Methods, compositions and apparatus for localized delivery of compounds are provided. In certain embodiments, radiation force is used to direct carriers to a target site, and additional radiation is used to fragment the localized carriers, releasing associate compounds. Ultrasound radiation is preferred as the source for radiation force and for fragmentation. Also encompassed are embodiments in which targeting and fragmentations are combined with imaging of the treatment site. Alternate embodiments are disclosed in which compounds are locally delivered without use of carriers.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is defined by the following claims, and nothing in this section should be taken as a limitation on those claims. Disclosed herein are methods, compositions, and apparatus for targeted delivery of compounds and carriers using radiation force.

Accordingly one aspect of the invention includes methods of using radiation force to target a carrier to a site. In one aspect, the radiation force is generated using ultrasonic radiation. In another aspect, the carrier is engineered to carry a compound such as a drug payload. In another aspect, the invention includes methods in which carriers are fragmented at the site. Yet other aspects of the invention include methods that combine imaging with the above methods, as well as methods that include administering agents or radiation to affect tissue permeability or otherwise alter cell physiology at the site.

In one embodiment, the carrier includes a molecule to further improve targeting. In a preferred embodiment, the carrier is acoustically active. Exemplary embodiments include liquid and solid contrast agents containing entrapped gas, although the invention also may be practiced using a carrier having a liquid core. Any carrier may be used, provided there exists an acoustic mismatch between the carrier and the surrounding tissue or liquid. Carriers having a liquid core are preferred for targeted delivery of water-soluble agents.

In a preferred variation of the invention, targeting is accomplished using radiation force to concentrate a carrier along a vessel wall. In another preferred variation, targeting is accomplished using radiation force to reduce carrier velocity within a vessel.

In addition, the invention provides methods of targeted delivery of compounds without carriers by altering tissue permeability or cell physiology at a target site by administering agents or radiation to affect tissue permeability or otherwise modulate cell physiology at the site. In preferred embodiments the tissue comprises a vessel or a tumor. In another preferred embodiment, the administered radiation is ultrasonic radiation. In yet other preferred embodiments, the agents modulate bradykinin receptor activity or P-gp activity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Briefly, and as described in more detail below, described herein are methods, compositions and apparatus for improving the efficacy and diminishing the toxicity of administered compounds. The improvements are realized by using radiation force, such as that produced by, e.g., ultrasonic radiation, to concentrate carriers at target sites, such as along vessel walls, within tumors, or at other predetermined sites. Vessels, as used herein, include any of the various tubes in which bodily fluids circulate as known in the art, e.g., veins, arteries, venules, arterioles, capillaries, and lymphatics. Once carriers have been concentrated at the target site, the carriers optionally may be disrupted to promote extravasation of carrier fragments or otherwise promote release of a compound associated with the carrier. Preferably, disruption is achieved by insonating the carrier at a frequency and pressure sufficient to fragment the carrier. The details of the parameters required to manipulate a carrier by radiation force are described further within, and with respect to a model useful for predicting carrier behavior.

Several features of the current approach should be noted. Combinations and subcombinations of various approaches involving use of radiation force to affect carrier localization or velocity in a targeted and predetermined way, fragmentation of the carrier, imaging of the carrier or of target sites, application of agents or radiation to affect tissue permeability or physiology all are contemplated to be within the scope of the present invention. In addition, the invention contemplates use of techniques to improve the specificity and reduce the toxicity of compounds by formulation with carriers. In preferred aspects of these methods of the invention, ultrasonic radiation is used to modulate vessel permeability in a targeted region, thereby promoting extravasation and absorption of the administered compound. The compounds used in this aspect of the invention may comprise any therapeutic or diagnostic substance including, by way of example but not limitation, small molecules, peptides, nucleic acids, and synthetic and semi-synthetic analogues thereof.

Advantages of this approach are numerous. Among the advantages are improved specificity and reduced toxicity for administered compounds, and improved treatment outcomes for subjects in need of treatment for a wide variety of medical conditions, especially cancers, cardiovascular diseases, and inflammatory disorders such as rheumatoid arthritis and Crohn's disease.

The invention is useful for diagnostic and or therapeutic applications in which it is beneficial to administer a compound such as, e.g., a physiologically-active compound, with or without a carrier for the purpose of diagnosing and/or treating a medical condition.

Methods and Apparatus of the Invention

Background

Microcapsule drug delivery. Many oncologic drugs are toxic to normal tissues in addition to tumor cell lines. Paclitaxel, a common chemotherapeutic drug, must be solubilized in cremophore because of its low water solubility. This is undesirable as cremophore is also highly toxic. This systemic toxicity makes it desirable to deliver the antitumor agent directly to the affected area. Unger et al. Invest. Radiol. 33(12):886-892 (Dec. 1998) have demonstrated that paclitaxel can be suspended in a drug delivery capsule with an oil shell and that local delivery of paclitaxel can be effective against brain tumors. This drug delivery capsule is a microbubble, on order of several microns in diameter, and has a multiple layer shell that contains the compound. The mechanism of action of current microcapsule drug delivery vehicles such as Acoustively Active Lipospheres (AAL, ImaRx Therapeutics) includes injection into the bloodstream, followed by disruption at the site of interest using a high-intensity acoustic pulse. This disruption causes the contents of the capsule (the drug) to be delivered at the site of interest. Currently, this technology is in clinical trials.

Additionally, many new anti-angiogenic drugs are under development, including nine drugs currently in clinical trials that act directly on endothelial cells. These drugs inhibit endothelial cell-specific signaling or endothelial cell function, with a resulting effect on the tumor. Since a single endothelial cell supplies nutrients to many tumor cells, the inhibition of activity in a single endothelial cell has a great effect. With microcapsule drug delivery, a greater dose of drug can be delivered to endothelial cells near the tumor. In addition, investigators have shown that ultrasound in conjunction with microbubbles can result in capillary rupture in rats, with resulting extravasation of erythrocytes into the interstitial space. See, e.g., Skyba, et al., Circulation 98(4):290-293 (July 1998); Price, et al., Circulation 98(13): 1264-1267 (September 1998). Microbubble drug delivery carriers may cause increased endothelial permeability and at the same time release a chemotherapeutic agent and so have the potential to be a powerful therapeutic tool. See, e.g., Wheatley, et al., Mat. Res. Soc. Symp. Proc. 550:113-118 (1999). Additionally, researchers have shown the utility of microbubble agents for gene delivery. See, e.g., Wickline and Lanza, J. Cell. Biochem. Suppl. 39:90-97 (2002).

Ultrasound Radiation Force--Ultrasound produces a radiation force that is exerted upon objects in a medium with an acoustic impedance different than that of the medium. An example is a microbubble in blood, although, as one of ordinary skill will recognize, ultrasound radiation forces also may be generated on non-gaseous carriers. We have shown the ability of radiation force to concentrate microbubbles in-vitro and in-vivo. Dayton, et al., Ultrasound in Med. & Biol., 25(8):1195-1201(1999). An ultrasound transducer pulsing at 5 MHz center frequency, 10 kHz pulse repetition frequency ("PRF"), and 800 kPa peak pressure, has been shown to concentrate microbubbles against a vessel wall in-vivo, and reduce the velocity of these flowing agents an order of magnitude. However, to this date, the application of radiation to concentrate drug delivery carriers has not been demonstrated, nor have the combined effects of radiation force-induced concentration and carrier fragmentation.

Sonoporation--The mechanical effects of ultrasound (with and without microbubbles) to alter the permeability of cells and vessels, termed sonoporation, has now been well established. Application of ultrasound with specific acoustic parameters causes increases in cell permeability.

Ultrasonically-disrupted microcapsule drug delivery is a new idea, which is still in research trials. Initial results are promising, and this technology has the potential for significant clinical impact. Acoustic radiation force is known to act on particles in a fluid, and has recently been shown by the inventors to concentrate drug delivery carriers. The potential to concentrate drug delivery capsules at the site of interest before disruption, as described by this invention, provides a significant increase in the therapeutic efficacy of ultrasound-disrupted drug carriers. The application of ultrasound for sonoporation further contributes to the therapeutic delivery of targeted carriers such as microbubbles.

In one aspect, the invention provides ultrasound radiation force to enhance effectiveness of carriers such as acoustically active vesicles and other particles useful as carriers in the practice of the invention. Radiation force is used to "push" or concentrate carriers along the wall of a vessel. In small blood vessels, particles such as cells or carriers tend to flow along the center of the vessel, rather than along the sides. By concentrating the carriers along the vessel wall, a larger percentage of a carrier-associated compound is delivered to or through the endothelium, especially upon carrier rupture, vibration or fragmentation (generically referred to herein as fragmentation).

Additionally, the invention encompasses use of radiation force to assist delivery of targeted carriers. Targeted carriers have an adhesion mechanism incorporated into the capsule wall that is specific for a molecular signature of disease expressed on the endothelium. Since available adhesion mechanisms work on the distance of nanometers, it is important to localize the drug delivery vehicles along the vessel wall in order for such adhesion to occur. Radiation force produced perpendicular to or against the direction of flow reduces the velocity of particles flowing in a fluid. Thus, in another aspect the invention uses radiation force to assist targeted carrier delivery, since slower moving particles have a greater opportunity to interact with adhesion mechanisms on an endothelial or other surface.

The invention further encompasses use of radiation force in cooperation with ultrasonic imaging, to allow a user to observe the area being treated, and optionally with sonoporation, to increase permeability of cells in the target area. Also described in this proposal is a system specifically designed to deliver microcapsule delivery vehicles with ultrasound.

In one aspect, the invention uses ultrasound and a carrier to enhance delivery of a drug or other agent at the desired site in the following preferred manners:

1. Ultrasound e.g., at center frequencies about 0.1 MHz-40 MHz, and at a low acoustic pressure e.g., at about 20 kPa-6 MPa, and a long pulse length (e.g., about >10 cycles) or a short pulse length (e.g., about <10 cycles) and high pulse repetition frequency (e.g., about >500 Hz) to produce radiation force and concentrate carriers. The specific parameters will depend on the choice of carrier, as detailed further below, and can be readily determined by ordinarily skilled artisans having the benefit of this disclosure.

2. Ultrasound e.g., at about 0.1 MHz-40 MHz, and at a low acoustic pressure e.g., at about 20 kPa-6 MPa and a long cycle length (e.g., about >10 cycles) or a short cycle length (e.g., about <10 cycles) and high pulse repetition frequency (e.g., about >500 Hz) to produce radiation force and reduce the flow velocity of carriers. Again, the specific parameters chosen depend on the choice of carrier, as detailed further below, and can be readily determined by ordinarily skilled artisans having the benefit of this disclosure.

3. An ultrasonic pulse sequence of the above description followed by short pulses (e.g., about <10 cycles) of high acoustic pressure e.g., about 0.3 MPa to 20 MPa, which disrupts carriers, once they are concentrated by radiation force. As noted, the specific parameters chosen depend on the choice of carrier, as detailed further below, and can readily be determined by ordinarily skilled artisans having the benefit of this disclosure.

4. A combination of ultrasonic transducers, specifically designed for production of acoustic radiation force according to the description of 1 or 2 or 3, supra or any combination.

5. A single ultrasonic transducer, specifically designed for production of acoustic radiation force according to the description of 1 or 2 or 3, supra or any combination.

6. An ultrasonic system designed for simultaneous drug delivery with radiation force and imaging.

7. An ultrasonic system designed for simultaneous vasoporation and drug delivery with radiation force.

8. Any combination of the above techniques.

In preferred embodiments of the invention, a subject in need of diagnosis or treatment receives an injection of carriers, preferably loaded with a compound. Preferably the subject is mammalian, and more preferably is human. The compound preferably comprises a therapeutic agent such as, e.g., a drug, nucleic acid, or other therapeutic agent. An ultrasound transducer may be simultaneously, or immediately thereafter be positioned over the site of delivery such as, e.g., a tumor, or an inflamed joint, or a vascular lesion. The pulse sequence of the ultrasound scanner produces bursts of radiation force to displace flowing carriers to the walls of blood vessels at the desired site. Interspersed with radiation force generating pulses, are high-acoustic pressure destructive pulses that rupture the carriers at the targeted site, releasing the drug at the targeted site.

The mechanical effects of ultrasound (with and without microbubbles) to alter the permeability of cells and vessels have now been well established. In addition, targeted drug delivery vehicles and acoustically-activatable vehicles have been developed and characterized, with a model developed to predict their behavior. An ultrasound system that implements these developments is provided by the present invention to realize the benefits of the methods of the invention.

In one aspect, the invention provides for a system to combine imaging and drug delivery. The system comprises the following components:

1. The system is capable of sweeping imaging frames through a three dimensional volume. Imaging frames should consist of typical clinical center frequencies (e.g., about 2-20 MHz), and typical acoustic pressures (e.g., mechanical index or MI<1.9).

2. In addition, the system interleaves imaging pulses with therapeutic pulses. These therapeutic pulses can take several forms:

a. For vehicles that contain gas as well as an oil or liquid, the use of a lower frequency pulse (e.g., with a center frequency of about 0.1 MHz-20 MHz) can be applied to fragment the vehicle. The advantage of this fragmentation is that particles small enough to easily extravasate from the vasculature are created, or alternatively the small particles may be pinocytosed. This process is repeated throughout the three dimensional region of interest. This process preferably is repeated each time the vasculature re-fills with the carrier. Usually, the time required for re-filling is on the order of about 5-20 seconds. The process preferably is repeated until the total volume of injected vehicles has been delivered to the desired site. This time can be determined using the imaging pulses described above.

b. For vehicles that include a targeting mechanism such as a ligand or predetermined charge distribution or are susceptible to radiation force, the therapeutic system has the ability to apply this force to bring the carrier ligand or charges into contact with the cells of interest. In order to accomplish this goal, either the imaging or therapeutic array transmits a sequence of low intensity (for example <800 kPa) long (for example, >10 cycles) pulse train to each area within the three dimensional volume. This radiation force sequence preferably is interleaved with imaging pulses, and preferably precedes the therapeutic pulses. The typical center frequency of operation for the therapeutic pulses will be on the order of from about 100 kHz to about 40 MHz, and more preferably from about 1 MHz-20 MHz.

c. To further deliver a drug to a region of interest, a therapeutic sequence that creates "vasoporation" is transmitted while microbubble-based or other compounds fill the vasculature. In this sequence, therapeutic pulses with a center frequency between about 0.1 MHz-5.0 MHz, and more preferably from about 0.75 MHz-1.5 MHz are applied to each region within the therapeutic volume at an intensity from about 0.1 MPa-10.0 MPa, and more preferably from about 0.75 MPa-2 MPa. These therapeutic pulses preferably are interleaved with the imaging pulses. Subsequent to or concurrently with the application of these vasoporation pulses, a drug that extravasates through this altered vasculature is administered, alone, or in association with a carrier.

The invention thus contemplates a system to carry out imaging along with therapeutic strategies described in a, b, or c either separately or in combination. The system provided by the present invention therefore includes the following aspects:

1. Transducer--a combined imaging and therapeutic transducer is provided. In one embodiment, the transducer uses an interface strategy such as is used for a 1.5 D array with the center array used for imaging and the outer arrays used for the therapeutic pulses. Such an arrangement is described in, e.g., U.S. Pat. No. 5,558,092, the entire disclosure of which is hereby incorporated by reference in its entirety. One implementation of this transducer 101 is diagrammed in FIG. 1 (see Original Patent) and comprises an inner imaging array 102 and an outer therapeutic array 103 for which the elements are expected, in preferred embodiments to be larger, and with a different spacing.

2. The transducer may be scanned mechanically to treat and or image the required three dimensional target site. Scanning may be accomplished manually, or automatically using computer guided robotics, as is well known to ordinarily skilled practitioners.

3. The ultrasound system timing is adjusted such that both imaging and therapeutic pulse sequences can be transmitted.

Further modifications to parameters such as, e.g., the duty cycle, pulse length, acoustic pressure, and center frequency may be altered by the practitioner or system depending on the flow rate of blood vessels at the desired site, the depth of the region of interest, and the specific properties of the carrier vehicle.

Compositions Useful for Practicing the Invention

Compositions comprising carriers and compounds are especially useful for practice of the present invention. In preferred embodiments, the carriers are acoustically active, and the compounds are therapeutically active. Such carriers and compounds are well known to those of skill in the art, and may be selected without undue experimentation by skilled practitioners having the benefit of this disclosure. Representative examples of useful compositions are described below.

Liquid and solid contrast agents containing entrapped gas are well known in the art and are useful for practice of the instant invention. See, e.g., U.S. Pat. Nos. 4,235,871; 4,265,251; 4,442,843; 4,533,254; 4,572,203; 4,657,756; 4,681,199; 5,088,499; 5,147,631; 5,228,446; 5,271,928; 5,380,519; 5,413,774; 5,527,521; 5,531,980; 5,547,656; 5,558,094; 5,573,751; 5,585,112; 5,620,689; 5,715,824; 5,769,080; EP 0 122 624; EP 0 727 225; WO 96/40285; and WO 99/65467, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. Microbubbles provided by these contrast agents act as sound wave reflectors due to the acoustic differences between the gas microbubble and surrounding liquid.

Compounds can be linked to or dissolved within carrier lipid coatings, or deposited in subsurface oil layers, or trapped within the carriers themselves.

U.S. Pat. No. 5,190,766 to Ishihara (incorporated herein by reference in its entirety for all purposes) teaches compositions and manufacturing techniques for selecting or producing and using a microcapsule or a particle containing a liquid/sol that has an acoustic impedance greatly different from the acoustic impedance of the ambience in which the drug is released and having acoustic characteristics such as the resonance frequency and the scattering/absorption characteristics that facilitate the use of the drug carrier in the ambience in which the drug is released. The patent describes methods and apparatus for loading an ultrasound contrast agent with a drug, administering the agent by injection into a vessel, imaging via ultrasound the accumulation of the injected agent, and promoting release of drug from the agent at a localized site through application of focused ultrasound energy at a frequency designed to induce resonance within the agent.

ALBUNEX.RTM. (Molecular Biosystems Inc., San Diego, Calif.) is another composition useful for practicing the instant invention. ALBUMIN is the generic name for ALBUNEX. ALBUNEX is an ultrasound contrast agent used in echocardiography and in other areas, it consists of microspheres of which more than 95% have a diameter in the range 1-10 microns. Methods to adsorb a compound of interest onto the protein coating of ALBUNEX are well known to ordinarily skilled practitioners.

Other carriers useful for practicing the invention include commercial sources of microbubbles and associated methods for loading drugs (hydrophobic or hydrophilic) exemplified by Optison (Molecular Biosystems), Imagent (Aliance Pharmaceuticals), DMP-115 (ImaRx Pharmaceutical), and BR1 (Brasco Imaging); as well as the acoustically active liposomes composed of small nongaseous multilamellar lipid vesicles (Alkan-Onyuksel, et al., J. Pharm. Sci 85:486-490 (1996) incorporated herein by reference in its entirety for all purposes), and acoustically active lipospheres (ImaRx Therapeutics).

U.S. patent application Publication US 2002/0102215 A1 to Klaveness et al. (incorporated herein by reference in its entirety) discloses targetable diagnostic and/or therapeutically active agents comprising gas-filled microbubbles stabilized by monolayers of film-forming surfactants, optionally coupled or linked to a vector having affinity for a target site or structure within the body, and teaches incorporation of therapeutic compounds encapsulated in the interior of the microbubbles or attached to or incorporated in the stabilizing membranes.

Hollow polymeric contrast agents also are useful for practicing the invention and may be formed by microencapsulating a solid core of ammonium carbonate which is then removed by decomposition and freeze-drying. Suitable polymers preferably are FDA approved and susceptible to in vivo degradation such as, e.g., poly D,L(lactide-co-glycolide) (PLGA). Spray drying, coacervation and solvent extraction methods may be used. Ideally, the resulting particles have a mean particle size on the order of less than or equal to 10 .mu.m. Compounds may be loaded onto the capsules by adsorption. Such methods are described in more detail in Wheatley, El-Sherif, et al., Mat. Res. Soc. Symp. Proc. Vol. 550:113-118 (1999), the entire disclosure of which is hereby incorporated by reference for all purposes.

Temperature activated gaseous precursor-filled microspheres useful for the practice of the invention are described in U.S. Patent Publication No. US 2003/0039613 A1 to Unger et al. Similar disclosures are found in U.S. Pat. No. 6,554,989 B1 to Unger et al., and in U.S. Pat. No. 6,416,740 B1 to Unger; formation of gas-filled lipid bilayers useful for practice of invention is described in U.S. Pat. No. 6,146,657 to Unger, et al. U.S. Pat. No. 5,770,222 to Unger et al. teaches therapeutic drug delivery systems comprising gas-filled microspheres comprising a therapeutic compound, along with methods for employing them in therapeutic drug delivery applications. U.S. Pat. No. 5,770,222 also teaches methods of and apparatus for preparing liposomes, including liposomes having encapsulated drugs, that are suitable for practice of the present invention. The entire disclosures of the publications and patents cited in this paragraph are hereby incorporated in their entirety for all purposes.

U.S. Patent Publication No. US 2003/0039613 A1 to Unger, et al. (incorporated herein by reference in its entirety for all purposes) also teaches procedures to adjust particle size, including extrusion, filtration, sonication, homogenization, employing a laminar stream of a core of liquid introduced into an immiscible sheath of liquid, extrusion under pressure through pores of defined size, and similar methods.

Particle sizes useful for practice of the present invention will vary depending on the makeup of a carrier. In general, particles on the order of 10 .mu.m or less in diameter are preferred. Described below is a model that is useful for guiding the skilled practitioner on selecting frequencies, pressures, and other parameters, based on the size and physical properties of the carriers. Particle size may be determined using, e.g., a Model 770A Accusizer particle sizer (Particle Sizing Systems, Santa Barbara, Calif.). Especially useful for practice of the invention are particles that comprise an oil having a kinematic viscosity at 37.degree. C. between about 1 mm.sup.2/sec and about 100 mm.sup.2/sec, or between about 10 mm.sup.2/sec and about 80 mm.sup.2/sec, or between about 20 mm.sup.2/sec and 60 mm.sup.2/sec. Kinematic viscosity can be measured using a device such as a KV5000 Kinematic Viscosity Bath available from Koehler Instrument Co., Inc. (Bohemia, N.Y.).

As described above, the present invention also may be practiced with carriers comprising targeting moieties designed to assist in the targeting of the carrier to a site. Such targeting moieties are well known in the art, and may be selected and incorporated into the carriers without undue experimentation by ordinarily skilled practitioners having the benefit of this disclosure. Exemplary teachings in the prior art relating to targeting moieties are provided below.

Methods suitable for coupling targeting moieties to carriers can be found in Hermanson, "Bioconjugate Techniques," Academic Press: New York, 1996; and in "Chemistry of Protein Conjugation and Cross-linking" by S. S. Wong, CRC Press, 1993, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. Other suitable methods are taught in paragraphs 66 through 130 of U.S. Patent Application Publication U.S. 2002/0102215 A1 to Klaveness et al. Specific coupling methods include, but are not limited to, the use of bifunctional linkers, carbodiimide condensation, disulfide bond formation, and use of a specific binding pair, where one member of the pair is on the targeting agent and the other is on the carrier, e.g., a biotin-avidin interaction, see, e.g., Dayton et al. J. Acoust. Soc. Am. 112(5):2183-2192 (Nov. 2002), and references 10 through 14 cited in the bibliography (Dayton et al., and internal references 10 through 14 are hereby incorporated by reference in their entirety for all purposes).

The use of charged phospholipids are advantageous in that they contain functional groups such as carboxyl or amino that permit linking of targeting moieites, if desired, by way of linking units.

Suitable compositions for practicing embodiments of the invention using targeted carriers include those having an avidin biotin bridge to target an antigen as taught by Lindner and Kaul, Echocardiography 18(4):329-337 (2001). The initial step comprises administration of a biotinylated monoclonal antibody against the antigen followed by administration of avidin, and then administration of an emulsion of microbubbles containing a biotinylated phospholipid. Avidin forms a bridge between a surface expressing the antigen and biotinylated microbubbles.

MRX-408 manufactured by ImaRx Pharmaceutical Corp., Tucson, Ariz., USA is another suitable composition for practicing the invention. MRX-408 is a lipid-shelled microbubble having an oligopeptide sequence conjugated to microbubble surface which is recognized by the RGD-binding site of platelet IIB/IIIa receptors. The peptide is conjugated to the microbubble surface through a molecular spacer, polyethylene glycol, which allows a greater number of ligand-receptor pairs. See Lindner and Kaul, Echocardiography at 330.

Also suitable are microbubbles and acoustically active microemulsion agents that have been formulated with monoclonal antibodies that recognize ICAM-1 conjugated to their surface, such as those described by Lindner and Kaul, Echocardiography at 332, referencing internal refs. 19, 20, incorporated herein by reference in their entirety for all purposes. These formulations may include as therapeutic compounds, inhibitors of endothelial cell adhesion molecules such as ICAM-1 and proinflammatory cytokines for use in treating inflammatory disorders such as rheumatoid arthritis, and Crohn's disease. See Lindner and Kaul, Echocardiography at 333, and internal refs. 21, 23. Exemplary compounds comprise anti-ICAM-1; CD54 antibody--for rheumatoid arthritis); anti-TNF monoclonal antibody with methotrexate (also for rheumatoid arthritis); and chimeric monoclonal antibody cA2 to TNF for Crohn's disease.

Also suitable are immunoliposomes specific for tumors containing cytotoxic agents along with monoclonal antibodies against tumor-associated antigens conjugated to their surface such as described in Lindner and Kaul, Echocardiography at 333, and internal refs. 26, 27, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. Specific examples include e.g., tumor antigen p185, encoded by the HER-2 protooncogene expressed on surface of certain breast, lung, and ovarian carcinomas. Immunoliposomes containing doxorubicin formulated with an Fab' against extracellular domain of p185 conjugated to surface are described in internal ref. 28 of Lindner & Kaul, Echocardiography (2001), the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Suitable targeting moieties and methods for their attachment to carriers also are listed in U.S. Patent Application Publication No. US 2002/0071843 A1 to Li et al., in, e.g., paragraphs 0109 through 0116, and in paragraphs 157 through 159, and in paragraphs 131-145 of U.S. Patent Application Publication U.S. 2002/0102215 to Klaveness, et al. the entire disclosures of which are incorporated by reference in their entirety for all purposes.

Other suitable targeting moieties include aptamers, and peptidomimetics.

Multivalent binding can be useful to enhance avidity and reduce "off-rates" so that binding persists long enough to permit imaging at convenient times after delivery of the agent. Polyvalent binding is possible with the use of more than one ligand type per carrier, or with mixtures of ligand-carrier constructs directed at different targets.

The invention may be practiced using a wide variety of different compounds, including therapeutic compounds having widely varying molecular weights, chemical composition, oil/water partition coefficient, etc. Exemplary compounds and carriers include bilayer-shelled microbubbles that contain concentrated drug between an inner and outer shell especially useful for packaging nonamphilic drugs into acoustically active microbubbles, as taught by Lindner & Kaul, Echocardiography at 331. Also contemplated within the scope of useful compounds for practicing the invention are nucleic acids, including mRNA, cDNA, genomic DNA, antisense, and RNAi, any of which may further comprise semi-synthetic backbones or synthetic nucleic acids to modify stability or specificity.

Myocardial transfection in vivo by acoustic destruction of gene-laden microbubbles has recently been reported. See, e.g., Lindner, Am. J. Cardiol. 90(suppl):72J-80J (2002), and internal ref. 36; see also Lindner (2002) internal reference 40 showing oligonucleotides can bind to the surface of albumin-dextrose microbubbles, Lindner (2002) internal reference 41 showing microbubbles containing antisense oligonucleotides against c-myc proto-oncogene attenuating carotid neointimal hyperplasia post balloon catheter injury in pigs. Each of the reported compositions is useful for practice of the invention. Each of these references is hereby incorporated by reference in its entirety for all purposes.

Also useful in practicing the invention are stabilized gaseous microbubble contrast agents that have demonstrated potential for use as transfection agents by incorporating DNA directly into the bubble shell or interior, as described in Unger, et al. Invest. Radiol. 32:723-727 (1997); Shohet, et al. Circulation 101:2554-2556 (2000); reflective liposomes useful for specifically targeting endothelial integrins as described in Lanza, et al. J. Am. Coll. Cardiol. 19(3 Suppl A): 114A (1992); Demos, et al. J. Am. Coll. Cardiol. 33:867-875 (1999) and others described in Wickline and Lanza, J. Cellular Biochemistry Supplement 39:90-97 (2002), each of which is incorporated by reference in its entirety for all purposes.

Additional exemplary compounds are listed in U.S. Patent Publication No. 2003/0039613 A1 to Unger et al. (incorporated herein by reference in its entirety for all purposes) in, e.g., paragraphs 0156 through 0172 and include antineoplastic agents, hormones, anti-helmintics, antimalarials, and antituberculosis drugs; biologicals; viral vaccines; aminoglycosides; thyroid agents; cardiovascular products; glucagon; blood products; biological response modifiers; antifungal agents; vitamins; anti-allergic agents; circulatory drugs; metabolic potentiators; antivirals; anti-anginals; anticoagulants; antibiotics; antiinflammatories; antirheumatics; narcotics; opiates; cardiac glycosides; neuromuscular blockers; sedatives; local anesthetics; radioactive particles or ions; monoclonal antibodies; genetic material; and prodrugs.

Pharmaceutical Compositions of the Invention

Methods for treatment of various diseases are also encompassed by the present invention. Said methods of the invention include administering a therapeutically effective amount of a carrier and a compound, or, in alternate embodiments, of a compound without a carrier. The carriers and compounds useful for practicing the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to the compounds and optional carrier, a pharmaceutically acceptable excipient, bulking agent, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the compound. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. In the practice of the invention, preferred administration routes include, e.g., intravascularly, intralymphatically, parenterally, subcutaneously, intramuscularly, intranasally, intrarectally, intraperitoneally, interstitially, into the airways, orally, topically, intratumorly. See, e.g., Unger, et al. U.S. Patent Publication No. US 2003/0039613 A1 at paragraph 0202.

Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.

Whether it is a polypeptide, antibody, nucleic acid, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to a subject, administration is preferably in a "therapeutically effective amount" or "prophylactically effective amount" (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dose, timing, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980 (incorporated herein by reference for all purposes).

A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Radiation Sources and Parameters

The relationship between carrier translation, center frequency, pressure, pulse length and fundamental or harmonic resonance frequencies of insonified carriers are described in, e.g., Dayton, et al. J. Acoust. Soc. Am. 112 (5):2183-2192 (Nov. 2002) (incorporated herein by reference in its entirety for all purposes.) Further teachings about these relationships are set forth in the Examples below.

Ultrasound systems useful for practicing the invention include the phased system array (HDI c000cv, Advanced Technologies Laboratories) for delivering ultrasound and imaging, the system described in U.S. Pat. No. 5,558,092, to Unger, et al., and may include external application, preferred for skin and other superficial tissues, but for deep structures, application of sonic energy via interstitial probes or intravascular ultrasound catheters may be preferred.

The physics governing imaging, fragmentation, and steering (as by, e.g., radiation force) are well understood by ordinarily skilled practitioners having the benefit of this disclosure. For example, it is well known that harmonic emissions may be generated from insonated vesicles (usually at 2.times. frequency of incident therapeutic ultrasonic waves), and that such harmonic emissions are useful for, e.g., imaging. As described in U.S. Pat. No. 5,770,222 to Unger, et al., the microspheres useful for practicing the present invention have a peak resonant frequency of between about 0.5 MHz and about 10 MHz. Of course, the peak resonant frequency of gas-filled microspheres will vary depending on the diameter and, to some extent, the elasticity or flexibility of the microspheres, with the larger and more elastic or flexible microspheres having a lower resonant frequency than the smaller and less elastic or flexible microspheres.

The fragmentation or rupturing of microsphere carriers useful for practicing the invention is easily carried out by applying ultrasound of a certain frequency to the region of the subject where therapy is desired, after the carriers have been administered to or have otherwise reached that region. When ultrasound is applied at a frequency corresponding to the peak resonant frequency of the compound containing gas-filled microsphere carriers, the microspheres rupture and release their contents.

The peak resonant frequency can be determined by the ordinarily skilled practitioner either in vivo or in vitro, but preferably in vivo, by exposing the microsphere carriers to ultrasound, receiving the reflected resonant frequency signals and analyzing the spectrum of signals received to determine the peak, using conventional means. The peak, as so determined, corresponds to the peak resonant frequency (or second harmonic, as it is sometimes termed).

Gas-filled microsphere carriers will also rupture when exposed to non-peak resonant frequency ultrasound in combination with a higher intensity (wattage) and duration (time). This higher energy, however, results in greatly increased heating, which may not be desirable. By adjusting the frequency of the energy to match the peak resonant frequency, the efficiency of rupture and therapeutic release is improved, appreciable tissue heating does not generally occur (frequently no increase in temperature above about 2.degree. C.), and less overall energy is required. Thus, application of ultrasound at the peak resonant frequency, while not required, is most preferred.
 

Claim 1 of 51 Claims

1. A method for localized delivery of a compound, comprising: administering a carrier, said carrier comprising a therapeutic compound, wherein the carrier is selected from the group consisting of an acoustically active liposphere and a gas-filled agent; concentrating said carrier by exposing said carrier to an ultrasound radiation force generated by an ultrasound wave at a first frequency and pressure combination, thereby locally delivering said therapeutic compound, wherein said ultrasound wave at said first frequency and pressure combination concentrates and displaces said administered carrier, but does not rupture said carrier; and rupturing said concentrated carrier by insonating said concentrated carrier with an ultrasound wave at a second frequency and pressure combination.

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