RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 17/743,056, filed May 12, 2022, which is a continuation in part of International Application No. PCT/US2021/057961, filed Nov. 3, 2021, which claims the benefit of priority to U.S. Provisional Application No. 63/109,193, filed on Nov. 3, 2020. The entire contents of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 28, 2023, is named 132075-00102_SL.xml and is 11,360 bytes in size.

BACKGROUND

Currently, the administration of medicines to treat disease (particularly in cancer treatment) is typically a regimen where the dosage is based on such factors as patient weight or body surface area. This methodology leads to a treatment philosophy of administering the maximum tolerated dose of a therapeutic agent (or combination of agents), rather than taking into consideration relevant factors such as, but not limited to, how much of a therapeutic agent(s) is actually delivered to the target tissue (which may be a cell, e.g., tumor cell), or more relevantly what amount is required at the target in order to have a therapeutic effect. Even if the amount of an agent needed to have a therapeutic effect is known, there may be other factors that affect the ability to deliver said amount to the target, such as, but not limited to, anatomical, physiological, mechanical, chemical, biological, etc. For some agents (e.g., viruses, nucleic acids, etc.), the body's natural immune system may actually inhibit effective delivery. For some agents, mechanical or chemical processes (e.g., efflux pumps, interstitial fluid pressure, reactive oxygen species (ROS), bacteria, etc.) may inhibit effective therapy.

While drug discovery and development is beginning to adapt to new technologies, the actual treatment of cancer (and other diseases) itself is still stuck in an analog age. If a patient has a disease, one standard methodology is to prescribe a maximum tolerated dose of drug(s) that has been shown to have a clinical effect for 50 percent of treated patients from a clinical trial. The administration of many traditional or novel drugs is still performed in the same way since the 17th century, e.g., using a syringe to inject a specific dose of drugs intravenously, then waiting to see what clinical effect occurs in a specific patient, if any. Currently, dosage of drugs is typically either a fixed dose, or is determined by a patient's weight, body surface area, or other gross measures. Even when, in the case of cancer, the genetic profile of a patient's tumor has been identified and a specific mix of drugs is prescribed, the dosage remains fixed, or is based on some gross measure. Especially with systemically administered therapeutics, the amount of the therapeutic agent that actually reaches its intended target is a small fraction of the total injected dose, due in part because the therapeutic agents rely on the circulatory system to passively deliver the molecules to the target while the body's natural defense mechanisms may prevent or inhibit the process.

Pharmacokinetics (PK) describes how the body works on a particular drug. It is separated in time along four steps common to all drugs:

- 1. Absorption. How the drug gets into the systemic plasma (oral, IV, Infusion etc.)
- 2. Distribution. How the drug gets from systemic circulation into the target tissue.
- 3. Metabolism. How cells process the presence of the therapeutic.
- 4. Excretion. How tissue and the circulation remove the agent or its metabolites from the body.

Each drug has a therapeutic window defined by the range of concentration in plasma that provides safe and effective therapy. Too little drug and it will be ineffective. Too much drug in the system and adverse effects will be encountered, even if the underlying disease is treated.

The therapeutic window can be quantified in terms of a therapeutic index, represented by TD₅₀/ED₅₀, where TD₅₀is the toxic dose where the plasma concentration causes 50% of patients to have adverse effects, and ED₅₀, where the plasma concentration is effective in 50% of patients.

This tradeoff in effective versus toxic levels is common in the field of chemotherapy, where cytotoxic agents take their toll on normal tissue in addition to cancer cells. Even with the latest immunotherapy approaches, sometimes the greater opportunity for curative effects comes with even more risks for serious and life threatening systemic immune responses to other organs not affected by the tumor that is being targeted. The wider the therapeutic window and higher the therapeutic index, the easier the drug is to safely administer and the higher likelihood that it will be effective for more patients.

Pharmacodynamics (PD), on the other hand, describes how a specific drug works on the body. Its steps are described by:

- 1. Affinity: A measure of how avidly a drug binds to a receptor.
- 2. Efficacy: Measure of the magnitude of effect once the drug is bound.
- 3. Potency: Measure of the quantity of drug needed to produce maximal effect.

If a drug can be delivered and maintained at its target for a longer period of time in higher concentrations, it improves its ability to bind receptors and to provide a longer duration of effect at the same plasma concentration.

Typically a drug has a PK profile well described during its drug discovery and development stages. This detailed experimental data is also critical to dosing selection for clinical trials and is a key component of regulatory agency submissions for approval. A key design goal of pharmaceutical developers is to maximize absorption and distribution of a therapeutic agent, as more active agent at the target tissue will increase potency and efficacy. There are pharmacological solutions to this optimization. One example is nab-paclitaxel, where the generic drug paclitaxel was made significantly more efficacious and less toxic by encapsulating it within an albumin bound nanoparticle.

There are also many drug delivery technologies that attempt to overcome distribution limitations of a drug's PK by direct injection into tumors, drug eluting beads or iontophoresis. Each of these approaches has limitations including the need to change the method of administration of a drug and requiring invasive procedures. These drug delivery methods are suboptimal with one drug, but are even more unwieldy for combination oncology therapy that requires coordination of drugs with different methods of administration at varying schedules. Treatment regimens may consist of a combination of daily oral tablets, weekly infusions or monthly monoclonal antibody (mAb) injections and are increasingly becoming more prevalent in cancer care.

If a drug, at a specific blood concentration, can be delivered more optimally to the target—and does not have limited binding sites—then the therapeutic window could be shifted to a lower blood concentration at which the minimum therapeutic effect can be achieved. This widens the therapeutic window at equal blood concentrations. If desired, the blood concentration can be reduced through a lower dose and achieve the same therapeutic window with fewer adverse events.

If, for a given blood concentration, the therapeutic effect can be improved for more patients due to better distribution pharmacokinetics, then the slope of the dose response curve for the therapeutic effect achieves a steeper slope, which in turn increases both the potency and efficacy of a drug.

Even with the advent of personalized medicine, clinicians are still administering therapeutic agents with doses that are standardized across all patients, with dosage typically dependent on patient weight or body mass. While the specific combination of agents may be based on, say a patient's tumor genetic profile, the dosage of each agent is generally not personalized to a particular patient. There is a need to change the paradigm of treatment instead to a specific patient, rather than relying on standard formulas that may or may not apply to an individual patient. For example, if it has been previously determined that a certain level of a therapeutic delivered to a target (e.g., tumor) has a clinical effect, then a major goal should be to deliver the required amount, versus delivering a maximum tolerated dose. This should be an improvement to current treatments, as administering a large amount results in much of the therapeutic agent being delivered off-target and presenting unintended effects.

There is a further need to provide a means to protect certain therapeutics while in circulation from degradation or from an immune response, to actively target the delivery of therapeutics to improve biodistribution to the target tissue, and to do so in a simple way, e.g., at the time of administration (rather than at the manufacturing stage to obviate the need to change manufacturing processes), all to improve the efficacy or the safety (or both) profile of therapeutic treatments.

There is also a further need to account for conditions where a patient has biological characteristics that may impede or inhibit effective treatment of a standard dose of a therapeutic. A major goal should be to adjust for the biological characteristics to allow for effective treatment rather than continuing to administer a dose based on, say just a patient's weight, body mass or surface area.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein an at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers), and/or bound to the surface (e.g., the exterior or interior surface, of the carriers) for delivery to a subject. In addition, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, to be co-administered with compositions comprising at least one active agent to a subject. Furthermore, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers), and/or bound to the surface (e.g., the exterior or interior surface, of the carriers), to be co-administered with compositions comprising at least one free active agent, e.g., at least one active agent that is not associated with the plurality of carriers, to a subject.

The present invention also provides methods of treating a disease or condition, for example, cancer, and methods of targeted delivery of an active agent to, e.g., a target tissue, such as an organ, a tissue, or a tumor site, in a subject, using the carrier based compositions of the invention. The present invention further provides devices and methods for preparing the compositions of the present invention. In particular, the inventors of the present invention have surprisingly discovered that by associating or co-administering the active agent with the carriers, e.g., microbubbles, the level of the active agent required to achieve a therapeutic effect is substantially lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated or co-administered with carriers such as microbubbles, e.g., by systemic administration without being associated or co-administered with carriers.

Accordingly, in one aspect, the present invention provides, a composition comprising a plurality of carriers, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers and by systemic administration.

In some embodiments, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers.

In some embodiments, the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers.

In some embodiments, wherein the carrier comprises a shell and a core.

In some embodiments, (i) the shell comprises a lipid, a polymer, a lipopolymer, a protein, or combination thereof; and/or (ii) the core comprises a gas.

In some embodiments, the lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 4-dimethylaminochalcone (DMAC), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearyol-sn-glycero-3-phosphocoline (DSPC), and 1,2-distearyol-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-PEG2000).

In some embodiments, the lipid is DMPC.

In some embodiments, the core comprises nitrogen, air, a perfluorocarbon gas, or combination thereof.

In some embodiments, the perfluorocarbon gas is selected from the group consisting of perfluorohexane, sulfur hexafluoride, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane.

In some embodiments, the perfluorocarbon gas is perfluorohexane.

In some embodiments, the core comprises a combination of perfluorohexane and nitrogen gas.

In one aspect, the present invention provides a composition comprising a plurality of carriers, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers and by systemic administration.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers.

In some embodiments, the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers.

In some embodiments, the at least one active agent is associated with the plurality of carriers by being encapsulated within the plurality of carriers.

In some embodiments, the at least one active agent is associated with the plurality of carriers by binding to the interior or exterior surface of the plurality of carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 99%, about 0.001% to about 75%, about 0.001% to about 50%, about 0.005% to about 50%, about 0.005% to about 25%, or about 0.0001% to about 25% of the level of the active agent required to achieve a therapeutic effect, optionally, a substantially equivalent therapeutic effect, when administered without being associated with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 0.005%, about 0.005% to about 0.1%, about 0.005% to about 10%, about 0.005% to about 20%, about 10% to about 50%, or about 50% to about 99% of the level of the active agent required to achieve a therapeutic effect, optionally a substantially equivalent therapeutic effect, when administered without being associated with carriers.

In some embodiments, the level of the active agent associated with the plurality of carriers is about 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 99% of the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, optionally, by systemic administration without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the same level of active agent without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the active agent without being associated with carriers required to achieve a substantially equivalent therapeutic effect.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers elicits an immunostimulatory effect.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers does not elicit an immunosuppressive effect.

In some embodiments, the at least one active agent is selected from the group consisting of a protein, an antibody, a small molecule, a virus, an antibiotic, a radionuclide, a peptide, a nucleic acid, a gene, a vector or a plasmid encoding a gene, and a component of a gene editing system.

In some embodiments, the at least one active agent comprises a cytokine, and/or a vector or plasmid encoding a gene encoding the cytokine.

In some embodiments, the cytokine is selected from a group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, interferon, GM-CSF and TNF-alpha.

In some embodiments, the cytokine is IL-2.

In some embodiments, the level of IL-2 associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 10 pg/mL to about 100 pg/mL, about 100 pg/mL to about 100 μg/mL, about 100 pg/mL to about 100 ng/mL, about 100 ng/mL to about 1 μg/mL, about 1 μg/ml to about 10 μg/mL, about 10 μg/mL to about 100 μg/mL, about 200 ng/mL to about 500 ng/mL, about 500 ng/mL to about 2 μg/mL, or about 2 μg/mL to about 5 μg/mL.

In some embodiments, the at least one active agent comprises a monoclonal antibody, and/or a vector or plasmid comprising a gene encoding the monoclonal antibody.

In some embodiments, the monoclonal antibody is selected from the group consisting of bevacizumab, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, rituximab, cetuximab and trastuzumab.

In some embodiments, the monoclonal antibody is bevacizumab.

In some embodiments, the level of bevacizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL.

In some embodiments, the monoclonal antibody is pembrolizumab.

In some embodiments, the level of pembrolizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL.

In some embodiments, the at least one active agent comprises one or more components of a gene editing system.

In some embodiments, the at least one active agent comprises one or more components of a CRISPR system.

In some embodiments, the at least one active agent comprises a cDNA and/or mRNA encoding a Cas9 protein, a guide RNA, and/or a ribonucleoprotein (RNP) complex.

In some embodiments, the composition comprises a virus that is an oncolytic virus, for example, wherein the oncolytic virus is encapsulated within the carrier.

In some embodiments, the oncolytic virus is selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, poliovirus, measles virus, polioviruses, poxvirus, Newcastle disease virus, reovirus, coxsackievirus, vesicular stomatitis virus, Zika virus, RNA virus, and DNA virus.

In some embodiments, the composition comprises a viral vector comprising a gene encoding the active agent.

In some embodiments, the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus.

In some embodiments, the carrier is a microbubble, optionally selected from the group consisting of Levovist™, Imagent®, Optison®, Sonazoid®, BR38, and SonoVue®.

In some embodiments, wherein the composition, or the preparation thereof, is free of an inactivation agent for the active agent.

In some embodiments, the composition, or the preparation thereof, is free of a viral inactivating agent.

In some embodiments, wherein the composition is free of a binding agent or a binding ligand.

In a particular embodiment, the present invention provides a composition comprising a conjugation, covalently or non-covalently, of an active agent and a carrier formulated from a combination of a lipid (e.g., 9.2 mg DMPC), a starch (e.g., 75 mg hydroxyethyl starch), a poloxamer (e.g., 2.1 mg poloxamer 288), sodium chloride (e.g., 75 mg), buffer (e.g., 36 mg sodium phosphate), at least one gas (e.g., 17% v/v perflexane vapor in nitrogen). In a preferred embodiment, the agent is a nucleic acid. DMPC, as a zwitterionic lipid (rather than a cationic lipid) with a net neutral charge, has a headgroup containing a positively charged choline group and negatively charged phosphate group.

In various embodiments of the foregoing aspects of the invention, the compositions further comprise free lipids. The free lipids may comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total lipid content in the composition. In a particular embodiment, the free lipids are in a high energy state before reconstitution of the carrier. “High energy” state of lipids can refer to a state in which the bonds of the lipids have been broken by high velocity, high temperature spray drying. Upon reconstitution in an aqueous diluent, the high energy state lipids immediately (in milliseconds) form lower energy structures such as sheets, rods and liposomes so that their hydrophobic tails are not exposed to water molecules.

Accordingly, in certain embodiments, the free lipids form a second plurality of carriers upon exposure to an aqueous solution. The second plurality of carriers may include a liposome, a lipid rod, a lipid disc and/or a lipid sheet. In a particular embodiment, the free lipids form a unilamellar liposome. In another embodiment, the free lipids form a multilamellar liposome. In certain embodiments the at least one active agent is encapsulated in, bound to, associated with or interacted with the second plurality of carriers.

In a further embodiment, the first plurality of carriers comprises microbubbles, for example, comprising (i) a shell comprising a lipid, a polymer, a lipopolymer, a protein, or combination thereof; and/or (ii) a core comprising a gas. In a particular embodiment, the shell comprises a lipid, and wherein the lipid is DMPC; and the core comprises at least one of perfluorohexane and nitrogen gas. In a particular embodiment, the plurality of free lipids comprises DMPC.

In another aspect, the present invention provides a composition wherein the carriers form fragments within the body upon inertial cavitation of the carrier. For example, the carrier is formed of a shell, e.g., at least one lipid, and a gas core, e.g., a perfluorohexane or perfluorocarbon gas with or without nitrogen, and upon inertial cavitation, breaks into fragments within the body. The active agent may be associated with or bound to the carriers prior to administration of the composition, or may bind or associate with the fragments of the carriers in vivo upon inertial cavitation. In various embodiments, (i) the shell may comprise (but is not limited to) at least one lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core may comprise a perfluorohexane gas with or without nitrogen or other gases; or (ii) the shell may comprise a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core may comprise a gas comprising a perfluorocarbon gas, with or without nitrogen or other gases.

In this aspect, the compositions are a prodrug where active agent is encapsulated in a carrier, such that upon administration, a micrometer scale drug/carrier complex is converted to a nanometer scale drug suitable for lipid fusion, lipid mediated poration, or receptor-mediated endocytosis for release of the agent into the cytosol or nucleus of a targeted cell. Alternatively, the composition is a prodrug where administration of a carrier and a co-administered agent (i.e., unencapsulated) results in a micrometer scale carrier (e.g., microbubble) converted into nanometer scale particles (for example, as a result of inertial cavitation) which are subsequently complexed with the agent in vivo whether by affinity or self-assembly. The resulting nanoscale carrier/drug complex (e.g., microbubble fragment/agent complex) associates with the cellular membrane enabling the release or endocytosis of the agent into the cell. The effect of vascular permeabilization due to sonoporation during the micrometer scale (microbubble phase) combined with the creation of nanometer scale particles (post-inertial cavitation phase) complexed with at least one agent allows for increased local concentration of nanoscale lipid particles at the target tissue (including cells).

Alternatively in another aspect, the composition comprises (i) a plurality of carriers, e.g., microbubbles, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, and (ii) a plurality of free lipids (such as a free lipid in solution or a free lipid suspension of other structures). Active agent may be encapsulated within, bound to, associated with or interacted with the free lipids in the composition prior to administration and/or in vivo. In a particular embodiment, upon exposure to an aqueous solution, the free lipids may form a second plurality of carriers, to which the active agent may be encapsulated, bound to, associated with or interacted with. In one embodiment, the first plurality of carriers comprises microbubbles, for example, formed of a shell comprising a lipid, a polymer, a protein or combination thereof; and a core comprising a gas. In an exemplary embodiment, the shell is formed of the lipid DMPC and the core comprises at least one of a perfluorohexane and nitrogen gas. Alternatively or in combination, the free lipids are DMPC. The association of the active agent with the free lipids similarly serves as nanometer scale drug complexes enhancing the release or endocytosis of the agent into the cell, as described above.

In some embodiments, the level of active agent associated with the plurality of nanometer scale particle carriers/free lipids/fragments created after inertial cavitation (or from free lipids) is about 0.001 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL, as measured in target tissue by biopsy.

In some embodiments, the nanometer scale particles with an agent associated therewith formed are unilamellar complexes. In some embodiments, the nanometer scale particles with an agent associated therewith formed are multilamellar complexes. In a preferred embodiment, the particles are composed of at least one lipid. The lipid may be cationic, zwitterionic or anionic. The lipid may be a combination of at least two lipids, including but not limited to, a cationic lipid and a zwitterionic lipid. In the case where the active agent is a nucleic acid, for example DNA, mRNA, or ribonucleoprotein (RNP), the created nanometer scale particle may be a hexagonal structure with lipid elongated micelles arranged on a hexagonal lattice and DNA rods arranged on a honeycomb lattice in the interstices between the lipid micelles. The particle may be a lamellar structure with alternating lipid layers and DNA monolayers between the lipid layers. Alternatively, the particle may resemble an inverted hexagonal structure with lipid inverted micelles coating the DNA arranged on a hexagonal lattice (Gaspar 2020).

One advantage of the present invention is the extension of time in which the agent may be in circulation or present with the target (e.g., cell). In the case of a gas-filled microbubble carrier, the time in circulation for the microbubble is typically measured in minutes as eventually the gas core is diffused out, or when inertial cavitation is induced the gas is released instantaneously. In the present invention, the nanometer scale particles no longer contain gas thereby enabling the particles to remain in circulation beyond the typical time period of the original microbubbles. Furthermore, when the particle (along with its associated agent) reaches its target cell and binds with the cell membrane, at least one molecule of the associated agent may be released into the cytoplasm or nucleus. Upon particle fusion with the cellular membrane, cytosol enzymes may interact to release the active agent while avoiding endosomal or lysosomal elimination or degradation.

Another advantage of a preferred embodiment of the present invention is its principal constituent of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC is a zwitterionic lipid and is a chemical synthesis of phosphatidylcholine (PC). PC is present on the outer leaflet of all mammalian cell and intracellular membranes, compared to phosphatidylethanolamine or phosphatidylserine, which are present on the inner membrane leaflet. Accordingly, in embodiments of the present invention in which the carrier is formed of a PC, e.g., DMPC, or embodiments where the composition includes free PC lipids, e.g., DMPC, uptake of active agent across the cell membrane may be enhanced. Specifically, the use of DMPC may result in fusion of the DMPC lipid with the cell membrane to enable the agent to enter the cytosol or nucleus.

Another embodiment of the present invention includes the association of a DMPC lipid carrier with a conjugated cationic lipid and agent, and the resultant nanometer scale fragment comprised of DMPC/cationic lipid/agent created by inertial cavitation. Such a nanometer scale DMPC/cationic lipid/agent complex fragments migrate out of the vasculature to interact with the target tissue or cell, either through pores created by sonoporation or leaky vasculature.

In one aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In another aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In another aspect, the present invention provides a method for treating a disease by administering a composition as described herein, wherein the carriers form fragments within the body upon inertial cavitation of the carrier. For example, the carrier is formed of a shell, e.g., at least one lipid, and a gas core, e.g., a perfluorohexane or perfluorocarbon gas with or without nitrogen, and upon inertial cavitation, breaks into fragments within the body. The active agent may be bound to, associated with or interacted with the fragments prior to administration of the composition, and/or may bind to, associate with or interacted with the fragments of the carriers in vivo upon inertial cavitation. In various embodiments, (i) the shell may comprise (but is not limited to) at least one lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core may comprise a perfluorohexane gas with or without nitrogen or other gases; or (ii) the shell may comprise a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core may comprise a gas comprising a perfluorocarbon gas, with or without nitrogen or other gases.

In this aspect of the methods for treatment of a disease, the compositions are a prodrug where active agent is encapsulated in a carrier, such that upon administration, a micrometer scale drug/carrier complex is converted to a nanometer scale drug suitable for lipid fusion, lipid mediated poration, or receptor-mediated endocytosis for release of the agent into the cytosol or nucleus of a targeted cell. Alternatively, the composition is a prodrug where administration of a carrier and a co-administered agent (i.e., unencapsulated) results in a micrometer scale carrier (e.g., microbubble) converted into nanometer scale particles (for example, as a result of inertial cavitation) which are subsequently complexed with the active agent in vivo whether by affinity or self-assembly. The resulting nanoscale carrier/drug complex (e.g., microbubble fragment/agent complex) associates with the cellular membrane enabling the release or endocytosis of the active agent into the cell. The effect of vascular permeabilization due to sonoporation during the micrometer scale carrier (microbubble phase) combined with the creation of nanometer scale particles (upon inertial cavitation) complexed with at least one agent allows for increased local concentration of nanoscale lipid particles at the target tissue (including cells).

Alternatively, in another aspect, methods of treating a disease are providing, the method comprising administering a composition comprises (i) a plurality of carriers, e.g., microbubbles, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, and (ii) a plurality of free lipids (such as a free lipid in solution or a free lipid suspension of other structures). Active agent may be encapsulated within, bound to associated with or interacted with the free lipids in the composition prior to administration and/or in vivo. In a particular embodiment, upon exposure to an aqueous solution, the free lipids may form a second plurality of carriers, to which the active agent may be encapsulated, bound to, associated with or interacted with. In one embodiment of the method, the first plurality of carriers comprises microbubbles, for example, formed of a shell comprising a lipid, a polymer, a protein or combination thereof; and a core comprising a gas. In an exemplary embodiment, the shell is formed of the lipid DMPC and the core comprises at least one of a perfluorohexane and nitrogen gas. Alternatively or in combination, the free lipids are DMPC. The association of the active agent with the free lipids similarly serves as nanometer scale drug complexes enhancing the release or endocytosis of the agent into the cell, as described above. The effect of vascular permeabilization due to sonoporation during the micrometer scale (microbubble phase) carriers combined with (a) the creation of nanometer scale particles of such micrometer scale carriers (upon inertial cavitation) complexed with at least one agent, and (b) the co-administration of nanometer scale particles (e.g., from the free lipids) complexed with at least one agent allow for increased local concentration of nanoscale lipid particles at the target tissue (including cells). In one aspect, the present invention provides a method of reducing tumor growth in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and applying an ultrasound energy to the subject, thereby reducing tumor growth in the subject.

In another aspect, the present invention provides a method of reducing tumor growth in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; and applying an ultrasound energy to the subject, thereby reducing tumor growth in the subject.

In one aspect, the present invention provides a method of delivering an active agent to a target site in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and applying an ultrasound energy at the target site in the subject, thereby delivering the active agent to the target site in the subject.

In another aspect, the present invention provides a method of delivering an active agent to a target site in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; and applying an ultrasound energy at the target site in the subject, thereby delivering the active agent to the target site in the subject.

The target site is an organ, a tissue, or a tumor site in the subject.

In some embodiments, the methods further comprise administering to the subject a composition comprising at least one free active agent, i.e., an active agent that will not be associated or encapsulated within a carrier, e.g., a microbubble.

In one aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, administering to the subject a second composition comprising at least one active agent, wherein the level of the at least one active agent co-administered with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being co-administered with carriers, and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In one aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; administering to the subject a second composition comprising at least one active agent, and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In one aspect, the present invention provides a method of reducing tumor growth in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, administering to the subject a second composition comprising at least one free active agent, wherein the level of the at least one active agent co-administered with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being co-administered with carriers, and applying an ultrasound energy to the subject, thereby reducing tumor growth in the subject.

In one aspect, the present invention provides a method of reducing tumor growth in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; administering to the subject a second composition comprising at least one active agent, and applying an ultrasound energy to the subject, thereby reducing tumor growth in the subject.

In one aspect, the present invention provides a method of delivering an active agent to a target site in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, administering to the subject a second composition comprising at least one active agent, wherein the level of the at least one active agent co-administered with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being co-administered with carriers, and applying an ultrasound energy at the target site in the subject, thereby delivering the active agent to the tumor site in the subject.

In one aspect, the present invention provides a method of delivering an active agent to a target site in a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; administering to the subject a second composition comprising at least one active agent, and applying an ultrasound energy to the subject, thereby delivering an active agent to a target site in the subject.

In some embodiments, the target site is an organ, a tissue or a tumor site in the subject.

In some embodiments, the level of the at least one active agent co-administered with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being co-administered with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers by systemic administration.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers.

In some embodiments, the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers.

In some embodiments, applying the ultrasound energy induces cavitation of the carriers and/or enhances delivery of the active agent within the subject.

In some embodiments, the level of the at least one active agent associated or co-administered with the plurality of carriers is about 0.001% to about 99%, about 0.001% to about 75%, about 0.001% to about 50%, or about 0.001% to about 25% of the level of the active agent required to achieve a therapeutic effect, optionally, a substantially equivalent therapeutic effect, when administered without being associated or co-administered with carriers.

In some embodiments, the level of the at least one active agent associated with or co-administered with the plurality of carriers is about 0.001% to about 0.1%, about 0.1% to about 10%, about 0.1% to about 20%, about 10% to about 50%, or about 50% to about 99% of the level of the active agent required to achieve a therapeutic effect, optionally a substantially equivalent therapeutic effect, when administered without being associated or co-administered with carriers.

In some embodiments, the level of the active agent associated with the plurality of carriers is about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 99% of the level of the active agent required to achieve a therapeutic effect when administered without being associated or co-administered with carriers, optionally, by systemic administration without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the same level of active agent without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the active agent without being associated with carriers required to achieve a substantially equivalent therapeutic effect.

In some embodiments, the level of the at least one active agent associated or co-administered with the plurality of carriers elicits an immunostimulatory effect.

In some embodiments, the level of the at least one active agent associated or co-administered with the plurality of carriers does not elicit an immunosuppressive effect.

In some embodiments, the at least one active agent is selected from the group consisting of a protein, an antibody, a small molecule, a virus, an antibiotic, a radionuclide, a peptide, a nucleic acid, a gene, a vector or a plasmid encoding a gene, and a component of a gene editing system.

In some embodiments, the at least one active agent comprises a cytokine, and/or a vector or plasmid encoding a gene encoding the cytokine.

In some embodiments, the cytokine is selected from a group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, interferon, GM-CSF and TNF-alpha.

In some embodiments, the cytokine is IL-2.

In some embodiments, the level of IL-2 associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 10 pg/mL to about 100 pg/mL, about 100 pg/mL to about 100 ug/mL, about 100 pg/mL to about 100 ng/mL, about 100 ng/mL to about 1 μg/mL, about 1 μg/ml to about 10 μg/mL, about 10 μg/mL to about 100 μg/mL, about 200 ng/mL to about 500 ng/mL, about 500 ng/mL to about 2 μg/mL, or about 2 μg/mL to about 5 μg/mL.

In some embodiments, the at least one active agent comprises a monoclonal antibody, and/or a vector or plasmid comprising a gene encoding the monoclonal antibody.

In some embodiments, the monoclonal antibody is selected from the group consisting of bevacizumab, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, rituximab, cetuximab and trastuzumab.

In some embodiments, the monoclonal antibody is bevacizumab.

In some embodiments, the level of bevacizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL.

In some embodiments, the monoclonal antibody is pembrolizumab.

In some embodiments, the level of pembrolizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL.

In some embodiments, the at least one active agent comprises one or more components of a gene editing system.

In some embodiments, the at least one active agent comprises one or more components of a CRISPR system.

In some embodiments, the at least one active agent comprises a cDNA and/or mRNA encoding a Cas9 protein, a guide RNA, and/or a ribonucleoprotein (RNP) complex.

In some embodiments, the at least one active agent is an oncolytic virus, for example, encapsulated within the carrier. In some embodiments, the oncolytic virus is selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, poliovirus, measles virus, polioviruses, poxvirus, Newcastle disease virus, reovirus, coxsackievirus, vesicular stomatitis virus, Zika virus, RNA virus, and DNA virus.

In some embodiments, the composition comprises a viral vector comprising a gene encoding the active agent.

In some embodiments, the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus.

In some embodiments, the carrier is a microbubble, optionally selected from the group consisting of Levovist™, Imagent®, Optison®, Sonazoid®, BR38, and SonoVue®.

In some embodiments, the disease or condition is a non-cancer disease or condition.

In some embodiments, the disease or condition is cancer.

In some embodiments, the cancer is selected from a group consisting of pancreatic cancer, liver cancer, colorectal cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, breast cancer and brain cancer.

In some embodiments, the method further comprises administering an additional therapeutic agent.

In some embodiments, the additional agent is an anti-PD-1 agent, anti-PD-L1 agent, or an anti-CTLA-4 agent.

In various embodiments of the foregoing methods of the invention, the compositions further comprise free lipids. The free lipids may comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total lipid content in the composition. In a particular embodiment, the free lipids are in a high energy state before reconstitution of the carrier. In certain embodiments, the free lipids form a second plurality of carriers upon exposure to an aqueous solution. The second plurality of carriers may include a liposome, a lipid rod, a lipid disc a lipid sheet and a combination thereof. In a particular embodiment, the free lipids form a unilamellar liposome. In another embodiment, the free lipids form a multilamellar liposome. In certain embodiments, the at least one active agent is encapsulated in, bound to, associated with or interacted with the second plurality of carriers.

In a further embodiment of the foregoing methods, the first plurality of carriers comprises microbubbles, for example, comprising (i) a shell comprising a lipid, a polymer, a lipopolymer, a protein, or combination thereof; and/or (ii) a core comprising a gas. In a particular embodiment, the shell comprises a lipid, and wherein the lipid is DMPC; and the core comprises at least one of perfluorohexane and nitrogen gas. In a particular embodiment, the plurality of free lipids comprises DMPC.

In one aspect, the present invention provides a method for reducing systemic toxicity of an active agent upon administration into a subject, the method comprising providing a composition comprising a plurality of carriers, associating the active agent with the plurality of carriers, administering the plurality of carriers associated with the active agent to the subject, and applying an ultrasound energy to the subject, thereby reducing the systemic toxicity of the active agent.

In one aspect, the present invention provides a method for reducing innate immune response against an active agent upon administration into a subject, the method comprising providing a composition comprising a plurality of carriers, associating the active agent with the plurality of carriers, administering the plurality of carriers associated with the active agent to the subject, and applying an ultrasound energy to the subject, thereby reducing the innate immune response against the active agent.

In one aspect, the present invention provides a method for reducing systemic toxicity of an active agent upon administration into a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, administering to the subject a second composition comprising the active agent, and applying an ultrasound energy to the subject, thereby reducing the systemic toxicity of the active agent.

In one aspect, the present invention provides a method for reducing innate immune response against an active agent upon administration into a subject, the method comprising administering to the subject a first composition comprising a plurality of carriers, administering to the subject a second composition comprising the active agent, and applying an ultrasound energy to the subject, thereby reducing the innate immune response against the active agent.

In some embodiments, the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof.

In some embodiments, associating the active agent with the plurality of carriers comprises encapsulating the active agent within the plurality of carriers.

In some embodiments, associating the active agent with the plurality of carriers comprises binding the active agent to the interior or exterior surface of the plurality of carriers.

In one aspect, the present invention provides a method of preparing a carrier-active agent complex, the method comprising reconstituting the carrier in a first solution, reconstituting the active agent in an at least second solution, and mixing the active agent in the first solution with the carrier in the second solution to create a carrier-active agent complex in a carrier-active agent complex solution, thereby preparing the carrier-active agent complex.

In one aspect, the present invention provides a method of preparing a carrier-active agent complex, the method comprising reconstituting the carrier in a solution comprising the active agent to create a carrier-active agent complex, thereby preparing the carrier-active agent complex.

In one aspect, the present invention provides a method of preparing a carrier-active agent complex, the method comprising reconstituting the carrier in a first solution, providing the active agent in a second solution, and mixing the carrier in the first solution with the active agent in the second solution to create a carrier-active agent complex in a carrier-active agent complex solution, thereby preparing the carrier-active agent complex.

In some embodiments, the method further comprises removing the free active agent from the carrier-active agent complex solution after preparation of the carrier-active agent complex.

In some embodiments, the active agent is encapsulated within the carrier to create the carrier-active agent complex.

In some embodiments, the active agent is bound to the interior or exterior surface of the carrier to create the carrier-active agent complex.

In some embodiments, the free active agent is removed from the carrier-active agent complex solution after preparation of the carrier-active agent complex by a separation method selected from a group consisting of centrifugation, flotation, filtration, chromatography, vortexing and precipitation.

In some embodiments, the carrier-active agent complex is prepared without modifying the surface of the carrier.

In some embodiments, the carrier-active agent complex is prepared in the absence of a binding agent.

In some embodiments, the carrier-active agent complex is prepared in the absence of an inactivating agent.

In some embodiments, the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus.

In some embodiments, the carrier is a microbubble, optionally selected from the group consisting of Levovist™, Imagent®, Optison®, Sonazoid®, BR38, and SonoVue®.

In one aspect, the present invention provides a kit comprising (a) a plurality of microbubbles, (b) an active agent, and (c) a device for incubation of the plurality of microbubbles and the active agent.

In some embodiments, the plurality of microbubbles is present in a vial or a syringe in suspension, or in the form of lyophilized powder.

In some embodiments, the lyophilized powder of the plurality of microbubbles are reconstituted in a solution.

In some embodiments, the solution is a sterile water, a saline solution, or a solution comprising an active agent.

In some embodiments, the active agent is present in a vial or a syringe in suspension, or in the form of lyophilized powder.

In some embodiments, the lyophilized powder of the active agent is reconstituted in a solution.

In some embodiments, the solution is a sterile water, a saline solution, or a solution comprising a plurality of microbubbles.

In some embodiments, the device allows for isolation of microbubbles of different sizes.

In some embodiments, the device is an apparatus comprising: a housing having a base portion and an upper portion, the upper portion having a sealable access that allows placement and withdrawal of a fluid filed container; and a motor in the base portion having multiple modes of operation, a first mode operable to impart a first rotational motion to the container to mix two or more components held in the container, and a second mode operable to impart a second rotational motion to the container to generate a vortex therein to separate the first component from the second component.

In one aspect, the present invention provides an apparatus comprising: a housing having a base portion and an upper portion, the upper portion having a sealable access that allows placement and withdrawal of a fluid filed container; and a motor in the base portion having multiple modes of operation, a first mode operable to impart a first rotational motion to the container to mix two or more components held in the container, and a second mode operable to impart a second rotational motion to the container to generate a vortex therein to separate the first component from the second component.

In some embodiments, at least one of the two or more components comprises a plurality of microspheres.

In some embodiments, the plurality of microspheres comprise a plurality of carriers, wherein each of the plurality of carriers comprises a shell and a core.

In some embodiments, the vortex causes the plurality of microspheres to float inwardly away from walls of the container.

In some embodiments, the plurality of microspheres comprises a plurality of buoyant, pressure-sensitive particles, and wherein the liquid vortex causes the plurality of buoyant, pressure-sensitive particles to travel to the walls of the containers and precipitate.

In some embodiments, a spin speed of the motor is greater than 0 and less than 7,580 revolutions per minute (RPM).

In some embodiments, spin time to separate the first component from the second component is greater than 0 and less than 15 minutes.

In some embodiments, the motor is further configured such that the generated vortex is along a vertical axis of the container.

In some embodiments, the container is a syringe.

In some embodiments, the apparatus further comprise a connection collar to support the container, wherein the connection collar connects the container to the motor.

In some embodiments, the apparatus further comprises a holder to hold a second container for incubation and/or mixing, the holder includes a heater to heat a liquid mixture in the second container, and the motor is operable in the first mode to generate waves and/or gentle swirling in the liquid mixture via at least one of a seesaw motion, a back and forth rotating motion, a swing motion, a shaking motion, and a gentle rotation motion.

In some embodiments, the second container is a vial.

In some embodiments, the apparatus further comprises a controller configured to control the motor to switch between modes.

In some embodiments, the second container is placed in a horizontal position.

In one aspect, the present invention describes compositions of matter and methods that affect the therapeutic window of a drug, or combination of at least two drugs. As shown in FIG. 1, the present invention describes a drug (or combination of at least two drugs) that lowers the blood concentration at which a therapeutic effect can be achieved, thus widening the therapeutic window (Circled 1). The present invention allows a user to reduce blood concentration via a lower dose to reduce adverse effects without losing any therapeutic effect (Circled 2), thus providing the same therapeutic window with lower toxicity. By lowering the dose at which a threshold effect can be achieved, the duration of effect is increased which means the time during which a therapeutic agent concentration can be sustained at the target tissue is significantly widened (Circled 3).

In FIG. 2, the present invention enables incrementally higher doses to have more therapeutic effect for more people, thus rotating the top of the curve to the left (Circled 1), thereby increasing efficacy of a therapeutic agent. Also, incrementally lower doses can have increased therapeutic effect for more people, thus rotating the bottom of the curve to the right. The present invention lowers the dose at which for 50% of patients the agent will be effective, which shifts the ED curve left (Circled 2), thus widening the therapeutic index by increasing potency.

The present invention describes novel compositions comprising a plurality of osmotically stabilized, neutral charged, biphasic carriers, e.g., microbubbles, under specific and controllable sonication conditions, that can enable systemically delivery of therapeutic agents to cross targeted tissue barriers to achieve a therapeutic effect, even at doses and plasma concentrations that are at (or below) doses currently approved for clinical use or thought to be effective. The present invention is capable of fundamentally changing the pharmacokinetic profile of a drug and its distribution characteristics, thereby increasing potency and efficacy of drugs, while minimizing toxicity or adverse events.

The present invention includes delivering a clinically relevant level (which may include a microdose) of therapeutics into the target tissue of a body of a living being, gathering data about the therapeutic agent being administered, gathering data about devices used, gathering data about the patient (e.g., biological, physiological, anatomical, chemical, genetic or morphological conditions), and gathering data about events prior to, during or post-treatment, using machine learnings or artificial intelligence to adapt, modify or improve the diagnosis, treatment or theranosis of disease or condition.

The present invention further discloses a method and process of first determining the relevant amount of a therapeutic agent required to be delivered to a target (for example, but not limited to, cell, tumor, tissue, organ, area, volume, location, etc.) that has been previously determined to have a desired therapeutic effect, then delivering such desired therapeutic amount to the target. Distinct from present methods of delivering a dose of a therapeutic agent that is, for example, a maximum tolerated dose (MTD) of which a very small percentage actually reaches the intended target, the present invention administers the therapeutic level needed and delivers a significant amount to the target.

Furthermore, the present invention consists of novel therapeutic compositions, combinations, conjugates, or complexes that can be mixed onsite just prior to administration and are delivered in clinically relevant levels into the target tissue of a body of a living being. Such inventions may include therapeutics that are co-administered or encapsulated into a drug delivery vehicle (which may be, but not limited to, a microbubble, nanoparticle, nanodroplet, micelle, liposome, exosome, cell, virus, or other carrier). The invention discloses compositions that include therapeutic agents of various sizes, types, classes, or properties.

The present invention provides methods of combining two or more drugs at the time of administration (of which one may be a microbubble), the combination of which results in improved efficacy or biodistribution to a target compared to the administration of each drug alone. The present invention discloses various methods of encapsulation or binding of therapeutic agents into or onto a drug delivery vehicle at the time of administration as well as various methods of administering such compositions, combinations, conjugates, or complexes to improve a therapeutic agent's efficacy or safety profile.

Additionally, the present invention includes the use of devices, equipment, therapeutic complexes, software, and analytical tools, to improve the diagnosis, therapy or theranosis of disease or conditions. Such devices, equipment, therapeutic complexes, software, and analytical tools may be currently commercially available, added to those commercially available, or may be proprietary.

There is a need to improve upon the efficacy or safety (or both) of therapeutic agents. Although the present invention discloses improvements to the efficacy or safety of oncology agents, particularly immuno-oncology agents, the present invention discloses methods and compositions that are not intended to be limited to oncology, nor to chemical or biological agents.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 depicts the change of a drug concentration in blood over time.

FIG. 2 depicts an exemplary dose response curve of a drug.

FIG. 3 is a schematic of the present invention where one or more agents 101a, 101b, 101c, etc., are associated with a carrier 100 to form a complex or combination.

FIG. 4 is a schematic of the present invention where one or more agents 101a, 101b, 101c, etc., are associated with each other prior to being joined with a carrier 100 to form a complex or combination.

FIG. 5 is a schematic outlining the basic steps in the methods of the present invention.

FIGS. 6A-F depict various methods of the present invention relating to the timing or sequence and use of a carrier in the presence or absence of an encapsulated agent.

FIG. 7 shows the amount of IL-2 encapsulated in Imagent microbubbles by Human IL-2 Quantikine ELISA assay.

FIG. 8 depicts inhibition of tumor growth in MC38 mouse model of colon cancer using encapsulated IL-2 drug.

FIG. 9 shows the mean percentage inhibition data of FIG. 8.

FIG. 10 presents data from a human cytokine and chemokine analysis.

FIG. 11 depicts the tumor volume growth in mice treated with anti-PD-1 antibody.

FIG. 12 depicts an increase in the tumor CD8+/Treg ratio in mice treated with microbubbles co-administered with anti-PD-1 antibody.

FIG. 13 is a schematic depicting the syringe method for making the compositions of the invention.

FIG. 14 is a schematic depicting the vial method for making the compositions of the invention.

FIG. 15 depicts the amount of IL-2 encapsulated within the Imagent microbubbles prepared using the syringe method.

FIG. 16 depicts an exemplary ultrasound image for use in the methods of the invention.

FIG. 17 depicts an exemplary ultrasound image for use in the methods of the invention.

FIG. 18 depicts an exemplary data flow for use in the methods of the invention.

FIG. 19 is a schematic depicting a network effect of the methods of the invention.

FIG. 20 depicts mAb-encoding plasmid DNA constructs in Imagent microbubbles.

FIG. 21 depicts the overall protocol for cynomolgus monkey study.

FIG. 22 depicts the overall protocol for the delivery of Crisper/Cas9 DNA, mRNA, and ribonucleoprotein (RNP) targeting the Col1a1 locus by sonoporation.

FIG. 23 depicts the knock-in model for overexpression of a gene from the Col1a1 locus.

FIG. 24 is a schematic depicting that sonoporation-enhanced delivery of STING agonist, MSA-1, induced robust immune modulation and tumor regression.

FIGS. 25A-C depict a dose-escalation study of IV administered MSA-1. C57BL/6 female mice were inoculated on the right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells and when tumor volumes reached 100 mm³, treatments of PBS (vehicle), 0.3, 1, 3, and 10 mpk of MSA-1 were administered. (×1) indicates single injection on day 1; (×3) indicates injections on day 1, 4, and 7. FIG. 25A depicts the tumor volume in mice receiving the indicated treatments. Tumor volumes were measured at indicated times until animals reached a tumor volume of 2000 mm³. Volume measures are presented as mean tumor volume ±SEM. The number of mice that achieved CR is indicated. n=9-10 mice. FIG. 25B depicts the percent survival of mice receiving the indicated treatments. Log-rank (Mantel-Cox) test was used to compute statistical significance. *p<0.05; **p<0.01; ***p<0.001. FIG. 25C depicts the percentage body weight change in mice receiving the indicated treatments. Body weight measures are presented as mean percent body weight change ±SEM.

FIGS. 26A-F depict the correlation between the level of MSA-1 in the tumor and plasma with the anti-tumor efficacy of MSA-1. C57BL/6 mice were inoculated on the right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells and when tumor volumes reached 100 mm³, treatments of 0.3, 1, 3, and 10 mpk single dose of MSA-1 were administered. FIGS. 26A-B depict the tumor and plasma concentrations of MSA-1 10 min post-administration, shown as mean±SD. FIGS. 26C-F depict the tumor and plasma IL-6, TNF-α, IFN-α, and IFN-β cytokine levels 4 hr post-dosing, shown as mean±SD. Statistics calculated using one-way ANOVA followed by Tukey's test. *p<0.05; ***p<0.001. n=3 mice.

FIGS. 27A-C depict the anti-tumor efficacy behavior when combining MSA-1 at either 1 mpk or 0.3 mpk with sonoporation. C57BL/6 female mice were inoculated on the right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells and when tumor volumes reached 100 mm³, treatments were administered. FIG. 27A depicts the tumor volume in mice receiving the indicated treatments. Tumor volumes were measured at indicated times until animals reached a tumor volume of 2000 mm³. Volume measures are presented as mean tumor volume ±SEM. The number of mice that achieved CR is indicated. n=8-10 mice. FIG. 27B depicts the percent survival of mice receiving the indicated treatments. Log-rank (Mantel-Cox) test was used to compute statistical significance. *p<0.05; **p<0.01; ****p<0.0001. FIG. 27C depicts the percentage body weight change in mice receiving the indicated treatments. Body weight measures are presented as mean percent body weight change ±SEM.

FIGS. 28A-L depict that sonoporation drastically enhanced tumor local concentration of MSA-1 and resulted in substantially elevated cytokine production. C57BL/6 female mice were inoculated on the right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells and when tumor volumes reached 100 mm³, treatments of 1 mpk MSA-1, color-coded orange, and 1 mpk MSA-1 plus sonoporation, color-coded blue, were administered. FIGS. 28A-B depict the violin plot of tumor and plasma concentrations of MSA-1 20 min post-administration. FIGS. 28C-D depict the Violin plot of tumor and plasma concentrations of MSA-1 60 min post-administration. FIGS. 28E-H depict the violin plot of plasma IL-6, TNF-α, IFN-α, and IFN-β cytokine levels 4 hr post-dosing. FIGS. 28I-L depict the violin plot of tumor IL-6, TNF-α, IFN-α, and IFN-β cytokine levels 4 hr post-dosing. Statistics calculated using a two-tailed t-test. *p<0.05. n=5 mice.

FIGS. 29A-L depict that sonoporation enhanced MSA-1's immune activation mechanisms while reduced its immune suppression mechanisms. C57BL/6 female mice were inoculated on the right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells and when tumor volumes reached 100 mm³, treatments of PBS (vehicle), PBS plus sonoporation, 1 mpk MSA-1, 1 mpk MSA-1 plus ultrasound, and 1 mpk MSA-1 plus sonoporation were administered. Five days post-treatment, mice were euthanized and tumors were collected for immunophenotyping. FIGS. 29A-F depict selective immune cells from the lymphoid lineage presented as a percentage of CD45⁺ cells. FIGS. 29G-L depict selective immune cells from the myeloid lineage as a percentage of CD45⁺ cells. Statistics calculated using a one-way ANOVA followed by Dunnett's test referencing the vehicle. *p<0.05. **p<0.01; ***p<0.001; ****p<0.0001. n=6 mice.

FIGS. 30A-B depict that MSA-1 in combination with sonoporation eradicated distantly located non-ultrasound-treated tumors. C57BL/6 female mice were inoculated on both the left and right flank with 0.5×10⁶MC38 mouse colon adenocarcinoma cells each and when both tumors reached an average of 100 mm³, indicated treatments were administered. Ultrasound was only applied to the primary tumor. 1 mpk MSA-1 IT was injected into the primary tumor. FIG. 30A depicts the primary tumor volume in mice receiving the indicated treatments. Primary tumor volumes were measured at indicated times until animals reached a combined tumor volume of 2000 mm³. Volume measures are presented as mean tumor volume ±SEM. FIG. 30B depicts the distant tumor volume in mice receiving the indicated treatments. Distant tumor volumes were measured at indicated times until animals reached a combined tumor volume of 2000 mm³. Volume measures are presented as mean tumor volume ±SEM. n=9 mice.

FIG. 31 depicts the number of CD8⁺ and GranB⁺CD8⁺ T cells in MC38 in vivo tumor model upon receiving an intravenous dose of STING agonist and microbubbles. Animals were exposed to 0.531 MI ultrasound with or without the high 1.304 MI pulses.

FIG. 32A depicts the mean concentration of bevacizumab-awwb as measured in the tumors upon receiving the indicated treatments. FIG. 32B depicts the tumor weights in animals receiving the indicated treatments.

FIG. 33 depicts a reverse angle centrifuge rotor for use in the methods of the present disclosure.

FIG. 34 illustrates an apparatus for performing incubation, spin separation, and mixing of the present disclosure.

FIG. 35 illustrates an example connection collar and an example holder of the present disclosure.

FIG. 36 illustrates an example connection collar in greater detail.

FIG. 37 illustrates an example holder in greater detail of the present disclosure.

FIG. 38 illustrates an example spinning syringe of the present disclosure.

FIG. 39 depicts a device for use in the phase separation step of the microbubbles.

FIG. 40 illustrates example components of on-site eIL-2 preparation of the present disclosure.

FIG. 41 illustrates a flowchart for an example method for on-site eIL-2 preparation using the components in FIG. 40.

FIG. 42 illustrates a diagram for on-site eIL-2 preparation using the components in FIG. 40.

FIG. 43A is a schematic of a cryo TEM testing protocol. FIG. 43B is a cryo transmission election microscopy (TEM) image showing a nanoscale carrier of the present invention. FIG. 43C is a histogram for object 43Ba in FIG. 43B. FIG. 43D is a histogram for the object 43Bb in FIG. 43B. FIG. 43E is a histogram for object 43Bc in FIG. 43B.

FIG. 44A is a cryo TEM image showing various liposomal objects of the present invention after microbubble implosion. FIG. 44B is a histogram for object 44a in FIG. 44A. FIG. 44C is a histogram for object 44b in FIG. 44A.

FIG. 45 is a cryo TEM image showing the presence of DNA and a liposome.

FIG. 46 is a cryo TEM image showing various lipid objects or structures after processing the microbubble infranatant using an emulsion technique.

FIG. 47 is a cyro TEM image showing various lipid objects present in the carrier infranatant.

FIG. 48A-C are cryo TEM images showing various lipid objects present in the carrier infranatant.

FIGS. 49A-B are cryo TEM images of the infranatant from the carrier reconstituted as per manufacturer's FDA labeling and instructions.

FIG. 50A is a schematic of a cryo TEM testing protocol. FIGS. 50B-E are cryo TEM images of the infranatant from the carrier reconstituted with a vaccine solution containing lipid nanoparticles (LNPs) encapsulating messenger RNA (mRNA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein an at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers), and/or bound to the surface (e.g., the exterior or interior surface, of the carriers) for delivery to a subject. In addition, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, to be co-administered with compositions comprising at least one active agent to a subject. Furthermore, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein an at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers), and/or bound to the surface (e.g., the exterior or interior surface, of the carriers), to be co-administered with compositions comprising an at least one free active agent, wherein the at least one active agent is not associated with the plurality of carriers, to a subject.

The present invention also provides methods of treating a disease or condition, and methods of targeted delivery of an active agent, e.g., to a target tissue, e.g., an organ, a tissue, a tumor site, in a subject, using the carrier based compositions of the invention. Furthermore, the present invention provides methods for improving efficacy of therapeutics, e.g., for treating a disease or disorder in a subject by identifying or determining or selecting a target characteristic, determining the amount of a therapeutic agent to be delivered to reach the target characteristic, and delivering the desired amount of active agent to reach the target characteristic.

In particular, the inventors of the present invention have surprisingly discovered that by associating or co-administering the active agent with carriers, the level of the active agent required to achieve a therapeutic effect is substantially lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated or co-administered with carriers, e.g., by systemic administration without being associated or co-administered with carriers.

The compositions of the invention are protected and/or targeted. Creating the compositions just prior to administration allows for the rapid use of different combinations of active agents that could benefit from protection or targeting, or both, since the active agent (i.e., particularly an FDA approved agent) and the delivery vehicle, (i.e., particularly an FDA approved microbubble, such as Imagent) have known safety and toxicology profiles, are manufactured at scale, and able to be mixed into a complex just prior to or at the time of administration. This provides significant advantages when compared to reformulating an agent at the research and development or manufacturing stage to provide a means of protection in circulation or targeting, or both protection and targeting, such as by pegylation or through the use of nanoparticles, which may introduce issues related to manufacturing, biocompatibility, safety or efficacy of the carrier or the therapeutic agent.

The present invention also discloses devices and methods related to the preparation of the compositions of the invention after the manufacturing process and at the point of care (for example, but not limited to, in the hospital, outpatient center, medical office or facility, pharmacy, bedside, patient home, etc.), and methods for treating a disease or disorder in a subject using the compositions of the invention. The present invention further describes means to change the amount of active agent (e.g., concentration) that is attached or bound to said carrier. Additionally, the present invention relates to the creation of complexes consisting of multiple agents, or different classes of agents, with a carrier.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entireties for any purpose.

I. Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

The term “active moiety” as used herein refers to the molecule or ion, excluding those appended portions of the molecule that cause the drug to be an ester, salt (including a salt with hydrogen or coordination bonds), or other noncovalent derivative (such as a complex, chelate, or clathrate) of the molecule, responsible for the physiological or pharmacological action of the drug substrate.

The term “active pharmaceutical ingredient” or “API” as used herein refers to any substance or mixture of substances intended to be used in the manufacture of a drug product and that, when used in the production of a drug, becomes an active ingredient in the drug product. Such substances are intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease or to affect the structure and function of the body. Also see, e.g., International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q7, Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients).

The term “administration” as used herein refers to any method or process of delivering a diagnostic, therapeutic or theranostic substance into a patient and may include, but is not limited to, parenteral and non-parenteral routes, such as intravenous, subcutaneous, intramuscular, intracranial, intranodal, intratumoral, intralesional, intraperitoneal, directly into an organ, transdermal, or oral.

The term “agent” as used herein refers to a therapeutic agent or an agent that is intended to create directly or indirectly a therapeutic effect, whether by itself or in combination with a delivery vehicle having, or intended to have a beneficial effect on a patient. The agent can be, but is not limited to, a small molecule, a protein, a peptide, a monoclonal or polyclonal antibody, a nucleic acid, a plasmid, RNA, DNA, or a cell, a gene, a vector or a plasmid encoding a gene, or a component of a gene editing system.

The term “free active agent”, as used herein, refers an active agent that will not be associated or encapsulated within a carrier, e.g., a microbubble, upon contacting with a carrier, e.g., a microbubble.

The term “substantially equivalent therapeutic effect” of an active agent, as used herein, refers to an effect that is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent. The therapeutic effect may be assessed by a variety of art recognized techniques to assess, for example, efficacy, bioavailability, etc. For example, a therapeutic effect may be a reduction in tumor size or growth.

The term “subject” as used herein refers to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, or a chimpanzee), or a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse). In an embodiment, the subject is a human.

The term “carrier” as used herein refers to a structure either inside or outside of a cell consisting of a substance enclosed by an envelope or shell. The inside of the carrier may be a liquid, gas or solid, while the shell may be a lipid. An example of a carrier is a microbubble that contains a gas enclosed in a biocompatible shell. Other examples of a carrier are a nanoparticle, nanodroplet, vacuole, lysosome, exosome, endosome, micelle or cell, and may be constructed from of biological, chemical, or synthetic materials. The term “vehicle” as used herein is interchangeable or synonymous with the term “carrier.”

The term “co-administered” or “co-administration” as used herein refers to administered at approximately the same time as another agent, or while another agent is in circulation (which may be hours to days to weeks after an agent is administered).

The term “complex” as used herein refers to the formation of a combination product using the active pharmaceutical ingredient (API) and companion delivery vehicle while preserving the integrity of the API.

The term “data” as used herein refers to information relating to a patient's gender, race, height, weight, age, condition, medication regimen, demographics, or genetic profile. Data may also be related to an organ, tissue, gas, solid, fluid, cell, particle, molecule, tumor, microenvironment, stroma, vasculature, treatment target, or non-treated organ, tissue, gas, solid, fluid, cell, particle, molecule, tumor, microenvironment, stroma, vasculature. Data on properties or characteristics of an organ, tissue gas, solid, fluid, cell, particle, molecule, tumor, microenvironment, stroma, vasculature, etc. may include location, proximity to other tissues, volume, area, dimensions, heterogeneity, fibrosis, chemical, electrical, biological, physical, anatomical, histological, pathological, genetic, genomic, proteomic, transcriptomic, spatial, volumetric, temperature, energy, bioeffect, reaction, stiffness, elasticity, density, permeability of tissue, vascularity, vascular density, size or duration of a vascular opening, viscosity, or flow rate. The data may be related to, but not limited to, the therapeutic agent, delivery vehicle, or drug/drug complex, such as PD, PK, AUC, MTD, molecular weight, chemical composition, event sequence, temporal patterns of the agent-target/disease/pathogen dynamics, flow rate, biodistribution, injected dose, drug interactions, date and time of administration, date and lot number of manufacture, name of manufacturer, amount of free agent, amount of therapeutic agent within or on delivery vehicle, amount of carrier, amount of microbubble. Data may include information on instrumentation, device or equipment used before, during, or after treatment, such as model number, serial number, lot number, manufacture information, measure of power, energy, mechanical index, peak negative pressure, radiation force, force, frequency, pulse length, duration, duty cycle, frame rate, transmission time, receive time, attenuation, displacement, action, reaction, mechanical, physical, effect, bioeffects, transmit or receive data, input or output data created as a result of the use of such instrument, device or equipment. Data may also include time of day, or duration of treatment, hospital or treatment location or setting information, or user or operator information (such as user name, age, title, number of times such user has used said device, etc.). The data may be related to, but not limited to, events that occurred prior to, during, or after the administration of an agent and may include clinical outcomes data.

The term “delivery vehicle” as used herein refers to a transporter that carries a therapeutic agent, whether attached to some element on the surface, attached directed to the surface, embedded onto the surface, or contained partially or completely within the surface, or within the interior of the vehicle.

The term “device” as used herein refers to a device, equipment, instrument, processor, sensor, externally or internally applied, emitting, transmitting or receiving a signal, energy or data and used as part of treatment of the present invention. The device may include, but is not limited to a, physical, mechanical, digital, ultrasonic, electrical, or magnetic instrument.

The term “associated” or “association” is used herein to mean the enclosure of an active agent within the interior of, or the attachment or binding onto the interior or exterior surface of a carrier or a delivery vehicle, e.g., a microbubble. Such association, e.g., encapsulation within the delivery vehicle or bound with the delivery vehicle, is intended either to protect the agent while in circulation within a body or to enable the active targeting of the delivery of said agent, or both protect and target.

The term “interacted” as used herein may refer to a temporary or transient binding of the active agent to the surface of the lipid.

The term “FDA” is used herein to mean the Food and Drug Administration of the United States Department of Health and Human Services or any successor agency thereof performing similar functions.

The term “Imagent®” or “Imagent” as used herein refers to that certain microbubble as listed in the NDA 21-191. Imagent also may be used herein interchangeably with carrier when referring to the use of microbubbles.

The term “microbubble” as used herein refers to a microsphere comprising a biocompatible shell surrounding at least one biocompatible gas.

The term “NDA” as used herein refers to a New Drug Application (as more fully described in 21 CFR 314.50 et seq. or its successor regulation), or any amendment or supplement thereto, with respect to NDA 21-191 approved by the FDA.

II. Compositions of the Invention

The present invention provides compositions comprising a plurality of carrier, e.g., microbubbles, wherein an at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers) and/or bound to the surface (e.g., the exterior or interior surface, of the carriers) for delivery to a subject, e.g., a target site, e.g., an organ, a tissue, or a tumor site in the subject. In addition, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, to be co-administered with compositions comprising at least one active agent to a subject, e.g., to a target tissue such as an organ, a tissue, a tumor site, in a subject. Furthermore, the present invention provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein an at least one active agent is associated with the plurality of carriers, for example, encapsulated within the plurality of carriers (e.g., between the gas/shell interface and/or within the gas of the carriers), and/or bound to the surface (e.g., the exterior or interior surface, of the carriers), to be co-administered with compositions comprising an at least one free active agent, wherein the at least one active agent is not associated with the plurality of carriers, to a subject.

In particular, the inventors of the present invention have surprisingly discovered that by associating or co-administering the active agent with carriers, the level of the active agent required to achieve a therapeutic effect is substantially lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated or co-administered with carriers, e.g., by systemic administration without being associated or co-administered with carriers.

Accordingly, in one aspect, the present invention provides a composition comprising a plurality of carriers, wherein an at least one active agent is associated with the plurality of carriers for delivery to a subject, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers and by systemic administration.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers. As used herein, the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers. In another aspect, the present invention provides a composition comprising a plurality of carriers, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof.

In some embodiments, the carrier releases a nanoparticle, nanodroplet, vacuole, lysosome, exosome, endosome, micelle or cell that includes at least one active agent and a lipid. Non-limiting examples of a lipid include 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or 4-dimethylaminochalcone (DMAC), or dipalmitoyl phosphatidylcholine (DPPC), or 1,2-distearyol-sn-glycero-3-phosphocoline (DSPC), 1,2-distearyol-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-PEG2000), lipofectamine, or any other cationic or helper lipids.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers and by systemic administration.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers. As used herein, the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers.

The at least one active agent may be associated with the plurality of carriers by being encapsulated within the plurality of carriers, or the at least one active agent is associated with the plurality of carriers by binding to the interior or exterior surface of the plurality of carriers.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 99%, about 0.001% to about 75%, about 0.001% to about 50%, about 0.005% to about 50%, about 0.005% to about 25%, or about 0.0001% to about 25% of the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, optionally, a substantially equivalent therapeutic effect, and optionally, by systemic administration without being associated with carriers.

In some embodiments, the level of the active agent associated with the plurality of carriers is about 0.001% to about 0.005%, about 0.005% to about 0.1%, about 0.005% to about 10%, about 0.005% to about 20%, about 10% to about 50%, or about 50% to about 99% of the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, optionally, a substantially equivalent therapeutic effect, optionally, a substantially equivalent therapeutic effect, optionally, by systemic administration without being associated with carriers. In some embodiments, the level of the active agent associated with the plurality of carriers is about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 99% of the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, optionally, a substantially equivalent therapeutic effect, optionally, by systemic administration without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the same level of active agent without being associated with carriers.

In some embodiments, administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the active agent without being associated with carriers required to achieve a substantially equivalent therapeutic effect.

In some embodiments, the level of the at least one active agent associated with the plurality of carriers elicits an immunostimulatory effect. In some embodiments, the level of the at least one active agent associated with the plurality of carriers does not elicit an immunosuppressive effect.

A. Carriers

In some embodiments, the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus.

In some embodiments, the carrier is a microbubble. Any microbubbles known in the art may be used for the compositions and methods of the present invention. Exemplary commercially available, FDA-approved microbubbles include, but are not limited to, Levovist™ (Schering AG), Imagent® (Vesselon), Optison® (GE Healthcare), Sonazoid® (GE Healthcare), SonoVue® (GE Healthcare), Lumason® (GE Healthcare), and BR38 (Bracco Suisse SA).

In one embodiment, the microbubble comprises a shell and a core.

In one embodiment, the microbubble shell comprises a polymer. Polymers suitable for use in the microbubbles for use in the compositions and methods of the present invention include, but are not limited to polyvinyl alcohol (PVA), polyethylene glycol (PEG), poly(iso-butylcyanoacrylate), and Poly(lactic-co-glycolic acid) (PLGA), poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS).

In another embodiment, the microbubble shell comprises a lipid. In one embodiment, the lipid is a phospholipid. Examples of lipids useful in the microbubbles for use in the compositions and methods of the present invention include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 4-dimethylaminochalcone (DMAC), dipalmitoyl phosphatidylcholine (DPPC), 1,2-distearyol-sn-glycero-3-phosphocoline (DSPC), and 1,2-distearyol-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-PEG2000). In one embodiment, the microbubble shell comprises DMPC.

In one embodiment, the microbubble shell comprises a lipopolymer.

In another embodiment, the microbubble shell comprises a protein. Exemplary protein useful in the microbubbles for use in the compositions and methods of the present invention include, but are not limited to, albumin.

In another embodiment, the microbubble shell comprises a surfactant. Examples of surfactants useful in the microbubbles for use in the compositions and methods of the present invention include, but are not limited to, Span 60 and Tween 80.

In a further embodiment, the shell comprises one or more of the following: a polymer, a lipid, a lipopolymer, a protein, a surfactant, or a combination thereof.

The core of the microbubbles for use in the compositions and methods of the present invention comprises a gas. In one embodiment, the gas is an inert gas. In another embodiment, the gas is nitrogen. In another embodiment, the gas is air. In still another embodiment, the gas is a perfluorocarbon gas. Exemplary perfluorocarbon gases for use in the microbubbles for use in the compositions and methods of the present invention include, but are not limited to, perfluorohexane, sulfur hexafluoride, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, and octafluoropropane. In one embodiment, the gas is perfluorohexane. In some embodiments, the core comprises a combination of perfluorohexane and nitrogen gas.

In some embodiments, the carriers, e.g., the microbubbles, comprise a core comprising a perfluorocarbon gas and/or nitrogen, which are surrounded by a shell that comprises lipids. In some embodiments, the carriers, e.g., the microbubbles, comprise a core comprising a perfluorocarbon gas and/or nitrogen, which are surrounded by a shell that comprises DMPC. In another embodiment, the carriers, e.g., the microbubbles comprise a core comprising a perfluorohexane gas and/or nitrogen, which are surrounded by a shell that comprises DMPC (e.g., Imagent® perflexane microspheres, as described in NDA 21-191).

A preferred embodiment of the present invention describes the use of a carrier, such as a microbubble with the following properties: ultra-thin, flexible shell for low surface tension properties; highly insoluble inert gas to prevent dissolution and allow longer circulation times; nitrogen gas to osmotically stabilize the microbubble in the bloodstream; neutral charge so as not to activate the complement system or bind to charged branches of large therapeutic agents; and, biphasic size distribution that can be optimized for greater drug delivery efficiency. Ultrasound pulses generated by commercially available ultrasound systems (or specialized ultrasound system designs for certain clinical applications) deliver appropriate levels of ultrasound to a specific tissue to activate the microbubble, whether or not at least one therapeutic agent is encapsulated therein, for the purpose of permeabilizing vessel walls.

Research has suggested that the efficiency of permeabilization may be associated with the total volume of microbubbles rather than their specific average diameter. It has also been postulated that the concentration of microbubbles can affect drug delivery efficiency. The total efficiency factor, therefore, for a given ultrasound exposure, that may describe microbubble enabled drug delivery could be

N×V×A×T=Efficiency

where N=the number of microbubbles per unit dose, V=total volume of microbubbles per unit dose, A is the ultrasound energy absorption characteristics of the microbubble and T=the total time for circulation in the target vessel. If microbubbles are no longer present in the vessel, ultrasound alone, at safe, low intensity levels, may not benefit drug delivery to any significant degree.

The number of microbubbles available in a dose that can be administered will be dependent upon the body's ability to accommodate higher concentrations of microbubbles in circulation at any given time. A microbubble with a positive or negative charge is likely to be recognized by the body's immune system as a foreign body and the complement system would be activated to remove it. Microbubbles carrying a neutral charge, e.g., Imagent, have been shown experimentally to have little complement activation and to have zero absorption by Kupffer Cells in the liver, potentially allowing a much higher concentration of microbubbles to be infused with minimal adverse effects.

In some embodiments, for an optimal volume parameter, Imagent has a volume weighted average diameter of 6 microns so that a large percentage of the volume of gas will be made up of larger diameter microbubbles. This is achieved by the novel manufacturing process where preformed starch molds of the bubbles are created within the lyophilized powder which, when reconstituted, produced larger diameter microbubbles without the need for additional size isolation steps.

Increased acoustic absorption efficiency is provided for with the ultra-thin, ultra-low surface tension low carbon chain phospholipid DMPC shell that allows the microbubble to optimally absorb the maximum amount of ultrasound energy upon sonication that is far greater than comparable longer chain phospholipids, or stiffer albumin protein shells. Imagent microbubbles have a very thin (˜3 nm) and malleable, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), lipid shell and contain a mixture of nitrogen and vapor perfluorohexane within the core. The soft-shelled microbubble has a malleable shell that stabilizes the core in vivo above its crystalline phase transition temperature (˜24° C.). For a given input ultrasound energy, the Imagent microbubble will expand and contract with greater diameter distensions without incurring destruction or inertial cavitation. Osmotically stabilized microbubbles have a thinner shell that permits larger amplitude oscillations allowing them to be subject to greater radiation forces at their peak resonant frequency and for stable cavitation to incur maximal relaxation pressures on the vessel walls for increased sonoporation at a given acoustic input intensity. Additionally, the thin, flexible shell allows for larger Imagent microbubbles to be “squeezed” through smaller diameter capillaries and achieve more contact with vessel walls for maximal sonoporation closer to the targeted regions of interest. This large displacement stable cavitation may provide for more than 20% greater permeabilization for a given input of ultrasound energy compared to other carriers with more stiff shells. The absorption properties of the thin shell will enable stable cavitation at a lower mechanical index threshold so that a larger percentage of available microbubbles in circulation achieve a therapeutic stable cavitation level. When inertial cavitation is induced, Imagent's thin, low surface tension shell may have a lower microjet velocity than stiffer shell microbubbles, which may provide a greater safety profile for higher mechanical index sonications in a therapeutic context.

To extend circulation life, in addition to the neutral charge, the Imagent microbubble possesses other properties to substantially reduce the dissolution impact of the aqueous bloodstream. The interior gas used is a perfluorohexane that has a very low solubility index. For each CF₂group added to a fluorocarbon gas, the solubility in water is decreased approximately by an order of 8.0. So microbubbles that use fluoropropanes (3 CF₂groups) and fluorobutanes (4 CF₂groups) will have a far faster dissolution time constant in blood than the Imagent perfluorohaexane (6 CF₂groups). A benefit of the lengthened circulation time and neutral charge of Imagent would be its persistence in the liver and spleen. As increased numbers of Imagent microbubbles collect in the liver or spleen, additional sonication of those organs (or others in which Imagent persists) may provide benefits of priming T-Cells within those organs that would provide systemic benefits potentially well beyond the time of the sonoporation treatment.

The thin DMPC shell may permit larger amplitude oscillations when subjected to ultrasound, but could lack the ability to retain the core material driven out by the Laplace pressure (heightened internal pressure due to curvature and surface tension) of the microbubble. To avoid the dissolution of the Imagent microbubble, nitrogen is added to the gas core so that its partial pressure is equivalent to that of human tissue and blood. Imagent includes nitrogen to osmotically stabilize with the blood nitrogen gas concentration thus structurally reducing the blood pressure surface tension on the microbubble. Balancing the partial pressures of nitrogen within and outside of the microbubble creates an osmotically stabilized microbubble capable of retaining the perfluorohexane core for longer. An osmotically stabilized microbubble will persist in the blood stream longer as its original size distribution enables longer and multiple sonication treatments during a single bolus injection.

The microbubbles may be uniform in size or the plurality of microbubbles may comprise microbubbles of different sizes. In one embodiment, the microbubbles are from about 1 μm to about 10 μm in diameter. In one embodiment, the microbubbles are from about 2 μm to about 8 μm in diameter. In one embodiment, the microbubbles are from about 1 μm to about 5 μm in diameter. In another embodiment, the microbubbles are from 1 μm to about 4 μm in diameter. In another embodiment, the microbubbles are from about 1 μm to about 3 μm in diameter. In a further embodiment, the microbubbles are from about 1 μm to about 2 μm in diameter. In one embodiment, the microbubbles are about 1 μm in diameter. In another embodiment, the microbubbles are about 4.5 μm in diameter.

In one embodiment, the plurality of microbubbles has a diameter distribution having multiple peaks. In one embodiment, the microbubbles have two peaks in their diameter distribution. In one embodiment, the microbubbles, e.g., Imagent, have two peaks in its diameter distribution—one peak corresponding to a diameter of about 1 μm and a second peak corresponding to a diameter of about 4.5-5 μm. Although there are a large number of 1 μm bubbles in a given volume of Imagent, the vast majority of the volume of bubbles is represented by the 5 μm distribution. The efficiency of sonoporation is directly related to microbubble size and number present in the region of interest. An embodiment of the present invention may apply a bifrequency ultrasound pulse sequence to optimally stimulate the microbubbles to achieve stable cavitation. The smaller diameter microbubbles will more efficiently cavitate with an ultrasound frequency pulse range of 2.5 to 4.0 MHz whereas the larger diameter will more efficiently cavitate with an ultrasound frequency pulse range of 0.5 MHz to 2.0 MHz.

Due to their smaller gas volume, the smaller diameter microbubbles will circulate for the shortest period of time at which point there is insufficient gas volume to keep the bubble intact with the surface pressure from the bloodstream. Therefore, an embodiment of the present invention may use initial sonication pulses optimized for the smaller diameter microbubbles with a higher frequency sonication. After the initial higher frequency pulses are completed for a specified duration (for example, approximately 10 to 25% of the total sonication time), the remaining sonication time may be either automatically or manually switched to the lower frequency more optimized for the larger diameter microbubbles in the biphasic distribution. As an example, approximately 75% to 90% of the sonication time may be optimized for the larger diameter microbubbles that last longer in circulation and represent the largest volume weighted component to the microbubble dose.

Transient inertial cavitation may provide beneficial sonoporation capabilities. It may be a desired objective of the present invention to take advantage of this effect while minimizing risk for longer sonication exposures at peak negative pressures that induce widespread inertial cavitation. The present invention describes transient increases of acoustic intensity that initiate widespread microbubble destruction, for example on the order of a fraction of one second up to two or four seconds duration, can be periodically interposed during the sonication that otherwise predominantly activates stable cavitation. For example, during a five-minute sonication period, the ultrasound system can either manually or through automated software transiently increase the mechanical index of pulses above 1 MPa at minutes 1, 2, 3 and 4 that will ensure all microbubbles within the field will burst due to inertial cavitation. New circulating microbubbles will replace those that are destroyed within 5-10 seconds so that the continuing sonication at the lower acoustic intensity can continue to stably cavitate microbubbles in proximity to a target tissue and continue the sonoporation effect until the next inertial cavitation burst or until the sonication is complete. As the total sonication time is adjusted to optimize for specific tumor types or for a specific combination of agents, the intervals of high mechanical index bursts can be increased or decreased. For example, during a 5-minute total treatment time the bursts may be scheduled at intervals of 10 seconds, 30 seconds, 1 minute or 1.5 minutes. For a 10-minute total sonication time, the bursts may be scheduled at 2, 4, 6 and 8 minutes.

The combination of either stable or inertial cavitation of circulating microbubbles in a blood vessel has also been shown to activate an immune response to attract T Cell CD8+cytotoxic T-lymphocytes (killer T Cells), CD4+ and or dendritic cells to adhere to the vessel wall at the point of sonoporation treatment and to be absorbed to the tissue in significantly higher concentrations. The larger diameter distensions allowed by the Imagent's thin shell, low solubility gas and osmotic stabilization with nitrogen may create a greater immune response for a given sonication energy and duration. In addition, the transient inertial cavitation pulses may increase this immune response.

In order to attract a greater T Cell immune response one preferred method would be to have intermittent high MI flashes where the ultrasound intensity would transiently reach a level to destroy microbubbles in the field of view. While high MI flashes have been used in diagnostic imaging for re-perfusion studies, in therapeutic drug delivery high MI flashes may preferentially activate an immune response at the site of tumor to attract the adhesion and absorption of more CD8+ cytotoxic T Cells, as well as create Reactive Oxygen Species that may be beneficial in a hypoxic tumor microenvironment.

In one embodiment, an MC38 in vivo colon tumor model was treated with an intravenous (IV) dose of STING agonist and Imagent microbubbles, and was exposed to 2.2 MHz ultrasound with only stable cavitation at 0.531 MI for 5 minutes. A separate arm of the animal was treated with the same 0.531 MI ultrasound for 5 minutes but with periodic 1100 ms ultrasound bursts of 1.304 MI administered every 10 s. FIG. 31 demonstrates that the increase of CD8⁺ and GranB⁺CD8⁺ was significantly higher than the sonoporation arm without the high MI pulses. These cytotoxic lymphocytes are correlated with beneficial anti-tumor response mechanisms across many cancers.

By encapsulating an agent into or onto the carrier (e.g., a microbubble) and inducing cavitation of the carrier, the present invention describes a mixture of substances intended to be used in the manufacture (e.g., the mixing process) of a drug product and that, when used in the production of a drug, becomes an active ingredient in the drug product and is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease or to affect the structure and function of the body. As a non-limiting example of the present invention, the encapsulation of IL-2 into or onto Imagent microbubbles describes a mixture of the two drugs that furnishes pharmacological activity and affects the structure and function of the body. In this case, the IL-2 when released from the Imagent (upon inertial cavitation) drug furnishes pharmacological activity while the Imagent drug (upon stable cavitation or inertial cavitation) affects the structure and function of the vascular or cellular membrane that is in contact or the vicinity of said Imagent microbubbles.

The use of experimental microbubbles in the clinic may be limited due to the addition of components that may elicit unintended biological effects or may involve significant or complicated processing steps that may introduce variability not conducive to achieving desired clinical results.

FIG. 3 illustrates a preferred embodiment of the present invention where one or more agent(s), etc., are combined with a carrier to form a complex, conjugate or combination. The agents may be used as a diagnostic, therapeutic, theranostic, mechanical, chemical, biological, genetic, synthetic, or other molecule. The agent may be a manufactured molecule, such as, but not limited to, a finished and packaged cancer drug that is ready to be administered to a patient, or may be a component used to aid in the formation of the complex (e.g., linker, ligand, etc.). As a non-limiting example of the present invention, an agent could consist of a solution of IL-2 that is mixed with a microbubble (carrier) to form a combination product. Such mixing could be performed using sterile products and closed fluid transfer processes just prior to administration, rather than at a manufacturing facility.

The present invention is particularly advantageous when the therapeutic molecule or compound has a safety or toxicity profile that could be improved to benefit a patient, has a low minimum inhibitory concentration measure, or has a low therapeutic index. Also, the present invention is particularly advantageous when, in order to improve clinical effect, greater concentrations of the therapeutic molecule or compound should be delivered to the desired target tissue.

The carriers, e.g., microbubbles, for use in the compositions and methods of the present invention can be modified at or near the time of administration without the addition of chemicals or ingredients (other than the solution containing the at least one active agent) during reconstitution or preparation to create a different version of the carrier. For example, during the phase separation step (e.g., flotation or centrifugation) since microbubbles having different sizes will float at different speeds, the infranatant (which may contain smaller microbubbles that take longer to float) may be expressed from the syringe of microbubbles, so that primarily larger microbubbles are present in the supernatant. Alternatively, the smaller microbubbles may be collected from the infranatant and used to deliver an encapsulated agent, as the smaller microbubbles collectively have greater surface area than the larger microbubbles and can therefore encapsulate more agent per a given volume of microbubbles, compared to the amount of agent per volume in the larger microbubbles. Using a differential centrifugation technique, larger diameter microbubbles float at different speeds (i.e., slower) compared to smaller diameter microbubbles (Feshitan 2009). Larger microbubbles may be more efficient at opening larger pores in tissues and therefore may be advantageous especially for the delivery of larger molecules, such as mAbs.

The present invention uses either the time period for a given method of phase separation, or a novel short axis syringe centrifugation method to create size isolated microbubbles at or near the time of administration. Centrifugation (or flotation) may be stopped at a certain point in time before substantially all of the microbubbles have floated to the supernatant layer. This would leave some smaller microbubbles still in the infranatant. As larger microbubbles (compared to smaller microbubbles) may be advantageous for sonoporation, the larger diameter microbubbles then could be isolated during the reconstitution or preparation phase by expressing the infranatant containing smaller microbubbles. Compared to the use of large and expensive centrifuge devices required to separate large microbubbles as described in Feshitan (2009), the use of a simple, low cost, single use device for the phase separation step and other processing steps in the present invention provides significant benefit. In the case of Imagent, the microsphere size distribution is noted in its package insert (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2002/21-191_Imagent_Approv.pdf) where 78.8% are <3 μm, while 21.0% are between 3-10 μm in diameter. By isolating the larger Imagent microbubbles and expressing out many of the smaller microbubbles in the infranatant, the final administered dose of larger sized Imagent microbubbles may be injected and advantageous for sonoporation. Additionally, the larger microbubbles may be used to encapsulate a therapeutic agent or may be used without encapsulating an agent. In the case of Imagent, the number of larger microbubbles is less than the number of smaller microbubbles in each reconstituted suspension. Therefore, in order to ensure that a sufficient number of larger microbubbles is made available in the syringe for administration, multiple units of larger Imagent microbubbles (after using the processing steps as disclosed herein) may be combined into the final suspension, so that the target number of larger Imagent microbubbles is attained. Combining two or more units of larger microbubbles (or smaller microbubbles if desired) may be helpful also to increase the number of encapsulated drugs in the carrier.

Additional phase separation steps similar to the ones described above may be used to further isolate the smaller microbubbles in the infranatant layer. For example, after an initial phase separation step, once the infranatant has been expressed from the reconstituted or prepared microbubbles the infranatant layer, centrifugation or flotation may be used again to separate the smaller microbubbles from the infranatant. Additionally, the smaller microbubbles may be used to encapsulate a therapeutic agent or may be used without encapsulating an agent.

B. Active Agents

The compositions of the present invention comprise a plurality of carriers, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, optionally, by systemic administration without being associated with carriers.

Exemplary active agent may include, but are not limited to, a cytokine, e.g., interleukin-2 (IL-2); a monoclonal antibody, e.g., bevacizumab; an antibiotic, e.g., ciprofloxacin; a small molecule, e.g., gemcitabine; a nucleic acid, e.g., messenger ribonucleic acid (mRNA), a DNA, a gene, or a vector or plasmid comprising a gene, e.g., a gene encoding a protein, a cytokine, an antibody and/or a peptide; a gene editing or gene therapy system; an inhibitor, e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4; an agonist, e.g., a STING agonist or angiotensin II receptor agonist); a binding agent, e.g., a ligand, cross-linker; or immune receptor, e.g., T cell immunoglobulin and ITIM domain (TIGIT). In some embodiments, the composition further comprises a virus, e.g., an oncolytic virus (OV), e.g., a multitargeted OV.

As used herein, the term “association” or “associated with” refers to the enclosure or encapsulation of a therapeutic agent within the interior of, or the attachment or binding onto the interior or exterior surface of a delivery vehicle. In some embodiments, the active agent is encapsulated within the carriers, e.g., between the gas/shell interface and/or within the gas of the carriers. In other embodiments, the active agent is bound and/or attached to the interior or exterior surface of the carriers. Such association of active agents with the carriers provides several benefits, including protecting a molecule while in blood circulation from either degradation (e.g., a nucleic acid), metabolism, inactivation or immunogenicity (e.g., an oncolytic virus). Association with the carriers, e.g., encapsulated within the carriers, also can benefit the targeted deposition of a molecule to tissue, particularly when the encapsulated drug is released only at or near the intended tissue.

Encapsulation or attachment of at least one therapeutic agent into or onto a carrier, e.g., microbubble, creates a drug/drug combination, drug/drug conjugate, carrier/drug conjugate, or a drug/drug complex. Other classes of active agents or drugs or molecules (whether within a specific class or in combination with a different class) may also be encapsulated, including but not limited to, small molecules, proteins, DNA, RNA, genes, fragments of genes, interferons, poly(ADP-ribose) polymerase inhibitors, aptamers, polyclonal antibodies, antibody drug conjugates, bispecifics, nanoparticles, liposomes, micelles, silica particles, metallic particles, nanobots, antibiotics, molecules, repurposed agents, or compositions up to or exceeding approximately 4 megadaltons in molecular weight. The encapsulation process may be performed either at the time of administration (e.g., by the clinician, nurse, pharmacist or technician), or near the time of administration (e.g., at the pharmacy), or at any point prior to the time of administration (e.g., including but not necessarily at the manufacturing or distribution stage).

Cytokines

The active agent for use in the compositions and methods of the invention comprises a cytokine, and/or a vector or plasmid encoding the cytokine. Exemplary cytokines may include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, interferon, GM-CSF or TNF-alpha.

Aldesleukin (brand name Proleukin®, Prometheus Laboratories Inc., San Diego, CA, USA) is a human recombinant interleukin-2 agent that is FDA approved to treat certain solid tumor cancer patients. Aldesleukin is a highly purified protein with an approximate molecular weight of 15.3 kDa. Aldesleukin has been shown to have a beneficial clinical effect for a subset of metastatic melanoma and renal cell carcinoma patients, but also is very toxic. It would be advantageous to be able to encapsulate into a microbubble carrier and to release therapeutically relevant amounts of aldesleukin in a controlled manner to allow such released aldesleukin to extravasate out of the vasculature into the desired target tissue. By encapsulating an amount (in a microbubble carrier) that is less than the clinical dose of 600,000 International Units (IU)/kg (0.037 mg/kg) of aldesleukin (https://www.proleukin.com), the present invention describes the delivery of therapeutically relevant rIL-2 doses at levels expected to have a lower toxicity profile.

In safety and efficacy studies leading to its eventual FDA approval and as described in its package insert, Proleukin was “given by 15-minute intravenous infusion every 8 hours for up to 5 days (maximum of 14 doses). No treatment was given on days 6 to 14 and then dosing was repeated for up to 5 days on days 15 to 19 (maximum of 14 doses). These 2 cycles constituted 1 course of therapy. Patients could receive a maximum of 28 doses during a course of therapy. In practice >90% of patients had doses withheld. Doses were withheld for specific toxicities . . . ” Furthermore, the Proleukin package insert stated that there is a “Lack of efficacy with low dose [systemically administered] Proleukin regimens.” In a 65-patient clinical study with metastatic renal cell carcinoma, it was determined that two low doses Proleukin regimens were ineffective. The Proleukin regimens administered 18,000,000 international units (IU) as a single subcutaneous injection, daily for 5 days during week 1; then 9×10⁶IU days 1-2, and 18×10⁶IU days 3-5, weekly for an additional 3 weeks (n=40) followed by a 2-week rest or 5 weeks (n=25) followed by a 3 week rest, for a maximum of 3 or 2 treatment cycles, respectively. (See https://www.proleukin.com/.)

Local delivery is generally considered as a more effective approach than systemic delivery for most therapeutic agents. In addition, given the complexities associated with intratumoral injections and the systemic toxicity of high dose IL-2, there is a clear need to deliver therapeutically relevant but low dose amounts of IL-2 systemically that can provide reduced systemic toxicity.

The present invention is a novel drug/drug complex of a cytokine (e.g., IL-2, or preferably a recombinant Human IL-2 (rIL-2)) and a carrier (e.g., a microbubble, or preferably Imagent). In summary, using the Syringe Method (as described below) and reconstituting vials of rIL-2 (1 mg) and Imagent separately, then after centrifugation to separate the microbubbles from the infranatant and expressing out the infranatant leaving the “cream” layer of microbubbles, rIL-2 was added to the syringe. The syringe containing rIL-2 and Imagent was then placed in an incubation bath, further centrifuged and the suspension washed to eliminate as much “free” or un-encapsulated rIL-2 from the syringe for injection of primarily only Imagent encapsulated rIL-2 creating a novel drug/drug complex herein referred to as SonoLeukin™, or “eIL-2”. Clinically relevant levels of rIL-2 encapsulated in Imagent (after wash step) were confirmed using human IL-2 Quantikine ELISA Kits (R&D Systems, Minneapolis, MN, USA).

In the present invention, a surprising and unexpected in vivo result was the signal of efficacy with low dose systemically administered rIL-2 shown in an MC38 colon cancer tumor animal model. The present invention describes encapsulated low concentrations of rIL-2 (GenScript®, Piscataway, NJ, USA) in the Imagent microbubbles (the combination referred to as “SonoLeukin™”). Novel drug/drug complexes were formed and used in an MC38 colon cancer tumor model to show the effect of SonoLeukin on tumor volume growth in mice (C57BL/6J mice, Jackson Labs, Bar Harbor, ME, USA). In an in vivo study also was shown the effect of eIL-2 co-administered with an RMP1-14 monoclonal antibody (aPD-1) that reacts with mouse PD-1 (Bio X Cell, West Lebanon, NH, USA) (aPD-1).

Using separate sterile vials of rIL-2 and Imagent microbubbles, the present invention combines two drugs (by Vial Method, as described below) into a single vial for injection without requiring the addition of linkers, ligands, binders, additives, or other chemical means. While the relative amount of rIL-2 encapsulated in the Imagent microbubbles may be low (<50% of starting rIL-2 concentration, relative amount encapsulated in the present invention ranging preferably from 0.01-5.0% or more), of particular relevance is the absolute amount of rIL-2 molecules encapsulated in the microbubble carrier and how much of IL-2 is actually delivered to the target tissue (e.g., tumor).

An embodiment of the present invention consisting of microdoses of encapsulated IL-2 injected systemically alongside anti-PD-1 monoclonal antibody (Group 7) exhibited a benefit above and beyond either the monoclonal antibody or encapsulated IL-2 alone. Key measures such as, but not limited to, G-CSF, GM-CSF, IFNγ, IL-12 and IP-10 were unexpectedly shown to be affected, suggestive of a clinical benefit.

Additional cytokines for use in the compositions of the invention may include IL-10, IL-12, IL-15, or IL-23, or TNF-alpha.

In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprise a vector or a plasmid comprising a gene encoding any one of the cytokines, e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-23, TNF-alpha, G-CSF, GM-CSF, IFNγ.

Monoclonal Antibodies (mAbs)

In one embodiment, the active agent for use in the compositions of the invention comprises antibodies, e.g., monoclonal antibodies, and/or a vector or plasmid comprising a gene encoding the antibody, e.g., a gene encoding the light chain of the antibody, a gene encoding the heavy chain of the antibody, and/or a gene encoding the heavy and light chain of the antibody. Exemplary antibodies may include, but are not limited to, bevacizumab, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, rituximab, cetuximab or trastuzumab. In some embodiments, the active agent for use in the compositions and methods of the present invention comprise Bevacizumab, or a vector or a plasmid encoding the light chain and heavy chain of Bevacizumab. In some embodiments, the active agent for use in the compositions and methods of the present invention comprise Pembrolizumab, or a vector or a plasmid encoding the light chain and heavy chain of Pembrolizumab.

Bevacizumab: Bevacizumab (brand name Avastin®, Genentech, Inc., South San Francisco, CA, USA) is a recombinant humanized monoclonal IgG1 antibody that acts as a vascular endothelial growth factor inhibitor and has an approximate molecular weight of 149 kDa. Per the manufacturer, bevacizumab binds VEGF and prevents the interaction of VEGF to its receptors (Flt1 and KDR) on the surface of endothelial cells, thereby acting to inhibit or prohibit angiogenesis. Avastin is administered as an intravenous infusion over 30-90 minutes and is dosed between 5-15 mg/kg every 2-3 weeks, either as a monotherapy or in combination with other therapeutic agents, depending on clinical indication.

According to the Avastin package insert (https://www.gene.com/download/pdf/avastin_prescribing.pdf), serious and sometimes fatal adverse events may occur when patients received Avastin, including but not limited to, gastrointestinal perforations (0.3-3.0%), Grade 3-5 hemorrhagic events (0.4-7.0%), serious or fatal pulmonary hemorrhage (4-31%), Grades 3-5 Arterial Thromboembolic Events (5%), Grades 3-4 Venous Thromboembolic Events (11%), Grades 3-4 hypertension (5-18%), proteinuria (20%), and ovarian failure (34%). Meanwhile, documentation provided by the manufacturer to the European Medicines Authority (https://www.ema.europa.eu/en/medicines/human/EPAR/avastin) stated “dose reduction for adverse reactions [of Avastin] is not recommended. If indicated, therapy should either be permanently discontinued or temporarily suspended.” Given the high rate of adverse events associated with Avastin, there exists a clear need to improve the safety and efficacy profile of bevacizumab.

The present invention is a novel drug/drug complex of a monoclonal antibody (e.g., bevacizumab) and a carrier (e.g., a microbubble, or preferably Imagent). In summary, using the Syringe Method (as described below) and reconstituting vials of bevacizumab (100 mg) and Imagent separately, then after centrifugation to separate the microbubbles from the infranatant and expressing out the infranatant leaving the “cream” layer of microbubbles, bevacizumab was added to the syringe. The syringe containing bevacizumab and Imagent was then placed in an incubation bath, further centrifuged and the suspension washed to eliminate as much “free” or un-encapsulated bevacizumab from the syringe for injection of primarily only Imagent encapsulated bevacizumab creating a novel drug/drug complex herein referred to as “eBEV”. Bevacizumab was encapsulated in Imagent (after wash step) as confirmed by using human VEGF Quantikine ELISA Kits (R&D Systems, Minneapolis, MN, USA).

In the present invention, a surprising and unexpected in vitro result was the ability to encapsulate clinically relevant levels of bevacizumab with commercially available carriers (e.g., microbubbles, such as, but not limited to, Imagent) without modifying the surface of carrier or adding binding agents or ligands. Testing in vivo was expected to demonstrate an increased delivery of bevacizumab to a tumor microenvironment using either eBEV alone or a combination of eBEV and free bevacizumab, compared to intravenous injection of bevacizumab alone.

The present invention uses a manufactured carrier, Imagent, and without adding any chemicals or molecules at the manufacturing stage, adding a monoclonal antibody to create a microbubble drug conjugate having sufficient therapeutic agent encapsulated onto the carrier and delivered to the tissue target to have a clinically meaningful beneficial effect. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprise a vector or a plasmid comprising a gene encoding the antibody, e.g., Bevacizumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the light chain of the antibody, a gene encoding the heavy chain of the antibody, and/or a gene encoding the heavy and light chain of the antibody, e.g., Bevacizumab.

Pembrolizumab: Pembrolizumab (brand name Keytruda®, Merck & Co., Kenilworth, NJ, USA) is a programmed death receptor-1 (PD 1)-blocking humanized monoclonal IgG4 kappa antibody and has an approximate weight of 149 kDa. Per the manufacturer, pembrolizumab binds to the PD-1 receptor and blocks the receptor's interaction with PD-L1 and PD-L2, releasing PD-1 pathway-mediated anti-tumor immune response. Pembrolizumab is administered as an intravenous infusion over 30 minutes and is dosed between 200 or 400 mg every 3 or 6 weeks, either as a monotherapy or in combination with other therapeutic agents, depending on clinical indication.

While immune checkpoint inhibitors (ICI) are approved for use in a number of solid tumors and patients, depending on the clinical indication, as many as 40-85% of treated patients do not respond to treatment. Certain patients may be more likely to respond to ICI treatment if more ICI molecules or ICI activated cells are present in the TME, for example those whose “cold” tumors are turned “hot”. In the present invention, an ICI may be co-administered with a carrier or may be encapsulated within a carrier in order to improve the biodistribution of the ICI within the TME or to the tumor cells.

The present invention used Imagent with acoustic exposures from a standard diagnostic ultrasound system and was able to significantly benefit tumor inhibition and tumor immune environment by increasing the key measure of CD8+/Treg ratio in the tumor in combination with anti-PD-1 checkpoint inhibitor at standard doses. The fact that tumor inhibition with sonoporation was equivalent to and or better than checkpoint inhibition alone demonstrated that the sonoporation effect does not diminish the activity of the checkpoint inhibitor.

The present invention contemplates a carrier (e.g., Imagent) used to encapsulate antibodies (monoclonal or polyclonal) or vectors or plasmids encoding the antibodies, e.g., the light and heavy chains of the antibodies, such as, but not limited to, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, bevacizumab, rituximab, cetuximab, trastuzumab. Additionally, a microbubble may be used to cause sonoporation when co-administered with an antibody. Furthermore, a microbubble encapsulated drug (other than an antibody) may be used to cause sonoporation when co-administered with an agent different from the encapsulated agent.

An advantage of encapsulation would be a reduction in side effects as a smaller amount of the antibody would be delivered since the encapsulation efficiency of microbubbles is <100% of the agent being bound or encapsulated. In situations where the carrier (whether a drug is encapsulated or not into the carrier) is co-administered with an antibody, sonoporation would lead to greater biodistribution of the antibody to the target, thereby reducing the amount of antibody going off target and the toxicity related to antibody dose. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprise a vector or a plasmid comprising a gene encoding the antibody, e.g., Pembrolizumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the light chain of the antibody, a gene encoding the heavy chain of the antibody, and/or a gene encoding the heavy and light chain of the antibody, e.g., Pembrolizumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the heavy chain of Pembrolizumab comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the light chain of Pembrolizumab comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the heavy chain of Pembrolizumab comprising the amino acid sequence of SEQ ID NO: 1, and a vector or a plasmid comprising a gene encoding the light chain of Pembrolizumab comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the light chain and heavy chain of Pembrolizumab are encoded in a single plasmid or a single vector. In some embodiments, the light chain and heavy chain of Pembrolizumab comprises the amino acid sequence of SEQ ID NO: 5.

Ipilimumab: In some embodiments, the monoclonal antibody is Ipilimumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprise a vector or a plasmid comprising a gene encoding the antibody, e.g., Ipilimumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the light chain of the antibody, a gene encoding the heavy chain of the antibody, and/or a gene encoding the heavy and light chain of the antibody, e.g., Ipilimumab. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the heavy chain of Ipilimumab comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the light chain of Ipilimumab comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the carriers, e.g., microbubbles, e.g., Imagent, comprises a vector or a plasmid comprising a gene encoding the heavy chain of Ipilimumab comprising the amino acid sequence of SEQ ID NO: 3, and a vector or a plasmid comprising a gene encoding the light chain of Ipilimumab comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain and heavy chain of Ipilimumab are encoded in a single plasmid or a single vector. In some embodiments, the light chain and heavy chain of Ipilimumab comprises the amino acid sequence of SEQ ID NO: 6.

Gene Editing System

In some embodiments, the active agents for use in the compositions and methods of the present invention comprise one or more components of a gene editing system. Components of gene editing systems may be used in a variety of contexts including but not limited to gene editing. For example, such components may be used to target agents that physically modify, genetically modify, and/or epigenetically modify any gene sequences.

Exemplary gene editing systems include the clustered regulatory interspaced short palindromic repeat (CRISPR) system, zinc finger nucleases (ZFNs), and Transcription Activator-Like Effector-based Nucleases (TALEN). ZFNs, TALENs, and CRISPR-based methods are described, e.g., in Gaj et al. Trends Biotechnol. 31.7(2013):397-405; CRISPR methods of gene editing are described, e.g., in Guan et al, Application of CRISPR-Cas system in gene therapy: Preclinical progress in animal model. DNA Repair 2016 July 30 [Epub ahead of print]; Zheng et al, Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. BioTechniques, Vol. 57, No. 3, September 2014, pp. 115-124.

CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpf1) to cleave foreign DNA. In a typical CRISPR/Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence. The crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid. The crRNA/tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence must generally be adjacent to a “protospacer adjacent motif (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcus thermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3), and 5′-NNNGATT (Neisseria meningiditis). Some endonucleases, e. g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5′-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5′ from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpf1 system requires only the Cpf1 nuclease and a crRNA to cleave the target DNA sequence. Cpf1 endonucleases, are associated with T-rich PAM sites, e. g., 5′-TTN. Cpf1 can also recognize a 5′-CTA PAM motif. Cpf1 cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5′ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3′ from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e. g., Zetsche et al. (2015) Cell, 163:759-771.

A variety of CRISPR associated (Cas) genes or proteins can be used in the present invention and the choice of Cas protein will depend upon the particular conditions of the method.

Specific examples of Cas proteins include class II systems including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, the site-specific targeting moiety includes a sequence targeting polypeptide, such as an enzyme, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram positive bacteria or a gram negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus, (e.g., a S. pyogenes, a S. thermophilus) a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter. In some embodiments nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, may be introduced into a cell, zygote, embryo, or animal, e.g., to allow for recognition and modification of sites comprising the same, similar or different PAM motifs. In some embodiments, the Cas protein is modified to deactivate the nuclease, e.g., nuclease-deficient Cas9, and to recruit transcription activators or repressors, e.g., the co-subunit of the E. coli Pol, VP64, the activation domain of p65, KRAB, or SID4X, to induce epigenetic modifications, e.g., histone acetyltransferase, histone methyltransferase and demethylase, DNA methyltransferase and enzyme with a role in DNA demethylation (e.g., the TET family enzymes catalyze oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidative derivatives).

For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least about 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1 at least about 16 nucleotides of gRNA sequence is needed to achieve detectable DNA cleavage.

Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut the target DNA but interferes with transcription by steric hindrance. dCas9 can further be fused with a heterologous effector to repress (CRISPRi) or activate (CRISPRa) expression of a target gene. For example, Cas9 can be fused to a transcriptional silencer (e.g., a KRAB domain) or a transcriptional activator (e.g., a dCas9-VP64 fusion). A catalytically inactive Cas9 (dCas9) fused to FokI nuclease (“dCas9-FokI”) can be used to generate DSBs at target sequences homologous to two gRNAs. See, e. g., the numerous CRISPR/Cas9 plasmids disclosed in and publicly available from the Addgene repository (Addgene, 75 Sidney St., Suite 550A, Cambridge, MA 02139; addgene.org/crispr). A “double nickase” Cas9 that introduces two separate double-strand breaks, each directed by a separate guide RNA, is described as achieving more accurate genome editing by Ran et al. (2013) Cell, 154: 1380-1389.

CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1.

In some embodiments, the active agents for use in the compositions and methods of the present invention comprise one or more components of a CRISPR system described herein. In some embodiments, the active agent comprises a vector a plasmid comprising a cDNA encoding a Cas9 protein. In some embodiments, the active agent comprises a vector a plasmid comprising a guide RNA. In some embodiments, the active agent comprises a vector a plasmid comprising a cDNA encoding a gene of interest or fragment thereof. In some embodiments, the active agent comprises a vector a plasmid comprising a cDNA encoding a Cas9 protein, a guide RNA, and/or a gene of interest or fragment thereof. In some embodiments, the active agent comprises a synthetic mRNA encoding a Cas9 protein. In some embodiments, the active agent comprises a synthetic mRNA encoding a target protein. In some embodiments, the active agent comprises a synthetic mRNA encoding a Cas9 protein and a target protein. In some embodiments, the active agent comprises a Cas9/guide RNA ribonucleoprotein (RNP) complex.

In some embodiments, the active agents for use in the compositions and methods of the invention include, for example, nucleases, physical blockers, epigenetic recruiters, e.g., a transcriptional enhancer or a transcriptional repressor, and epigenetic CpG modifiers, e.g., a DNA methylase, a DNA demethylase, a histone modifying agent, or a histone deacetylase, and combinations of any of the foregoing.

In some embodiments, the one or more components of the gene editing system are included in an adenoviral or AAV vectors. In some embodiments, the active agents include an adenoviral or AAV vector encoding Cas9, a guide RNA and/or a template DNA.

Oncolytic Viruses

The compositions of the present invention may further comprise a virus, e.g., an oncolytic virus. Oncolytic viruses (OV) are modified to infect and replicate to kill cancer cells. Adenoviruses are used as an OV because of their high cell transduction and their safety profile. A multi-targeting conditionally replicating adenovirus that displays enhanced oncolysis while maintaining expression of immunotherapeutic agents has been shown to lyse tumor cells effectively at low titers (Dobbins 2015).

The present invention is a novel drug/drug complex of an OV (e.g., adenovirus, and preferably an Ad5/3-RGD-D24) and a carrier (e.g., a microbubble, or preferably Imagent). An advantage of encapsulating an Ad5/3-RGD-D24 into a microbubble is protecting the OV from immune response while in the vasculature until the application of ultrasound energy to induce cavitation and sonoporation, thereby releasing the OV into the TME. Via sonoporation, encapsulating the Ad5/3-RGD-D24 into Imagent and inactivating the free OV prior to administration, low amounts of virus particles would be delivered into the TME and given OV's effectiveness at low titers, the complex would still be effective against tumors. Efficacy of the Ad5/3-RGD-D24 OV in the present invention can be enhanced by loading certain transgenes (such as, but not limited to, p53, GM-CSF, immune checkpoint inhibitors, cytokines, other transgenes) into the OV prior to encapsulation by the microbubble.

Significant challenges to OV therapy may be overcome by the present invention of a drug/drug complex, namely enabling more efficient extravasation of the OV to the tumor microenvironment via sonoporation, reduced innate immune response from antiviral immunity, preexisting circulating antibodies, or complement proteins.

Other oncolytic virus complexes of the present invention include, but are not limited to, talimogene laherparepvec (T-VEC®), type 1 herpes simplex virus, poliovirus, measles virus, Newcastle disease virus, reovirus, coxsackievirus, vesicular stomatitis virus, Zika virus, RNA virus, DNA virus, etc.

C. eDrug+Co-Administered CAR T

Chimeric antigen receptor T cells (CAR T cells) are an emerging immunotherapy class of agents that have shown tremendous efficacy in hematological malignancies, but to date have shown limited efficacy against solid tumors. Exogenous IL-2 has been shown to enhance the survival and proliferation of adoptive T cells.

The present invention combines encapsulated agents, e.g., cytokines such as IL-2, to enhance CAR T therapy. By targeting the delivery of agents to modify the TME or to stimulate immune cells including T cells, natural killers or dendritic cells, the present invention provides important advantages to improve clinical outcomes. In the case of IL-2 or other potent but highly toxic agents, improving the safety profile or the biodistribution of such agents at the target tissue can not only provide single agent benefit, but also synergistic or additive benefit.

D. eDrug A+eDrug B, or e(Drug A+Drug B)

The present invention also provides compositions comprising a plurality of carriers, e.g., microbubbles, wherein at least one, e.g., two, three, four or more, active agents are associated with, e.g., encapsulated within the carriers, and/or bound to the surface of the carriers. In some embodiments, one of the active agents may comprise a therapeutic agent, e.g., chemotherapeutic, and the other active agent may comprise an antibiotic agent, e.g., ciprofloxacin. An example of the present invention combines different classes of agents either into, along with a carrier, or both within and along with a carrier. For example, a combination of separately administered chemotherapy (gemcitabine) and an antibiotic has been shown to have benefit in a mouse model of pancreatic cancer (Geller 2017). In the case of antibiotics, long-term treatment may lead to the emergence of antibiotic-resistant bacterial strains, and the use of antibiotics may also affect the response to cancer treatments (Bashiardes 2017).

Alternatively, the present invention describes a dosing of a patient using an encapsulated therapeutic agent and another separate encapsulated agent (both of which may be delivered through the same syringe, or through separate syringes).

The present invention describes the encapsulation of one or more agents, each of which may be in the same class of drug or may be in different classes of drugs (e.g., small molecules, proteins, antibodies, nucleic acids, viruses, etc.). For example, two chemotherapy agents may be encapsulated (e.g., gemcitabine and abraxane) into a carrier. Multiple agents of the same class may be encapsulated into a carrier, for example, oxaliplatin, folinic acid, irinotecan and fluorouracil (representing an encapsulated version of FOLFIRINOX). Or a chemotherapy agent (e.g., gemcitabine) and an antibiotic agent (e.g., ciprofloxacin) may be encapsulated into a carrier. Alternatively, for example, a TME modulating agent may be encapsulated along with an immunostimulatory agent. Additionally, for example, a monoclonal antibody and a small molecule, or a bispecific or multispecific drug may be encapsulated into a carrier with or without an additional agent.

The present invention also contemplates combining treatment of an encapsulated (or unencapsulated) carrier with a different agent. Such combination may be administered at or around the same time (generally within 24 hours of each other), may be administered on different schedules (for example, Drug A administered at time 0 (to) and eDrug B administered either before (t₋₁) or after (t₊₁) Drug A. Another dosing schedule may be Drug A and eDrug B are co-administered on day 0 (d₀), eDrug B is then administered on a subsequent day (d₊₁), Drug A and eDrug B are again co-administered on yet another subsequent day (d₊₂), and the dosing cycle is repeated (or amended). The drugs may be administered on the same day or on different days, or in any combination of days.

In certain embodiments of the present invention, an encapsulated agent(s) may be administered as often, more often, or less often than a different agent. Alternatively, in the present invention, a carrier (without an encapsulated agent) may be administered as often, more often, or less often than a different agent. A consideration of the present invention dictating the frequency and specific dosing days of either the encapsulated agent or the (unencapsulated) carrier may be the circulation time of the agent used in combination with either the encapsulated agent or the unencapsulated carrier. For example, a monoclonal antibody such as aPD-1 may stay in a patient's circulatory system for several weeks, while encapsulated IL-2 may stay in circulation for only a matter of minutes or hours. Even though the encapsulated IL-2 has a short circulatory duration, the sonoporation effect of the present invention will permeabilize the vasculature allowing any circulating aPD-1 (that may have been administered at a time pre-dating the administration of the encapsulated IL-2) to reach the target tumor tissue. Alternatively, an unencapsulated carrier (typically also having a short circulatory duration) may be administered as often, more often, or less often than a different agent, such as aPD-1.

E. Bioeffects

A carrier (e.g., microbubble) under the influence of an energy source (e.g., ultrasound) may elicit bioeffects leading to significant anticancer immune response and significantly suppressed tumor progression (Liu, 2012). In contrast, rather than observing the bioeffects, the present invention describes the use of a carrier under the influence of an energy source to create bioeffects, such as, but not limited to, ROS at desired levels. The measurement of the desired ROS levels in vivo may be conducted using imaging or the inclusion of protein-based indicators. For example, by administering a greater concentration of microbubbles and using certain ultrasound parameters to direct energy to the target area, an increase in ROS to a targeted level may be reached, thereby eliciting the desired bioeffect. For example, it may be beneficial for anti-tumor activity if reactive oxygen species levels could be manipulated to modify the TME (Weinberg 2019). Bioeffects, such as, but not limited to, ROS may be affected whether the carrier is delivered with or without a therapeutic agent. In the case of co-administration of the carrier with another drug, certain bioeffects can be attained or inhibited by manipulation of such factors as, but not limited to, type of carrier used, device operating parameters, amount of carrier introduced into the target area or vicinity, duration of treatment, etc.

A contributing factor to chemotherapy toxicity may be the generation of excess ROS production in normal tissue. The present invention describes a means to increase (or decrease) the level or range of desired bioeffects to both a target level and location. In other words, if for example, the desired level of ROS production within a TME is x, then providing certain ultrasound operating parameters along with the presence of microbubbles in the tumor vasculature would induce the target level of ROS in the TME.

If a target level or range of bioeffects such as, but not limited to, ROS, Heat-shock protein 60 (hsp60), calcium ions, CD8+ T cell recruitment, CD8+/Treg ratio is desired at a certain tissue target, then under the present invention certain carrier or ultrasound parameters may be used in combination to achieve such desired level or range at such location. In particular, conventional diagnostic ultrasound probes, e.g., sector arrays, linear arrays, curvilinear arrays, may be used to induce the level or range of bioeffects desired for a therapeutic use of a carrier or microbubble. The target bioeffects achieved at the desired tissue may result from such factors as, but not limited to, specific cavitation states at specific intervals, microbubble concentrations, ultrasound frequencies, duty cycles, pulse lengths, peak negative pressures, mechanical indices, etc.

Encapsulation of an agent provides several benefits, including protecting a molecule while in blood circulation from either degradation (e.g., a nucleic acid), metabolism, inactivation or immunogenicity (e.g., an oncolytic virus). Encapsulation also can benefit the targeted deposition of a molecule to tissue, particularly when the encapsulated drug is released only at or near the intended tissue.

Targeted deposition of the therapeutic agent may be enhanced through the use of an energy source that is both aimed at the target tissue and interacts with the carrier only when the carrier is present within the energy field. For example, when a therapeutic molecule is encapsulated in a carrier (e.g., a microbubble) and the carrier is activated (e.g., stable or inertial cavitation) using an energy source (e.g., ultrasound), certain barriers (e.g., vessel walls, cell membranes, etc.) may become permeable as a result of mechanical shear forces from cavitation imparted onto the barrier surfaces (“sonoporation”). Additionally, certain bioeffects also may occur as a result of sonoporation. In the case of encapsulated agents, the therapeutic agent is released from the microbubble upon inertial cavitation, and since either stable or inertial microbubble cavitation is known to cause sonoporation, the released agent can pass through the permeabilized tissue into either the cells or tissue. In the case of therapeutic agents co-administered (and not encapsulated in the microbubbles), stable and inertial cavitation of the microbubbles still cause sonoporation of the ultrasound targeted barriers thereby allowing the freely circulating therapeutic agents to have a pathway into the cells or tissues by virtue of the permeabilized barrier.

III. Devices and Methods for Producing the Compositions of the Invention

The present disclosure provides methods for producing the compositions taught herein. In some embodiments, a vial of a carrier (e.g., a microbubble, such as Imagent) is supplied or reconstituted (if necessary) as per its manufacturer's instructions (e.g., Imagent is reconstituted with SWFI) and at least one drug, Drug A, is mixed with the carrier in suspension (herein referred to as “Syringe Method”). Alternatively, a vial of a carrier (e.g., a microbubble, such as Imagent) is instead reconstituted with a sterile solution of at least one drug, Drug A, rather than with SWFI and mixed (herein referred to as “Vial Method”). Various processes of forming complexes and combinations are herein disclosed. In some embodiments of the present disclosure, a complex is formed through the combination of two or more drugs, one of which is a microbubble (e.g., but not limited to, a perflexane gas in a lipid microbubble shell such as the microbubble referenced in FDA NDA 21-191) acting as the carrier, while (at least one) therapeutic agent is another drug. Examples are described in FIGS. 13-15, and 40-42.

Syringe Method

FIG. 13 is a schematic depicting a syringe method for making the compositions of the present disclosure. As shown in FIG. 13, in the syringe method, carrier MCC101 (e.g., Imagent) is reconstituted or provided as per manufacturer's instructions or packaging. For example, if carrier MCC101 is Imagent, then sterile water for injection (SWFI) is injected into the vial MCC102 to reconstitute Imagent powder (not shown). Carrier MCC101 is withdrawn into a syringe MCC103. Once carrier MCC101 suspension is in the syringe MCC103, the microbubbles are separated using any of a variety of means, e.g., centrifugation, spinning, filtration, gravity, etc. after which infranatant is expressed out of syringe MCC103 leaving a layer of primarily carrier MCC101. In some embodiments, after the phase separation step, diluent (not shown) may be added into the syringe MCC103 to re-suspend carrier MCC101. At least one drug, e.g., Drug X MCC101a, is drawn into syringe MCC103 so that Drug X MCC101a can be mixed with carrier MCC101 using any number of physical, mechanical, chemical, electrical means, e.g., incubation, stirring, agitation, shaking, rocking, rolling, amalgamation, linkers, binding agents, charges, etc. For example, the mix step using incubation may consist of placing the syringe in a water bath or incubation plate at 40° C. (or higher or lower) for up to 60 minutes (or more) with regular intervals of agitation.

The starting amount or concentration of Drug X MCC101a, Drug Y (not shown), Drug Z (not shown), etc. may be manipulated (e.g., higher, lower or the equal concentration of Drug X MCC101a than the amount that would be used as an agent separate from the present invention) such that, before drawing the diluent containing Drug X MCC101a into syringe MCC103, the desired concentration of Drug X MCC101a after the creation of the drug/drug complex is achieved. Differing amounts of carrier MCC101 and Drug X MCC101a, phase separation methods, mixing parameters all may influence the amount of agent encapsulated in the carrier.

Use of phase separation techniques provides the ability to express substantially all or a portion of infranatant, leaving desired levels of encapsulated drug (or the carrier without encapsulating any drug) remaining in microbubble suspension. One or more separation methods (e.g., centrifugation, flotation, filtration, chromatography, precipitation, spinning, vortexing, or other separation methods) may be utilized in one or more steps of the present disclosure as a means to separate the carrier (whether loaded with therapeutic agents or not) from the infranatant (or any “free” drug, i.e., drug not encapsulated or attached to the carrier). One advantage of separating the carrier (e.g., microbubble) is that, in the case of microbubbles, they will float to become the top layer (“supernatant”) in a liquid solution, whereupon the supernatant becomes a high concentration with which to use to encapsulate a therapeutic agent (or to deliver a high concentration of non-drug loaded microbubbles (per milliliter). Another advantage of separating the supernatant of microbubbles is that the amount of therapeutic agent encapsulated into the microbubbles and the amount of free drug (i.e., therapeutic agent not encapsulated into the microbubbles) can be quantified using, for example, an ELISA kit as in the case of IL-2.

After phase separation and a wash or inactivation step, a higher concentration of the carrier (with or without encapsulated agents) could be injected to achieve the desired therapeutic effect. For example, after normal preparation of a microbubble, a phase separation step would create a layer of microbubbles in the supernatant. Once the desired amount of microbubbles has been separated, the infranatant could be discarded by, e.g., being expressed out of the syringe containing the phase separated microbubbles. Prior to injection into a patient, the microbubbles could be resuspended in an isotonic solution (such as sterile saline) in an amount less than the amount of diluent indicated in the normal reconstitution process. For example, Imagent is reconstituted from a powder with 10 mL of SWFI; in the present disclosure, after phase separation, it may be desirable to resuspend Imagent in the isotonic solution with less than 10 mL prior to injection. Furthermore, Imagent microbubbles in supernatant from more than one syringe may be combined together prior to adding a diluent, or with small amounts of isotonic solution in each syringe before combining multiple syringes prior to injection. In certain instances, free drug in the mixture solution may be inactivated, eradicated, filtered or expressed out, bound to non-carrier components, e.g., antibodies, free lipids, etc., or eliminated from the solution to leave only or primarily encapsulated drug. Alternatively, such free drug that may be bound to non-carrier components, e.g., antibodies, free lipids, etc., may be injected along with the encapsulated drug.

To increase the amount of therapeutic agent that could be encapsulated into a carrier, the present disclosure may use at least one portion of a therapeutic agent, but likely two or more portions, per portion of a carrier. In some embodiments, depending on the starting agent concentration, used a ratio of multiple vials (e.g., 2 vials) of a therapeutic agent (IL-2) per 1 vial of Imagent. The higher the starting concentration of a manufactured agent, the fewer finished vials needed to mix with each vial of Imagent. If the therapeutic agent needs reconstitution and the amount of diluent may be reduced (without affecting the safety profile of the agent), then the reduced diluent used would result in a higher starting concentration of the agent that could be combined with the carrier for encapsulation. As described below in the “Vial Method”, to further increase the amount of IL-2 available for encapsulation into Imagent, each 1 mg vial of IL-2 (Genscript) was reconstituted with a small amount of SWFI, in this case 1.5 mL of SWFI was injected into each IL-2 vial and mixed. Then the contents of 2 vials of IL-2, each containing 1.5 mL of IL-2 in solution were withdrawn and injected into 1 vial of Imagent for a total solution of 3 mL for reconstitution of Imagent.

Vial Method

FIG. 14 is a schematic depicting a vial method for making the compositions of the present disclosure. As shown in FIG. 14, a method for encapsulating an agent into a carrier is herein referred to as the Vial Method. In this method, the carrier MCC201 is reconstituted using a solution containing Drug X MCC201a in the vial MCC202 substituting the agent solution for the fluid normally used to reconstitute the carrier MCC201. The amount and concentration of Drug X MCC201a used to reconstitute carrier MCC201 may be varied, depending on the desired amount of Drug X MCC201a to be delivered to the target tissue. Once the carrier MCC201 has been reconstituted using Drug X MCC201a, the solution is mixed by any of a variety of methods, including but not limited to such methods as described in the Syringe Method to enable Drug X MCC201a to bind or become encapsulated in the carrier MCC201. After the mix step, the encapsulated agent solution may be withdrawn using a syringe MCC203 so that a phase separation step is conducted (alternatively the phase separation step could be conducted in the vial MCC202). After the phase separation step is completed, the infranatant containing much of the free agent is expressed out of the syringe MCC203 leaving a layer of encapsulated Drug X MCC201a in the syringe MCC203 (any free drug not encapsulated in the reconstituted carrier may be further processed or concentrated and reintroduced into the syringe or vial containing the encapsulated carrier and mixed together to further increase the amount of drug encapsulated). With the layer of encapsulated Drug X MCC201a remaining in the syringe MCC203, an isotonic solution may be added into the syringe MCC203 prior to administration to assist in the injection of the encapsulated Drug X MCC201.

In the embodiment of encapsulated IL-2, the reconstituted vials of Imagent using a diluent containing IL-2 were then placed on an incubation shaker for a period of time, in this case for 60 minutes. After incubation, the vials were cooled in room temperature water for 5 minutes. After cooling, 2.0 mL of the Imagent/IL-2 drug/drug complex (SonoLeukin) were withdrawn into a syringe from the Imagent vial and the syringe was set at an approximately 75-degree angle for about 60-75 minutes to allow the microbubbles to float. After the flotation phase separation step, the infranatant was expressed out of the SonoLeukin syringe, with roughly 100 μL injected into a separate, marked tube for assay (each tube marked “AI1”, “BI1”, or “CI1”). Then 4 mL of sterile saline was added to the SonoLeukin syringe to resuspend the microbubbles (and providing a “partial” wash), after which 100 μL was injected into a separate, marked tube for assay (each tube marked “AMB1”, “BMB1”, or “CMB1”). Then the SonoLeukin syringe was again set at an approximately 75-degree angle for 60 minutes to allow the SonoLeukin to float to the top of the syringe. The infranatant was expressed out of the SonoLeukin syringe, with roughly 100 μL injected into a separate, marked tube for assay (each tube marked “AI2”, “BI2”, or “CI2”). Finally, 1.0 mL of sterile saline was added to the SonoLeukin syringe (representing a full wash step) to prepare the complex for injection, of which 100 μL was injected into a separate, marked tube for assay (each tube marked “AMB2”, “BMB2”, or “CMB2”). The amount of a therapeutic agent, e.g., IL-2, encapsulated in a carrier (i.e., Imagent) using the Syringe Method were measured using an ELISA kit and tabulated in FIG. 15 (which depicts the amount of IL-2 encapsulated within the Imagent microbubbles prepared using the syringe method). As shown in FIG. 15, the target amount of IL-2 within the SonoLeukin complex had mean concentrations of 2710.5 ng/mL after the first partial wash (AMB1, BMB1, CMB1), and 486 ng/mL after the second wash step (AMB2, MBM2, CMB2), thereby exceeding a target concentration of 61 ng/mL of IL-2.

A similar process was used in a separate experiment to encapsulate bevacizumab into Imagent (“Sonovastin”) and data on the amount of bevacizumab was collected.

Any free Drug X (not encapsulated in carrier MCC201) may need to be washed or inactivated prior to administration into a patient. For example, prior to administration, it may be desirous to have only or mostly encapsulated IL-2 or other highly toxic or immunogenic agents (e.g., chemotherapeutics, STING agonists, viruses, genes, etc.) remaining in the solution to limit systemic toxicity of or immune response to the administered agent. With certain therapeutic agents, like nucleic acid constructs or certain repurposed drugs, where systemic toxicity may be of less concern (e.g., compared to chemotherapeutics, STING agonists, or IL-2), a wash step may be omitted or modified as some amount of free drug in the carrier suspension may be acceptable or even desirable. A partial wash step may be acceptable to substantially reduce the amount of free drug, rather than eliminating substantially all the free drug prior to administration. Alternatively, any free drug not encapsulated in the microbubbles could instead be processed further and recaptured such that those free drug molecules could be reintroduced into the syringe containing the microbubbles (whether the microbubbles contain encapsulated drugs or not) and mixed together to increase the amount of drug ultimately encapsulated into the microbubbles. For example, in the case of negatively charged nucleic acids, those plasmids in the infranatant (not encapsulated in the carrier) may also be bound to the positively charged surfaces of any free lipids remaining after the reconstitution of Imagent. Those plasmids not encapsulated in the carrier, but bound and encapsulated by free lipids may also be either left in the syringe along with the carrier encapsulated drugs, expressed or washed out of the syringe, or further processed and reintroduced back into the syringe containing the carrier encapsulated drugs.

IV. Methods of Improving Efficacy of Therapeutics

The present invention further provides methods for improving efficacy of therapeutics, e.g., methods for treating a disease or disorder, e.g., cancer or non-cancer disease or disorder, methods for reducing tumor growth, and methods for delivering an active agent to a target site, in a subject.

The inventors of the present invention have surprisingly discovered that by associating or co-administering the active agent with carriers, the level of the active agent required to achieve a therapeutic effect is substantially lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated or co-administered with carriers, e.g., by systemic administration without being associated or co-administered with carriers.

In one aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In another aspect, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the carriers comprise a shell and a core, wherein (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) the core comprises a perfluorohexane gas and/or nitrogen; (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; and applying an ultrasound energy to the subject, thereby treating the disease or condition in the subject.

In one aspect, the present invention provides a method of reducing tumor growth in a subject, the method comprising administering to the subject a composition comprising at least one active agent associated with a plurality of carriers, wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and applying an ultrasound energy to the subject, thereby reducing tumor growth in the subject.