Phospholipid compounds and formulations

Abstract

The present disclosure provides phospholipid-containing compounds, pharmaceutical compositions and microspheres that exhibit high affinity for mineralized metals. The disclosure also provides strategies for using the compounds, compositions and microspheres in the treatment of renal lithiasis or renal lithiasis, as well as methods of making and making the compounds and compositions.

Description

[0001] This divisional application is a divisional application of the patent application with the application number 202080098465.9 and the title "Phospholipid Compounds and Formulations" submitted by the present applicant on January 28, 2020. The entire content of the parent case is incorporated into this divisional application by reference. Background Art

[0002] Microspheres having a fluid core surrounded by a thin shell composed of lipids, proteins, and / or sugars have been used in medical and therapeutic products. These micron-sized gas-core particles - sometimes referred to as microparticles - have been administered systemically to increase the information content of echocardiograms. Microspheres can be particularly useful for making the left ventricle of the heart opaque, thus enabling echocardiograms to more precisely reveal the details of aortic valve surgery. The class of microsphere products designed to be used in this way for disease diagnosis is often referred to as ultrasound contrast agents (UCA). In the use of UCA, when using mechanical energy (e.g., the ultrasonic waves that generate echocardiograms) and always occurring related mechanical responses of the microspheres, their function generally does not depend on the tissue effects from the mechanical responses of the microspheres, and in fact, they are often engineered to minimize tissue effects.

[0003] Microspheres can also be used in therapies where the mechanical response of the microspheres with directly or indirectly related tissue effects is central. For example, it has been proposed that for certain cancers, microspheres can be used for the targeted delivery of chemotherapeutic agents. For targeted delivery, microsphere design generally requires a combination of tumor-specific ligands in the shell structure and a fluid-phase chemotherapeutic agent in the core. After administration, the tumor-specific ligands in the shell are expected to cause the microspheres to accumulate in and around the tumor. Then, mechanical energy can be used to open the microspheres, thereby releasing the chemotherapeutic agent from the core.

[0004] The microsphere mechanical effect can also be used in the treatment of pathological biomineralization, as described in US Publication No. 2013 / 0123781, which is incorporated herein by reference in its entirety. In these treatments, the microspheres are designed to produce a pressure effect similar to shock wave lithotripsy, where a powerful shock wave is focused on a stone in the kidney or ureter. In shock wave lithotripsy, a focused high-intensity pressure wave with a peak pressure exceeding 100 megapascals generates a cavitation effect that gradually erodes, pitts, and fragments the stone. In microsphere-based lithotripsy, the microspheres accumulated on the surface of the stone can exhibit a cavitation effect with a pressure similar to or greater than that in conventional shock wave lithotripsy, but the input mechanical energy is approximately two orders of magnitude lower (Pishchalnikov et al., 2018); from both the clinical workflow and patient outcome perspectives, the relatively weak input acoustic energy compared to conventional shock wave lithotripsy can have various advantages. The accumulation of microspheres on the stone surface can be promoted by introducing a bisphosphonate-like component into the microsphere shell.

[0005] The mechanical effects of microspheres can be used to treat a variety of medical conditions involving abnormal or obstructive masses, such as kidney stones, urinary tract stones, gallstones, blood clots, fibroids, cancerous tumors, and atherosclerotic plaques. The treatment method allows for minimally invasive treatment of medical conditions by destroying or reducing the mass without damaging healthy tissue and minimizing the pain, discomfort, and risks associated with surgery or other invasive treatments.

[0006] Given the wide therapeutic applications of microspheres, efforts have been made to develop improved components for therapeutic microspheres. Despite these efforts, there remains a need in the art for safe and therapeutically effective improved phospholipid compounds. Additionally, it is important to develop phospholipid compounds that can be produced in large quantities and are of high quality for the therapeutic and commercial use of therapeutic microspheres. Summary of the Invention

[0007] The present disclosure provides compounds, pharmaceutical compositions, and microsphere particles (or microspheres or particles) that are hydrophobic and exhibit a high affinity for calcium and other metals in their mineralized forms, including biomineralization. In addition, the present disclosure provides methods for manufacturing and preparing the compounds, compositions, and microsphere particles, as well as methods for using them to treat medical conditions involving abnormal or obstructive masses, such as nephrolithiasis or kidney stone disease. The methods described herein allow for the large-scale production of high-quality microspheres, thereby enabling the therapeutic and commercial use of therapeutic microspheres.

[0008] Accordingly, in a first aspect, the present disclosure provides a compound of formula IV

[0009]

[0010] or a salt, isomer, or salt of an isomer thereof, wherein:

[0011] p is from 10 to 30;

[0012] q is from 1 to 100;

[0013] C is selected from:

[0014]

[0015] B is selected from: a covalent bond and ethyl;

[0016] A is selected from: a covalent bond, acyl, acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonamino, aminosulfonyl, amidino, and carboxylate; and

[0017] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxyl ester, phthalimido, SO3H, and PO3H.

[0018] In some embodiments, p is 14 or 16, and q is from 38 to 50.

[0019] In some embodiments, the compound of formula IV is the compound of formula I

[0020]

[0021] or a salt, isomer or salt of an isomer thereof, wherein: n is from 10 to 30, and

[0022] m is from 1 to 100.

[0023] In some embodiments, n is 14 or 16, and m is from 38 to 50. In some embodiments, the compound of formula I is the compound of formula Ia

[0024]

[0025] or a salt, isomer or salt of an isomer thereof.

[0026] In some embodiments, the compound of formula I is the compound of formula Ib

[0027]

[0028] In a second aspect, the present disclosure provides a pharmaceutical composition comprising: a compound of formula IV

[0029]

[0030] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0031] wherein:

[0032] p is from 10 to 30,

[0033] q is from 1 to 100,

[0034] C is selected from:

[0035]

[0036] B is selected from: a covalent bond and ethyl;

[0037] A is selected from: covalent bond, acyl group, acylamino group, aminoacyl group, acyloxy group, thioacyl group, aminocarbonyl group, aminoacylcarbonyloxy group, aminothiocarbonyl group, aminocarbonylamino group, aminothiocarbonylamino group, aminocarbonyloxy group, aminosulfonyloxy group, aminosulfonamino group, aminosulfonyl group, amidino group, amide and carboxylic acid ester; and

[0038] X is selected from: hydrogen, silyl group, acyl group, aminoacyl group, thioacyl group, aminocarbonyl group, aminoacylcarbonyloxy group, aminothiocarbonyl group, aminosulfonyl group, amidino group, substituted sulfonyl group, substituted sulfinyl group, carboxyl ester, phthalimido group, OH, SO3H and PO3H.

[0039] In some embodiments, p is 14 or 16, and q is from 38 to 50.

[0040] In some embodiments, the composition further comprises:

[0041] a compound of formula II

[0042]

[0043] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0044] a compound of formula III

[0045]

[0046] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0047] wherein:

[0048] t is from 10 to 30,

[0049] y is from 1 to 100, and

[0050] z is from 10 to 30.

[0051] In some embodiments, t is 14 or 16, z is 14 or 16, and y is from 38 to 50. In some embodiments, the compound of formula IV is the compound of formula I

[0052]

[0053] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof, wherein: n is from 10 to 30, and

[0054] m is from 1 to 100.

[0055] In some embodiments, n is 14 or 16, and m is from 38 to 50.

[0056] In some embodiments, the compound of formula I is a compound of formula Ia

[0057]

[0058] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof.

[0059] In some embodiments, the compound of formula Ia is a compound of formula Ib

[0060]

[0061] In some embodiments, the composition further comprises a fluid having a standard boiling point below 30 °C and optionally at least one pharmaceutically acceptable excipient. In some embodiments, the fluid is a gas at body temperature. In some embodiments, the fluid has low solubility in aqueous solution. In some embodiments, the fluid is air, nitrogen, argon, carbon dioxide (CO2), sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof. In some embodiments, the fluorinated C 1-6 alkane is selected from octafluoropropane, n-decafluorobutane and dodecafluoropentane.

[0062] In some embodiments, the composition comprises 0.01 - 5 mol% of the compound of formula IV or formula I or a salt, isomer or salt of an isomer thereof. In some embodiments, the composition comprises 5 - 9.9 mol% of the compound of formula II or a salt, isomer or salt of an isomer thereof. In some embodiments, the composition comprises 80 - 95 mol% of the compound of formula III or a salt, isomer or salt of an isomer thereof. In some embodiments, the composition comprises no more than 5 mol% of the compound of formula IV or formula I or a salt, isomer or salt of an isomer thereof. In some embodiments, the composition comprises no more than 10 mol% of the compound of formula II or a salt, isomer or salt of an isomer thereof. In some embodiments, in some embodiments, the composition comprises no more than 95 mol% of the compound of formula III or a salt, isomer or salt of an isomer thereof.

[0063] In some embodiments, the composition comprises an ammonium salt of the compound of formula IV or formula I. In some embodiments, the composition comprises an ammonium salt of the compound of formula II.

[0064] In some embodiments, the compound of formula IV or formula I has a molecular weight of 1,500 to 5,000 daltons. In some embodiments, the compound of formula II has a molecular weight of 1,500 to 5,000 daltons. In some embodiments, the compound of formula III has a molecular weight of 500 to 2,000 daltons.

[0065] In some embodiments, the molar ratio of the compound of Formula IV or Formula I to the compound of Formula II ranges from 1:100 to 1:1. In some embodiments, the molar ratio of the compound of Formula II to the compound of Formula III ranges from 1:20 to 1:8.

[0066] In some embodiments, the compound of Formula Ia is the compound of Formula Ib

[0067]

[0068] The compound of Formula II is the compound of Formula IIb

[0069]

[0070] The compound of Formula III is the compound of Formula IIIa

[0071] And

[0072] The composition comprises a fluid, wherein the fluid is n-decafluorobutane.

[0073] In some embodiments, the average molecular weight of the compound of Formula Ib is about 3200 daltons. In some embodiments, the average molecular weight of the compound of Formula IIb is about 2800 daltons. In some embodiments, the average molecular weight of the compound of Formula IIIa is about 790 daltons.

[0074] In some embodiments, the composition comprises 0.01 - 5 mol% of the compound of Formula Ib. In some embodiments, the composition comprises 5 - 9.9 mol% of the compound of Formula IIb. In some embodiments, the composition comprises 80 - 95 mol% of the compound of Formula IIIa. In some embodiments, the composition comprises no more than 5 mol% of the compound of Formula Ib. In some embodiments, the composition comprises no more than 10 mol% of the compound of Formula IIb. In some embodiments, the composition comprises no more than 95 mol% of the compound of Formula IIIa.

[0075] In some embodiments, the molar ratio of the compound of Formula Ib to the compound of Formula IIb ranges from 1:100 to 1:1. In some embodiments, the molar ratio of the compound of Formula IIb to the compound of Formula IIIa ranges from 1:20 to 1:8.

[0076] In some embodiments, the composition is capable of forming microspheres in the presence of water. In some embodiments, the composition comprises microspheres. In some embodiments, the microspheres comprise the compound of Formula Ib, the compound of Formula IIb, and the compound of Formula IIIa.

[0077] In some embodiments, the microspheres have an average diameter of from about 0.5 microns to about 10 microns. In some embodiments, the microspheres have an average diameter of from about 1 micron to about 5 microns.

[0078] In some embodiments, the composition comprises trehalose and povidone K12 (PLASDONE K12) as excipients. In some embodiments, the composition comprises microspheres present as a lyophilized dry powder or as an anhydrous concentrate.

[0079] In another aspect, the present disclosure provides a method of making microspheres, the method comprising:

[0080] (a) preparing a formulation for making microspheres, the formulation comprising: a compound of formula I

[0081]

[0082] or a salt, isomer or salt of an isomer thereof;

[0083] a compound of formula II

[0084]

[0085] or a salt, isomer or salt of an isomer thereof;

[0086] a compound of formula III

[0087]

[0088] or a salt, isomer or salt of an isomer thereof;

[0089] optionally at least one pharmaceutically acceptable excipient; and water,

[0090] wherein:

[0091] n is from 10 to 30,

[0092] m is from 1 to 100,

[0093] t is from 10 to 30,

[0094] y is from 1 to 100,

[0095] z is from 10 to 30, and

[0096] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxyester, phthalimido, OH, SO3H and PO3H.

[0097] In some embodiments, the method further comprises:

[0098] (b) Combine the formulation of step (a) with a fluid having a standard boiling point below 30 °C in a container; and

[0099] (c) Agitate the container containing the formulation and the fluid from step (b) to obtain microspheres.

[0100] In some embodiments, the method further comprises:

[0101] (d) Treat the microsphere solution from step (c) to extend its shelf life or expand the range of environmental conditions for storage.

[0102] In some embodiments, the method further comprises:

[0103] (e) Optionally under vacuum, stopper the container containing the microspheres from step (c) or (d).

[0104] In some embodiments, the formulation of step (a) is filtered before step (b). In some embodiments, the treatment step (d) is freeze-drying the microsphere solution.

[0105] In some embodiments, the container is a unit dose container. In some embodiments, the fluid is n-decafluorobutane. In some embodiments, the headspace of the capped container is filled with n-decafluorobutane.

[0106] In some embodiments, the average diameter of the microspheres from step (c) is about 0.1 to 1000 μm. In some embodiments, the average diameter of the microspheres from step (c) is about 0.1 to 100 μm. In some embodiments, the average diameter of the microspheres from step (c) is about 0.1 to 30 μm. In some embodiments, the average diameter of the microspheres from step (c) is about 0.7 to 10 μm.

[0107] In some embodiments, the treatment step (d) is freeze-drying the microsphere solution and the loss of microspheres during freeze-drying does not exceed 25%. In some embodiments, the loss of microspheres during freeze-drying does not exceed 15% or does not exceed 10%.

[0108] In some embodiments, the molecular weight of the compound of formula I is from 1,500 to 5,000 daltons. In some embodiments, the molecular weight of the compound of formula II is from 1,500 to 5,000 daltons. In some embodiments, the molecular weight of the compound of formula III is from 500 to 2,000 daltons.

[0109] In some embodiments, the molar ratio of the compound of formula I to the compound of formula II in the formulation is in the range of 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula II to the compound of formula III in the formulation is in the range of 1:20 to 1:8.

[0110] In some embodiments, the microspheres from step (c) comprise 0.01 - 5 mol% of the compound of formula I. In some embodiments, the microspheres from step (c) comprise 5 - 9.9 mol% of the compound of formula II. In some embodiments, the microspheres from step (c) comprise 80 - 95 mol% of the compound of formula III. In some embodiments, the microspheres from step (c) comprise no more than 5 mol% of the compound of formula I. In some embodiments, the microspheres from step (c) comprise no more than 10 mol% of the compound of formula II. In some embodiments, the microspheres from step (c) comprise no more than 95 mol% of the compound of formula III.

[0111] In some embodiments, the compound of formula I is the compound of formula Ib; the compound of formula II is the compound of formula IIb; and the compound of formula III is the compound of formula IIIa.

[0112] In some embodiments, the average molecular weight of the compound of formula Ib is about 3200 daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2800 daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 790 daltons.

[0113] In some embodiments, the microspheres from step (c) comprise 0.01 - 5 mol% of the compound of formula Ib. In some embodiments, the microspheres from step (c) comprise 5 - 9.9 mol% of the compound of formula IIb. In some embodiments, the microspheres from step (c) comprise 80 - 95 mol% of the compound of formula IIa.

[0114] In some embodiments, the microspheres from step (c) comprise no more than 5 mol% of the compound of formula Ib. In some embodiments, the microspheres from step (c) comprise no more than 10 mol% of the compound of formula IIb. In some embodiments, the microspheres from step (c) comprise no more than 95 mol% of the compound of formula IIIa.

[0115] In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb in the microspheres from step (c) is in the range of 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula IIb to the compound of formula IIIa in the microspheres from step (c) is in the range of 1:20 to 1:8.

[0116] In yet another aspect, the present disclosure provides a unit dose container comprising: a therapeutically effective amount of the pharmaceutical composition provided herein. In some embodiments, the compound of formula I is the compound of formula Ib. In some embodiments, the compound of formula II is the compound of formula IIb. In some embodiments, the compound of formula III is the compound of formula IIa. In some embodiments, the fluid is air, CO2, sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof. In some embodiments, the fluorinated C 1-6 alkane is selected from octafluoropropane, n-decafluorobutane, and dodecafluoropentane. In some embodiments, the fluid is n-decafluorobutane. In some embodiments, the composition comprises trehalose and povidone K12 as excipients. In some embodiments, the composition further comprises a buffer solution, wherein the buffer solution is phosphate. In some embodiments, the buffer solution comprises calcium- and magnesium-free saline.

[0117] In some embodiments, the composition is capable of forming microspheres in the presence of water. In some embodiments, the composition comprises microspheres. In some embodiments, the microspheres are present as a lyophilized dry powder or as an anhydrous concentrate. In some embodiments, the container is prepared by the method provided herein. In some embodiments, the container is sealed by a crimp-top cap equipped with a septum. In some embodiments, the container is airtight.

[0118] In one embodiment, the present disclosure provides a kit comprising: at least one unit dose container as described above, and instructions for using the kit.

[0119] In some embodiments, the unit dose container comprises trehalose and povidone K12 as excipients. In some embodiments, the kit further comprises a container containing an aqueous solution. In some embodiments, the aqueous solution is sterile water. In some embodiments, the aqueous solution is a saline solution ready for perfusion. In some embodiments, the kit further comprises a syringe. In some embodiments, the kit further comprises a needle equipped for the syringe. In some embodiments, the kit comprises a needleless syringe with a sharp tip. In some embodiments, the needle or sharp tip is capable of piercing the septum cap.

[0120] In some embodiments, the kit further comprises a container containing a fluid having a standard boiling point below 30°C, optionally wherein the container is a syringe. In some embodiments, the fluid is air, nitrogen, argon, CO2, sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof. In some embodiments, the fluorinated C 1-6 alkane is selected from octafluoropropane, n-decafluorobutane, and dodecafluoropentane. In some embodiments, the fluid is n-decafluorobutane.

[0121] In some embodiments, the kit further comprises at least one gel pad and ultrasound gel. In some embodiments, the kit further comprises a device selected from a device and a vented bottle adapter.

[0122] Another aspect of the present disclosure provides a method for reconstituting microspheres, wherein the method comprises:

[0123] (a) adding a sufficient amount of water or saline solution to the microspheres within a container of the present disclosure,

[0124] (b) optionally, adding a volume of a fluid having a standard boiling point below 30 °C to the container of step (a); and

[0125] (c) optionally shaking the container of step (a) or step (b).

[0126] In some embodiments, the method further comprises the step of filling the container with a gas before step (a). In some embodiments, the amount of water or saline solution does not exceed 100 milliliters. In some embodiments, the amount of water or saline solution added is sufficient to produce a homogeneous mixture comprising reconstituted microspheres. In some embodiments, the shaking of the container lasts no more than 180 seconds.

[0127] In yet another aspect, the present disclosure provides a method for treating urolithiasis, the method comprising: administering to a subject suffering from urolithiasis an effective amount of a pharmaceutical composition, microspheres or reconstituted microsphere solution disclosed herein such that the microspheres contact a urinary tract stone, and directing energy to the urinary tract stone within the subject's body at a frequency that excites the fluid within the microspheres.

[0128] In some embodiments, the reconstituted microsphere solution is administered through a catheter into the ureter of the subject. In some embodiments, the energy is in the form of electromagnetic, acoustic, microwave, photon or other forms. In some embodiments, the energy is ultrasound.

[0129] In some embodiments, the ultrasonic energy is in the frequency range of 100 kilohertz (kHz) to 2 megahertz (MHz). In some embodiments, the ultrasonic energy is associated with a peak pressure in the range of 0.1 MPa to 10 MPa. In some embodiments, the energy is applied for a sufficient amount of time to fragment the urinary tract stone.

[0130] In some embodiments, the amount of time does not exceed 100 minutes. In some embodiments, the amount of time does not exceed 90 minutes. In some embodiments, the amount of time does not exceed 80 minutes. In some embodiments, the amount of time does not exceed 70 minutes. In some embodiments, the amount of time does not exceed 60 minutes. In some embodiments, the amount of time does not exceed 50 minutes. In some embodiments, the amount of time does not exceed 40 minutes. In some embodiments, the amount of time does not exceed 30 minutes. In some embodiments, the amount of time does not exceed 25 minutes. In some embodiments, the amount of time does not exceed 20 minutes. In some embodiments, the amount of time does not exceed 15 minutes. In some embodiments, the amount of time does not exceed 10 minutes.

[0131] In some embodiments, the applied energy causes a volume change of the reconstituted microspheres or other cavitation effects of the microspheres. In some embodiments, cavitation of the microspheres results in a change in the pressure gradient and other mechanical effects in the urinary tract stone surrounding the reconstituted microspheres. In some embodiments, the change in the pressure gradient and other mechanical effects can fragment the urinary tract stone. In some embodiments, the subject is a human.

[0132] In one aspect, the present disclosure provides a reconstituted microsphere solution for the treatment of urolithiasis, wherein an effective amount of the microsphere solution is administered to a subject to bring the microspheres into contact with the urinary tract stone; and energy is directionally applied to the urinary tract stone in the subject's body at a frequency that excites the fluid within the microspheres.

[0133] In some embodiments, the reconstituted microsphere solution is administered into the ureter of the subject through a catheter. In some embodiments, the energy is in the form of electromagnetic, acoustic, microwave, photon, laser, or other forms. In some embodiments, the energy is ultrasonic waves.

[0134] In some embodiments, the ultrasonic energy is in the frequency range of 100 kilohertz (kHz) to 2 megahertz (MHz). In some embodiments, the ultrasonic energy is associated with a peak pressure in the range of 0.1 MPa to 10 MPa.

[0135] In some embodiments, the energy is in the form of a laser. In some embodiments, the laser energy has a wavelength in the infrared range of 1000 nm to 2500 nm. In some embodiments, the laser energy can evaporate the liquid in the lumen, thereby generating associated sound waves. In some embodiments, the laser energy has a frequency in the range between 1 kHz and 1 MHz.

[0136] In some embodiments, energy is applied for a sufficient amount of time to fragment urinary calculi. In some embodiments, the amount of time does not exceed 100 minutes. In some embodiments, the amount of time does not exceed 90 minutes. In some embodiments, the amount of time does not exceed 80 minutes. In some embodiments, the amount of time does not exceed 70 minutes. In some embodiments, the amount of time does not exceed 60 minutes. In some embodiments, the amount of time does not exceed 50 minutes. In some embodiments, the amount of time does not exceed 40 minutes. In some embodiments, the amount of time does not exceed 30 minutes. In some embodiments, the amount of time does not exceed 25 minutes. In some embodiments, the amount of time does not exceed 20 minutes. In some embodiments, the amount of time does not exceed 15 minutes. In some embodiments, the amount of time does not exceed 10 minutes.

[0137] In some embodiments, the applied energy causes a volume change of the reconstituted microspheres or other cavitation effects of the microspheres. In some embodiments, cavitation of the microspheres results in a change in the pressure gradient in the urine around the reconstituted microspheres and other mechanical effects. In some embodiments, the change in the pressure gradient and other mechanical effects are capable of fragmenting urinary calculi. In some embodiments, the subject is a human.

[0138] The present invention also includes the following items:

[0139] 1. A compound of formula IV

[0140]

[0141] or a salt, isomer or salt of an isomer thereof, wherein:

[0142] p is from 10 to 30;

[0143] q is from 1 to 100;

[0144] C is selected from:

[0145]

[0146] B is selected from: a covalent bond and ethyl;

[0147] A is selected from: a covalent bond, acyl, acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyl, amidino and carboxylate; and

[0148] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxylate ester, phthalimido, SO3H and PO3H.

[0149] 2. The compound according to item 1, wherein:

[0150] p is 14 or 16, and

[0151] q is from 38 to 50.

[0152] 3. The compound according to item 1 or 2, wherein the compound of formula IV is a compound of formula I

[0153]

[0154] or a salt, isomer or salt of an isomer thereof, wherein:

[0155] n is from 10 to 30, and

[0156] m is from 1 to 100.

[0157] 4. The compound according to item 3, wherein

[0158] n is 14 or 16, and

[0159] m is from 38 to 50.

[0160] 5. The compound according to item 3 or 4, wherein the compound of formula I is a compound of formula Ia

[0161]

[0162] or a salt, isomer or salt of an isomer thereof.

[0163] 6. The compound according to item 5, wherein the compound of formula I is a compound of formula Ib

[0164]

[0165] 7. A pharmaceutical composition, the pharmaceutical composition comprising:

[0166] A compound of formula IV

[0167]

[0168] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0169] wherein:

[0170] p is from 10 to 30,

[0171] q is from 1 to 100,

[0172] C is selected from:

[0173]

[0174] B is selected from: a covalent bond and ethyl;

[0175] A is selected from: a covalent bond, acyl, acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonamido, aminosulfonyl, amidino, amide, and carboxylate ester; and

[0176] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxylate ester, phthalimido, OH SO3H, and PO3H.

[0177] 8. The pharmaceutical composition according to item 7, wherein

[0178] p is 14 or 16, and

[0179] q is from 38 to 50.

[0180] 9. The pharmaceutical composition according to item 7 or 8, wherein the composition further comprises: a compound of formula II

[0181]

[0182] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0183] a compound of formula III

[0184]

[0185] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof;

[0186] wherein:

[0187] t is from 10 to 30,

[0188] y is from 1 to 100, and

[0189] z is from 10 to 30.

[0190] 10. The pharmaceutical composition according to item 9, wherein:

[0191] t is 14 or 16,

[0192] z is 14 or 16, and

[0193] y is from 38 to 50.

[0194] 11. The pharmaceutical composition according to any one of items 7 - 10, wherein the compound of formula IV is the compound of formula I

[0195]

[0196] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof, wherein:

[0197] n is from 10 to 30, and

[0198] m is from 1 to 100.

[0199] 12. The pharmaceutical composition according to item 11, wherein

[0200] n is 14 or 16, and

[0201] m is from 38 to 50.

[0202] 13. The pharmaceutical composition according to item 12, wherein the compound of formula I is the compound of formula Ia

[0203]

[0204] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof.

[0205] 14. The pharmaceutical composition according to item 13, wherein the compound of formula Ia is the compound of formula Ib

[0206]

[0207] 15. The pharmaceutical composition according to any one of items 7 - 14, wherein the composition further comprises a fluid having a standard boiling point below 30 °C and optionally at least one pharmaceutically acceptable excipient.

[0208] 16. The pharmaceutical composition according to item 15, wherein the fluid is a gas at body temperature.

[0209] 17. The pharmaceutical composition according to item 15 or 16, wherein the fluid has low solubility in aqueous solution.

[0210] 18. The pharmaceutical composition according to any one of items 15 - 17, wherein the fluid is air, nitrogen, argon, carbon dioxide (CO2), sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof.

[0211] 19. The pharmaceutical composition according to item 18, wherein the fluorinated C 1-6 alkane is selected from octafluoropropane, n - decafluorobutane and dodecafluoropentane.

[0212] 20. The pharmaceutical composition according to any one of items 7 - 19, wherein the composition comprises 0.01 - 5 mol% of the compound of formula IV or formula I or a salt, isomer or salt of an isomer thereof.

[0213] 21. The pharmaceutical composition according to any one of items 9 - 20, wherein the composition comprises 5 - 9.9 mol% of the compound of formula II or a salt, isomer or salt of an isomer thereof.

[0214] 22. The pharmaceutical composition according to any one of items 9 - 21, wherein the composition comprises 80 - 95 mol% of the compound of formula III or a salt, isomer or salt of an isomer thereof.

[0215] 23. The pharmaceutical composition according to any one of items 7 - 22, wherein the composition comprises no more than 5 mol% of the compound of formula IV or formula I or a salt, isomer or salt of an isomer thereof.

[0216] 24. The pharmaceutical composition according to any one of items 9 - 23, wherein the composition comprises no more than 10 mol% of the compound of formula II or a salt, isomer or salt of an isomer thereof.

[0217] 25. The pharmaceutical composition according to any one of items 9 - 24, wherein the composition comprises no more than 95 mol% of the compound of formula III or a salt, isomer or salt of an isomer thereof.

[0218] 26. The pharmaceutical composition according to any one of items 7 - 25, wherein the composition comprises an ammonium salt of the compound of formula IV or formula I.

[0219] 27. The pharmaceutical composition according to any one of items 9 - 26, wherein the composition comprises an ammonium salt of the compound of formula II.

[0220] 28. The pharmaceutical composition according to any one of items 7 - 27, wherein the molecular weight of the compound of formula IV or formula I is 1,500 to 5,000 daltons.

[0221] 29. The pharmaceutical composition according to any one of items 9 - 28, wherein the molecular weight of the compound of formula II is 1,500 to 5,000 daltons.

[0222] 30. The pharmaceutical composition according to any one of items 9 - 29, wherein the molecular weight of the compound of formula III is 500 to 2,000 daltons.

[0223] 31. The pharmaceutical composition according to any one of items 7 - 30, wherein the molar ratio of the compound of formula IV or formula I to the compound of formula II is in the range of 1:100 to 1:1.

[0224] 32. The pharmaceutical composition according to any one of items 9 - 30, wherein the molar ratio of the compound of formula II to the compound of formula III is in the range of 1:20 to 1:8.

[0225] 33. The pharmaceutical composition according to any one of items 7 - 32, wherein:

[0226] the compound of formula Ia is the compound of formula Ib

[0227]

[0228] the compound of formula II is the compound of formula IIb

[0229]

[0230] the compound of formula III is the compound of formula IIIa

[0231] and

[0232] the composition contains a fluid, and the fluid is n - decafluorobutane.

[0233] 34. The pharmaceutical composition according to item 33, wherein the average molecular weight of the compound of formula Ib is about 3200 daltons.

[0234] 35. The pharmaceutical composition according to item 33 or 34, wherein the average molecular weight of the compound of formula IIb is about 2800 daltons.

[0235] 36. The pharmaceutical composition according to any one of items 33 - 35, wherein the average molecular weight of the compound of formula IIIa is about 790 daltons.

[0236] 37. The pharmaceutical composition according to any one of items 33 - 36, wherein the composition contains 0.01 - 5 mol% of the compound of formula Ib.

[0237] 38. The pharmaceutical composition according to any one of items 33 - 37, wherein the composition contains 5 - 9.9 mol% of the compound of formula IIb.

[0238] 39. The pharmaceutical composition according to any one of items 33 - 38, wherein the composition contains 80 - 95 mol% of the compound of formula IIIa.

[0239] 40. The pharmaceutical composition according to any one of items 33 - 39, wherein the composition comprises no more than 5 mol% of the compound of formula Ib.

[0240] 41. The pharmaceutical composition according to any one of items 33 - 40, wherein the composition comprises no more than 10 mol% of the compound of formula IIb.

[0241] 42. The pharmaceutical composition according to any one of items 33 - 41, wherein the composition comprises no more than 95 mol% of the compound of formula IIIa.

[0242] 43. The pharmaceutical composition according to any one of items 9 - 37, wherein the molar ratio of the compound of formula Ib to the compound of formula IIb is in the range of 1:100 to 1:1.

[0243] 44. The pharmaceutical composition according to any one of items 9 - 30, wherein the molar ratio of the compound of formula IIb to the compound of formula IIIa is in the range of 1:20 to 1:8.

[0244] 45. The pharmaceutical composition according to any one of items 7 - 44, wherein the composition is capable of forming microspheres in the presence of water.

[0245] 46. The pharmaceutical composition according to any one of items 7 - 44, the pharmaceutical composition comprising microspheres.

[0246] 47. The pharmaceutical composition according to any one of items 45 - 46, wherein the microspheres comprise the compound of formula Ib, the compound of formula IIb, and the compound of formula IIIa.

[0247] 48. The pharmaceutical composition according to any one of items 45 - 47, wherein the microspheres have an average diameter of about 0.5 microns to about 10 microns.

[0248] 49. The pharmaceutical composition according to item 48, wherein the microspheres have an average diameter of about 1 micron to about 5 microns.

[0249] 50. The pharmaceutical composition according to any one of items 7 - 49, wherein the composition comprises trehalose and povidone K12 as excipients.

[0250] 51. The pharmaceutical composition according to any one of items 7 - 50, wherein the composition comprises microspheres present as a lyophilized dry powder or as an anhydrous concentrate.

[0251] 52. A method for manufacturing microspheres, the method comprising:

[0252] (f) Preparing a preparation for manufacturing microspheres, the preparation comprising:

[0253] A compound of formula I

[0254]

[0255] or a salt, isomer or salt of an isomer thereof;

[0256] A compound of formula II

[0257]

[0258] or a salt, isomer or salt of an isomer thereof;

[0259] A compound of formula III

[0260]

[0261] or a salt, isomer or salt of an isomer thereof;

[0262] Optionally at least one pharmaceutically acceptable excipient; and water,

[0263] wherein:

[0264] n is from 10 to 30,

[0265] m is from 1 to 100,

[0266] t is from 10 to 30,

[0267] y is from 1 to 100,

[0268] z is from 10 to 30, and

[0269] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxyester, phthalimido, OH, SO3H and PO3H.

[0270] 53. The method according to item 52, the method further comprising:

[0271] (g) Combining the preparation of step (a) with a fluid having a standard boiling point below 30 °C in a container; and

[0272] (h) Agitating the container containing the preparation and the fluid from step (b) to obtain microspheres.

[0273] 54. The method according to item 53, the method further comprising:

[0274] (i) Treat the microsphere solution from step (c) to extend its shelf life or expand the range of environmental conditions for storage.

[0275] 55. The method according to item 53 or 54, the method further comprising:

[0276] (j) Optionally under vacuum, stopper the container containing the microspheres from step (c) or (d).

[0277] 56. The method according to any one of items 52 - 55, wherein the formulation of step (a) is filtered before step (b).

[0278] 57. The method according to any one of items 54 - 56, wherein the treatment step (d) is freeze - drying the microsphere solution.

[0279] 58. The method according to any one of items 53 - 57, wherein the container is a unit - dose container.

[0280] 59. The method according to any one of items 53 - 58, wherein the fluid is n - decafluorobutane.

[0281] 60. The method according to any one of items 53 - 59, wherein the headspace of the capped container is filled with n - decafluorobutane.

[0282] 61. The method according to any one of items 53 - 60, wherein the average diameter of the microspheres from step (c) is about 0.1 to 1000 μm.

[0283] 62. The method according to item 61, wherein the average diameter of the microspheres from step (c) is about 0.1 to 100 μm.

[0284] 63. The method according to item 62, wherein the average diameter of the microspheres from step (c) is about 0.1 to 30 μm.

[0285] 64. The method according to item 63, wherein the average diameter of the microspheres from step (c) is about 0.7 to 10 μm.

[0286] 65. The method according to any one of items 55 - 64, wherein the treatment step (d) is freeze - drying the microsphere solution and the loss of microspheres during freeze - drying does not exceed 25%.

[0287] 66. The method according to item 65, wherein the loss of the microspheres during freeze - drying does not exceed 15% or does not exceed 10%.

[0288] 67. The method according to any one of items 52 - 66, wherein the molecular weight of the compound of formula I is from 1,500 to 5,000 Daltons.

[0289] 68. The method according to any one of items 52 - 67, wherein the molecular weight of the compound of formula II is from 1,500 to 5,000 Daltons.

[0290] 69. The method according to any one of items 52 - 68, wherein the molecular weight of the compound of formula III is from 500 to 2,000 Daltons.

[0291] 70. The method according to any one of items 52 - 69, wherein the molar ratio of the compound of formula I to the compound of formula II in the formulation is in the range of 1:100 to 1:1.

[0292] 71. The method according to any one of items 52 - 70, wherein the molar ratio of the compound of formula II to the compound of formula III in the formulation is in the range of 1:20 to 1:8.

[0293] 72. The method according to any one of items 52 - 71, wherein the microspheres from step (c) contain 0.01 - 5 mol% of the compound of formula I.

[0294] 73. The method according to any one of items 52 - 72, wherein the microspheres from step (c) contain 5 - 9.9 mol% of the compound of formula II.

[0295] 74. The method according to any one of items 52 - 73, wherein the microspheres from step (c) contain 80 - 95 mol% of the compound of formula III.

[0296] 75. The method according to any one of items 52 - 74, wherein the microspheres from step (c) contain no more than 5 mol% of the compound of formula I.

[0297] 76. The method according to any one of items 52 - 75, wherein the microspheres from step (c) contain no more than 10 mol% of the compound of formula II.

[0298] 77. The method according to any one of items 52 - 76, wherein the microspheres from step (c) contain no more than 95 mol% of the compound of formula III.

[0299] 78. The method according to any one of items 47 - 77, wherein:

[0300] the compound of formula I is a compound of formula Ib;

[0301] The compound of formula II is a compound of formula IIb; and

[0302] The compound of formula III is a compound of formula IIIa.

[0303] 79. The method according to item 78, wherein the average molecular weight of the compound of formula Ib is about 3200 Daltons.

[0304] 80. The method according to item 78 or 79, wherein the average molecular weight of the compound of formula IIb is about 2800 Daltons.

[0305] 81. The method according to any one of items 78 - 80, wherein the average molecular weight of the compound of formula IIIa is about 790 Daltons.

[0306] 82. The method according to any one of items 78 - 81, wherein the microspheres from step (c) contain 0.01 - 5 mol% of the compound of formula Ib.

[0307] 83. The method according to any one of items 78 - 82, wherein the microspheres from step (c) contain 5 - 9.9 mol% of the compound of formula IIb.

[0308] 84. The method according to any one of items 78 - 83, wherein the microspheres from step (c) contain 80 - 95 mol% of the compound of formula IIa.

[0309] 85. The method according to any one of items 78 - 84, wherein the microspheres from step (c) contain no more than 5 mol% of the compound of formula Ib.

[0310] 86. The method according to any one of items 78 - 85, wherein the microspheres from step (c) contain no more than 10 mol% of the compound of formula IIb.

[0311] 87. The method according to any one of items 78 - 86, wherein the microspheres from step (c) contain no more than 95 mol% of the compound of formula IIIa.

[0312] 88. The method according to any one of items 78 - 87, wherein the molar ratio of the compound of formula Ib to the compound of formula IIb in the microspheres from step (c) is in the range of 1:100 to 1:1.

[0313] 89. The method according to any one of items 78 - 88, wherein the molar ratio of the compound of formula IIb to the compound of formula IIIa in the microspheres from step (c) is in the range of 1:20 to 1:8.

[0314] 90. A unit - dose container, the unit - dose container comprising: a therapeutically effective amount of the pharmaceutical composition according to any one of items 7 - 51.

[0315] 91. The container according to item 90, wherein the compound of formula I is a compound of formula Ib.

[0316] 92. The container according to item 90 or 91, wherein the compound of formula II is a compound of formula IIb.

[0317] 93. The container according to any one of items 90 - 92, wherein the compound of formula III is a compound of formula IIa.

[0318] 94. The container according to any one of items 90 - 93, wherein the fluid is air, CO2, sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof.

[0319] 95. The container according to item 94, wherein the fluorinated C 1-6 alkane is selected from octafluoropropane, n - decafluorobutane, and dodecafluoropentane.

[0320] 96. The container according to any one of items 90 - 95, wherein the fluid is n - decafluorobutane.

[0321] 97. The container according to any one of items 90 - 96, wherein the composition comprises trehalose and povidone K12 as excipients.

[0322] 98. The container according to any one of items 90 - 97, wherein the composition further comprises a buffer solution, and the buffer solution is phosphate.

[0323] 99. The container according to item 98, wherein the buffer solution comprises calcium - and magnesium - free saline.

[0324] 100. The container according to any one of items 90 - 99, wherein the composition is capable of forming microspheres in the presence of water.

[0325] 101. The container according to any one of items 90 - 99, wherein the composition comprises microspheres.

[0326] 102. The container according to item 100 or item 101, wherein the microspheres are present as a lyophilized dry powder or as an anhydrous concentrate.

[0327] 103. The container according to any one of items 90 - 102, wherein the container is prepared by the method according to any one of items 52 - 89.

[0328] 104. The container according to any one of items 90 - 103, wherein the container is sealed by a crimped top lid equipped with a diaphragm.

[0329] 105. The container according to any one of items 90 - 104, wherein the container is airtight.

[0330] 106. A kit, the kit comprising:

[0331] At least one unit - dose container according to any one of items 90 - 105,

[0332] And instructions for using the kit.

[0333] 107. The kit according to item 106, wherein the unit - dose container contains trehalose and povidone K12 as excipients.

[0334] 108. The kit according to any one of items 106 - 107, wherein the kit further comprises a container containing an aqueous solution.

[0335] 109. The kit according to item 108, wherein the aqueous solution is sterile water.

[0336] 110. The kit according to item 108, wherein the aqueous solution is a sterile solution ready for perfusion.

[0337] 111. The kit according to any one of items 106 - 110, wherein the kit further comprises a syringe.

[0338] 112. The kit according to item 111, wherein the kit further comprises a needle for the syringe.

[0339] 113. The kit according to any one of items 106 - 111, wherein the kit comprises a needle - free syringe with a sharp tip.

[0340] 114. The kit according to item 112 or 113, wherein the needle or the sharp tip is capable of piercing the septum cap.

[0341] 115. The kit according to any one of items 106 - 114, wherein the kit further comprises a container containing a fluid with a standard boiling point below 30 °C, optionally wherein the container is a syringe.

[0342] 116. The kit according to item 115, wherein the fluid is air, nitrogen, argon, CO2, sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof.

[0343] 117. The kit according to item 116, wherein the fluorinated C 1-6 alkane is selected from octafluoropropane, n-decafluorobutane, and dodecafluoropentane.

[0344] 118. The kit according to any one of items 105 - 117, wherein the fluid is n-decafluorobutane.

[0345] 119. The kit according to any one of items 105 - 118, wherein the kit further comprises at least one gel pad and ultrasonic gel.

[0346] 120. The kit according to any one of items 105 - 119, wherein the kit further comprises a device selected from devices and vented bottle adapters.

[0347] 121. A method for reconstructing microspheres, wherein the method comprises:

[0348] (d) adding a sufficient amount of water or saline solution to the microspheres in the container according to any one of items 90 - 105,

[0349] (e) optionally, adding a fluid with a standard boiling point below 30 °C in a certain volume to the container in step (a); and

[0350] (f) optionally shaking the container in step (a) or step (b).

[0351] 122. The method according to item 121, wherein the method further comprises a step of filling the container with gas before step (a).

[0352] 123. The method according to item 121 or 122, wherein the amount of water or the saline solution does not exceed 100 milliliters.

[0353] 124. The method according to item 123, wherein the amount of water or the saline solution added is sufficient to produce a homogeneous mixture containing the reconstructed microspheres.

[0354] 125. The method according to any one of items 121 - 124, wherein the shaking of the container lasts no more than 180 seconds.

[0355] 126. A method for treating urolithiasis, the method comprising:

[0356] Administering an effective amount of the pharmaceutical composition according to any one of items 7 - 51, the microspheres manufactured according to any one of items 52 - 89, or the reconstituted microsphere solution according to any one of items 121 - 125 to a subject suffering from urolithiasis so that the microspheres contact the urinary tract stone, and

[0357] Directing energy to the urinary tract stone in the body of the subject at a frequency that excites the fluid within the microspheres.

[0358] 127. The method according to item 126, wherein the reconstituted microsphere solution is administered into the ureter of the subject through a catheter.

[0359] 128. The method according to item 126 or 127, wherein the energy is in the form of electromagnetic, acoustic, microwave, photon, or other forms.

[0360] 129. The method according to any one of items 126 - 128, wherein the energy is ultrasonic waves.

[0361] 130. The method according to item 129, wherein the ultrasonic energy is in the frequency range of 100 kilohertz (kHz) to 2 megahertz (MHz).

[0362] 131. The method according to item 129 or 130, wherein the ultrasonic energy is associated with a peak pressure in the range of 0.1 MPa to 10 MPa.

[0363] 132. The method according to any one of items 126 - 131, wherein the energy is applied for a sufficient amount of time to fragment the urinary tract stone.

[0364] 133. The method according to item 132, wherein the amount of time does not exceed 100 minutes.

[0365] 134. The method according to item 132, wherein the amount of time does not exceed 90 minutes.

[0366] 135. The method according to item 132, wherein the amount of time does not exceed 80 minutes.

[0367] 136. The method according to item 132, wherein the amount of time does not exceed 70 minutes.

[0368] 137. The method according to item 132, wherein the amount of time does not exceed 60 minutes.

[0369] 138. The method according to item 132, wherein the amount of time does not exceed 50 minutes.

[0370] 139. The method according to item 132, wherein the amount of time does not exceed 40 minutes.

[0371] 140. The method according to item 132, wherein the amount of time does not exceed 30 minutes.

[0372] 141. The method according to item 132, wherein the amount of time does not exceed 25 minutes.

[0373] 142. The method according to item 132, wherein the amount of time does not exceed 20 minutes.

[0374] 143. The method according to item 132, wherein the amount of time does not exceed 15 minutes.

[0375] 144. The method according to item 132, wherein the amount of time does not exceed 10 minutes.

[0376] 145. The method according to any one of items 126 - 144, wherein the applied energy causes a volume change of the reconstituted microspheres or other cavitation effects of the microspheres.

[0377] 146. The method according to any one of items 126 - 145, wherein cavitation of the microspheres causes a pressure gradient change and other mechanical effects in the urinary calculus around the reconstituted microspheres.

[0378] 147. The method according to any one of items 126 - 146, wherein the pressure gradient change and other mechanical effects are capable of fragmenting the urinary calculus.

[0379] 148. The method according to any one of items 126 - 147, wherein the subject is a human.

[0380] 149. The reconstituted microsphere solution according to any one of items 121 - 125, which is used for the treatment of urolithiasis,

[0381] wherein an effective amount of the microsphere solution is administered to the subject to bring the microspheres into contact with the urinary calculus; and

[0382] energy is directionally applied to the urinary calculus in the subject's body at the frequency of the fluid in the microspheres.

[0383] 150. The reconstituted microspheres for the use according to item 149, wherein the reconstituted microsphere solution is administered into the ureter of the subject through a catheter.

[0384] 151. The reconstituted microspheres for the use according to item 149 or 150, wherein the energy is in the form of electromagnetic, acoustic, microwave, photon, laser or other forms.

[0385] 152. Reconstructed microspheres for use according to any one of items 149 - 151, wherein the energy is ultrasonic waves.

[0386] 153. Reconstructed microspheres for use according to the use described in item 149, wherein the ultrasonic energy is in the frequency range of 100 kHz to 2 MHz.

[0387] 154. Reconstructed microspheres for use according to the use described in item 152 or 153, wherein the ultrasonic energy is associated with a peak pressure in the range of 0.1 MPa to 10 MPa.

[0388] 155. Reconstructed microspheres for use according to any one of items 149 - 151, wherein the energy is in the form of a laser.

[0389] 156. Reconstructed microspheres for use according to the use described in item 155, wherein the laser energy has a wavelength in the infrared range of 1000 nm to 2500 nm.

[0390] 157. Reconstructed microspheres for use according to the use described in item 155, wherein the laser energy can evaporate the liquid in the lumen of the tube, thereby being able to generate relevant sound waves.

[0391] 158. Reconstructed microspheres for use according to the use described in item 155, wherein the laser energy has a frequency in the range between 1 kHz and 1 MHz.

[0392] 159. Reconstructed microspheres for use according to any one of items 149 - 158, wherein the energy is applied for a sufficient amount of time to fragment the urinary calculus.

[0393] 160. Reconstructed microspheres for use according to the use described in item 159, wherein the amount of time does not exceed 100 minutes.

[0394] 161. Reconstructed microspheres for use according to the use described in item 160, wherein the amount of time does not exceed 90 minutes.

[0395] 162. Reconstructed microspheres for use according to the use described in item 161, wherein the amount of time does not exceed 80 minutes.

[0396] 163. Reconstructed microspheres for use according to the use described in item 162, wherein the amount of time does not exceed 70 minutes.

[0397] 164. Reconstructed microspheres for use according to the use described in item 163, wherein the amount of time does not exceed 60 minutes.

[0398] 165. A reconstituted microsphere for use according to the use described in item 164, wherein the amount of time does not exceed 50 minutes.

[0399] 166. A reconstituted microsphere for use according to the use described in item 165, wherein the amount of time does not exceed 40 minutes.

[0400] 167. A reconstituted microsphere for use according to the use described in item 166, wherein the amount of time does not exceed 30 minutes.

[0401] 168. A reconstituted microsphere for use according to the use described in item 167, wherein the amount of time does not exceed 25 minutes.

[0402] 169. A reconstituted microsphere for use according to the use described in item 168, wherein the amount of time does not exceed 20 minutes.

[0403] 170. A reconstituted microsphere for use according to the use described in item 169, wherein the amount of time does not exceed 15 minutes.

[0404] 171. A reconstituted microsphere for use according to the use described in item 170, wherein the amount of time does not exceed 10 minutes.

[0405] 172. A reconstituted microsphere for use according to any one of the uses described in items 149 - 171, wherein the applied energy causes a volume change of the reconstituted microsphere or other cavitation effects of the microsphere.

[0406] 173. A reconstituted microsphere for use according to any one of the uses described in items 149 - 172, wherein cavitation of the microsphere causes a change in the pressure gradient in the urine around the reconstituted microsphere and other mechanical effects.

[0407] 174. A reconstituted microsphere for use according to any one of the uses described in items 149 - 173, wherein the change in the pressure gradient and other mechanical effects can cause urinary calculi to fragment.

[0408] 175. A reconstituted microsphere for use according to any one of the uses described in items 149 - 174, wherein the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0409] These and other features, aspects, and advantages of the present invention will be better understood in conjunction with the following description and drawings, wherein:

[0410] Figure 1 A cross-sectional schematic diagram of an exemplary microsphere attached to a mineralized material 105 is shown, including a microsphere shell constituent 120 that promotes accumulation, a microsphere lipid shell 110, and a microsphere fluid core 115.

[0411] Figure 2It is a block diagram showing exemplary steps for synthesizing the bisphosphonate-PEG-lipid provided in the present disclosure.

[0412] Figure 3 It is a block diagram showing exemplary steps for preparing bisphosphonate-PEG-lipid to produce the microspheres provided in the present disclosure.

[0413] Figure 4 It shows the size distribution of the liquid phase of the bisphosphonate-PEG-lipid mixture measured by dynamic light scattering (DLS).

[0414] Figure 5 It shows samples of a bisphosphonate-PEG-lipid composition with a gas headspace (left) and a microsphere preparation of the composition described herein (right), illustrating the qualitative differences between the two preparations.

[0415] Figure 6 It is a graph showing the change of the microsphere accumulation assay results over time. The graph shows that compared with the microspheres lacking bisphosphonate-PEG-lipid in the shell, the microspheres incorporating the bisphosphonate-PEG-lipid of the present disclosure have a higher microsphere accumulation rate on the mineralized surface.

[0416] Figure 7 It shows the size distribution of the microspheres measured by electrozone sensing. The number of particles (y-axis) in each diameter bin (x-axis) is provided.

[0417] Figure 8 It shows the 1 1H-NMR spectrum of the intermediate PEG2DA.

[0418] Figure 9 It shows the 1 1H-NMR spectrum of the intermediate 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2)-COOH.

[0419] Figure 10 It shows the 31 31P-NMR spectrum of the intermediate 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2)-COOH.

[0420] Figure 11 It shows the 1 1H-NMR spectrum of the compound of formula Ib.

[0421] Figure 12 It shows the 31 31P-NMR spectrum of the compound of formula Ib.

[0422] Figure 13 The gas chromatogram showing the headspace analysis of n-perfluorobutane is presented.

[0423] Figure 14 The ³¹P-NMR spectrum of a liposomal formulation containing the compound 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K, the compound of formula IIb) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, the compound of formula IIIa) is presented. 31 ³¹P-NMR spectrum. Detailed Description

[0424] Definition

[0425] Unless defined otherwise herein, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0426] The term "subject" refers to any mammal, including humans and mammals such as those of veterinary and research interest, including but not limited to: apes, bovines, equines, canines, felines, and rodents.

[0427] The term "treating" a disorder or disease or "treatment" of a disorder or disease refers to taking measures to relieve the symptoms of the disorder or disease, such as shrinking, destroying, or removing an abnormal or obstructive mass such as a kidney stone, urinary calculus, biliary calculus, blood clot, fibroma, cancerous tumor, and atherosclerotic plaque, or otherwise obtaining some beneficial or desired result for the subject, including a clinical result. Any beneficial or desired clinical result can include, but is not limited to, alleviating or improving one or more symptoms of the disorder or disease; reducing the severity of the disease; delaying or slowing the progression of the disease; improving, ameliorating, or stabilizing the disease state; or other beneficial results.

[0428] The term "effective amount" means an amount sufficient to produce the desired effect.

[0429] The term "sufficient amount" means an amount sufficient to produce the desired effect.

[0430] The term "therapeutically effective amount" is an amount effective to improve or alleviate the symptoms of a disease.

[0431] The term "alleviate" one or more symptoms (and grammatical equivalents of this expression) means reducing the severity or frequency of the one or more symptoms, or eliminating the one or more symptoms.

[0432] As used herein, the term "cavitation effect" refers to an effect sufficient to cause the shrinkage or destruction of abnormal or obstructive masses in the body, such as kidney stones, urinary tract stones, biliary tract stones, blood clots, fibroids, cancerous tumors, and atherosclerotic plaques.

[0433] As used herein, the term "PL2kS" refers to the compound 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000-alendronate] (ammonium salt), which is also disclosed as the compound of Formula Ib herein.

[0434] As used herein, the term "DSPE-PEG2K" refers to the compound 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), which is also disclosed as the compound of Formula IIb herein.

[0435] As used herein, the term "DSPC" refers to the compound 1,2-distearoyl-sn-glycero-3-phosphocholine, which is also disclosed as the compound of Formula IIIa herein.

[0436] The practice of the present invention includes the use of conventional techniques of organic chemistry, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the skill of the art.

[0437] In this application, many technical names will be mentioned. All numerical names, such as pH, temperature, time, concentration, and weight, including their respective ranges, are approximate values and can generally vary in increments of 0.1, 1.0, or 10.0 (+) or (-) as appropriate. All numerical names can be understood to be preceded by the term "about". The reagents described herein are exemplary, and their equivalents may be known in the art.

[0438] The compounds used in the present invention may have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereoisomers, geometric isomers, positional isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention.

[0439] The compounds of the present invention may also contain non-natural proportions of atomic isotopes at one or more atoms constituting such compounds. For example, the compounds may be radiolabeled with radioactive isotopes such as but not limited to tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). All isotopic variants of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0440] Stereoisomers (also known as optical isomers) of a compound include all chiral, diastereoisomeric, and racemic forms of the structure, unless a specific stereochemistry is explicitly indicated. Thus, the compounds used in the present technology include optical isomers enriched or resolved at any or all of the asymmetric atoms, as will be apparent from the description. Both racemic and diastereoisomeric mixtures, as well as individual optical isomers, can be separated or synthesized to be substantially free of their enantiomeric or diastereoisomeric counterparts, and these stereoisomers are all within the scope of the present technology.

[0441] The compounds of the present technology can exist as solvates, especially hydrates. Hydrates may form during the manufacture of the compound or a composition containing the compound, or may form over time due to the hygroscopicity of the compound. The compounds of the present technology can also exist as organic solvates, including DMF, ether, and alcohol solvates, etc. The identification and preparation of any specific solvate are within the skill of the ordinary person in synthetic organic chemistry or medicinal chemistry.

[0442] Unless otherwise specified, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group and then naming the adjacent functional group closer to the point of attachment.

[0443] Stereoisomers (also known as optical isomers) of a compound include all chiral, diastereoisomeric, and racemic forms of the structure, unless a specific stereochemistry is explicitly indicated. Thus, the compounds used in the present technology include optical isomers enriched or resolved at any or all of the asymmetric atoms, as will be apparent from the description. Both racemic and d or l enriched stereoisomeric mixtures, as well as individual optical isomers, can be separated or synthesized to be substantially free of their enantiomeric or diastereoisomeric counterparts, and these stereoisomers are all within the scope of the present technology.

[0444] It should be understood that in all of the substituted groups defined above, polymers achieved by defining substituents that themselves have further substituents (e.g., a substituted aryl having a substituted aryl group as a substituent, where the substituent itself is substituted by a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substituents is three. That is, each of the above definitions is subject to the following constraint: each functional group (at one to three positions) is substituted and any and all of these substituents can be substituted once again (at one to three positions).

[0445] It should be understood that the above definitions are not intended to include non-permissible substitution patterns (e.g., a methyl group substituted by 5 fluorine groups). Such non-permissible substitution patterns are well known to those skilled in the art.

[0446] Throughout this application, the body text relates to various embodiments of the compounds, compositions, and methods of the present invention. The various embodiments described are intended to provide various illustrative examples and should not be construed as a description of alternative species. On the contrary, it should be noted that the descriptions of the various embodiments provided herein may have overlapping scopes. The embodiments discussed herein are merely illustrative and are not intended to limit the scope of the technology.

[0447] Compound

[0448] The present disclosure provides compounds that exhibit both hydrophobicity and high affinity for mineralized forms of calcium and other metals, including biominerals. The compounds can be used in a variety of applications that require both hydrophobicity and binding to metal-containing materials.

[0449] More specifically, in a first aspect, the present disclosure provides compounds of formula IV

[0450]

[0451] or salts, isomers, or salts of isomers thereof, wherein:

[0452] p is from 10 to 30;

[0453] q is from 1 to 100;

[0454] C is selected from:

[0455]

[0456] B is selected from: a covalent bond and ethyl;

[0457] A is selected from: a covalent bond, acyl, acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyl, amidino, and carboxylate ester; and

[0458] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxylate ester, phthalimido, OH, SO3H, and PO3H.

[0459] In some embodiments, p is from 12 to 28, from 14 to 26, from 14 to 24, from 14 to 22, from 14 to 20, from 16 to 20, or from 16 to 18. In some embodiments, p is 16. In some embodiments, q is from 1 to 100, from 5 to 90, from 10 to 80, from 15 to 70, from 20 to 60, from 25 to 50, from 30 to 50, from 40 to 50, or from 40 to 45. In some embodiments, q is 43. In some embodiments, q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49 or 50. In some embodiments, C is

[0460]

[0461] In some embodiments, the compound of formula IV is the compound of formula I

[0462]

[0463] or a salt, isomer or salt of an isomer thereof, wherein:

[0464] n is from 10 to 30,

[0465] m is from 1 to 100.

[0466] In some embodiments, n is from 20 to 25, about 12 to 20, 14 to 18, or 14 to 16. In some embodiments, n is 14 or 16. In some embodiments, m is from 1 to 90, from 5 to 80, from 10 to 70, from 20 to 60, from 30 to 50, or from 38 to 50. In some embodiments, m is 43. In some embodiments, q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49 or 50. In some embodiments, X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxyester, phthalimido, OH, SO3H and PO3H.

[0467] In some embodiments, the compound of formula I is the compound of formula Ia

[0468]

[0469] or a salt, isomer or salt of an isomer thereof.

[0470] In some embodiments, the compound of formula I is the compound of formula Ib

[0471]

[0472] Pharmaceutical composition

[0473] In a second aspect, the present disclosure provides a pharmaceutical composition comprising

[0474] a compound of formula IV

[0475]

[0476] or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof, wherein:

[0477] p is from 10 to 30;

[0478] q is from 1 to 100;

[0479] C is selected from:

[0480]

[0481] B is selected from: a covalent bond and ethyl;

[0482] A is selected from: a covalent bond, acyl, acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyl, amidino and carboxylate ester; and

[0483] X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxylate ester, phthalimido, SO3H and PO3H; and

[0484] optionally, a pharmaceutically acceptable excipient or diluent.

[0485] In some embodiments, p is 12 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 16 to 20, or 16 to 18. In some embodiments, p in the pharmaceutical composition is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the average value of p in the pharmaceutical composition is 12 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 16 to 20, or 16 to 18. In some embodiments, the average value of p in the pharmaceutical composition is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0486] In some embodiments, q is from 1 to 100, 5 to 90, 10 to 80, 15 to 70, 20 to 60, 25 to 50, 30 to 50, 40 to 50, or 40 to 45. In some embodiments, q is 43. In some embodiments, q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49 or 50. In some embodiments, the average value of q in the pharmaceutical composition is from 1 to 100, 5 to 90, 10 to 80, 15 to 70, 20 to 60, 25 to 50, 30 to 50, 40 to 50, or 40 to 45. In some embodiments, the average value of q in the pharmaceutical composition is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49 or 50.

[0487] In some embodiments of the pharmaceutical composition, the molecular weight of the compound of formula IV is from 1,500 to 5,000 Daltons. In certain embodiments, the molecular weight of the compound of formula IV is from 1,500 to 5,000 Daltons. In certain embodiments, the molecular weight of the compound of formula IV is from 2,000 to 4,500 Daltons. In certain embodiments, the molecular weight of the compound of formula IV is from 2,500 to 4,000 Daltons. In certain embodiments, the molecular weight of the compound of formula IV is from 3,000 to 3,500 Daltons.

[0488] In some embodiments, C is

[0489]

[0490] In some embodiments of the pharmaceutical composition, the compound of formula IV is the compound of formula I

[0491]

[0492] or a salt, isomer or salt of an isomer thereof, wherein:

[0493] n is from 10 to 30 and

[0494] m is from 1 to 100.

[0495] In some embodiments, n is from 10 to 50, from 20 to 25, about 12 to 20, 14 to 18, or 14 to 16. In some embodiments, n is 14 or 16. In some embodiments, n is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the average value of n in the pharmaceutical composition is from 10 to 50, from 20 to 25, 12 to 20, 14 to 18, or 14 to 16. In some embodiments, the average value of n in the pharmaceutical composition is 14 or 16. In some embodiments, the average value of n in the pharmaceutical composition is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0496] In some embodiments, m is from 1 to 150, from 1 to 140, from 1 to 130, from 1 to 120, from 1 to 110, from 1 to 100, from 1 to 90, 5 to 80, 10 to 70, 20 to 60, 30 to 50, or 38 to 50. In some embodiments, m is 43. In some embodiments, m is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or 50. In some embodiments, the average value of m in the pharmaceutical composition is from 1 to 150, from 1 to 140, from 1 to 130, from 1 to 120, from 1 to 110, from 1 to 100, from 1 to 90, 5 to 80, 10 to 70, 20 to 60, 30 to 50, or 38 to 50. In some embodiments, the average value of m in the pharmaceutical composition is 43. In some embodiments, the average value of m in the pharmaceutical composition is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or 50.

[0497] In some embodiments of the pharmaceutical composition, the molecular weight of the compound of Formula I is from 1,500 to 5,000 Daltons. In certain embodiments, the molecular weight of the compound of Formula I is from 2,000 to 4,500 Daltons. In certain embodiments, the molecular weight of the compound of Formula I is from 2,500 to 4,000 Daltons. In certain embodiments, the molecular weight of the compound of Formula I is from 3,000 to 3,500 Daltons.

[0498] In some embodiments, X is selected from: hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl, substituted sulfinyl, carboxylate ester, phthalimido, OH, SO3H, and PO3H.

[0499] In some embodiments, the compound of Formula I is a compound of Formula Ia

[0500]

[0501] or a salt, isomer or salt of an isomer thereof.

[0502] In some embodiments of the pharmaceutical composition, the average molecular weight of the compound of formula Ia is about 3500 Daltons. In some embodiments, the average molecular weight of the compound of formula Ia is about 3200 Daltons. In some embodiments, the average molecular weight of the compound of formula Ia is about 2500 Daltons. In some embodiments, the average molecular weight of the compound of formula Ia is about 2000 Daltons.

[0503] In some embodiments of the pharmaceutical composition, the compound of formula Ia is the compound of formula Ib

[0504]

[0505] In some embodiments, the compound of formula I is the compound of formula Ib

[0506]

[0507] In some embodiments of the pharmaceutical composition, the average molecular weight of the compound of formula Ib is about 3500 Daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 3200 Daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 2500 Daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 2000 Daltons.

[0508] In some embodiments, the chain lengths (16 and 43) in formula Ib are the average chain lengths of the compounds in the pharmaceutical composition.

[0509] In some embodiments of the pharmaceutical composition, the composition further comprises at least one additional phospholipid compound.

[0510] In some embodiments, the pharmaceutical composition further comprises

[0511] a compound of formula II

[0512]

[0513] or a pharmaceutically acceptable salt or isomer thereof.

[0514] In some embodiments, t is 10 to 30, 12 to 28, 14 to 25, 16 to 22, 16 to 20, or 18 to 20. In some embodiments, t is 14 or 16. In some embodiments, the average value of t in the pharmaceutical composition is 10 to 30, 12 to 28, 14 to 25, 16 to 22, 16 to 20, or 18 to 20. In some embodiments, the average value of t in the pharmaceutical composition is 14 or 16.

[0515] In some embodiments, y is 1 - 100, 5 to 90, 10 to 80, 20 to 70, 40 to 60, or 40 to 50. In some embodiments, y is 38 to 50. In some embodiments, the average value of y in the pharmaceutical composition is 1 - 100, 5 to 90, 10 to 80, 20 to 70, 40 to 60, or 40 to 50. In some embodiments, the average value of y in the pharmaceutical composition is 38 to 50.

[0516] In some embodiments of the pharmaceutical composition, the molecular weight of the compound of formula II is 1,500 to 5,000 Daltons. In certain embodiments, the molecular weight of the compound of formula II is 2,000 to 4,500 Daltons. In certain embodiments, the molecular weight of the compound of formula II is 2,500 to 4,000 Daltons. In specific embodiments, the molecular weight of the compound of formula II is 3,000 to 3,500 Daltons.

[0517] In some embodiments of the pharmaceutical composition, the compound of formula II is a compound of formula IIa

[0518]

[0519] or a pharmaceutically acceptable salt or isomer thereof.

[0520] In some embodiments of the pharmaceutical composition, the average molecular weight of the compound of formula IIa is about 3000 Daltons. In some embodiments, the average molecular weight of the compound of formula IIa is about 2800 Daltons. In some embodiments, the average molecular weight of the compound of formula IIa is about 2600 Daltons. In some embodiments, the average molecular weight of the compound of formula IIa is about 2400 Daltons. In some embodiments, the average molecular weight of the compound of formula IIa is about 2200 Daltons. In some embodiments, the average molecular weight of the compound of formula IIa is about 2000 Daltons.

[0521] In some embodiments, the chain lengths (16 and 43) in formula IIa are the average chain lengths of the compounds in the pharmaceutical composition.

[0522] In some embodiments of the pharmaceutical composition, the compound of formula II is a compound of formula IIb

[0523]

[0524] In some embodiments of the pharmaceutical composition, the average molecular weight of the compound of formula IIb is about 3000 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2800 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2600 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2400 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2200 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2000 Daltons.

[0525] In some embodiments, the chain lengths (16 and 43) in formula IIb are the average chain lengths of the compounds in the pharmaceutical composition.

[0526] In some embodiments, the pharmaceutical composition further comprises

[0527] a compound of formula III

[0528]

[0529] or a pharmaceutically acceptable salt or isomer thereof. In some embodiments, z is from 10 to 30, 10 to 25, 12 to 20, 15 to 20, or 16 to 18. In some embodiments, z is 14 or 16. In some embodiments, the average value of z in the pharmaceutical composition is from 10 to 30, 10 to 25, 12 to 20, 15 to 20, or 16 to 18. In some embodiments, the average value of z in the pharmaceutical composition is 14 or 16.

[0530] In some embodiments of the pharmaceutical composition, the molecular weight of the compound of formula III is from 500 to 2,000 Daltons. In certain embodiments, the molecular weight of the compound of formula III is from 750 to 1,750 Daltons. In certain embodiments, the molecular weight of the compound of formula III is from 1,000 to 1,500 Daltons. In certain embodiments, the molecular weight of the compound of formula III is from 1,100 to 1,400 Daltons. In certain embodiments, the molecular weight of the compound of formula III is from 1200 to 1,300 Daltons.

[0531] In some embodiments of the pharmaceutical composition, the compound of formula III is a compound of formula IIIa

[0532]

[0533] In some embodiments of the pharmaceutical composition, the average molecular weight of the compound of formula IIIa is about 840 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 790 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 750 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 700 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 650 Daltons.

[0534] In some embodiments, the chain length (16) in formula IIIa is the average chain length of the compounds in the pharmaceutical composition.

[0535] In some embodiments, the pharmaceutical composition comprises no more than 5 mol% of the compound of formula IV or a salt thereof. As used herein, mol% refers to the percentage of the number of molecules of a given lipid compound (e.g., a compound of formula IV, formula I, formula II, formula III or a salt, isomer or salt of an isomer thereof) relative to the total number of molecules of all lipid compounds within the pharmaceutical composition. Similarly, in the context of microspheres, as used herein, mol% refers to the percentage of the number of molecules of a given lipid compound (e.g., a compound of formula IV, formula I, formula II, formula III or a salt, isomer or salt of an isomer thereof) relative to the total number of molecules of all lipid compounds within the microsphere. In certain embodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of formula IV or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of formula IV or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 3 mol% of the compound of formula IV or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 2 mol% of the compound of formula IV or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 1 mol% of the compound of formula IV or a salt thereof.

[0536] In some embodiments, the pharmaceutical composition comprises 0.1 - 5 mol% of the compound of formula IV or a salt thereof. In some embodiments, the pharmaceutical composition comprises 0.1 - 10, 0.1 - 5, 0.5 - 5, 1 - 5, 1 - 4, 1 - 3, 2 - 3, 2 - 4, or 3 - 5 mol% of the compound of formula IV or a salt thereof.

[0537] In some embodiments, the composition comprises 0.01 - 5 mol% of the compound of formula I or a salt thereof. In certain embodiments, the composition comprises 0.1 - 5 mol% of the compound of formula I or a salt thereof. In some embodiments, the pharmaceutical composition comprises 0.1 - 10, 0.1 - 5, 0.5 - 5, 1 - 5, 1 - 4, 1 - 3, 2 - 3, 2 - 4, or 3 - 5 mol% of the compound of formula I or a salt thereof.

[0538] In some embodiments, the pharmaceutical composition comprises no more than 5 mol% of the compound of formula I or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of formula I or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 3 mol% of the compound of formula I or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 2 mol% of the compound of formula I or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 1 mol% of the compound of formula I or a salt thereof.

[0539] In some embodiments, the pharmaceutical composition comprises 0.01 - 5 mol% of the compound of formula Ia or Ib. In certain embodiments, the pharmaceutical composition comprises 0.1 - 5 mol% of the compound of formula Ia or Ib. In certain embodiments, the composition comprises 0.5 - 4.5 mol% of the compound of formula Ia or Ib. In certain embodiments, the composition comprises 1.0 - 4.0 mol% of the compound of formula Ia or Ib. In certain embodiments, the composition comprises 1.5 - 3.5 mol% of the compound of formula Ia or Ib. In certain embodiments, the composition comprises 2.0 - 3.0 mol% of the compound of formula Ia or Ib.

[0540] In some embodiments, the composition comprises no more than 5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 4.5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 4.0 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 3.5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 3.0 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 2.5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 2.0 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 1.5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 1.0 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 0.5 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 0.1 mol% of the compound of formula Ia or Ib. In some embodiments, the composition comprises no more than 0.05 mol% of the compound of formula Ia or Ib.

[0541] In some embodiments, the composition comprises 5 - 10 mol% of the compound of formula II or a salt thereof.

[0542] In some embodiments, the pharmaceutical composition comprises no more than 10 mol% of the compound of formula II or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 8 mol% of the compound of formula II or a salt thereof. In certain embodiments, the composition comprises no more than 6 mol% of the compound of formula II or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of formula II or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 3 mol% of the compound of formula II or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 2 mol% of the compound of formula II or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 1 mol% of the compound of formula II or a salt thereof.

[0543] In some embodiments, the pharmaceutical composition comprises 5-10 mol% of the compound of formula II or a salt thereof. In some embodiments, the pharmaceutical composition comprises 5-20, 5-15, 5-10, 5-9.9, 5-9.6, 5-9, 5-9, 7-9, or 8-9 mol% of the compound of formula II or a salt thereof.

[0544] In some embodiments, the composition comprises 5.0-10.0 mol% of the compound of formula IIa or IIb. In certain embodiments, the composition comprises 5.5-9.5 mol% of the compound of formula IIa or IIb. In certain embodiments, the composition comprises 6.0-9.0 mol% of the compound of formula IIa or IIb. In certain embodiments, the composition comprises 6.5-8.5 mol% of the compound of formula IIa or IIb. In certain embodiments, the composition comprises 7.0-8.0 mol% of the compound of formula IIa or IIb.

[0545] In some embodiments, the composition comprises no more than 10 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 9.5 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 9.0 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 8.5 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 8.0 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 7.5 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 7.0 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 6.5 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 6.0 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 5.5 mol% of the compound of formula IIa or IIb. In some embodiments, the composition comprises no more than 5.0 mol% of the compound of formula IIa or IIb.

[0546] In some embodiments, the composition comprises 80 - 95 mol% of the compound of formula III or a salt thereof. In some embodiments, the pharmaceutical composition comprises 70 - 99, 75 - 98, 75 - 97, 80 - 95 mol% of the compound of formula III or a salt thereof.

[0547] In some embodiments, the pharmaceutical composition comprises no more than 95 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 85 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 75 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 65 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 55 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 45 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 35 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 25 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 15 mol% of the compound of formula III or a salt thereof. In certain embodiments, the pharmaceutical composition comprises no more than 5 mol% of the compound of formula III or a salt thereof.

[0548] In some embodiments, the composition comprises 80 - 95 mol% of the compound of formula IIIa. In certain embodiments, the composition comprises 82.5 - 92.5 mol% of the compound of formula IIIa. In certain embodiments, the composition comprises 85 - 90 mol% of the compound of formula IIIa. In certain embodiments, the composition comprises 87.5 - 92.5 mol% of the compound of formula IIIa.

[0549] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:1000 to 1:1. In certain embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:500 to 1:1. In certain embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:100 to 1:1. In certain embodiments, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:90 to 1:5. In certain embodiments, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:80 to 1:10. In certain embodiments, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:70 to 1:20. In certain embodiments, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:60 to 1:30. In certain embodiments, the molar ratio of the compound of formula IV to the compound of formula II ranges from 1:50 to 1:40.

[0550] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:1000 to 1:1. In certain embodiments of the pharmaceutical composition, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:500 to 1:1. In certain embodiments of the pharmaceutical composition, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:100 to 1:1. In certain embodiments, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:90 to 1:5. In certain embodiments, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:80 to 1:10. In certain embodiments, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:70 to 1:20. In certain embodiments, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:60 to 1:30. In certain embodiments, the molar ratio of the compound of formula I to the compound of formula II ranges from 1:50 to 1:40.

[0551] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:1000 to 1:1. In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:500 to 1:1. In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:90 to 1:5. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:80 to 1:10. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:70 to 1:20. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:60 to 1:30. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa ranges from 1:50 to 1:40.

[0552] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:1000 to 1:1. In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:500 to 1:1. In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:90 to 1:5. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:80 to 1:10. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:70 to 1:20. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:60 to 1:30. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:50 to 1:40.

[0553] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula II to the compound of formula III ranges from 1:20 to 1:8. In certain embodiments, the molar ratio of the compound of formula II to the compound of formula III ranges from 1:18 to 1:10. In certain embodiments, the molar ratio of the compound of formula II to the compound of formula III ranges from 1:16 to 1:12. In certain embodiments, the molar ratio of the compound of formula II to the compound of formula III ranges from 1:15 to 1:13.

[0554] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IIa to the compound of formula IIIa ranges from 1:20 to 1:8. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa ranges from 1:18 to 1:10. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa ranges from 1:16 to 1:12. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa ranges from 1:15 to 1:13.

[0555] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IIb to the compound of formula IIIa ranges from 1:20 to 1:8. In some embodiments, the molar ratio of the compound of formula IIb to the compound of formula IIIa ranges from 1:18 to 1:10. In some embodiments, the molar ratio of the compound of formula IIb to the compound of formula IIIa ranges from 1:16 to 1:12. In some embodiments, the molar ratio of the compound of formula IIb to the compound of formula IIIa ranges from 1:15 to 1:13.

[0556] In some embodiments, the pharmaceutical composition comprises a compound of formula IV, optionally a compound of formula I or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof; a compound of formula II or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof; and a compound of formula III or a pharmaceutically acceptable salt, isomer or salt of an isomer thereof.

[0557] In some embodiments, the pharmaceutical composition comprises an ammonium salt of at least one of the compounds of formula IV, formula I, formula II and formula III.

[0558] In some embodiments, the pharmaceutical composition comprises a homogeneous population of compounds having the same carbon chain length. In some embodiments, the pharmaceutical composition comprises a heterogeneous population of lipid compounds having carbon chains of different lengths. In such embodiments, the chain length represented as a variable or a specific number in formula IV, formula I, formula Ia, formula Ib, formula II, formula IIa, formula IIb, formula III or formula IIIa refers to the average number of links of the lipid composition present in the pharmaceutical composition.

[0559] In some embodiments, the composition further comprises a fluid having a standard boiling point below 30 °C.

[0560] In typical embodiments, the fluid is a gas at the temperature and pressure present in the mammalian urinary tract. In preferred embodiments, the fluid responds to various forms of energy, such as electromagnetic energy, acoustic energy, microwave energy, photon energy or others. In certain embodiments, the fluid has low solubility in aqueous solution and is air, nitrogen, argon, CO2, sulfur hexafluoride, fluorinated C1-6 Alkanes or combinations thereof.

[0561] In some embodiments of the pharmaceutical composition, the fluid is a perfluorocarbon. In some embodiments, the fluid is selected from octafluoropropane, n-decafluorobutane, n-perfluoropropane, tetradecafluorohexane, and dodecafluoropentane. In a particular embodiment, the fluid is n-decafluorobutane (also known as perfluorobutane).

[0562] In some embodiments of the pharmaceutical composition, the compound of Formula IV, which is typically the compound of Formula I; the compound of Formula II; the compound of Formula III; and a liquid having a standard boiling point below 30 °C can be assembled into microspheres. Thus, in some embodiments, the pharmaceutical composition comprises microspheres, the microspheres comprising a lipid shell surrounding a fluid having a standard boiling point below 30 °C, wherein the lipid shell comprises the compounds of Formulas I, II, and III.

[0563] Any method known in the art can be used to measure the average diameter of the microspheres. In some embodiments, the average diameter is measured by electrical zone sensing, such as using a Coulter counter. In some embodiments, when measured by electrical zone sensing, the microspheres have an average diameter of about 0.1 to about 10 microns, or 0.1 to 10 microns. In certain embodiments, the microspheres have an average diameter of about 0.2 to about 9.5 microns. In certain embodiments, the microspheres have an average diameter of about 0.3 to about 9.0 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 8.5 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 8.0 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 7.5 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 7.0 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 6.5 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 6.0 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 5.5 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 5 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 4 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 3 microns. In certain embodiments, the microspheres have an average diameter of about 0.5 to about 2 microns. In certain embodiments, the microspheres have an average diameter of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, or 5 microns.

[0564] In some embodiments, the pharmaceutical composition comprising the microspheres is a lyophilized powder or a dried concentrate.

[0565] In other embodiments, the pharmaceutical composition comprising the microspheres further comprises an aqueous medium, such as water, saline, or a buffered salt solution. In certain embodiments, the pharmaceutical composition comprises a phosphate buffered solution lacking calcium and magnesium ions. In a particular embodiment, the composition further comprises phosphate buffered saline.

[0566] In some embodiments, the pharmaceutical composition comprising the microspheres, whether in lyophilized, dry concentrate, or liquid formulation, further comprises one or more of trehalose and povidone K12 as excipients. Povidone K12 is a commercially available product.

[0567] Method for manufacturing bisphosphonate-PEG-lipid compounds

[0568] In another aspect, the present disclosure provides a method for preparing a bisphosphonate-PEG-lipid compound.

[0569] Figure 2 An exemplary method for preparing a bisphosphonate-PEG-lipid compound is illustrated. The method comprises the following steps: (i) synthesizing a polyethylene glycol compound in diacid form by oxoammonium-catalyzed oxidation; (ii) synthesizing a PEG-lipid by NHS esterification and chromatography; and (iii) synthesizing a bisphosphonate-PEG-lipid by NHS esterification and chromatography.

[0570] In the first step, the synthesis method comprises an oxidation reaction, in which the polyethylene glycol compound is contacted with an oxidant for a sufficient amount of time to produce a dicarboxylic acid analogue of the polyethylene glycol compound. In some embodiments, the oxidation reaction is an oxoammonium-catalyzed oxidation reaction. In some embodiments, the oxidant is NaClO2.

[0571] The synthesis method further comprises a subsequent step, in which the resulting PEG in diacid form is esterified to a lipid, and the esterification reaction is mediated by N-hydroxysuccinimide. In some embodiments, the resulting PEG-lipid is purified by chromatography.

[0572] The synthesis method further comprises a further step, in which a bisphosphonate-PEG-lipid is synthesized by an esterification reaction. In some embodiments, the esterification reaction is mediated by N-hydroxysuccinimide. In some embodiments, the bisphosphonate-PEG-lipid is purified by chromatography.

[0573] In some embodiments, the bisphosphonate-PEG-lipid is a compound of formula IV. In some embodiments, the bisphosphonate-PEG-lipid is a compound of formula I. In some embodiments, the bisphosphonate-PEG-lipid is a compound of formula Ia. In some embodiments, the bisphosphonate-PEG-lipid is a compound of formula Ib.

[0574] Method for preparing PEG-lipid mixture

[0575] In another aspect, the present disclosure provides a method for preparing an assemblable phospholipid composition that comprises a fluid having a standard boiling point below 30°C. The method comprises the steps of: (a) blending PEG-lipids, including bisphosphonate-PEG-lipids, (b) homogenizing, and (c) filtering.

[0576] In a typical embodiment, step (a) comprises blending a bisphosphonate-PEG-lipid, a compound of formula II, and a compound of formula III.

[0577] In a typical embodiment, the bisphosphonate-PEG-lipid is a compound of formula IV. In some embodiments, the compound of formula IV is a compound of formula 1. In a particular embodiment, the compound of formula I is a compound of formula Ia. In a particular embodiment, the compound of formula I is a compound of formula Ib.

[0578] In some embodiments, the compound of formula II is a compound of formula IIa. In some embodiments, the compound of formula II is a compound of formula IIb. In some embodiments, the compound of formula III is a compound of formula IIIa.

[0579] In step (b), the blended mixture of PEG-lipids is homogenized.

[0580] In step (c), the homogenized mixture is filtered.

[0581] Method for manufacturing microspheres

[0582] In yet another aspect, the present disclosure provides a method for manufacturing microspheres, the method comprising: (a) preparing a PEG-lipid mixture as set forth in the above section, the mixture optionally further comprising at least one pharmaceutically acceptable excipient; and combining with water; (b) combining the PEG-lipid mixture of step (a) in a container with a fluid having a standard boiling point below 30°C; and (c) agitating or otherwise imparting energy to the container from step (b) containing the PEG-lipid mixture and the fluid to obtain microspheres. In some embodiments, the microspheres obtained from step (c) are provided for therapeutic use. In such a case, the microspheres can be in a liquid composition. The liquid composition can be in a container.

[0583] In some embodiments, the method further comprises step (d): treating the composition containing the microspheres to extend its shelf life or expand the range of environmental conditions for storage. In one embodiment, step (d) involves lyophilizing the composition containing the microspheres, but other methods known in the art can also be used to extend the shelf life of the microsphere solution or expand the range of environmental conditions for storage.

[0584] In some embodiments, the method further comprises filling a composition comprising the microspheres from step (c) or (d) into a container. In some embodiments, the method further comprises performing certain processing steps in the container. In some embodiments, the method further comprises the step of plugging the container comprising the microspheres from step (c) or (d).

[0585] In some embodiments, the method further comprises aseptic processing.

[0586] In some embodiments, the phospholipid composition of step (a) further comprises a compound of formula II or a salt thereof. In some embodiments, the phospholipid composition of step (a) further comprises a compound of formula III or a salt thereof. In some embodiments, the phospholipid composition of step (a) further comprises both a compound of formula II or a salt thereof and a compound of formula III or a salt thereof.

[0587] In some embodiments, the compound of formula I or a salt thereof used in the method is a compound of formula Ia or a compound of formula Ib. In some embodiments, the compound of formula II or a salt thereof used in the method is a compound of formula IIa or a compound of formula IIb. In some embodiments, the compound of formula III or a salt thereof used in the method is a compound of formula IIIa.

[0588] In some embodiments, the phospholipid composition of step (a) is filtered before step (b).

[0589] In some embodiments, the phospholipid composition is transferred to a container after step (a) and before step (b). In some embodiments, some or all of steps (a)-(d) are carried out by continuous processing.

[0590] In some embodiments, the lyophilized microspheres are present as a lyophilized dry powder or as an anhydrous concentrate.

[0591] In some embodiments, the container can be plugged while under vacuum.

[0592] In some embodiments, the lyophilized microspheres are more stable than the microsphere solution. In some embodiments, the lyophilized microspheres have a longer shelf life than the microsphere solution.

[0593] In some embodiments, the container is a unit dose container.

[0594] In some embodiments, the fluid is n-decafluorobutane.

[0595] In some embodiments, the capped container is filled with n-decafluorobutane.

[0596] In some embodiments, the phospholipid composition of step (a) comprises liposomes having an average diameter of from 40 nanometers to 5000 nanometers. In some embodiments, the phospholipid composition of step (a) comprises liposomes having an average diameter of from 100 nanometers to 1000 nanometers, from 200 nanometers to 900 nanometers, from 300 nanometers to 800 nanometers, from 400 nanometers to 700 nanometers, from 500 nanometers to 600 nanometers, from 100 nanometers to 200 nanometers, or from 10 nanometers to 100 nanometers. In some embodiments, the average diameter of the liposomes in the phospholipid composition of step (a) is about 50 nanometers.

[0597] In some embodiments, the method produces energy-responsive microspheres. For example, the microspheres generated by the method can collapse in response to the application of energy. In some embodiments, the energy is in the form of electromagnetic, acoustic, microwave, photon, or other forms. The collapse of the microspheres can release energy, such as mechanical energy.

[0598] In some embodiments, the average diameter of the microspheres before lyophilization is about 0.1 micrometer to 1000 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 0.1 micrometer to 100 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 0.1 micrometer to 30 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 0.7 micrometer to 10 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 0.5 micrometer to 15.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 10.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 8.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 7.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 6.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 5.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 4.0. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 3.0 micrometers. In some embodiments, the average diameter of the microspheres before lyophilization is about 1.0 micrometer to 2.0 micrometers.

[0599] In some embodiments, the method includes step (d), which involves lyophilizing a composition comprising microspheres, and the loss of microspheres during lyophilization does not exceed 50%. In some embodiments, the loss of microspheres during lyophilization does not exceed 40%. In some embodiments, the loss of microspheres during lyophilization does not exceed 30%. In another embodiment, the loss of microspheres during lyophilization does not exceed 25%. In some embodiments, the loss of microspheres during lyophilization does not exceed 20%. In some embodiments, the loss of microspheres during lyophilization does not exceed 15%. In some embodiments, the loss of microspheres during lyophilization does not exceed 10%.

[0600] In some embodiments, the molecular weight of the compound of formula I is from 1,500 to 5,000 Daltons. In some embodiments, the molecular weight of the compound of formula I is from 2,000 to 5,000 Daltons. In some embodiments, the molecular weight of the compound of formula I is from 2,000 to 4,500 Daltons. In some embodiments, the molecular weight of the compound of formula I is from 2,500 to 4,000 Daltons. In some embodiments, the molecular weight of the compound of formula I is from 3,000 to 3,500 Daltons.

[0601] In some embodiments, the molecular weight of the compound of formula II is from 1,500 to 5,000 Daltons. In some embodiments, the molecular weight of the compound of formula II is from 2,000 to 4,500 Daltons. In some embodiments, the molecular weight of the compound of formula II is from 2,500 to 4,000 Daltons. In some embodiments, the molecular weight of the compound of formula II is from 3,000 to 3,500 Daltons.

[0602] In some embodiments, the molecular weight of the compound of formula III is from 500 to 2,000 Daltons. In some embodiments, the molecular weight of the compound of formula III is from 750 to 1,750 Daltons. In some embodiments, the molecular weight of the compound of formula III is from 1,000 to 1,500 Daltons. In some embodiments, the molecular weight of the compound of formula III is from 1,100 to 1,400 Daltons. In some embodiments, the molecular weight of the compound of formula III is from 1200 to 1,300 Daltons.

[0603] In some embodiments, the microspheres obtained from step (c) contain 0.01 - 5 mol% of the compound of formula I. In some embodiments, the microspheres contain 0.05 - 5 mol% of the compound of formula I. In some embodiments, the microspheres contain 0.1 - 5 mol% of the compound of formula I. In some embodiments, the microspheres contain 0.5 - 4.5 mol% of the compound of formula I. In some embodiments, the microspheres contain 1.0 - 4.0 mol% of the compound of formula I. In some embodiments, the microspheres contain 1.5 - 3.5 mol% of the compound of formula I. In some embodiments, the microspheres contain 2.0 - 3.0 mol% of the compound of formula I.

[0604] In some embodiments, the microspheres obtained from step (c) contain 5 - 9.9 mol% of the compound of formula II. In some embodiments, the microspheres contain 5.5 - 9.5 mol% of the compound of formula II. In some embodiments, the microspheres contain 6.0 - 9.0 mol% of the compound of formula II. In some embodiments, the microspheres contain 6.5 - 8.5 mol% of the compound of formula II. In some embodiments, the microspheres contain 7.0 - 8.0 mol% of the compound of formula II.

[0605] In some embodiments, the microspheres obtained from step (c) contain 80 - 95 mol% of the compound of formula III. In some embodiments, the microspheres contain 82.5 - 92.5 mol% of the compound of formula III. In some embodiments, the microspheres contain 85 - 90 mol% of the compound of formula III. In some embodiments, the microspheres contain 87.5 - 92.5 mol% of the compound of formula III.

[0606] In some embodiments, the microspheres obtained from step (c) contain no more than 5 mol% of the compound of formula I. In some embodiments, the microspheres obtained from step (c) contain no more than 4.5 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 4.0 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 3.5 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 3.0 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 2.5 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 2.0 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 1.5 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 1.0 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 0.5 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 0.1 mol% of the compound of formula I. In some embodiments, the microspheres contain no more than 0.05 mol% of the compound of formula I.

[0607] In some embodiments, the microspheres obtained from step (c) contain no more than 10 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 9.5 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 9.0 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 8.5 mol% of the compound of formula II. In another embodiment, the microspheres contain no more than 8.0 mol% of the compound of formula II. In another embodiment, the microspheres contain no more than 7.5 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 7.0 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 6.5 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 6.0 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 5.5 mol% of the compound of formula II. In some embodiments, the microspheres contain no more than 5.0 mol% of the compound of formula II.

[0608] In some embodiments, the microspheres obtained from step (c) contain no more than 95 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 92.5 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 90 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 87.5 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 85.0 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 82.5 mol% of the compound of formula III. In some embodiments, the microspheres contain no more than 80.0 mol% of the compound of formula III.

[0609] In some embodiments, the average molecular weight of the compound of formula Ib in the microspheres of step (c) is about 3500 daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 3200 daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 2500 daltons. In some embodiments, the average molecular weight of the compound of formula Ib is about 2000 daltons.

[0610] In some embodiments, the average molecular weight of the compound of formula IIb in the microspheres of step (c) is about 3000 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2800 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2600 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2400 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2200 Daltons. In some embodiments, the average molecular weight of the compound of formula IIb is about 2000 Daltons.

[0611] In some embodiments, the average molecular weight of the compound of formula IIIa in the microspheres of step (c) is about 840 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 790 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 750 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 700 Daltons. In some embodiments, the average molecular weight of the compound of formula IIIa is about 650 Daltons.

[0612] In some embodiments, the microspheres obtained from step (c) contain 0.01 - 5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 0.05 - 5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 0.1 - 5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 0.5 - 4.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 1.0 - 4.0 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 1.5 - 3.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain 2.0 - 3.0 mol% of the compound of formula Ib.

[0613] In some embodiments, the microspheres obtained from step (c) contain 5 - 9.9 mol% of the compound of formula IIb. In some embodiments, the microspheres contain 6.0 - 9.0 mol% of the compound of formula IIb. In some embodiments, the microspheres contain 6.5 - 8.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain 7.0 - 8.0 mol% of the compound of formula IIb.

[0614] In some embodiments, the microspheres obtained from step (c) contain 80 - 95 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain 82.5 - 92.5 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain 85 - 90 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain 87.5 - 92.5 mol% of the compound of formula IIIa.

[0615] In some embodiments, the microspheres obtained from step (c) contain no more than 5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 4.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 4.0 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 3.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 3.0 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 2.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 2.0 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 1.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 1.0 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 0.5 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 0.1 mol% of the compound of formula Ib. In some embodiments, the microspheres contain no more than 0.05 mol% of the compound of formula Ib.

[0616] In some embodiments, the microspheres obtained from step (c) contain no more than 10 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 9.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 9.0 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 8.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 8.0 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 7.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 7.0 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 6.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 6.0 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 5.5 mol% of the compound of formula IIb. In some embodiments, the microspheres contain no more than 5.0 mol% of the compound of formula IIb.

[0617] In some embodiments, the microspheres obtained from step (c) contain no more than 95 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 92.5 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 90 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 87.5 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 85.0 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 82.5 mol% of the compound of formula IIIa. In some embodiments, the microspheres contain no more than 80.0 mol% of the compound of formula IIIa.

[0618] In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa in the microspheres obtained from step (c) is in the range of 1:1000 to 1:1. In some embodiments of the microspheres, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:500 to 1:1. In some embodiments of the microspheres, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:90 to 1:5. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:80 to 1:10. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:70 to 1:20. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:60 to 1:30. In some embodiments, the molar ratio of the compound of formula Ia to the compound of formula IIa is in the range of 1:50 to 1:40.

[0619] In some embodiments of the pharmaceutical composition, the molar ratio of the compound of formula IIa to the compound of formula IIIa is in the range of 1:20 to 1:8. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa is in the range of 1:18 to 1:10. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa is in the range of 1:16 to 1:12. In some embodiments, the molar ratio of the compound of formula IIa to the compound of formula IIIa is in the range of 1:15 to 1:13.

[0620] In some embodiments of the microspheres, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:1000 to 1:1. In some embodiments of the microspheres, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:500 to 1:1. In some embodiments of the microspheres, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:100 to 1:1. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:90 to 1:5. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:80 to 1:10. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:70 to 1:20. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:60 to 1:30. In some embodiments, the molar ratio of the compound of formula Ib to the compound of formula IIb ranges from 1:50 to 1:40.

[0621] Unit dosage form

[0622] In a fourth aspect, the present disclosure provides a unit dose container comprising a therapeutically effective amount of the pharmaceutical composition described herein.

[0623] In some embodiments, the unit dose container comprises a phospholipid composition, wherein the phospholipid composition comprises a compound of formula I, a compound of formula II, and a compound of formula III and at least one pharmaceutically acceptable excipient. In some embodiments, the unit dose container further comprises a fluid having a standard boiling point below 30 °C.

[0624] In some embodiments, the unit dose container comprises microspheres, wherein the microspheres comprise a compound of formula I, a compound of formula II, a compound of formula III, a fluid having a standard boiling point below 30 °C, and at least one pharmaceutically acceptable excipient. The microspheres can be obtained by the methods described herein related to the methods of manufacturing microspheres.

[0625] In some embodiments of the unit dose form, the compound of formula I is a compound of formula Ia or Ib.

[0626] In some embodiments of the unit dose form, the compound of formula II is a compound of formula IIa or IIb.

[0627] In some embodiments of the unit dose form, the compound of formula III is a compound of formula IIIa.

[0628] In some embodiments, the fluid having a standard boiling point below 30 °C is a gas at body temperature. In some embodiments, the fluid is air, CO2, sulfur hexafluoride, fluorinated C1-6 Alkanes or combinations thereof.

[0629] In some embodiments, the fluid is n-decafluorobutane.

[0630] In some embodiments, the composition of the unit dose container further comprises trehalose and povidone K12 as excipients. In some embodiments, the unit dose container further comprises a phosphate buffer solution free of calcium and magnesium ions.

[0631] In some embodiments, the composition of the unit dose container is capable of forming microspheres in the presence of water. In some embodiments, the composition of the unit dose container is a liquid composition comprising microspheres.

[0632] In some embodiments, the microspheres of the unit dose container are present as a lyophilized dry powder or as an anhydrous concentrate. In some embodiments, the lyophilized dry powder is a lyophilized product comprising microspheres. In some embodiments, the composition of the unit dose container is a product obtained by reconstituting a lyophilized preparation comprising microspheres.

[0633] In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 10 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 9.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 9.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 8.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 8.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 7.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 7.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 6.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 6.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 5.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 5.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 4.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 4.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 3.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 3.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 2.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 2.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 1.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 1.0 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.5 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.4 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.3 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.2 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.1 wt%. In some embodiments, the residual water content of the lyophilizate comprising the microspheres does not exceed 0.01 wt%.

[0634] In some embodiments, the composition of the unit dose container is prepared by any of the methods described herein.

[0635] In some embodiments, the unit dose container is sealed with a crimped top lid equipped with a septum. In some embodiments, the unit dose container is airtight.

[0636] Kit

[0637] In another aspect, the present disclosure provides a kit comprising at least one unit dose container as described herein and instructions for using the kit.

[0638] In some embodiments, the composition within each of the at least one unit dose container is capable of forming microspheres in the presence of water.

[0639] In some embodiments, the unit dose container further comprises trehalose and povidone K12 as pharmaceutically acceptable excipients.

[0640] In some embodiments, the kit further comprises a container containing an aqueous solution. In some embodiments, the aqueous solution is sterile water. In some embodiments, the aqueous solution is a saline solution ready for perfusion.

[0641] In some embodiments, the kit further comprises a syringe. In some embodiments, the kit further comprises a needle. In some embodiments, the kit further comprises a needleless syringe having a sharp tip (e.g., ), wherein the needleless syringe is capable of piercing a stopper.

[0642] In some embodiments, the kit further comprises a container of a fluid, wherein the fluid is air, nitrogen, argon, CO2, sulfur hexafluoride, fluorinated C 1-6 alkane or a combination thereof. In some embodiments, the container is a syringe.

[0643] In some embodiments of the kit, the fluorinated C 1-6 alkane is selected from octafluoropropane, n-decafluorobutane, and dodecafluoropentane.

[0644] In some embodiments of the kit, the fluid is n-decafluorobutane.

[0645] In some embodiments, the kit further comprises a gel pad or an ultrasound gel.

[0646] In some embodiments, the kit further comprises a device selected from a device and a vented vial adapter.

[0647] Reconstitution method

[0648] In another aspect, the present disclosure provides a method of reconstituting microspheres prepared according to any of the methods described herein. In some embodiments, a lyophilized product composed of microspheres is reconstituted. In some embodiments, the reconstitution method comprises adding a sufficient amount of sterile water or saline solution to the microsphere solution described in the above section of the container. The method may further comprise adding a volume of gas to the container and shaking the container. Specifically, in some embodiments, the method of reconstituting microspheres comprises:

[0649] (a) adding a sufficient amount of sterile water or saline solution to the microspheres within the container;

[0650] (b) optionally adding a volume of gas to the container of step (a); and

[0651] (c) optionally shaking the container of step (b).

[0652] In some embodiments of the reconstitution method, the amount of sterile water or saline solution does not exceed 1000 mL. In some embodiments of the reconstitution method, the amount of sterile water or saline solution does not exceed 750 mL. In some embodiments of the reconstitution method, the amount of sterile water or saline solution does not exceed 500 mL. In some embodiments of the reconstitution method, the amount of sterile water or saline solution does not exceed 200 mL. In some embodiments of the reconstitution method, the amount of sterile water or saline solution does not exceed 100 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 90 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 80 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 70 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 60 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 50 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 40 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 30 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 20 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 10 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 5 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 1 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 0.5 mL. In some embodiments, the amount of sterile water or saline solution does not exceed 0.3 mL.

[0653] In some embodiments, the amount of sterile water or saline solution added is sufficient to produce a homogeneous mixture comprising reconstituted microspheres.

[0654] In some embodiments of the reconstitution method, the shaking of the container lasts no more than 180 seconds. In some embodiments, the shaking of the container lasts no more than 160 seconds. In some embodiments, the shaking of the container lasts no more than 140 seconds. In some embodiments, the shaking of the container lasts no more than 120 seconds. In some embodiments, the shaking of the container lasts no more than 100 seconds. In some embodiments, the shaking of the container lasts no more than 80 seconds. In some embodiments, the shaking of the container lasts no more than 60 seconds. In some embodiments, the shaking of the container lasts no more than 40 seconds. In some embodiments, the shaking of the container lasts no more than 20 seconds. In some embodiments, the shaking of the container lasts no more than 10 seconds. In some embodiments, the shaking of the container lasts no more than 5 seconds.

[0655] In some embodiments of the reconstitution method, the shaking of the container lasts no more than 160 seconds. In some embodiments, the shaking of the container lasts at least 140 seconds. In some embodiments, the shaking of the container lasts at least 120 seconds. In some embodiments, the shaking of the container lasts at least 100 seconds. In some embodiments, the shaking of the container lasts at least 80 seconds. In some embodiments, the shaking of the container lasts at least 60 seconds. In some embodiments, the shaking of the container lasts at least 40 seconds. In some embodiments, the shaking of the container lasts at least 20 seconds. In some embodiments, the shaking of the container lasts at least 10 seconds. In some embodiments, the shaking of the container lasts at least 5 seconds.

[0656] Treatment method

[0657] In a last aspect, the present disclosure provides a method of treating a medical condition involving an abnormal or obstructive mass. In some embodiments, the medical condition includes kidney stones, urinary tract stones, biliary tract stones, blood clots, fibroids, cancerous tumors, and atherosclerotic plaques. In some embodiments, the subject has urolithiasis.

[0658] In some embodiments, the method includes: administering to a subject having the medical condition an effective amount of the reconstituted microsphere solution as described herein to contact the microspheres with the abnormal or obstructive mass (e.g., urinary tract stones, kidney stones, biliary tract stones, blood clots, fibroids, cancerous tumors, and atherosclerotic plaques); and directing energy to the abnormal or obstructive mass in the subject's body at a frequency that excites the fluid within the microspheres.

[0659] In some embodiments, the reconstituted microsphere solution is administered through a catheter into the ureter of the subject.

[0660] In some embodiments, the energy applied is in the form of electromagnetic, acoustic, microwave, photon, laser, or other forms.

[0661] In some embodiments, the applied energy is ultrasonic. In some embodiments, the applied energy is acoustic energy.

[0662] In some embodiments, the acoustic energy is ultrasonic energy in the frequency range of 100 kilohertz (kHz) to 2 megahertz (MHz). In some embodiments, the acoustic energy is ultrasonic energy in the frequency range of 200 kilohertz (kHz) to 1.5 megahertz (MHz). In some embodiments, the acoustic energy is ultrasonic energy in the frequency range of 300 kilohertz (kHz) to 1.2 megahertz (MHz). In some embodiments, the acoustic energy is ultrasonic energy in the frequency range of 500 kilohertz (kHz) to 1 megahertz (MHz).

[0663] In some embodiments, the applied ultrasonic energy is associated with a peak pressure in the range of 0.1 MPa to 10 MPa. In some embodiments, the applied ultrasonic energy is associated with a peak pressure in the range of 1 MPa to 10 MPa. In some embodiments, the applied ultrasonic energy is associated with a peak pressure in the range of 2 MPa to 9 MPa. In some embodiments, the applied ultrasonic energy is associated with a peak pressure in the range of 4 MPa to 8 MPa. In some embodiments, the applied ultrasonic energy is associated with a peak pressure in the range of 5 MPa to 7 MPa.

[0664] In some embodiments, the applied energy is generated by intraluminal energy from a solid-state pulsed laser.

[0665] In some embodiments, the wavelength of the laser energy is in the range of 1000 nm to 2500 nm. In some embodiments, the wavelength of the laser energy is in the range of 2050 nm to 2150 nm. In some embodiments, the evaporation of the liquid phase under the action of the laser energy is associated with a moving phase boundary that produces a pressure effect in the liquid phase.

[0666] In some embodiments, the laser energy has a frequency in the range between 1 kHz and 1 MHz. In some embodiments, the laser energy has a frequency in the range between 1 kHz and 1 MHz, between 10 kHz and 500 kHz, between 50 kHz and 100 kHz, or between 10 kHz and 50 kHz.

[0667] In some embodiments, the energy is applied for a sufficient amount of time to fragment the urinary calculus.

[0668] In some embodiments, the amount of time for which energy is applied does not exceed 100 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 90 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 80 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 70 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 60 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 50 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 40 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 30 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 25 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 20 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 15 minutes. In some embodiments, the amount of time for which energy is applied does not exceed 10 minutes.

[0669] In some embodiments, the applied photon energy causes a change in the volume of the microspheres or other cavitation effects in the microspheres.

[0670] In some embodiments, cavitation of the microspheres results in a change in the pressure gradient and mechanical effects in the urinary calculus surrounding the reconstituted microspheres.

[0671] In some embodiments, the change in the pressure gradient and other mechanical effects can cause the urinary calculus to fragment.

[0672] In some embodiments, the subject is a human. In some embodiments, the subject is an animal.

[0673] Example

[0674] The following synthetic and biological examples are provided to illustrate the technology and should not be construed in any way as limiting the scope of the technology.

[0675] Unless otherwise indicated, all temperatures are in degrees Celsius.

[0676] Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed.

[0677] Any functionally equivalent methods are within the scope of the technology. Various modifications to the technology, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications fall within the scope of the appended claims.

[0678] In the following examples, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

[0679] aq. = aqueous

[0680] LC-MS = Liquid Chromatography-Mass Spectrometry

[0681] MS = Mass Spectrometry

[0682] THF = Tetrahydrofuran

[0683] NaHCO3 = Sodium Bicarbonate

[0684] DIEA = Diisopropylethylamine

[0685] MS = Mass Spectrometry

[0686] NaH = Sodium Hydride

[0687] o / n = Overnight

[0688] HATU = 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0689] r.t. = Room Temperature

[0690] LAH = Lithium Aluminum Hydride

[0691] DCM = Dichloromethane

[0692] DMF = Dimethylformamide

[0693] DMSO = Dimethyl Sulfoxide

[0694] equiv. = Equivalent

[0695] EtOAc = Ethyl Acetate

[0696] EtOH = Ethanol

[0697] g = Gram

[0698] h = Hour

[0699] HCl = Hydrochloric Acid

[0700] HPLC = High Performance Liquid Chromatography

[0701] HOAc = Acetic Acid

[0702] M = Mole

[0703] MeOH = Methanol

[0704] mg = Milligram

[0705] mL = Milliliter

[0706] mmol = Millimole

[0707] mp = Melting Point

[0708] m / z = mass-to-charge ratio

[0709] NaCl = sodium chloride

[0710] Na2CO3 = sodium carbonate

[0711] NMR = nuclear magnetic resonance

[0712] NaOH = sodium hydroxide

[0713] Na2SO4 = sodium sulfate

[0714] NHS = N-hydroxysuccinimide

[0715] ppm = parts per million

[0716] TLC = thin layer chromatography

[0717] UV = ultraviolet

[0718] wt% = weight percentage

[0719] μM = micromole

[0720] General experimental details:

[0721] The final compound and phospholipid composition were confirmed by NMR. Recorded in CDCl3 (residual internal standard CHCl3 = δ 7.26), DMSO-d6 (residual internal standard CD3SOCD2H = δ 2.50), methanol-d4 (residual internal standard CD2HOD = δ 3.20) or acetone-d6 (residual internal standard CD3COCD2H = δ 2.05) or a mixture of CDCl3 and methanol-d4 1 1H-NMR, 31 31P-NMR, 13 13C-NMR spectra. The reported chemical shifts (δ) are given in parts per million (ppm), and the coupling constants (J) are in hertz (Hz). Spin multiplicities are reported as s = singlet, bs = broad singlet, bm = broad multiplet, d = doublet, t = triplet, q = quartet, p = pentuplet, dd = double doublet, ddd = double double doublet, dt = double triplet, td = triple doublet, tt = triple triplet, and m = multiplet.

[0722] Example 1: Synthesis of compound of formula Ib

[0723] Step 1 - Synthesis of PEG2DA

[0724] Dissolve PEG2000 in acetonitrile and warm to 40 °C. After dissolution, add an aqueous potassium phosphate buffer solution adjusted to pH ~ 7.5 with phosphoric acid, then add TEMPO. Then, simultaneously add aqueous solutions of sodium chlorite and sodium hypochlorite to the reaction mixture over 30 minutes to 1 hour. After addition is complete, stir the reaction at 40 °C for at least 24 - 48 hours. After the minimum stirring time, determine whether the reaction is complete by TLC analysis. When it is considered complete, cool the mixture to 0 °C and quench with an aqueous sodium thiosulfate solution. Then acidify the quenched solution to pH < 3 with an aqueous hydrochloric acid solution and extract the organic matter by partitioning between chloroform / methanol / water. Then remove the solvent from the organic phase via rotary evaporation and dry the material by azeotroping with toluene. Then purify the crude material by precipitation from toluene with diethyl ether. Figure 8 The purified PEG2DA is shown 1 in the 1H-NMR spectrum.

[0725] Step 2 - Synthesis of DSPE-PEG2-COOH

[0726] Dissolve PEG2DA in toluene and warm to 90 °C. Add CDI in portions to control the evolution of carbon dioxide. Once all solids are dissolved and all foaming has ceased, add solid DSPE to the reaction mixture, then add triethylamine. Stir the mixture at 90 °C for 12 - 24 hours, at which point check whether the reaction is complete by TLC. Once complete, quench the reaction mixture with 5 - 10 mL of methanol and cool to ambient temperature. Once cooled, dilute the solution with chloroform and partition with 1 M aqueous hydrochloric acid solution to remove imidazole, then perform a brine wash. Neutralize the mixture with ammonium hydroxide, then remove a large amount of solvent via rotary evaporation. Then chromatograph the crude mixture sequentially on a normal phase medium (chloroform / methanol / ammonium hydroxide gradient) and then on a reverse phase medium (methanol / water gradient), including washing the column with sodium chloride, to obtain the sodium salt of DSPE-PEG2-COOH. Figure 9 and 10 The 1H-NMR of DSPE-PEG2-COOH is shown 1 and 31 the 31P-NMR spectrum, respectively.

[0727] Step 3 - Synthesis of the compound of formula Ib

[0728] Before the reaction, alendronic acid was dissolved in tetrabutylammonium hydroxide (40 wt% aqueous solution) and water was removed by azeotropic distillation using toluene, followed by drying under vacuum. After preparing the alendronate-TBA, DSPE-PEG2-COOH was dissolved in an 85:15 anhydrous chloroform / anhydrous dimethylformamide mixture and warmed to 40 °C. N-Hydroxysuccinimide (NHS) was added to the solution, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), and the mixture was stirred at 40 °C for 4 - 6 hours. After the allotted time, the formation of DSPE-PEG2-COO-NHS ester was determined using 1 1H NMR spectroscopy (according to 1 1H-NMR analysis, typical reaction completion > 75%). Once the formation of DSPE-PEG2-COO-NHS ester was confirmed, the previously prepared alendronate-TBA salt was dissolved in anhydrous chloroform and added directly to the reaction vessel, followed by the addition of triethylamine. The reaction temperature was then raised to 50 °C and the mixture was allowed to stir at this temperature for 18 - 24 hours, at which point the completion of the reaction was determined by TLC. Once complete, the reaction mixture was partitioned three times with 1 M aqueous hydrochloric acid to remove the excess alendronate, and then washed twice with brine to remove the excess acid. The mixture was neutralized with ammonium hydroxide and then the bulk of the solvent was removed via rotary evaporation. Then the material was treated with 50WX8 hydrogen form ion exchange resin to remove the bulk of the tetrabutylammonium counterions. The resin was filtered and the bulk of the solvent was removed from the filtrate, and then the material was chromatographed on a reverse phase medium (methanol / water gradient), including washing the column with ammonium chloride, to obtain the compound of formula Ib. Figure 11 and Figure 12 respectively show the 1 1H-NMR and 31 31P-NMR spectra of the compound of formula Ib, which has the structure:

[0729]

[0730] The method for synthesizing the compound of formula Ib described herein is summarized as follows:

[0731]

[0732] Example 2: Synthesis of microspheres

[0733] The compound of formula Ib generated as described in Example 1 was combined with other lipid compositions – DSPE-PEG2k (compound of formula IIb)

[0734] and

[0735] DSPC (compound of formula IIIa)

[0736]

[0737] Mix with water. The 31 P-NMR spectrum ( Figure 14 ) shows three peaks, each corresponding to each lipid composition: the compound of formula Ib, DSPC or DSPE-PEG2k. The signal of each peak indicates the relative amounts of the compound of formula Ib ("DSPEPEG alendronate" at 28.37), DSPC ("DSPC" at 1363.68), and DSPE-PEG2k ("DSPE PEG" at 107.55) in the mixture.

[0738] The liquid phase of the mixture containing the compound of formula Ib, DSPC and DSPE-PEG2k is processed through a homogenizer with multiple passes until the desired diameter and PDI are obtained. The size distribution of the samples from each pass is analyzed using dynamic light scattering (DLS), and the results are provided in Figure 4 . The formulation is diluted in phosphate buffered saline and analyzed using Mobius (Wyatt).

[0739] n-Decafluorobutane (C4F 10 ) is added to the lipid mixture and the mixture is agitated for 1 - 2 minutes. Figure 5 Images of the lipid mixture before (left) and after (right) the addition and agitation steps of n-decafluorobutane are provided in

[0740] The particle number density and particle diameter of the microspheres are characterized by electrical zone sensing using a Beckman Coulter Multisizer 4e with a 30 μm aperture. The sample volume ranges from 1 to 10 μL, in 100 mL of Isoton II diluent. Figure 7 Exemplary results are provided in

[0741] Example 3: Accumulation assay

[0742] In a time-course study, the microspheres generated as described in Examples 1 and 2 are characterized using a Beckman Coulter Multisizer 4e while incubating with hydroxyapatite beads. The particle density of the microspheres at different time points is analyzed by the Multisizer. The change in the microsphere concentration is used to calculate the number of microspheres accumulated on the surface of the hydroxyapatite beads. A control microsphere sample without the compound of formula Ib is also analyzed.

[0743] As Figure 6As summarized, this experiment demonstrated that in the time-course study, microspheres of the compound of formula Ib significantly accumulated onto hydroxyapatite beads. The results indicate that the microspheres containing the compound of formula Ib have a high affinity for hydroxyapatite beads.

[0744] Example 4: Headspace analysis

[0745] A headspace sample was directly withdrawn from a container of a pharmaceutical composition of the compound of formula Ib and n-decafluorobutane using a manual gas chromatograph syringe. The headspace sample was immediately injected into the GC. Figure 13 The GC signals from this experiment are provided. It shows peaks indicative of n-decafluorobutane.

[0746] Example 5: Treatment of urolithiasis using microspheres with external acoustic energy

[0747] The microspheres generated as described in Examples 1 and 2 were administered to a patient diagnosed with urinary stones via a 5Fr catheter positioned by cystoscopy. The patient had presented with acute renal colic and was diagnosed with urolithiasis by CT scan. The cystoscope was removed after positioning the catheter in the affected side of the upper urinary tract of the patient and connecting a syringe containing the microsphere solution to the distal end of the catheter. Volumes of 0.5 - 1.0 mL of the microsphere solution were injected at multiple time intervals during a procedure lasting 20 to 90 minutes. Acoustic energy was applied at multiple time intervals during this procedure. The acoustic energy source used - a SmartSphere console with a treatment head designed to abut the skin of the patient's lower back or groin area - was designed to interact with the microspheres on or near the surface of the stone, resulting in stone erosion, pitting, and fragmentation, and has been shown to be safe in extensive studies in a porcine model. Many urinary stone patients have been successfully treated using this method.

[0748] Although the invention has been particularly shown and described in connection with preferred embodiments and various alternative embodiments, those skilled in the relevant art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention.

[0749] All references, issued patents, and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A compound of formula Ia or a salt thereof.

2. The compound according to claim 1, wherein the compound of formula Ia is a compound of formula Ib 3. A pharmaceutical composition comprising the compound according to claim 1 or 2.

Images