • the particle size of the discontinuous phase is 10 ⁇ ⁇ ⁇ 8 ⁇ ⁇
  • the film forming material is biocompatible
  • the core material is a gas, liquid, nano-scale biocompatible solid.
  • the HIFU additive is called a microbubble auxiliary;
  • the HIFU auxiliary of the present invention comprising a liquid encapsulating material is called a microparticle auxiliary, and the liquid is divided into two types: one is not produced at 38 to 100 °.
  • Liquid/gas phase change liquid and the other type is a liquid/gas phase change liquid at 38 to 100 ° C (that is, a liquid which is converted into a gas in an animal or a human body during HIFU treatment);
  • the HIFU adjuvant of the present invention composed of a biocompatible solid is called It is a plasmid auxiliary.
  • the film-forming material is contained in the auxiliary agent in an amount of 0.1 to 100 g/L, preferably 0.5 to 50 g/L, more preferably 0.5 to 20 g/L.
  • the film-forming material includes a lipid such as 3-sn-phosphatidylcholine (lecithin), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol-sodium salt, 1,2- Distearyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-sodium salt, 1,2-dipalmitoyl-sn-glycerol Alkyl-3-phosphatidylcholine, phosphatidylserine, hydrogenated phosphatidylserine, cholesterol, glycolipids; sugars, including, for example, glucose, fructose, sucrose, starch, and degradation products of different chain lengths;
  • the content of the gas in the auxiliary agent is 5 to 200 ml/L, preferably 20 to 150 ml/L, more preferably 20 to 100 ml/L.
  • the gas includes air, nitrogen, carbon dioxide, fluorocarbon hydrocarbon gases such as perfluoroethane, perfluoropropene, perfluorobutane, alkane gases such as butyl sulfonium, cyclobutane, pentane, hexanyl, hexafluorocarbon Sulfur and so on.
  • Microbubble-based contrast agents widely used in the field of ultrasound contrast can be used as HIFU therapeutic aids of the present invention.
  • the present invention accordingly also provides the use of a microbubble-like ultrasound contrast agent for use as an adjuvant in the present invention.
  • a particulate auxiliary agent when the liquid is a liquid which does not produce a liquid/gas phase change at 38 to 100 ° C, it includes water, a saturated fatty acid, an unsaturated fatty acid such as soybean oil, peanut oil, etc., and lipiodol, and the like.
  • the content in the auxiliary agent is 5 to 200 g/L, preferably 10 to
  • the liquid is a liquid which produces a liquid/gas phase change at 38 to 100 ° C, it includes ( 5 - 0 6 alkane such as n-pentamidine, isopentane, etc., and a fluorinated c 5 -c 12 hydrocarbon substance)
  • perfluoropentane, dihydrodecafluoropentane or the like is contained in the auxiliary agent in an amount of 5 to 200 ml/L, preferably 10 to 100 ml/L, more preferably 20 to 8.0 ml/L.
  • a fat emulsion for injection is a fat aqueous emulsion composed of refined soybean oil coated with a phospholipid film dispersed in water, and is suitable for intravenous injection.
  • emulsions are currently commercially available, including but not limited to Intmlipos® (fat emulsion injection), OMNILIPID® (fat emulsion injection), "fat emulsion (long chain)” or "national fat” Fat emulsion (medium chain and long chain compounding agent).
  • Such fat emulsions can also be used as HIFU therapeutic auxiliaries of the present invention.
  • the present invention also provides a corresponding The use of a fat emulsion as an adjuvant of the present invention.
  • nano-scale biocompatible solids include magnetic nano-scale biomaterials such as superparamagnetic nanoparticles (SPIO), nano-scale hydroxyapatite (HAP:), nano-scale calcium carbonate, etc.
  • SPIO superparamagnetic nanoparticles
  • HAP nano-scale hydroxyapatite
  • the diameter is generally from 1 to 500 nm, preferably from 1 to 200 nm, more preferably from 10 to 100 nm.
  • the above-mentioned nano-sized biocompatible solids themselves can also be used as an auxiliary agent of the present invention.
  • the present invention accordingly also provides the use of a nanoscale biocompatible solid for use as an adjuvant in the present invention.
  • the auxiliary agent may further contain an emulsifier, and the emulsifier is generally selected from the group consisting of monoethylene glycol mono c 16 . 18 fatty acid ester, diethylene glycol mono c 16 - 18 fatty acid ester, and diethyl Glycol di c 16 .
  • fatty acid ester triethylene glycol mono c 16 -18 fatty acid ester, sorbitan fatty acid ester (Span series) emulsifier, polysorbate (Tween series) emulsifier , polyethylene glycol monolaurate series emulsifier, polyoxyethylene laurate series emulsifier, 3-sn-phosphatidylcholine (lecithin), cholic acid and so on.
  • the emulsifier is present in an amount of from 5 to 150 g/L.
  • a stabilizer such as sodium carboxymethylcellulose (CMC-Na), glycerin or the like may be contained, and the content of the sodium carboxymethylcellulose is 0.01 to 10 g/L, preferably 0.05 to 0.6 g/L. More preferably, it is 0.1 to 0.3 g/L. The content of the glycerin is 5 to 100 g/L.
  • CMC-Na sodium carboxymethylcellulose
  • an inorganic or organic acid or a base may be used to adjust the pH of the auxiliary.
  • the pH of the auxiliary agent when the liquid is a liquid which does not cause liquid/gas phase change at 38 to 100 ° C, the pH of the auxiliary agent is adjusted to 7.0 to 9.0, preferably to 7.5 to 8.5.
  • the pH of the auxiliary agent when the liquid is a liquid/gas phase change liquid at 38 to 100 ° C, the pH of the auxiliary agent is adjusted to 7.0 to 9.0, preferably to 7.5 to 8.5.
  • the pH of the auxiliary is adjusted to 3.0 to 6.5, preferably to 50 to 6.0.
  • the film-forming material, the pre-encapsulated gas, the liquid or the solid, the emulsifier and the stabilizer are generally mixed and emulsified.
  • the particulate fat emulsion reference may be made to Chinese Patent Application No. 97128319.7 (invention name: fat emulsion containing reducing sugar and sterilization method thereof) or Chinese patent application No.02112860.X (invention name: fat for injection) A method of preparing a fat emulsion disclosed in an emulsion and a method of producing the same.
  • microbubble-based fluorocarbon emulsion For the microbubble-based fluorocarbon emulsion, reference may be made to Chinese Patent Application No. 96106566.4 (invention name: dextran albumin acoustic contrast agent containing perfluorocarbon and preparation method thereof), Chinese Patent Application No. 981 1901 1 .1 ( Title of Invention: An Ultrasound Diagnostic Contrast Agent and Preparation Method thereof) or Chinese Patent Application ZL 89100726.1 (Invention Name: Method for preparing microparticles and ultrasonic contrast agents for ultrasound contrast).
  • the HIFU therapeutic auxiliary preferably uses a biocompatible, degradable biological material such as a lipid as a film forming material, so that the auxiliary agent can be intravenously injected, smoothly passes through the blood circulation, and is quickly taken into the human body.
  • a biocompatible, degradable biological material such as a lipid as a film forming material
  • the invention also provides a method for increasing energy deposition in a target area during HIFU treatment, characterized in that the patient is injected with an effective dose of the HIFU of the present invention by intravenous rapid infusion or bolus injection 0 to 168 hours before HIFU treatment.
  • the effective dose will vary depending on the type of tumor, the weight of the patient, the location of the tumor, the volume of the tumor, and the like. However, the physician or pharmacist has the ability to determine the appropriate amount of injection for different patients.
  • the microbubble adjuvant may be selected in the range of 0.005 to 0.1 ml/kg body weight, preferably 0.01 to 0.05 ml/kg body weight.
  • the liquid when the liquid is a liquid which does not produce liquid/gas phase change at 38 to 100 ° C, it can be selected within the range of 0.01 to 5 ml/kg body weight, preferably 0.01 to 2.5 ml/kg. Selecting within the range of body weight; when the liquid is a liquid/gas phase change liquid at 38 to 100 ° C, it may be selected within the range of 0.005 to 0.1 ml/kg body weight, preferably 0.01 to 0.05 ml/kg body weight. Choose within the scope.
  • the plasmid-based adjuvant it is selected in the range of 0.1 to 10 ml/kg body weight, preferably in the range of 0.1 to 5 ml/kg body weight.
  • the invention also provides a method for screening HIFU adjuvants, comprising:
  • EEF energy efficiency factor
  • the invention further provides a method of treating a disease comprising administering a HIFU adjuvant to a patient prior to administering the HIFU to the patient to improve the ability of the HIFU treatment target to therapeutic ultrasound absorption.
  • Example 1-1 The liquid to be wrapped does not produce liquid/gas phase change at 38 ⁇ 100 °C.
  • Example 1-1-2 The colostrum was placed in a large test tube, and the colostrum was emulsified for 2 minutes at a power of 350 W to form a uniform emulsified lipiodol, which was then sterilized by passing through 100 Torr of steam for 30 minutes.
  • the pH was 7.5 to 8.5, and the amount of iodine was contained. 0.13 g/ml. Particle size ⁇ 1 ⁇ , osmotic pressure 350mosm/kg water.
  • Example 1-1-2 Example 1-1-4
  • Example 1-1-1 injectable soybean oil was used instead of iodized oil for injection as a core material, and lecithin for injection was used instead of egg yolk lecithin as a film-forming material, according to the following Table 1.
  • the following formulations have obtained the following HIFU therapeutic particulate adjuvants of the present invention.
  • the auxiliaries obtained are white emulsion liquids, which are suitable for intravenous injection in animals and humans. The corresponding parameters are shown in Table 1 below:
  • Injection soybean oil dosage 100g 200g 100g Injection lecithin dosage 12g 12g 12g glycerin for injection 22g 22g 16.7g water for injection (added) 1000ml 1000ml 1000ml pH value is about 8 8 8 particle size of discontinuous phase 0.
  • Example 1-2 Wrapped liquid at 38 ⁇ 100 °C Produced liquid/gas phase change
  • HAP Human Engineering Research Center of Sichuan University
  • lecithin for injection purchased from Shanghai Chemical Reagent Co., Ltd.
  • glycerin 1 ml of glycerin for injection
  • distilled water 1 ml of glycerin for injection
  • the mixture was placed in the vibrating chamber of the vibrometer, and the head of the vibrometer was placed 1.5 cm below the liquid level of the mixture, and the mixture was sonicated for 2 minutes at a vibration power of 400 W to form a uniform dispersion.
  • Stable milky white suspension The non-continuous phase of the auxiliary agent has a particle diameter of 10 to 1000 nm, and is mainly concentrated at 100 to 500 nm.
  • HIFU therapeutic plasmid adjuvants of the present invention were obtained according to the materials and ratios used in Table 3 below in accordance with the same procedures and procedures as described in Example ,-1, and the corresponding parameters are shown in Table 3 below:
  • Example III Nano-sized hydroxyapatite (HAP) was purchased from the Biomaterials Engineering Research Center of Sichuan University. It is a white powder with a particle size of 10 ⁇ 200nm and a normal distribution. The HAP was separately prepared into a milky white suspension having a concentration of 25 g/L and 50 g/L with 9% physiological saline, and shaken by an ultrasonic shaker at 600 W for 2 minutes before use to completely disperse uniformly. Test Example 1 The combination of the microparticle auxiliary agent obtained in Example 1-1-3 and the HIFU therapeutic apparatus was used in 50 New Zealand white rabbits (provided by the Animal Experimental Center of Chongqing Medical University), and the male and female were not limited, and the age of the month was about 3 months. The average score was divided into two groups. The body weight of rabbits in group A and group B was 2.22 ⁇ 0.21 kg and 2.24 ⁇ 0.19, respectively (P > 0.05).
  • JC type HIFU tumor treatment system mainly consists of five parts: adjustable power generator, B-super monitoring system, therapeutic probe, mechanical motion control system, treatment bed and acoustic coupling device.
  • the treatment head has a diameter of 150mm, a focal length of 150mm, an acoustic focal length of 2.3 x 2.4x26mm, a working frequency of 1 ⁇ , a circulating degassing water standard of gas content of ⁇ 3ppm, and an average sound intensity of 5500W/cm 2 .
  • the rabbit liver was pre-scanned with HIFU and B-ultrasound. Two layers of 2 cm interval and 2.0 cm depth of injury were selected. Group A was treated with left (middle/middle) rabbit liver as control leaf (administered to saline side), right side. (Right leaf) is the experimental leaf (the adjuvant prepared in Example 1-1-3, the administration side), and the B group is opposite to the A group.
  • the treatment depth is 2.0cm (the distance from the focus of the skin's outer side).
  • the HIFU injury was performed on the rabbit liver in the group or the right side (group B), and the gray scale change and treatment time of the target area were recorded. Then, the focus of the HIFU treatment machine was moved to the other side, and the auxiliaries prepared in Example 1-1-3 were used instead of the physiological saline input (infusion rate and time to the control leaves) for HIFU injury. The same rabbit had the same damage on both sides of the liver.
  • P is the total sound power (W) of the HIFU source;
  • t is the total treatment time (s);
  • It is the damage volume (mm 3 ).
  • Group A Group B Total P value Control leaf 7.09 ⁇ 4.11 6.67 ⁇ 3.13 6.87 ⁇ 3.60 > 0.5 * Experimental leaf 2.73 ⁇ 1.64 3.43 ⁇ 2 ⁇ 07 3, 10 ⁇ 1.89 >0.5 *
  • Test Example 1 The combination of the microparticle adjuvant prepared in Example 1-1-1 and the HIFU therapeutic apparatus was taken from 30 New Zealand white rabbits (provided by the Animal Experimental Center of Chongqing Medical University), weighing about 2 kg, randomly divided into experimental group and normal. Group, 15 in each group, each rabbit has two irradiation points. The normal group of white rabbits were given a physiological saline solution by rapid injection into the ear vein at a dose of 2.5 ml/kg.
  • the rabbits were given a rapid injection into the ear vein according to the amount of 2.5 ml/kg, and the emulsified lipiodol oil prepared in Example 1-1-1 was administered and rinsed with 1 ml of physiological saline to ensure that the drug completely entered the body.
  • the JC-type HIFU tumor treatment system manufactured by Chongqing Haifu (HIFU) Technology Co., Ltd.
  • Irradiation power is 220W
  • frequency is 1.0MHz
  • the irradiation time is preferably coagulation necrosis.
  • Measurement data were expressed as mean SD, using SPSS 10.0 for windows statistical software package, independent and paired t-test; count data using ⁇ 2 test.
  • the EEF of the experimental group and the normal group were compared as shown in Table 5 below: Table 5 Comparison of the EEF of the normal group and the EEF of the experimental group
  • Example 3 In Vitro Experimental Study of Microparticle Aids Prepared in Examples 1-1-3
  • CZF-1 type HIFU gynecological treatment instrument consists of three parts: power source, treatment head and circulating water. See Chinese invention patent No.01144259.X.
  • the experimental setting parameters are: power 4.05W, frequency 11 ⁇ , pulse 1000Hz.
  • Example 1-1-3 After intramuscular injection of anesthetized rabbits, the auxiliaries (experimental group) or physiological saline (control group) prepared in Example 1-1-3 were injected into the rabbit ear vein, and the infusion rate was 50 to 60 drops/min. Both are 20 minutes.
  • the rabbit was placed on the operating table on the supine position, and the mid-abdominal incision was taken about 4 to 5 cm long.
  • the entire abdominal wall was cut into the abdominal cavity layer by layer, exposed and gently pulled out of the liver.
  • Each rabbit's liver was damaged by 1 ⁇ 2 points in each period.
  • the injury period set in this experiment was 3s, 6s and 9s. After designing the damage point, the experiment is carried out according to the above experimental parameters. After the injury is completed, the rabbit liver is returned to the abdominal cavity, and the entire abdominal wall is sutured layer by layer.
  • the rabbits were sacrificed by excessive anesthesia the next day, and the liver was taken out and the diameter of the damaged tissue was measured and the EEF was calculated.
  • the data are expressed as the average SD, using SPSS 10.0 for windows Statistical software package, independent sample t-test, p ⁇ 0.05 for statistical significance.
  • the experimental results measured 21 lesions in each injury period of the control group, a total of 63 ( 21 X 3 ) points of tissue damage volume; experimental group 30 points per injury period, a total of 90 ( 30 X 3 ) points of tissue damage volume.
  • the EEF is calculated according to the formula, and the results are listed in Table 6 below:
  • n 3s 6s 9s control group 21 0.2749 ⁇ 0.2409 0.1783 ⁇ 0.0733 0.1846 ⁇ 0.0896 experimental group 30 0.1177 ⁇ 0.0609 0.1367 ⁇ 0.0613 0.1463 ⁇ 0.069
  • JC type HIFU tumor treatment system mainly consists of five parts: adjustable power generator, B-super monitoring system, therapeutic probe, mechanical motion control system, treatment bed and acoustic coupling device.
  • the diameter of the treatment head of the system is 150mm
  • the focal length is 150mm
  • the acoustic focal length is 2.3x2.4x26mm
  • the working frequency is 1 ⁇
  • the circulating degassing water standard is gas content ⁇ 3ppm
  • the average sound intensity is 5500W/cm 2 .
  • the transducer used in the experiment has a diameter of 150mm and the focal length is
  • the irradiation depth is 20mm, and the irradiation method is intermittently fixed, irradiated for 3 seconds, and stopped for 5 seconds.
  • the physiological saline was rapidly injected into the ear vein according to the amount of 0.02 ml/kg.
  • the rabbit liver was irradiated by HIFU, which was the control group.
  • the adjuvant prepared in Example 1-2-1 was administered by rapid injection into the ear vein according to the amount of 0.02 ml/kg.
  • HIFU irradiated the other plane of the same rabbit liver, which is true. Inspection team.
  • Irradiation is terminated after the gradation change occurs in the irradiation target area, and is irradiated for up to 20 seconds if no gradation change is observed.
  • the experimental yellow sheep was irradiated with a JC type HIFU tumor treatment system (manufactured by Chongqing Haifu (HIFU) Technology Co., Ltd.).
  • the experiment used a transducer with a diameter of 150 mm, a focal length of 135 mm, a frequency of 0.8 MHz, and a sound power (P) of 220 W.
  • the irradiation depth was 30 mm
  • the irradiation method was a discontinuous fixed point
  • the irradiation was 3 seconds
  • the stop was 5 seconds. All the sheep did not remove the ribs.
  • HIFU pre-scan before irradiation select the irradiation area, a total of 4 planes, a little irradiation on each plane, two-dimensional ultrasound observation of the intercostal space.
  • Physiological saline was administered by rapid injection into the ear vein according to the amount of 0.02 ml/kg. After 60 seconds, the liver of the sheep was irradiated by HIFU, and each sheep was irradiated at 2 points. This was the control group.
  • the adjuvant prepared in Example 1-2-1 was administered by rapid injection into the ear vein at a dose of 0.02 ml/kg. After 60 seconds, HIFU irradiation was also irradiated. Two points, this is the experimental group.
  • the irradiation target area has a gray scale change and is irradiated 4 to 5 times to end the irradiation. If no gray scale change is observed, the irradiation is performed for a maximum of 200 seconds.
  • the sheep were sacrificed 3 days later, and the volume of coagulative necrosis (V) in the liver was dissected. Calculate the energy efficiency factor according to the formula EE - ⁇ P ⁇
  • EEF EEF
  • T the irradiation time
  • n 0.7.
  • the experiment shows that HIFU damages goat liver without removing ribs. In the presence of fluorocarbon emulsion, the damage efficiency has undergone a qualitative change.
  • the experimental yellow sheep were irradiated with a JC type HIFU tumor treatment system (manufactured by Chongqing Haifu (HIFU) Technology Co., Ltd.).
  • the experimental transducer has a diameter of 150 mm, a focal length of 135 mm, a frequency of 0.8 MHz, and a sound power (P) of 220 W.
  • the irradiation depth is 20mm, the irradiation method is intermittent fixed point, irradiation for 3 seconds, stop for 5 seconds, all The sheep did not remove the ribs.
  • HIFU Before HIFU irradiation, pre-scan, select the irradiation area, select two planes for each of the upper and lower poles of the kidney, and irradiate a little on each plane, two-dimensional ultrasound observation. If the right rib is blocked, avoid it.
  • Physiological saline was administered by rapid injection into the ear vein according to the amount of 0.02 ml/kg. After 30 seconds, HIFU was spot-irradiated to test the kidney of the sheep, which was the control group.
  • the adjuvant prepared in Example 1-2-1 was administered by rapid injection into the ear vein at a dose of 0.02 ml/kg, and HIFU was irradiated 60 seconds later, which was an experimental group.
  • the irradiation target region has a gray scale change and then irradiates 3 to 4 times to end the irradiation. If no gray scale change is observed, the irradiation is performed for a maximum of 150 seconds.
  • the sheep were sacrificed 3 days later, and the volume of coagulative necrosis (V) in the liver was dissected.
  • Test Example 5 The combination of the plasmid-based auxiliary agent prepared in Example II-1 and the HIFU therapeutic apparatus was taken from 36 New Zealand white rabbits (provided by the Animal Experimental Center of Chongqing Medical University), weighing about 2 kg, and randomly divided into one control group and 2 One administration group, 12 in each group. In the control group, the rabbits were given a normal saline solution by rapid injection into the ear vein at a dose of 2 ml/kg.
  • Two drug-administered groups were given a rapid injection into the ear vein at a dose of 2 ml/kg to give the adjuvant prepared in Example II-1, and 1 ml of physiological saline was added to ensure that the drug completely entered the body. Irradiation was performed at 24 hours and 48 hours after the injection in each of the two administration groups.
  • the administration group irradiated 24 hours after the injection was referred to as the first administration group
  • the administration group irradiated 48 hours after the injection was referred to as the second administration group.
  • JC type HIFU tumor treatment system is mainly composed of adjustable power generator, B-super monitoring system, therapeutic probe, mechanical motion control system, treatment bed and acoustic coupling device.
  • the treatment head has a diameter of 150mm, a focal length of 150mm, an acoustic focal length of 2.3 x 2.4x26mm, a working frequency of 1 ⁇ , a circulating degassing water standard of ⁇ 3ppm, and an average sound intensity of 5500W/cm 2 .
  • the irradiation power was 220 W, the frequency was 1.0 MHz, the irradiation depth was 20 mm, and the irradiation time was 15 seconds.
  • the experimental animals were dissected and the volume of coagulative necrosis was calculated.
  • 1 control group and 2 administration groups formed certain coagulability in the liver of rabbits
  • the energy efficiency factor (EEF) required for the dead spot is shown in Table 7 below:
  • Test Example 6 In vivo experimental study of HIFU therapeutic plasmid adjuvant prepared in Example III 40 New Zealand white rabbits were obtained from the Animal Experimental Center of Chongqing Medical University, with an average body weight of 2.7 ⁇ 0.3 kg/head, regardless of male or female. They were randomly divided into 3 HAP experimental groups and 1 control group, with 10 in each group.
  • each rabbit in the HAP group was rapidly injected with different concentrations of HAP suspension prepared in Example III via the ear vein according to the amount of 2 ⁇ 3ml/kg, and was pushed in 5 seconds and given 1ml. Rinse with saline to ensure that the drug completely enters the body; in the control group, each rabbit was injected with 2 ml/kg of normal saline. Rabbits' right chest and abdomen 8% sodium sulfide hair removal, preoperative hypothermia new 0.2ml/kg intramuscular injection anesthesia, under sterile surgery, open the abdominal wall, fully exposed to the liver.
  • CZF-1 type HIFU gynecological treatment instrument (produced by Chongqing Haifu (HIFU) Technology Co., Ltd.) to carry out irradiation on rabbit liver.
  • CZF-1 type HIFU gynecological treatment instrument consists of three parts: power source, treatment head and circulating water. See Chinese invention patent No.01144259.X. The operating parameters are as follows: Frequency 9.85MHz, power 5W, focal length 4mm, treatment mode ⁇ with fixed-point irradiation. Each liver was irradiated in groups of 2 to 3, each group was 3 points, and the irradiation time was 10 seconds. After the operation, the incision was sutured. After 24 hours, the rabbits were sacrificed by rapidly injecting 10 ml of air into the ear vein, and the focal volume of the formed coagulative necrosis was measured, and the energy efficiency factor (EEF) was calculated.
  • EEF energy efficiency factor
  • the volume of the focal region formed by the HAP dose experimental group was significantly increased compared with the corresponding physiological saline group, and the required energy efficiency factor was significantly reduced. The difference was extremely significant (PO.001). Compared with the different nano-level HAP dosage groups, with the increase of HAP dosage, the volume of the focal length formed also increased significantly, and the required energy efficiency factor decreased significantly, and the difference was significant (P ⁇ 0.001). Table 8 below shows the comparison of focal volume and energy efficiency factors ( ⁇ s) for different HAP-focused ultrasound treatment groups.
  • HAP group 1 50mg/kg 30 153.1 ⁇ 41.8 0.24 ⁇ 0.05
  • HAP group 2 100mg/kg 25 223.2 ⁇ 55.1 0.19 ⁇ 0.01
  • HAP group 3 150mg/kg 21 287.7 ⁇ 47.9 0.13 ⁇ 0.00
  • the "n" in the table indicates the number of points irradiated.
  • nano-level HAP can significantly enhance the in vivo therapeutic effect of HIFU, and with the increase of HAP dosage, the enhanced therapeutic effect is stronger.
  • Test Example 7 In vitro study of plasmid-based adjuvants for HIFU treatment prepared in Example III 80 healthy New Zealand rabbits (provided by Animal Experimental Center of Chongqing Medical University), male or female, weighing 2.5 ⁇ 0.3 kg, fasting 24 hours before treatment Each rabbit was given a 25 g/L HAP milk white suspension prepared in Example III by rapid injection into the ear vein at a dose of 2 ml/kg, and washed with 1 ml of physiological saline. Hepatic HFIU scans were performed 24 hours (20 rats), 48 hours (25 rats), 72 hours (10 rats), and 168 hours (15 rats). The control group (10 rats) was given normal saline 2 ml/kg, followed by 24 hours.
  • HIFU liver scan Preoperative animals were treated with 8% sodium sulfide in the right chest and abdomen, and a new 0.2 ml/kg intramuscular anesthesia was fixed on the JC-A HIFU treatment device.
  • the JC-A high-intensity focused ultrasound tumor treatment system was developed by the Institute of Medical Ultrasound Engineering of Chongqing Medical University (the National Drug Administration has approved production, registration No. 99 No. 301032).
  • the system includes two parts of ultrasound real-time monitoring and positioning and treatment equipment.
  • Pick The circulating degassed water is used as an acoustic coupling agent, and its gas content is ⁇ 3 > ⁇ 10 6 .
  • the treatment parameters are: power 220W, frequency ⁇ , focal length 150mm, focal length 12mm, the treatment head can move freely in the X, ⁇ , ⁇ directions. .
  • the rabbit's chest and abdomen were immersed in the circulating degassed water.
  • the liver showed clear under the sputum, and the irradiation point was fixed.
  • the fixed treatment time was 15 seconds, the treatment depth was 20 mm, and each liver could be irradiated 1 to 2 points.
  • the animals were sacrificed by rapidly injecting 10 ml of air from the rabbit ear vein 24 hours after surgery.
  • the liver was taken out and the rabbit liver tissue was cut along the direction of the acoustic channel to show the largest lesion necrotic lesion.
  • the shape was observed and the size was measured (stained by TTC). For the boundary). Then calculate the EEF.
  • the nano-HAP group could form a larger coagulative necrosis than the control group (p ⁇ 0.05), and the energy-efficiency factor required for HIFU treatment was also significantly reduced, and 24 hours and 48 hours after administration.
  • the formation of the focal region necrosis volume is the largest, and the required energy efficiency factor is also the smallest, suggesting that this may be the best time for HIFU treatment after HAP medication.
  • the volume of necrotic foci formed gradually decreases.
  • the therapeutic effect of HIFU was better enhanced than before administration (p ⁇ 0.05). (See Table 9)
  • Time after treatment n mean treatment time (S) mean focal volume (mm 3 ) mean EEF control group 16 15 546.67 7.39 ⁇ 4.99
  • n is the actual number of irradiation points in the table.
  • nano-level HAP can significantly enhance the in vitro therapeutic effect of HIFU, and HIFU treatment at 48 to 72 hours after administration can produce the best enhancement effect on HIFU treatment.
  • Test Example 8 Combination of microbubble adjuvant and HIFU therapeutic apparatus for HIFU treatment Forty New Zealand white rabbits, weighing about 2 kg, were randomly divided into experimental group and normal group, with 20 rats in each group.
  • the control group was intravenously administered with physiological saline in an amount of 0.05 ml/kg.
  • the experimental group was rapidly injected with perfluoroblastic contrast agent (purchased from Ncapturing Hospital) through the ear vein at a dose of 0.05 ml/kg, and was given 1 ml of physiological saline to ensure that the drug completely entered the body.
  • irradiation was performed using a JC type HIFU tumor treatment system (manufactured by Chongqing Haifu (HIFU) Technology Co., Ltd.).
  • the irradiation power is 200W
  • the frequency is 1.0MHz
  • the irradiation depth is 20mm
  • the irradiation time is certain.
  • the amount of coagulative necrosis was measured and the EEF was calculated.
  • the measurement data were expressed as the mean SD, using the SPSS 10.0 for windows statistical software package, independent and paired t-test; the count data was analyzed by ⁇ 2 test. The results are shown in Table 10 below.
  • micro-foam HIFU adjuvant can significantly reduce the EEF of liver tissue damaged by HIFU.
  • the high-intensity focused ultrasound (HIFU) therapeutic adjuvant provided by the invention can significantly improve the acoustic environment of the target area, and can reduce the ultrasonic energy required to damage the target tissue (tumor and non-tumor tissue) per unit volume, so that the power is at a certain power. Under the circumstance, the tumor with deeper position and larger volume can be treated with high efficiency without damaging the normal tissue on the acoustic channel.
  • the use of the adjuvant of the present invention makes it possible to effectively perform HIFU treatment on liver tumor patients without removing the ribs on the patient's therapeutic acoustic channel.

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Description

一种高强度聚焦超声治疗用助剂及其筛选方法 技术领域
本发明涉及医药及医疗领域，具体地说，本发明涉及超声治疗领域， 更具体地说， 本发明涉及一种能够增加 HIFU 治疗时靶区组织能量沉积 的 HIFU治疗用助剂及其筛选方法。 背景技术
高强度聚焦超声 （High-intensity focused ultrasound, HIFU) 技术作 为一种治疗肿瘤和其他疾病的新方法已经得到临床的认可。 它通过聚焦 超声波， 在病灶上形成高强度、 连续超声能量， 从而产生瞬态高温效应 ( 65〜100°C ) 、 空化效应、 机械效应和声化学效应， 选择性地使病灶组 织凝固性坏死， 使肿瘤失去增殖、 浸润和转移的能力。
有研究表明， 超声波在人体组织传播的过程中， 其能量随传播距离 的增加呈指数级衰减（刘宝琴等人， 中国超声医学杂志， 2002， 18 ( 8 ) ：
565 - 568 ) 。 另外， 超声波在软组织中传播的能量衰减来自组织吸收、 散射、 折射、 衍射等， 其中最主要的能量损失来自于吸收和散射 （冯若， 王智彪主编， 实用超声治疗学， 北京： 中国科学技术文献出版社， 2002.14 ) 。 因此， 运用 HIFU技术治疗位置较深、 体积较大的肿瘤时， 由于能量的衰减必将导致到达靶区的能量偏低， 治疗效率下降， 治疗时 间延长。
当然， 为了提高治疗效率， 虽然可以单纯地加大超声换能器的发射 功率， 但是， 超声波传播通道上的正常组织被烧伤的可能性就大大增加 了。
另外， 目前临床上在运用 HIFU技术治疗声通道上有肋骨阻挡的肝 脏肿瘤时， 为了增加治疗靶区的能量沉积， 提高治疗速度和治疗效果， 常要在切除声通道上的肋骨后再治疗， 这有悖于 HIFU无创治疗的理念， 因而很难得到患者和医生的认可。
这些因素的存在必会在一定程度上影响和限制 HIFU作为一门临床 适用技术的推广和普及。 因此， 如何增加 HIFU治疗时靶区的能量沉积， 使得能高效率地治疗深部肿瘤而又不损伤声通道上的正常组织或在 不切除肋骨的情况下治疗被肋骨阻挡的肝脏肿瘤，就成为迫切需要解 决的技术问题。 发明内容
本发明的一个目的是提供一种能够增加 HIFU 治疗时靶区能量 沉积的高强度聚焦超声治疗用助剂。
本发明的另一个目的是提供高强度聚焦超声治疗用助剂的筛选 方法。
本发明的再一个目的是提供高强度聚焦超声治疗用助剂用于增 强 HIFU治疗疾病效果的用途。
为实现上述目的，本发明的一个技术方案提供了一种 HIFU治疗 用助剂，该助剂是一种能够在施用给生物体后有助于加强待 HIFU治 疗靶区吸收超声能量的物质， 即一种能降低单位体积的靶组织（肿瘤 和非肿瘤组织）被损伤所需的超声能量的物质。在本发明中， 对作为
HIFU治疗用助剂的物质的类型没有过多的限制， 只要该物质在施用 给靶组织后能改变该靶组织的声环境，促进靶组织对治疗性超声能量 的吸收和沉积， 从而使靶组织的能效因子 （energy efficiency factor, EEF ) 有效下降即可。 因而， 本发明的用于 HIFU治疗的助剂可以是 固体、 液体或气体。
此处所用的术语 "损伤" 是指肿瘤组织的生理状态发生实质性 改变，通常是指肿瘤组织发生凝固性坏死。所述单位体积的靶组织被 损伤所需的超声能量的多少，可用能效因子（ energy efficiency factor, EEF ) 来量化。 EEF = ， 单位为 J/mm3， 表示损伤单位体积的肿 瘤组织所需的超声能量。 式中 II表示 HIFU换能器聚焦系数， 它反映
HIFU换能器对超声能量汇聚的能力，取 η =0.7 ; P是 HIFU源总声功 率， 单位是瓦； t是治疗总时间， 单位是秒； V是损伤体积， 单位是 mm3。在一种物质施用给靶组织后， 如果靶组织的能效因子下降程度 越大， 该物质越适合用作本发明的 HIFU助剂。
本发明一个优选的实施方案为， HIFU治疗用助剂在施用给靶组 织后， 能使靶组织的 EEF下降， 从而该组织在施用 HIFU助剂前测 得的基础 EEF (即， EEF ( s ) )与该组织在施用 HIFU助剂后测得的 EEF (即， EEF (测 fl) ) 间的比值大于 1， 优选大于 2， 更优选大于 4。 对 于该比值的上限没有限制， 应该是越大越好。
按照本发明进一步优选的实施方案， HIFU治疗用助剂是一种能 以静脉注射、 动脉注射、 局部注射的给药方式施用的， 粒径范围在 10ηηι〜8μιη 的生物相容性的物质， 该物质在施用给靶组织后能使靶 组织的 EEF下降，从而该组织在施用 HIFU助剂前测得的基础 EEF (即 EEF ( ¾础）与该组织在施用 HIFU助剂后测得的 EEF (即， EEF (测 ) 间的比值大于 1， 优选大于 2， 更优选大于 4。 对于该比值的上限没 有限制， 应该是越大越好。
本发明的 HIFU助剂可以是被脂膜、蛋白膜或糖膜包裹的，也可 以是不被包裹的裸体形式。例如，对于网状内皮细胞丰富的组织如肝 脏、 脾脏和骨髓， 可以将 HIFU助剂类物质包裹在脂质膜中， 以改善 该助剂物质的靶向性。如果该 HIFU助剂本身在以静脉方式给药时不 会引起血管栓塞， 也可以采用不被脂膜、蛋白膜或糖膜包裹的裸体形 式。另外，为了使本发明的 HIFU助剂靶向特定的肿瘤组织如肝肿瘤、 肾肿瘤、骨肿瘤、 乳腺癌和子宫肌瘤等， 还可在该助剂中加入对所述 的肿瘤组织或病灶部位有特异亲和性的物质， 如识别肿瘤的抗体等。
本发明的一个优选的具体实施方案为， HIFU治疗用助剂包括由 成膜材料包裹芯构成的非连续相和水性介质构成的连续相，其中所述 非连续相均匀地分散在所述连续相中， 所述非连续相的粒径为 10ηπι〜8μηι， 所述成膜材料具有生物相容性， 所述芯材料采用气体、 液体、纳米级生物相容性固体。这种助剂适合于静脉给药。为了叙述 得清楚和简便，以下将由成膜材料包裹生物相容性气体构成的本发明
HIFU 助剂称为微泡类助剂； 将由成膜材料包裹液体构成的本发明 HIFU助剂称为微粒类助剂， 所述液体分为两类： 一类为在 38〜100 °〇不产生液 /气相变的液体， 另一类为在 38〜100°C产生液 /气相变的 液体（即可在 HIFU治疗时在动物或人体内转变为气体的液体） ； 将 由成膜材料包裹纳米级生物相溶性固体构成的本发明 HIFU助剂称 为质粒类助剂。
在上述技术方案中， 所述成膜材料在助剂中的含量为 0.1〜 100g/L, 优选为 0.5〜50g/L， 更优选为 0.5〜20g/L。 所述成膜材料包 括脂类， 如 3-sn-磷脂酰胆碱 （卵磷脂） 、 1,2-二棕榈酰基 -sn-甘油基 -3-磷脂酰甘油基 -钠盐、1,2-二硬脂酰基 -sn-甘油基 -3-磷脂酰胆碱、 1,2- 二棕榈酰基 -sn-甘油基 -3-磷脂酰酸-钠盐、 1,2-二棕榈酰基 -sn-甘油基 -3-磷脂酰胆碱、 磷脂酰丝氨酸、 氢化磷脂酰丝氨酸、 胆固醇、 糖脂； 糖类， 包括 (例如)葡萄糖、 果糖、 蔗糖、 淀粉及其不同链长的降解 产物； 蛋白类， 包括 （例如） 白蛋白、 球蛋白、 纤维蛋白原、 纤维蛋 白、 血红蛋白和楦物蛋白的不同链长的降解产物等。
在微泡类助剂的情况下， 气体在所述助剂中的含量为 5〜 200ml/L， 优选为 20〜150ml/L， 更优选为 20〜 100ml/L。所述气体包 括空气， 氮气， 二氧化碳， 氟碳烃类气体如全氟乙烷、 全氟丙垸、 全 氟丁垸， 烷烃类气体如丁垸、 环丁烷， 戊烷、 己垸、 六氟化硫等。
目前在超声造影领域中广泛使用的微泡类造影剂均可以作为本 发明的 HIFU治疗用助剂。 因而， 本发明还相应地提供了一种微泡类 超声造影剂用作本发明助剂的用途。
在微粒类助剂的情况下， 当所述液体为在 38〜100°C不产生液 / 气相变的液体时， 包括水， 饱和脂肪酸， 不饱和脂肪酸如大豆油、 花 生油等，以及碘油等，在所述助剂中的含量为 5〜200g/L，优选为 10〜
100g/L, 更优选为 20〜80g/L。 当所述液体为在 38〜100°C产生液 /气 相变的液体时， 包括 ( 5-06烷烃类物质如正戊垸、 异戊烷等， 以及氟 化 c5-c12烃类物质如全氟戊垸、 二氢十氟戊烷等， 在所述助剂中的 含量为 5〜200ml/L， 优选为 10〜100ml/L， 更优选为 20〜8.0ml/L。
例如， 注射用脂肪乳剂， 是一种脂肪的水乳剂， 由分散在水中 的磷脂膜包裹的精制大豆油构成，适于静脉注射。 目前这类乳剂可以 市售得到， 这些乳剂包括但不限于 Intmlipos® (脂肪乳注射液） 、 OMNILIPID® (脂肪乳注射液） 、 已列入国家基本药物的 "脂肪乳剂 (长链） "或 "脂肪乳剂 （中链及长链复合剂） "。 这类脂肪乳剂也 均可作本发明的 HIFU治疗用助剂。 因而， 本发明还相应地提供了一 种脂肪乳剂用作本发明助剂的用途。
在质粒类助剂的情况下， 纳米级生物相容性固体包括磁性纳米 级生物材料如超顺磁性纳米粒子 (SPIO)、 纳米级羟基磷灰石 (HAP：)、 纳米级碳酸钙等， 粒径一般为 l〜500nm， 优选为 l〜200nm， 更优选 为 10〜100nm。
此外， 上述的纳米级的生物相容性固体本身也可作为本发明的 助剂使用。因而，本发明还相应地提供了一种纳米级的生物相容性的 固体用作本发明助剂的用途。
在上述技术方案中， 所述助剂还可含有乳化剂， 该乳化剂一般 可选用单乙二醇单 c16.18脂肪酸酯、 二乙二醇单 c16-18脂肪酸酯、 二 乙二醇二 c16.18脂肪酸酯、 三乙二醇单 c16_18脂肪酸酯、 失水山梨醇 脂肪酸酯（司盘系列） 乳化剂、 聚山梨醇酯 （吐温系列）乳化剂、 聚 乙二醇单月桂酸酯系列乳化剂，聚氧乙烯月桂酸酯系列乳化剂、 3-sn- 磷脂酰胆碱 （卵磷脂） 、 胆酸等等。 该乳化剂的含量为 5— 150g/L。 另外， 也可包含稳定剂如羧甲基纤维素钠 （CMC-Na) 、 丙三醇等， 所述羧甲基纤维素钠的含量为 0.01〜10g/L， 优选为 0.05〜0.6g/L， 更优选为 0.1〜0.3g/L。 所述丙三醇的含量为 5〜100g/L。
在更为优选的技术方案中， 为了增加所述助剂的稳定性， 可以 使用无机或有机的酸、 碱来调节所述助剂的 pH值。 在微粒类助剂的 情况下， 当所述液体为在 38〜100°C不产生液 /气相变的液体时， 调 节该助剂的 pH值至 7.0〜9.0， 优选调至 7.5〜8.5。 在微粒类助剂的 情况下， 当所述液体为在 38〜100°C产生液 /气相变的液体时， 调节 该助剂的 pH值至 7.0〜9.0， 优选调至 7.5〜8.5。 在质粒类助剂的情 况下， 调节该助剂的 pH值至 3.0〜6.5， 优选调至 5 0〜6.0。
对于本发明的膜包裹类 HIFU 治疗用助剂的制备方法没有过多 的限制， 一般将成膜材料， 预被包裹的气体、 液体或固体， 乳化剂和 稳定剂等物质充分混合、 乳化制备。 例如， 对于微粒类脂肪乳而言， 可以参照中国专利申请 No.97182319.7 (发明名称： 含有还原糖的脂 肪乳剂及其灭菌方法） 或中国专利申请 NO.02112860.X (发明名称： 注射用脂肪乳剂及其制造方法）中公开的制备脂肪乳剂的方法。对于 微泡类氟碳乳剂而言， 可以参照中国专利申请 No.96106566.4 (发明 名称： 含有全氟化碳的右旋糖酐白蛋白声学造影剂及其制备方法)、 中国专利申请 No.981 1901 1 .1 (发明名称： 一种超声诊断造影剂及其 制备方法)或中国专利申请 ZL 89100726.1 (发明名称： 制备用于超声 造影的微粒和超声波造影剂的方法)。
本发明提供的 HIFU治疗用助剂优选以生物相容性的、可降解的 生物材料如脂类为成膜材料，使得该助剂能够经静脉注射，顺利通过 血液循环，并很快被人体内富含网状内皮细胞的组织所吞噬，一定时 间里能够大量沉积于人体组织内，从而显著增强了人体组织对声波的 吸收特性， 增加了 HIFU治疗时靶区组织的能量沉积， 最终可显著提 高临床 HIFU对肿瘤细胞的损伤效果。
本发明还提供了一种增加 HIFU治疗时靶区能量沉积的方法，其 特征在于， 在 HIFU治疗前 0〜168小时通过静脉连续快速滴注或团 注方式给患者注射有效剂量的本发明 HIFU治疗用助剂。所述的有效 剂量会随肿瘤类型、 患者的体重、肿瘤位置、肿瘤体积等因素有所变 化。 但是， 医师或药剂师有能力为不同的患者确定出合适的注射量。 例如， 对于微泡类助剂而言， 可在 0.005〜0.1ml/kg体重的范围内选 择， 优选在 0.01〜0.05ml/kg体重的范围内选择。 对于微粒类助剂而 言， 当所述液体为在 38〜100 °C不产生液 /气相变的液体时， 可在 0.01〜5ml/kg体重的范围内选择，优选在 0.01〜2.5ml/kg体重的范围 内选择； 当所述液体为在 38〜100 °C产生液 /气相变的液体时， 可在 0.005〜0.1ml/kg体重的范围内选择， 优选在 0.01〜0.05ml/kg体重的 范围内选择。 对于质粒类助剂而言， 在 0. 1〜10ml/kg 体重的范围内 选择， 优选在 0.1〜5ml/kg体重的范围内选择。
本发明还提供一种用于筛选 HIFU助剂的方法， 其包括：
(a)测定一种生物组织的能效因子 （EEF ) ， 得到 EEF ( 3 础） ；
(b)将一种候选物质施用给所述的生物组织；
(c)测定该生物组织在施用候选物质后的能效因子 （EEF ) ， 得 到 EEF (测 a )；
(d)将所测得的该组织的 EEF ( Mfl)与 EEF ( )进行比较判定，选 取 EEF ( 础）与 EEF (测 fi )的比值大于 1， 优选大于 2， 更优选 大于 4的候选物质。
本发明另外还提供了一种疾病的治疗方法， 包括在对患者施用 HIFU治疗前， 给患者施用 HIFU助剂以改善 HIFU治疗靶区对治疗 性超声波吸收的能力。 具体实施方式
下文以实施例的方式描述本发明的助剂的制备和一些物化参数， 以试 验例的方式显示本发明示例性助剂的技术效果。但是， 应当理解的是， 这些实施例和试验例都是示例性的， 无论如何不构成对本发明范围的 限制。
实施例 I HIFU治疗用微粒类助剂的制备
实施例 1-1 被包裹的液体在 38 ~ 100 °C不产生液 /气相变
实施例 1-1-1
分别称取注射用碘油（购自上海化学试剂公司） 4g、注射用蛋黄 卵磷脂（购自上海化学试剂公司） 0.6g、 注射用甘油 （购自上海化学 试剂公司） 1.25g， 混合， 加热至 70Ό使之溶解形成油相， 向油相中 加入含 1%(W/V)乳化剂 F-68 (购自 Sigma公司）的蒸馏水至 17.5ml， 剧烈振荡后形成初乳。将上述初乳置于大试管内， 在功率 350W下对 初乳进行超声乳化 2分钟， 形成均匀的乳化碘油， 再经 100Ό流通蒸 汽 30分钟消毒备用，其 pH值 7.5〜 8.5，含碘量 0.13g/ml。粒径 <1μιη， 渗透压 350mosm/千克水。 实施例 1-1-2 ~实施例 1-1-4
按照与实施例 1-1-1所述的相同的方法和步骤，使用注射用大豆 油代替注射用碘油作为芯材料，使用注射用卵磷脂代替蛋黄卵磷脂作 为成膜材料，按照下表 1所列的配比获得了下列本发明的 HIFU治疗 用微粒类助剂。所获得助剂为白色乳状液体，适用于动物及人体静脉 注射， 相应的参数如下表 1所示：
表 1 实施例 实施例 1-1-2 实施例 1-1-3
1-1-4 注射用大豆油在助剂
10 % 20 % 10 % 中的含量 （W/V)
注射用大豆油用量 100g 200g 100g 注射用卵磷脂用量 12g 12g 12g 注射用甘油用量 22g 22g 16.7g 注射用水 (加至） 1000ml 1000ml 1000ml pH值约为 8 8 8 非连续相的粒径 0.卜 2μιη 1〜 5 μιη 0.5〜2μπι 渗透压（mosm/千克水） 300 350 310 能量 MJ(kcal) 4.6 (1100) 8.4 (2000) 12.6 (3000) 实施例 1-2 被包裹的液体在 38 ~ 100°C产生液 /气相变
实施例 1-2-1
先将 3 % ( W/V) 的乳化剂 Pluronic F-68 (购自 Sigma公司） 、 0.5 % ( W/V) 蛋黄卵磷脂 （购自上海化学试剂公司） 、 5 % (V/V) 全氟戊垸 （购自 Sigma公司） 与蒸馏水混合共 1000ml, 在冰水浴条 件下， 10000转 /分钟剪切分散 5 分钟， 制成粗乳。 将该粗乳放入 4 Ό高压乳勾机中， 乳化 2次得到乳剂， 经 Ιμπι滤膜过滤得到颗粒小 于 Ιμηι的乳剂。 分装入 15ml瓶中， 再用 20KGY的钴 60辐照 10小 时， 颗粒浓度在 109/ml， 冷藏备用。 实施例 1-2-2
先将 6 % ( W/V) 的乳化剂 Pluronic F-68 (购自 Sigma公司） 、 1 % ( W/V) 蛋黄卵磷脂 （购自上海化学试剂公司） 、 10 % ( V/V) 全氟戊烷 （购自 Sigma公司） 与生理盐水混合共 1000ml， 在冰水浴 条件下， 10000转 /分钟剪切分散 5分钟， 制成粗乳。 将该粗乳放入 4 Ό高压乳匀机中， 乳化 2次得到乳剂， 经 Ιμιη滤膜过滤得到颗粒小 于 Ιμηι的乳剂。 分装入 15ml瓶中， 再用 20KGY的钴 60辐照 10小 时， 颗粒浓度在 109/ml， 冷藏备用。 实施例 1-2-3 ~实施例 1-2-6
按照与实施例 1-2-1所述的相同的方法和步骤，按照下表 2所使 用的材料和配比获得了本发明的 HIFU治疗用氟碳乳剂类助剂，相应 的参数如下表 2所示：
表 2
Figure imgf000011_0001
实施例 II HIFU治疗用质粒类助剂的制备
实施例 II-1
分别称取粒径范围为 l〜100nm的 HAP (购自四川大学生物材料 工程研究中心） 2.5g、 注射用卵磷脂 （购自上海化学试剂公司） 0.3g 和 CMC-Na (购自上海化学试剂公司） 0.3g混合，加入蒸馏水至 100ml。 混合均勾后， 用乙酸调节混合液的 pH值至 5.0。 将上述混合液置于 声振仪的声振室内， 声振仪的^;射头置于混合液的液面下 1.5cm处， 在声振功率 400W下对混合液声振 2分钟， 形成均勾分散、稳定的乳 白色混悬液。 所制得助剂非连续相的粒径为 10〜1000nm， 主要集中 在 100〜500nm。 实施例 II-2
分别称取粒径范围为 l〜100nm的 HAP (购自四川大学生物材料 工程研究中心） 2.5g、 注射用卵磷脂 （购自上海化学试剂公司） 0.3g 和注射用甘油 1ml混合， 加入蒸馏水至 100ml。 混合均匀后， 用乙酸 调节混合液的 pH值至 5.0。 将上述混合液置于声振仪的声振室内， 声振仪的发射头置于混合液的液面下 1.5cm处，在声振功率 400W下 对混合液声振 2分钟， 形成均匀分散、 稳定的乳白色混悬液。 所制得 助剂非连续相的粒径为 10〜1000nm， 主要集中在 100〜500nm。
实施例 11-3 ~实施例 II-5
按照与实施例 Π- 1所述的相同的方法和步骤， 按照下表 3所使 用的材料和配比获得了下列本发明的 HIFU治疗用质粒类助剂，相应 的参数如下表 3所示：
表 3
Figure imgf000012_0001
实施例 III 纳米级羟基磷灰石（HAP )购自四川大学生物材料工程研究中心， 为白色粉末， 粒径为 10〜200nm， 呈正态分布。 将 HAP用 9 %生理 盐水分别配成浓度为 25g/L， 50g/L的乳白色混悬液， 使用前经超声 振荡仪 600W振荡 2分钟， 使其完全分散均匀。 试验例 1 实施例 1-1-3制得的微粒类助剂和 HIFU治疗仪的联合使用 新西兰大白兔 50只 （重庆医科大学动物实验中心提供） ， 雌雄 不限， 月龄 3个月左右,平均分成 、 B两组， A、 B两组兔体重分别 为 2.22 ± 0.21kg和 2.24 ± 0.19 (P> 0.05 ) 。
肌肉注射麻醉大白兔，将其固定于 HIFU治疗床上，用 JC型 HIFU 肿瘤治疗系统（由重庆海扶 (HIFU)技术有限公司生产）对实验大白兔 进行辐照。 JC型 HIFU肿瘤治疗系统主要由可调功率发生器、 B超监 控系统、 治疗探头、机械运动控制系统、 治疗床和声耦合装置等五部 分组成。 该系统治疗头的直径为 150mm， 焦距 150mm， 声焦域 2.3 x2.4x26mm, 工作频率 1ΜΗζ， 循环脱气水标准为含气量 ^ 3ppm， 平均声强为 5500W/cm2。
采用 HIFU 随机带 B 超预扫描兔肝脏， 选择 2cm 的间隔及 2.0cm的损伤深度的两个层面， A组以左侧 (左 /中叶)兔肝脏为对照叶 (给予生理盐水侧） ， 右侧 (右叶)为实验叶 （给予实施例 1-1-3制得 的助剂， 给药侧） ， B组与 A组相反。 治疗深度均为 2.0cm (皮肤外 侧距焦点的距离） 。选定好肝脏层面后， 沿兔耳缘静脉输入生理盐水 ( 50〜60滴 /分钟）， 20分钟后行定点或直线方式（线长 l cm， 扫描， 速度 3mm/s )对左侧（A组）或右侧（B组）兔肝脏进行 HIFU损伤， 记录靶区灰度变化、治疗时间。然后将 HIFU治疗机焦点移至另一侧， 以实施例 1-1-3制得的助剂代替生理盐水静脉输入后 （输液速度及时 间同对照叶） 进行 HIFU损伤。 同一只兔肝脏两侧损伤方式相同。
辐照后 24小时解剖取材，测量兔肝脏损伤部位凝固性坏死区的 径线 （长、 宽、 厚） ， 以公式： ν=4/3 π χ 1^2长 X 1/2宽 X 1/2厚， 计 算凝固性坏死的大小。 按照公式 /V (J/mm3)计算 EEF (可用 能效因子， energy efficiency factor)， 并进行 A、 B组间和组内比较。 式中 ri表示 HIFU换能器聚焦系数， 它反映 HIFU换能器对超声能量 汇聚的能力， 取 η =0.7 ; P是 HIFU源总声功率 (W); t是治疗总时间 (s); V是损伤体积 (mm3)。 在一种物质施用给靶组织后， 如果靶组织 的能效因子下降的程度越大， 该物质越适合用作 HIFU助剂。 结果如 表 4所示： 表 4 对照叶的 EEF与实验叶的 EEF的比较
A组 B组 合计 P值 对照叶 7.09 ±4.11 6.67 ± 3.13 6.87±3.60 > 0.5 * 实验叶 2.73 ± 1.64 3.43 ±2·07 3, 10± 1.89 >0.5 *
Ρ值 < 0.001 <0.001 < 0.001 上述表 4的结果表明： 给予生理盐水在 Α， Β两组组间比较， 没有显著性差异； 给药本发明实施例 1-1-3制得的微粒类 HIFU助剂 在 A， B两组组间比较， 也没有显著性差异。 但是， 将对照叶和实验 叶的实验结果进行比较，无论是 A组还是 B组具有十分显著的差异。 在八、 B组合计的情况下， 实验叶较施用生理盐水的对照叶， EEF平 均下降 2.22倍。 试验例 1 实施例 1-1-1制得的微粒类助剂和 HIFU治疗仪的联合使用 取新西兰大白兔 30只 （重庆医科大学动物实验中心提供） ， 体 重 2kg左右， 随机分成实验组和正常组， 每组 15只， 每只大白兔设 两个辐照点。 正常组大白兔按 2.5ml/kg 的量经耳缘静脉快速注射给 予生理盐水。 实验组大白兔按 2.5ml/kg 的量经耳缘静脉快速注射给 予实施例 1-1-1制得的助剂乳化碘油， 并给予 lml生理盐水冲洗， 确 保药物完全进入体内。 1小时后， 使用 JC型 HIFU肿瘤治疗系统（重 庆海扶 (HIFU)技术有限公司生产）分别对实验组和正常组的大白兔肝 脏进行定点辐照。 辐照的功率为 220W, 频率为 1.0MHz, 辐照深度 为 20mm，辐照时间以出现凝固性坏死为好。计量资料用平均值士 SD 表示， 采用 SPSS 10.0 for windows统计软件包， 独立及配对 t检验； 计数资料用 χ2 检验。 比较实验组和正常组的 EEF, 如下表 5所示： 表 5 正常组的 EEF与实验组的 EEF的比较
组别 η EEF (x±s) (J/mm3) 正常组 30 31.05 ±2.68 实验组 30 7.16 ± 1 ·38* η表示福照点数。 *与对照组相比， Ρ<0,001。
上表结果表明： 实施例 1-1-1制得的乳化碘油可使 HIFU损伤肝 脏组织的 EEF明显降低。 试验例 3 实施例 1-1-3制得的微粒类助剂的体外实验研究
取新西兰大白兔 10只 （由重庆医科大学动物实验中心提供） , 雌雄不限， 月龄 3个月左右。 随机分成实验组 （给予实施例 1-1-3制 得的助剂） 与对照组 （给予生理盐水） ， 兔体重分别为 2.40 ±0.45kg 和 2.32± 0.08kg(P>0.5)。 实验前 24小时禁食。 采用 CZF-1型 HIFU 妇科治疗仪（由重庆海扶 (HIFU)技术有限公司生产）实施对兔肝脏的 辐照。 CZF-1型 HIFU妇科治疗仪由功率源、 治疗头和循环水三部分 组成， 参见中国发明专利 No.01144259.X。 本实验设置参数为： 功率 4.05W、 频率 11ΜΗζ、 脉冲 1000Hz。
肌肉注射麻醉大白兔后， 经兔耳缘静脉输入实施例 1-1-3制得的 助剂（实验组）或生理盐水 (对照组）， 输液速度均为 50〜60滴 /分钟， 输液时间均为 20分钟。
输液完成后约 1 小时， 将兔仰卧固定于手术台上， 取上腹部正 中切口约长 4〜5cm， 逐层切开腹壁全层进入腹腔， 暴露并轻轻牵出 肝脏。 每叶兔肝在每个时段损伤 1〜2个点， 本实验设定的损伤时段 为 3s， 6s, 9s三个时段。 设计好损伤点后即按上述实验参数进行实 验。 损伤结束后， 将兔肝送回腹腔， 逐层缝合腹壁全层。
次日过量麻醉处死兔， 取出肝脏照相及测量损伤组织的径线并 计算 EEF。 数据均用平均值士 SD表示， 采用 SPSS 10.0 for windows 统计软件包， 独立样本 t检验， p<0.05为统计有意义。 实验测得对照 组每个损伤时段各 21个点， 共计 63 ( 21 X 3 ) 个点的组织损伤体积； 实验组每个损伤时段 30个点， 共计 90 ( 30 X 3 ) 个点的组织损伤体 积。 根据公式计算出 EEF， 结果如下表 6所列：
表 6 对照组的 EEF值与实验组的 EEF值的比较
损伤时间
n 3s 6s 9s 对照组 21 0.2749 ± 0.2409 0.1783 ± 0.0733 0.1846±0.0896 实验组 30 0.1177 ± 0.0609 0.1367 ±0.0613 0.1463 ±0.069
P值 < 0.01 <0.05 >0.05 上表 6所列结果说明： 对照组在 3s， 6s, 9s时段的 EEF值分别 为实验组相应时段的 EEF值的 2.34倍， 1.30倍， 1.26倍。 总下降倍 数为： 1.59倍。 因 9s时差异已无显著性， 如不计算在内则 EEF下降 倍数为： 1.78倍。 试验例 4实施例 1-2-1制得的微粒类助剂和 HIFU治疗仪的联合使用 ( 1 )新西兰大白兔肝脏损伤实验
新西兰大白兔 20只 （重庆医科大学动物实验中心提供） ， 体重 2.21±0.56kg, 实验前一天脱去下胸部和上腹部的毛。 用 JC型 HIFU 肿瘤治疗系统（重庆海扶 (HIFU)技术有限公司生产）对实验大白兔进 行辐照。 JC型 HIFU肿瘤治疗系统主要由可调功率发生器、 B超监控 系统、 治疗探头、机械运动控制系统、 治疗床和声耦合装置等五部分 组成。 该系统治疗头的直径为 150mm， 焦距 150mm， 声焦域 2.3x2.4x26mm, 工作频率 1ΜΗζ， 循环脱气水标准为含气量 ^ 3ppm， 平均声强为 5500W/cm2。 实验采用的换能器直径为 150mm， 焦距为
135mm, 频率为 1.0MHz, 声功率（P)为 200W。 辐照深度为 20mm， 辐照方式为间断定点， 辐照 3秒， 停 5秒。 按 0.02ml/kg的'量经耳缘 静脉快速注射生理盐水， 60秒后 HIFU定点辐照实验兔肝脏， 此为 对照组。 按 0.02ml/kg的量经耳缘静脉快速注射给予实施例 1-2-1制 得的助剂， 60秒后 HIFU辐照同一兔肝脏的另一平面的点， 此为实 验组。辐照靶区出现灰度改变后结束辐照，如果未见灰度改变则最多 辐照 20秒。 3天后断颈处死实验兔， 解剖测量肝脏凝固性坏死体积 (V) 大小。 根据公式 EEF = ^ 计算能效因子 （EEF ) ， τ 为辐照 时间， n =0.7。 对照组 EEF 中位数为 6.0160， 实验组为 1.2505， Wilcoxon 符号秩和检验， Z=-2.485， P=0.013。 该实验结果表明， 注 射氟碳乳剂后 HIFU损伤兔肝脏的效率提高 4.81倍。
( 2 ) 山羊肝脏损伤实验
取南江黄羊 20只， 体重 22.25±4.5 1kg， 实验前当日脱去右胸部 和右腹部的毛。 用 JC型 HIFU肿瘤治疗系统 （由重庆海扶 (HIFU)技 术有限公司生产） 对实验黄羊进行辐照。 实验采用换能器直径为 150mm , 焦距为 135mm , 频率为 0.8MHz， 声功率 ( P )为 220W。 辐 照深度为 30mm， 辐照方式为间断定点， 辐照 3秒,停 5秒， 所有羊都 未切除肋骨。 HIFU辐照前先预扫描， 选择辐照区域， 共 4个平面， 每平面上辐照一点， 二维超声观察通肋间隙进行。 按 0.02ml/kg的量 经耳缘静脉快速注射给予生理盐水， 60秒后 HIFU定点辐照实验羊 肝脏， 每只羊辐照 2点， 此为对照组。 按 0.02ml/kg的量经耳缘静脉 快速注射给予实施例 1-2- 1制得的助剂， 60秒后 HIFU辐照， 也辐照 · 二点， 此为实验组。 辐照靶区出现灰度改变再辐照 4〜5次后结束辐 照， 如果未见灰度改变则最多辐照 200秒。 3天后杀死羊， 解剖测量 肝脏凝固性坏死体积 （V ) 大小。 根据公式 EE - ^P ^计算能效因子
(EEF) ， T为辐照时间， n =0.7。 对照组的 EEF中位数为无穷大， HIFU 加氟碳乳剂组的中位数为 5.1904。 Wilcoxon 符号秩和检验， P=0.004。实验说明在不切除肋骨的情况下， HIFU对山羊肝脏的损伤 在氟碳乳剂的存在下.， 损伤效率发生了质的改变。
( 3 ) 山羊肾脏损伤实验
取南江黄羊 20只， 体重 22.25±4.51kg， 实验前当日脱去右胸部 和右腹部的毛。 用 JC型 HIFU肿瘤治疗系统 （由重庆海扶 (HIFU)技 术有限公司生产） 对实验黄羊进行辐照。 实验釆用换能器直径为 150mm, 焦距为 135mm， 频率为 0.8 MHz, 声功率 （P ) 为 220W。 辐照深度为 20mm， 辐照方式为间断定点， 辐照 3秒， 停 5秒， 所有 羊都未切除肋骨。 HIFU辐照前先预扫描， 选择辐照区域， 肾上极和 下极各选一平面共 2个平面， 每平面上辐照一点， 二维超声观察。如 右肋骨阻挡则避开。 按 0.02ml/kg的量经耳缘静脉快速注射给予生理 盐水， 30秒后 HIFU定点辐照实验羊肾脏，此为对照组。按 0.02ml/kg 的量经耳缘静脉快速注射给予实施例 1-2-1制得的助剂， 60秒后 HIFU 辐照， 此为实验组。 辐照靶区出现灰度改变再辐照 3〜4次后结束辐 照， 如果未见灰度改变则最多辐照 150秒。 3天后杀死羊， 解剖测量 肝脏凝固性坏死体积 （V ) 大小。 根据公式 EEF = ^%计算能效因子 ( EEF ) ， T为辐照时间， n =0.7。 实验组的 EEF为 10.58±3.95， 对 照组为 486.37±215.41 o Wilcoxon秩和检验精确概率 P=0.008。 该实 验结果提示氟碳乳剂注入后， HIFU对正常山羊肾脏的损伤效率提高 了 40多倍。 试验例 5 实施例 II-1制得的质粒类助剂和 HIFU治疗仪的联合使用 取新西兰大白兔 36只 （重庆医科大学动物实验中心提供） ， 体 重 2kg左右， 随机分成 1个对照组和 2个给药组， 每组 12只。 对照 组大白兔按 2ml/kg的量经耳缘静脉快速注射给予生理盐水。 2个给 药组按 2ml/kg的量经耳缘静脉快速注射给予实施例 II- 1制得的助剂， 并给予 1ml生理盐水冲洗， 确保药物完全进入体内。 2个给药组分别 在注射后 24小时及 48小时进行辐照。 注射后 24小时进行辐照的给 药组称为第一给药组， 注射后 48小时进行辐照的给药组称为第二给 药组。 使用 JC型 HIFU肿瘤治疗系统 （由重庆海扶 （HIFU) 技术有 限公司生产）分别对 1个对照组和 2个给药组的家兔肝脏进行定点辐 照。 JC型 HIFU肿瘤治疗系统主要由可调功率发生器、 B超监控系统、 治疗探头、 机械运动控制系统、 治疗床和声耦合装置等五部分组成。 该系统治疗头的直径为 150mm，焦距 150mm，声焦域 2.3 x2.4x26mm， 工作频率 1ΜΗζ， 循环脱气水标准为含气量≤ 3ppm， 平均声强为 5500W/cm2。辐照的功率为 220W，频率为 1 .0MHz，辐照深度为 20mm， 辐照时间为 15秒。 辐照完成后， 解剖实验动物取材， 计算凝固性坏 死灶的体积。 1个对照组和 2个给药组在家兔肝脏形成一定凝固性坏 死灶所需的能效因子 （EEF ) 如下表 7所示:
表 7
组别 福照点数 V (mm3) EEF (J/mm3) 对照组 24 582.50±353.93 7.39±4.99 第一给药组 45 1281.56± 884.56 2.71 ± 1.29 第二给药组 17 1525.63 ± 1007.46 2.25 ± 1.61 从上表 7 中可以看出， 在相同条件下， 在注射后 24小时及 48 小时进行辐照， 辐照相同的时间， 给药组较对照组在 HIFU治疗过程 中形成凝固性坏死灶体积均明显增大，所需的能效因子明显减小。在 形成凝固性坏死灶体积和能效因子方面，给药组和对照组之间的差异 均具有显著性意义 P<0.05)。 试验例 6 实施例 III制得的 HIFU治疗用质粒类助剂的体内实验研究 新西兰大白兔 40只， 来源于重庆医科大学动物实验中心， 体重 平均 2.7±0.3kg/只， 雌雄不论。 随机分为 3个 HAP实验组和 1个对 照组组， 每组 10只。
HIFU治疗前 24小时， HAP组每只大白兔分别按 2〜3ml/kg的 量经耳缘静脉快速推注实施例 III制得的不同浓度 HAP混悬液， 5秒 内推完， 并给予 1ml 生理盐水冲洗， 确保药物完全进入体内； 对照 组每只家兔分别推注生理盐水 2ml/kg。 家兔右胸腹部 8 %硫化钠脱 毛， 术前速眠新 0.2ml/kg肌注麻醉， 于无菌手术下切开腹壁， 充分 暴露肝脏。
釆用 CZF-1型 HIFU妇科治疗仪 （由重庆海扶 (HIFU)技术有限 公司生产） 实施对兔肝脏的辐照。 CZF-1型 HIFU妇科治疗仪由功率 源、 治疗头和循环水三部分组成，参见中国发明专利 No.01144259.X。 工作参数如下： 频率 9.85MHz, 功率 5W， 焦距 4mm， 治疗方式釆用 定点辐照。 每个肝脏辐照 2〜3组， 每组 3点， 辐照时间为 10秒。术 后缝合切口， 24小时后经耳缘静脉快速推注空气 10毫升处死家兔， 测量所形成的凝固性坏死灶焦域体积， 并计算能效因子 （EEF) 。
釆用组间比较的 t检验及相关分析。 家兔肝脏在聚焦超声治疗结束后即可见靶区形成明显圆点状灰 白色凝固性坏死， 边界清楚。 在各组内， 在辐照相同的时间后， 不同
HAP 用量实验组经聚焦超声治疗后所形成的焦域体积均较相应的生 理盐水组明显增大， 所需能效因子明显缩小， 差异均具有极显著性意 义 （PO.001 ) 。 不同纳米级 HAP用量组内比较， 随着 HAP的用量 增加， 所形成的焦域体积亦随之明显增加，所需能效因子随之明显减 小， 差异均具有显著性意义 （P<0.001 ) 。 下表 8表示出了不同 HAP 用量聚焦超声治疗组的作用焦域体积及能效因子比较 ( ±s)。
¾ 8
药物 用量 n V/mm3 EEF
对照组 2ml/kg 30 95.3±21.6 0.39±0.09
HAP组 1 50mg/kg 30 153.1±41.8 0.24±0.05
HAP组 2 100mg/kg 25 223.2±55.1 0.19±0.01
HAP组 3 150mg/kg 21 287.7士 47.9 0.13±0.00
表中 "n" 表示辐照的点数。
从上述实验可以得出， 纳米级 HAP可以明显增强 HIFU的体内 治疗效果， 并随着 HAP用量增加， 增强治疗效果越强。
试验例 7 实施例 III制得的 HIFU治疗用质粒类助剂的体外实验研究 健康新西兰兔 80只 （重庆医科大学动物实验中心提供） ， 雌雄 不论， 体重 2.5±0.3kg， 治疗前 24小时禁食， 每只家兔按 2ml/kg的 量经耳缘静脉快速注射给予实施例 III制得的 25g/L的 HAP乳白色混 悬液， 并以生理盐水 lml冲洗。 分别于用药后 24小时 （20只） ， 48 小时 （25只） ， 72小时 （10只） 和 168小时 （15只） 进行 HFIU肝 脏扫描。 对照组 （10只） 给予生理盐水 2ml/kg， 之后 24小时时进行
HIFU肝脏扫描。术前动物右胸腹部 8 %硫化钠脱毛，速眠新 0.2ml/kg 肌注麻醉， 固定于 JC-A型 HIFU治疗仪上。
JC-A型高强度聚焦超声肿瘤治疗系统由重庆医科大学医学超声 工程研究所研制（国家药品监督管理局已批准生产， 注册号 99 第 301032号）。 该系统包括超声实时监控定位和治疗设备二大部分。 采 用循环脱气水作声耦合剂， 其含气量< 3 >< 10 6。 治疗参数为： 功率 220W, 频率 ΙΜΗζ, 焦距 150mm， 焦域长度 12mm， 治疗头可在 X， Υ， Ζ三个方向随意移动。 .
家兔胸腹部浸泡于循环脱气水中， 肝脏于 Β超下显示清楚， 定 位辐照点， 固定治疗时间为 15秒， 治疗深度为 20mm, 每个肝脏可 辐照 1〜2点。 然后于术后 24小时从兔耳缘静脉快速推入 10ml空气 处死动物， 取出肝脏， 沿声通道方向切开兔肝组织， 显示其最大损伤 坏死灶切面， 观察其形状并测量大小 （以 TTC染色为界） 。 然后计 算 EEF。
采用组间比较的 t检验及相关分析。
在相同的治疗条件下， 纳米级 HAP用药组较对照组可以形成更 大的凝固性坏死灶 （p<0.05 ) ， HIFU治疗所需的能效因子也明显縮 小， 且在用药后 24小时和 48小时后进行 HIFU扫描， 形成的焦域坏 死体积最大， 所需能效因子也最小， 提示此时可能是 HAP用药后进 行 HIFU治疗的最佳时机。 随着用药后时间的延长， 形成的坏死灶体 积则逐渐缩小。但即使在用药后 2周，也较用药前能更好的增强 HIFU 的治疗效果 （p<0.05 ) 。 （见表 9 )
表 9 用药后不同时间组与对照组之间的比较情况
用药后时间 n 平均治疗时间 （S ) 平均焦域体积（mm3 ) 平均 EEF 对照组 16 15 546.67 7.39±4.99
24小时 57 15.88 1291.56 2.68 ± 1.29
48小时 17 15.6 1525.63 2.32 ± 1.61
72小时 27 16.15 1153.26 3.28 ± 1.35
168小时 26 15 920 2.51 ±0.87 注： 表中 n为实际辐照点数。
从上述实验可以得出， 纳米级 HAP可以明显增强 HIFU的体外 治疗效果， 并且于用药后 48〜72小时进行 HIFU治疗能够产生对 HIFU治疗最好的增强效果。
试验例 8 HIFU治疗用微泡类助剂和 HIFU治疗仪的联合使用 取新西兰大白兔 40只， 体重 2kg左右， 随机分成实验组和正常 组， 每组 20只。 对照组按 0.05ml/kg的量静脉注射给予生理盐水。 实验组按 0.05ml/kg的量经耳缘静脉快速注射全氟显微泡造影剂 （购 自南方医院） ， 并给予 lml生理盐水冲洗， 确保药物完全进入体内。 1分钟后使用 JC型 HIFU肿瘤治疗系统 （重庆海扶 （HIFU) 技术有 限公司生产） 进行辐照。 辐照的功率为 200W, 频率为 1.0MHz, 辐 照深度为 20mm， 一定的辐照时间。 3天后取材， 测量凝固性坏死的 大小， 计算 EEF。 计量资料用平均值士 SD表示， 采用 SPSS 10.0 for windows统计软件包， 独立及配对 t检验； 计数资料用 χ2检验。 结 果如下表 10所示。
表 10 对照组的 EEF与实验组的 EEF的比较 组别 EEF ( J/mm3)
对照组 12.83 ± 10.99
实验组 2.70 ± 1.29*
与正常组相比， P< 0.
上述结果表明： 微泡类 HIFU助剂可使 HIFU损伤肝脏组织的 EEF明显降低。 产业上的实用性
本发明提供的高强度聚焦超声 （HIFU) 治疗用助剂能够显著改 善靶区的声环境， 能降低单位体积的靶组织（肿瘤和非肿瘤组织）被 损伤所需的超声能量，使得在一定功率下，可以高效率地治疗位置较 深、体积较大的肿瘤， 而又不损伤声通道上的正常组织。采用本发明 的助剂使得在不切除患者的治疗声通道上的肋骨的情况下对肝肿瘤 患者有效实施 HIFU治疗成为可能。
尽管结合优选实施例对本发明进行了说明， 但本发明并不局限 于上述实施例和附图， 应该理解， 在本发明构思的引导下， 本领域技 术人员可进行各种修改和改进， 所附权利要求概括了本发明的范围。

Claims

权 利 要 求 书
1. 一种高强度聚焦超声 （HIFU ) 治疗用助剂， 其特征在于， 该 助剂是在对患者实施 HIFU治疗前给患者施用的一种能使 HIFU治疗 靶区的 EEF下降的物质， 所述 EEF = "P ， 表示 HIFU有效治疗单位 体积的肿瘤所需的超声能量， 单位是 J/mm3， 其中 η =0.7 ; Ρ是 HIFU 源总声功率， 单位是瓦； t是 HIFU治疗的总时间， 单位是秒； V是 HIFU损伤的组织体积， 单位是 mm3 ;
将所述靶区在施用该助剂前的 EEF定为 EEF S ， 将所述靶区 在施用该 HIFU助剂后测得的 EEF定为 EEF ， 则 EEF (基础） /EEF
(测 a)的比率大于 1 。
2. 根据权利要求 1所述的助剂，其中 EEF (s¾) /EEF ( )的比率 大于 2。
3. 根据权利要求 2所述的助剂，其中 EEF ^^ /EEF ^fl)的比率 大于 4。
4. 根据权利要求 1一 3任一项所述的助剂， 其是静脉注射、动脉 注射、 局部注射用剂， 该助剂的粒径为 10ηηι〜 8μιη。
5. 根据权利要求 4所述的助剂， 该助剂包括由成膜材料包裹芯 构成的非连续相和水性介质构成的连续相，其中所述非连续相均勾地 分散在所述连续相中， 所述非连续相的粒径为 10ηιη〜8μηι， 所述成 膜材料具有生物相容性，所述芯材料采用气体、液体或纳米级生物相 容性固体。
6. 根据权利要求 5所述的助剂， 其中所述的成膜材料为脂类 蛋白类和糖类中的一种或多种物质。
7. 根据权利要求 6所述的助剂， 其中所述的脂类为磷脂， 选自 3-sn-磷脂酰胆碱、 1,2-二棕榈酰基 -sn-甘油基 -3-磷脂酰甘油基-钠盐、 1,2-二硬脂酰基 -sn-甘油基 -3-磷脂酰胆碱、 1,2-二棕榈酰基 -sn-甘油基 -3-磷脂酰酸-钠盐、 1,2-二棕榈酰基 -sn-甘油基 -3-磷脂酰胆碱、磷脂酰 丝氨酸和氢化磷脂酰丝氨酸。
8. 根据权利要求 5所述的助剂， 其中所述的气体选自空气、 氮 气， 二氧化碳、 氟碳烃气体和垸烃类气体。 9. 根据权利要求 8所述的助剂， 其为静脉注射用超声造影剂。
10. 根据权利要求 5所述的助剂，其中所述的液体选自 05-€6烷 烃、 5-< 12氟碳烃、 饱和脂肪酸、 不饱和脂肪酸和碘油。 11. 根据权利要求 10所述的助剂， 其为静脉注射用脂肪乳剂。
' 12. 根据权利要求 10所述的助剂， 其为静脉注射用乳化碘油。
13. 根据权利要求 10所述的助剂， 其为静脉注射用 C5-C12全氟 碳烃乳剂。
14. 根据权利要求 5所述的助剂，其中所述的固体选自磁性生物 材料、 羟基磷灰石和碳酸钙， 该固体的粒径为 l〜500nm。 15.根据权利要求 14所述的助剂，其中所述的固体为粒径在 1〜
200nm范围内的羟基磷灰石。
16. 根据权利要求 1一 15任一项所述的助剂，其中所述的靶区是 所述助剂经血液循环所到达的器官。
1177.. 一一种种增增加加 HHIIFFUU治治疗疗时时靶靶区区能能量量沉沉积积的的方方法法，， 其其特特征征在在于于，， 在在患患者者的的靶靶区区进进行行 HHIIFFUU治治疗疗前前 00〜〜116688小小时时，， 通通过过静静脉脉连连续续快快速速滴滴 注注或或团团注注方方式式给给患患者者注注射射有有效效剂剂量量的的权权利利要要求求 11一一 1155 任任一一项项所所述述的的 HHIIFFUU治治疗疗用用助助剂剂。。
1188.. 一一种种用用于于筛筛选选 HHIIFFUU助助剂剂的的方方法法，， 其其包包括括：：
((aa))对对给给定定的的组组织织辐辐照照高高强强度度聚聚焦焦超超声声（（HHIIFFUU))，，按按照照 FF
Figure imgf000025_0001
计算该组织的 EEF， 其单位为 J/mm3， 得到 EEF ( ) , 其中 η =0.7; Ρ是 HIFU源总声功率， 单位为瓦； t是 HIFU治疗的总时间， 单位 是秒； V是 HIFU损伤的组织体积， 单位是 mm3 ;
(b)将一种候选物质施用给所述的生物组织；
(c)测定该生物组织在施用该候选物质后的 EEF， 得到 EEF ,m m；
(d)将所测得的该组织的 EEF («fi)与 EEF ( a )进行比较判定， 选 取 EEF 础）与 EEF ( iafl)的比值大于 1的候选物质。
19. 根据权利要求 18 所述的方法， 其中将所测得的该组织的
EEF (测量）与 EEF (基础）进 ί丁比较判定， 选取 EEF ( 础）与 EEF (测 的比 率大于 2的候选物质。
20. 根据权利要求 18 所述的方法， 其中将所测得的该组织的
EEF (测≤]：)与 EEF (基础）进^亍比较判定， 选取 EEF 础）与 EEF (测 ）的比 率大于 4的候选物质。
PCT/CN2005/001367 2005-01-10 2005-08-31 Adjuvant pour un traitement par ultrasons focalises a haute intensite et son procede de depistage WO2006072198A1 (fr)

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