The present application claims priority from United Kingdom Patent Application No. GB1908716.2, filed Jun. 18, 2019, which is incorporated herein in its entirety.
The present application pertains to the field of pharmaceuticals. More particularly, the present application relates to colloidal drug aggregates, and methods of manufacture and uses thereof.
Some small-molecule drugs spontaneously self-assemble under aqueous conditions to form colloidal aggregates.1-3 The formation of these colloids is governed by a critical aggregation concentration (CAC) and generally occurs when water is added to a solution of drug in a water-miscible organic solvent.4 Although colloidal drug aggregates are best known for causing false hits in early drug discovery,5-7 recent efforts have aimed to stabilize these drug-rich particles for delivery.8-10
Development of stable colloidal drug aggregates has paradoxically created a new problem: colloid-associated drug does not permeate the cell membrane to interact with intracellular targets.11-12 One method to overcome this issue is to disrupt the particles to yield drug monomers.12, 13 Colloid dissolution has traditionally been accomplished by adding detergent; however, this strategy is more useful in vitro than in vivo, where toxic excipients are dose-limited.
A need remains for colloidal drug formulations that do not require chemical modification for aggregation and that minimize or avoid the use of toxic excipients for stabilization.
The above information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
An object of the present application is to provide stable colloidal drug aggregates and methods of manufacture and use thereof. In accordance with an aspect of the present application, there is provided an acid-responsive composition comprising: a colloidal aggregate of one or more drugs and a stabilizing agent, wherein the colloidal aggregate disrupts, dissolves or disassembles when the acid-responsive composition is in an acid environment having a pH of less than 7.4 (such as, a pH of less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4). In accordance with one embodiment, the acid-responsive composition comprises: a colloidal aggregate of one or more drugs and a stabilizing agent, wherein (a) at least one of the one or more drugs is an ionizable drug or ionizable drug analogue, wherein the conjugate acid of the ionizable drug or drug analogue has a pKa of at least 4, preferably at least 4.5 or at least 5, or at least 5.5, or at least 6, or at least 6.5; (b) the stabilizing agent is an acid-responsive stabilizing agent such that the stabilizing agent undergoes a morphological and/or functional change when pH is reduced to less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4; or (c) both (a) and (b).
In accordance with an embodiment of the present application, there is provided an acid-responsive composition comprising: a colloidal aggregate of one or more ionizable drugs or ionizable drug analogues; and a stabilizing agent, wherein the conjugate acid of the ionizable drug or drug analogue has a pKa of at least 4, preferably at least 4.5 or at least 5, or at least 5.5, or at least 6, or at least 6.5. In another embodiment of the present application, there is provided an acid-responsive composition comprising a colloidal aggregator, which is either an ionizable or non-ionizable drug or drug analogue; and an acid-responsive stabilizing agent that undergoes a morphological and/or functional change when the pH is reduced to less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4.
In certain embodiments the stabilizing agent is a protein (e.g. an antibody or antibody fragment), a polymer, a colloid-forming compound (e.g., vitamin E) or another colloid-forming drug (e.g., fulvestrant). In the example in which the stabilizing agent is another colloid-forming drug, the other colloid-forming drug is, optionally, not ionizable at a pH of 4 or less. Optionally, the stabilizing protein is IgG, trastuzumab, albumin, transferrin, or an attenuated bacterial toxin (such as attenuated diphtheria toxin or derivatives thereof). Suitable stabilizing polymers include polymeric surfactants, such as, but not limited to, UP80, PLAC-PEG, Brij 58, F127, Vitamin E-PEG, F68, or Brij L23.
In accordance with certain embodiments, the stabilizing agent compromises the integrity of plasma membranes in acidic environments (e.g., the stomach or a lysosome or an endosome of a cell), thereby allowing colloidal aggregates or their contents to be transported out of the acidic environment.
In accordance with certain embodiments, the colloidal aggregate is disrupted upon contact of the composition with an acid or introduction of the composition to an acidic environment (e.g., the stomach or a lysosome or an endosome of a cell), and the one or more drug or drug analogue is released as a result of the disruption of the colloid.
In accordance with certain embodiments, the colloidal aggregate comprises a targeting compound for delivery of the composition to a target site. The targeting compound can be, for example, a protein (e.g., transferrin), an antibody (e.g. trastuzumab), an attenuated bacterial toxin (such as attenuated diphtheria toxin or derivatives thereof), or another molecule that selectively binds to a cell receptor. Optionally, the targeting compound also functions as the stabilizing agent, or acts together with another stabilizing agent to aid in stabilization of the colloidal aggregate.
In a specific embodiment, the ionizable drug is lapatinib and the composition further comprises a stabilizing compound, which is fulvestrant, and a targeting compound, which is transferrin.
In another specific embodiment, the ionizable drug is lapatinib and the composition further comprises a stabilizing compound, which is a combination of fulvestrant and a polymeric surfactant (e.g., PLAC-PEG).
In accordance with certain embodiments, the colloidal drug aggregate composition comprises an ionizable drug analogue that comprises a drug molecule (e.g., sorafenib or fulvestrant) chemically modified to include an ionizable moiety. Optionally, the ionizable drug analogue is itself pharmaceutically active. Alternatively, the analogue functions as a prodrug such that following ionization and release from the colloid, the analogue is modified (e.g., by cleavage of the ionized moiety) in vivo to form the active drug molecule.
Another aspect of the present application provides a method of drug therapy comprising administration of the colloidal drug aggregate composition to a subject in need thereof.
For a better understanding of the application as described herein, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings and tables.
Table 1: Colloid-forming drugs with pKa's>5 show pH-dependent critical aggregation concentrations (CACs) between physiological pH 7.4 and endosomal pH 5.5. CACs were measured in pH-adjusted phosphate buffered saline (PBS) by dynamic light scattering (n=3, mean±95% CI) and pKa's were obtained from DrugBank.ca.
Table 2: Description of each variable and function used in calculation of the critical aggregation concentration (CAC). This calculation objectively applies a common approach for measuring the CAC where assays are used in which colloidal drug aggregates produce a signal and soluble drug molecules do not. For example, the light scattering intensity of a drug solution does not increase with increasing drug concentration until the CAC is reached, when colloidal particles begin to form. By increasing the concentration above the CAC, the number of colloids increases somewhat linearly, and the scattering intensity increases similarly. This behavior is reflected in Equation S4, which describes a line, the slope of which transitions from zero to positive once the CAC is reached.
Table 3: Plots of scattering intensity versus concentration that are used to calculate the CAC of each drug at pH 7.4 and 5.5.
Table 4: Formulations relating treatment name to final concentrations of each component. For imaging and flow cytometry experiments, CholEsteryl BODIPY 542/563 C11 was added before colloid formation to a final concentration of 500 nM.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
The term “comprising” as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and/or ingredient(s) as appropriate.
As used herein, the term “in need thereof” is used to refer to a judgment made by a physician or other caregiver (e.g., a veterinarian) that a subject requires or will benefit from treatment or preventative care. This judgment is made based on a variety of factors that are in the realm of the physician's or caregiver's expertise.
As used herein, “pharmaceutically acceptable” means approved or approvable by a regulatory agency of a federal or a state/provincial government or the corresponding agency in countries other than the United States or Canada, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, including in humans.
As used herein, the term “prodrug” will be understood to refer to a compound that, following administration to a subject, is metabolized into a pharmacologically active drug.
As used herein, the term “subject” refers to a human or a non-human animal (e.g., a mammal).
The present application provides a colloidal drug aggregate, and related composition, that is designed to enhance the effect of colloid-bound drug against its molecular target by being responsive to a change in environment as stimulus to trigger drug release from the stable colloids. The change in environment occurs without adding an exogenous reagent by exploiting an endogenous stimulus, such as, for example, the acidic environment of the stomach or of the endosomes and lysosomes of cells. Alternatively, in order to demonstrate this behaviour in the absence of a living organism, the colloidal drug aggregate composition can be triggered to release drug by the addition of an exogenous trigger, such as, for example, an acidic agent or solution.
The formation of some colloidal drug aggregates is pH-dependent, with the colloidal form of weakly basic drugs dissolving at low pH.15-17 This behaviour allows colloids that are stable outside the cell to be disrupted intracellularly by the acidic endo-lysosomal pathway.18-21 Colloidal drug aggregates stabilized with a targeting antibody have been shown to enter cancer cells via endocytosis,22 such that acidification within the cells can trigger release.
The present application provides a stable acid-responsive composition comprising a colloidal aggregate comprising one or more drugs, which facilitates acid-triggered release of the drug(s) following administration. Optionally, the stable colloidal aggregate is a targeted composition. The present application further provides methods of manufacture and use of such colloidal drug aggregates. Use of the stable, targeted colloidal drug aggregate-containing composition described herein results in an enhanced effect of the colloid-bound drug against its molecular target over non-colloidal controls and non-acid responsive colloidal controls, due to, for example, colloid endocytosis and subsequent endosomal escape.
Colloidal Drug Aggregates
Many drugs form colloidal drug aggregates in biologically relevant environments, including pharmaceutical excipients, cell culture media and simulated gastrointestinal fluids. Colloidal aggregates have unique nanostructures and are quite different from some other self-assembled drug nanoparticles such as nanocrystals. Colloids typically have a diameter in the range of from about 50 to about 1000 nm and form spontaneously by phase separation on addition to a liquid, such as water or an aqueous solution (the continuous phase). Aggregation is concentration dependent; at low concentrations the compound is fully solubilized and as the concentration increases to a “critical aggregation concentration” (or “CAC”) the compound spontaneously self-assembles, or aggregates, as a colloid. When the colloid is diluted, the colloid aggregate spontaneously disassembles and the compound returns to its solubilized form. The CAC of a compound is determined by a combination of intrinsic properties of the compound and extrinsic properties, such as, for example, temperature, nature of the continuous phase, and salt concentration. Also, the presence of solubilizing or stabilizing excipients can affect the CAC of a compound.
Non-limiting examples of drugs that form colloidal drug aggregates include clotrimazole, econazole, miconazole, sulconazole, intraconazole, celecoxib, mefenamic acid, oxaprozin, candesartan cilexetil, manidipine, nicardinpine, cinnarizine, glyburide, amiodarone, etravirine, delvirdine, nelfinavir, chlorpromazine, clofazimine, clopidogral sulfate, fenofibrate, meclizine, menatetrenone, pranlukast, raloxifene, triclabendazole, methylene blue, bexarotene, lapatinib, crizotinib, fulvestrant, nilotinib, sorafenib, vemurafenib, gefitinib, imatinib, pazopanib, cabozantinib, and siramesine.
In accordance with some embodiments, the colloidal drug aggregate composition of the present application comprises an ionizable drug. For example, the colloidal composition is formed from a colloidal aggregation of one or more ionizable drugs. At least one of the one or more ionizable drugs is basic, or weakly basic, such that the colloid is susceptible to dissolution in the presence of an acid or acidic environment. In particular, the protonated form of the ionizable drug in the colloidal formulation has a pKa of at least 4.0. Preferably, the pKa of the protonated form of the ionizable drug is at least 4.5, or at least 5.0, or at least 6, or at 6.5. Most dissolved drug molecules are positively charged at pH values below this pKa and uncharged at pH values greater than this pKa.
Suitable drugs for use in colloid drug aggregates as taught herein are drugs that solubilize in acidic environments and are colloids under physiologically neutral (e.g., approximately pH 7.4—slightly basic) conditions. An “acidic environment” is defined herein as an environment having a pH of less than 7.4. By ionizing at acidic pH, the drugs in colloids become soluble. The drug colloid aggregate comprising one or more ionizable drug is acid responsive, in that the colloid aggregate will dissolve or disassemble in upon contact with acid or introduction into an acidic environment, thereby releasing the drug from the colloid aggregate. The pH of the acid or acidic environment at which dissolution or disassembly occurs depends on the pKa of the ionizable drug in its protonated form.
Non-limiting examples of ionizable drugs that can be included in the colloidal drug aggregate composition of the present application include lapatinib, clotrimazole, nilotinib, pazopanib, and siramesine.
Drugs that form colloidal drug aggregates but that form colloids that do not respond to acid (e.g., sorafenib and fulvestrant), can be chemically modified by addition of an ionizable functional group to form an analogue that is responsive to acid. These drugs are either non-ionizable, or do not ionize in the presence of pH less than 4, until they have been chemically modified to include such an ionizable functional group. These drugs retain activity following chemical modification to include the ionizable functional group, or are metabolized in vivo to become biologically/pharmaceutically active. Accordingly, in accordance with some embodiments, the colloidal drug aggregate composition of the present application comprises an ionizable drug analogue that is responsive to acid.
Drug analogues useful in the formation of the colloidal drug aggregates as described herein, are drug molecules that have been chemically modified to include an ionizable functional group and that, as a result of the chemical modification, have a protonated form that has a pKa of at least 4.0. Preferably, the pKa of the protonated form of the ionizable drug analogue is at least 4.5, or at least 5.0, or at least 6, or at 6.5.
Suitable drug analogues for use in colloid drug aggregates as taught herein are drug analogues that solubilize in acidic environments and are colloids under physiologically neutral (e.g., approximately pH 7.4—slightly basic) conditions. By ionizing at acidic pH, the drug analogues in colloids become soluble. The drug colloid aggregate comprising one or more ionizable drug analogue is acid responsive, in that the colloid aggregate will dissolve or disassemble in upon contact with acid or introduction into an acidic environment, thereby releasing the drug analogue from the colloid aggregate. The pH of the acid or acidic environment at which dissolution or disassembly occurs depends on the pKa of the ionizable drug in its protonated form.
Non-limiting examples of suitable ionizable functional groups include amines, such as, primary amines, secondary amines, tertiary amines, imines, heterocyclic amines, aromatic amines. More specific examples of suitable amines include, hydroxylamine, hydrazine, imidazole, amine, and triazoles. Alternatively, an aggregator drug can be modified by chemical conjugation to an amine-containing molecule to form an analogue that is responsive to acid. Suitable amine-containing molecules include an amino nitrogen atom which is bound to three other atoms, at least one of which attaches the nitrogen atom to the aggregator molecule through a series of covalent bonds. Optionally, this amino nitrogen atom may be a part of one or more natural amino acids or derivatives thereof, including, but not limited to, β-alanine, N,N-dimethylglycine, N,N-dimethyl-β-alanine, histidine, serine, threonine, asparginine, glutamine, proline, alanine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, glycine, or valine. In this embodiment, the drug analogue exhibits similar activity to the unmodified drug, or the drug analogue functions as a prodrug that is metabolized in vivo to form an active drug molecule.
Non-limiting examples of drug analogues that can be included in the colloidal drug aggregate composition of the present application include a sorafenib analogue (e.g., an imidazole-containing sorafenib analogue), and a fulvestrant analogue (e.g., a glycine or valine modified fulvestrant, or a fulvestrant analogue bearing an amine derived from dimethylamine, imidazole, or morpholine).
The colloidal formulation additionally comprises a stabilizing agent or excipient. Most colloid-forming drugs aggregate at micromolar or sub-micromolar concentrations. Colloids formed without stabilizing excipients are often polydisperse and precipitate over several hours in physiological media, are unstable and, therefore, unsuitable for use according to the present invention. Excipients, including polymers, proteins, and other colloid-forming compounds, such as other drugs or azo-dyes, can control the size and stability of colloidal drug aggregates. In addition to stabilizing colloidal drug aggregates, proteins (e.g., antibodies) can also confer functionality to promote selective uptake by target cells.
In certain embodiments, the colloidal drug aggregate of the present application comprises a corona. As used herein, the term “corona” refers a surface-bound layer of adsorbed proteins and/or other stabilizing excipient(s), onto a colloidal aggregate, for example, resulting from the exposure of the aggregate to solution and media containing proteins and/or other stabilizing excipient(s). In one embodiment, in which the conditions for concentration and nature of the proteins and/or excipient(s) in the solution and media is known, the corona may be well defined and characterized. The corona formation and its characteristics may be influenced by various factors such as the surface chemistry of the nanoparticle, the colloidal aggregate size, and the composition of the continuous phase.
Proteins that can be used for the protein corona may be, but are not limited to, albumins, immunoglobulins, caseins, insulins, hemoglobins, lysozymes, α-2-macroglobulin, fibronectins, vitronectins, fibrinogens, lipases, transferrin, apolipoproteins, bacterial toxins, and the like. Proteins, peptides, enzymes, antibodies and combinations thereof, may also be used to stabilize the colloidal aggregates. Accodring to an embodiment, the use of novel antibodies or FDA-approved antibodies such as toclizumab, natalizumab, vedolizumab, alemtuzumab, cetuximab, daratumumab, ofatumumab, panitumumab, pertuzumab, trastuzumab, dinutuximab, obinutuzumab, ramucirumab, ipilumumab, nivolumab, pembrolizumab, brentuximab, catumaxomab, basilixumab, ibritomumab may be tailored to stabilize the colloidal aggregates. The target/indication for these FDA approved antibodies are provided in the table below.
In accordance with other embodiments, the acid-responsive colloidal drug composition comprises a combination of one or more non-ionizable, or ioniziable, drugs with an acid-responsive stabilizer. This acid-responsive stabilizer acts to disrupt the colloid or cellular membranes in an acidic environment. In particular, an acid-responsive stabilizing agent is one that undergoes a morphological and/or functional change when the pH is reduced to less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4.
Non-limiting examples of acid-responsive stabilizers include bacterial toxins, such as attenuated diphtheria toxin or variants thereof.
Accordingly, certain embodiments of the present application provide colloidal drug aggregate compositions that are useful in targeting a drug for treatment of a disease or disorder in a subject. In specific embodiments, the disease or disorder is selected from the diseases and disorders associated with the listed FDA-approved antibodies, as set out in the table above. This list is not exhaustive; therapeutic antibodies and targeting antibodies continue to be developed and can be incorporated in the colloidal drug aggregate compositions of the present application as a stabilizing agent, a targeting agent, or both.
In other embodiments, the colloidal drug aggregate composition of the present application comprises a stabilizing agent that is another aggregator drug, which may or may not be acid responsive. In a particular embodiment, the colloidal drug aggregate composition comprises an acid responsive (i.e., ionizable) drug and a non-acid responsive drug. In one example of this embodiment, the acid responsive drug is lapatinib and the non-acid responsive drug is fulvestrant.
Colloidal drug aggregate formulations of the present application can be formulated simply by dissolving a drug or drug analogue in a water-miscible solvent (e.g., DMSO) and adding water, or an aqueous solution, to the dissolved drug and allowing the spontaneous formation of colloids. One or more stabilizing agents are incorporated into either aqueous or organic phase prior to colloid formation, or to the mixture after colloid formation. For convenience, the formation of the colloidal drug aggregate is often done at ambient temperature, typically without the need for further mixing or stirring. The amount of the drug used will depend on the CAC for that drug under the formulation conditions used. Colloids are usually detectable within seconds of mixing the drug, or drug analogue, in the water-miscible solvent with the aqueous phase. In accordance with certain embodiments in which the colloidal drug aggregate formulation is for pharmaceutical use, the one or more stabilizing agents are cytocompatible. Also, as noted above, in certain embodiments the one or more stabilizing agents are acid-responsive.
In some instances, the pH of the colloid solution needs to be adjusted to facilitate colloid formation or for colloid stability. The pH can be adjusted using an adjusting agent that is an acid (e.g., HCl or citric acid) or a base (e.g. NaOH), as necessary. pH adjustment is typically performed by adding the pH adjusting agent as the final formulation step. Depending on the amount of pH adjusting agent required, it may be necessary to reduce the amount of water, or aqueous solution, used in the colloid formation.
Pharmaceutical Formulations and Use Thereof
The present application further provides pharmaceutical formulations for administration of a colloidal drug aggregate composition to a subject in need thereof. The pharmaceutical formulation of the present application comprises a colloidal drug aggregate, optionally in combination with a pharmaceutically acceptable carrier, diluent, excipient or medium, wherein the colloidal drug aggregate comprises one or more drugs and a stabilizing agent, and wherein the colloidal aggregate disrupts, dissolves or disassembles when the acid-responsive composition is in an acid environment.
In certain embodiments, the pharmaceutical composition comprises a combination of two or more colloidal drug aggregate compositions.
Suitable pharmaceutical compositions of the present application will generally include an amount of the one or more drugs to give a range of final concentrations, depending on the intended use.
In certain embodiments, the pharmaceutical compositions of the present application comprise a colloidal drug aggregate composition as the sole active component, alone or in combination with one or more desired pharmaceutically inactive additives, excipients, and/or components (e.g., polymers, fillers, carriers, excipients, diluents, disintegrating additives, lubricants, solvents, dispersants, absorption promoting additives, controlled release additives, anti-microbial additives, preservatives, sweetening additives, colorants, flavors, dyes, or the like), and no other active pharmaceutical ingredient(s).
In other embodiments, the pharmaceutical composition comprises a combination of two or more colloidal drug aggregate compositions as the active components, alone or in combination with one or more desired pharmaceutically inactive components as listed above.
In alternative embodiments, the pharmaceutical composition comprises one or more colloidal drug aggregate compositions in combination with one or more other therapeutic agent.
Accordingly, the present application further comprises a method of providing drug therapy to a subject by administering a pharmaceutical composition as described above. Such a method is particularly useful for delivering or targeting the one or more drugs in the pharmaceutical composition to an acidic target site of the patient (e.g., stomach, lysosomes endosomes, etc.).
In a specific embodiment, the pharmaceutical composition comprises a colloidal drug aggregate composition comprising lapatinib. Accordingly, the present application further comprises a method of treating cancer, such as breast cancer, comprising administration of a colloidal drug aggregate composition comprising lapatinib to a subject in need thereof. Optionally, the method additionally comprises administration of another chemotherapeutic agent before, after or simultaneous with administration of the colloidal drug aggregate composition comprising lapatinib.
In another specific embodiment, the pharmaceutical composition comprises a colloidal drug aggregate composition comprising lapatinib and fulvestrant. Accordingly, the present application further comprises a method of treating cancer, such as breast cancer, comprising administration of a colloidal drug aggregate composition comprising lapatinib and fulvestrant to a subject in need thereof. Optionally, the method additionally comprises administration of another chemotherapeutic agent before, after or simultaneous with administration of the colloidal drug aggregate composition comprising lapatinib and fulvestrant.
In another specific embodiment, the pharmaceutical composition comprises a colloidal drug aggregate composition comprising clotrimazole. Accordingly, the present application further comprises a method of treating a fungal infection, such as a fungal skin infection, comprising administration of a colloidal drug aggregate composition comprising clotrimazole to a subject in need thereof. Optionally, the method additionally comprises administration of another chemotherapeutic agent before, after or simultaneous with administration of the colloidal drug aggregate composition comprising clotrimazole.
In another specific embodiment, the pharmaceutical composition comprises a colloidal drug aggregate composition comprising sorafenib or an analogue of sorafenib. Accordingly, the present application further comprises a method of treating cancer, such as kidney cancer, liver cancer, or thyroid cancer, comprising administration of a colloidal drug aggregate composition comprising the sorafenib analogue to a subject in need thereof. Optionally, the method additionally comprises administration of another chemotherapeutic agent before, after or simultaneous with administration of the colloidal drug aggregate composition comprising the sorafenib analogue.
In another specific embodiment, the pharmaceutical composition comprises a colloidal drug aggregate composition comprising an analogue of fulvestrant. Accordingly, the present application further comprises a method of treating cancer, such as breast cancer, comprising administration of a colloidal drug aggregate composition comprising the fulvestrant analogue to a subject in need thereof. Optionally, the method additionally comprises administration of another chemotherapeutic agent before, after or simultaneous with administration of the colloidal drug aggregate composition comprising the fulvestrant analogue.
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.
Methods
Materials
Lapatinib and sorafenib were purchased from MedChemExpress. Nilotinib and pazopanib were purchased from Cedarlane. Fulvestrant was purchased from SelleckChem. Poly(D,L-lactide-co-2-methyl-2-carboxy-trimethylene carbonate)-graft-poly(ethylene glycol) (PLAC-PEG) was synthesized by ring-opening polymerization using a pyrenebutanol initiator to a molecular weight of 12 kDa and conjugated with an average of three 10 kDa PEG chains/backbone as previously described.43 Ultrapure polysorbate 80 (UP80) was purchased from NOF America Corporation. Clotrimazole, norethindrone, Dimethyl sulfoxide (DMSO), transferrin, EDTA, dodecane, lecithin, polyacrylic acid (PAA), methylcelluose, and hydroxypropylmethylcellulose (HPMC) were purchased from Sigma-Aldrich. Transferrin-Alexa Fluor 488 conjugate, RPMI 1640 cell culture medium, penicillin-streptomycin solution, trypsin-EDTA solution, Hank's balanced salt solution, PrestoBlue cell viability reagent, CholEsteryl BODIPY™ 542/563 C11, CellMask Green™ 1000× solution, and 7-AAD were purchased from Thermo Fisher Scientific. Fetal bovine serum and Dulbecco's phosphate buffered saline were purchased from Wisent Bio Products. Ultrapure Congo red was purchased from Enzo Life Sciences. HPLC grade acetonitrile and methanol were purchased from Caledon Laboratories. Mass spectrometry grade formic acid was purchased from Fluka.
Calculation of the Critical Aggregation Concentration (CAC)
CAC values are calculated by preparing colloidal formulations with different concentrations and measuring the scattering intensity (or fluorescence intensity when appropriate). The data is then fit to Equation S4, which was derived from first principles (Equations S1-S3), using the MATLAB curve fitting toolbox to obtain the critical aggregation concentration (CAC) under the conditions used. Table 2 describes the meaning of each variable;
Signal=SignalBL+m+H(Cagg−CACagg)·(Cagy−CACagg)
Colloidal Drug Aggregate Formulation
Colloidal drug aggregates were formulated as described previously.8 Briefly, the colloids formed spontaneously when water was added to drug that was dissolved in a water-miscible organic solvent (usually DMSO). Final colloid suspensions were typically made at a 1 mL scale with 1% (v/v) final DMSO concentration. First, solutions of drug, polymer, and dye were prepared in 10 μL DMSO at 100×the final concentration. For colloids formulated under serum-free conditions, 890 μL of double distilled water was then added, followed by 100 μL of 10×PBS (for experiments without cells) or 10×RPMI 1640 (for cell experiments). For colloids formulated under 10% (v/v) serum conditions, 800 μL of double distilled water was added, followed by 90 μL of 10×PBS or 10×media, and finally 100 μL of FBS. Transferrin stabilized colloids were prepared by supplementing the water added with a small amount of 5 mg mL−1 transferrin in PBS. When the pH of the colloid solution needed to be adjusted, the amount of water added was reduced by 10 μL, and 10 μL of aqueous citric acid was added as the last formulation step. The concentration of citric acid used depended on the desired final pH. In PBS, the concentration was 0.12 M for pH 6.5, 0.3 M for pH 5.5, 0.35 M for pH 5.2, and 0.5 M for pH 4.5. In PBS with 10% (v/v) FBS, the concentration was 0.15 M for pH 6.5, 0.35 M for pH 5.5, and 0.6 M for pH 4.5. For time-based studies, the colloid suspensions were incubated at 37° C. between time points.
Characterization by Dynamic Light Scattering
Colloid diameter, polydispersity index (dispersity), and scattering intensity were measured by dynamic light scattering (DLS) using a DynaPro Plate Reader II (Wyatt Technologies) that was optimized by the manufacturer for detection of colloidal aggregates (i.e. 100-1000 nm particles). The instrument was configured with a 60 mW 830 nM laser and detector angle of 158°. A 100 μL sample of each formulation was pipetted into a 96-well plate and measured with 20 acquisitions per sample.
Characterization of Zeta Potential
Colloids were prepared as described above, but with a single addition of 0.1 mM KCl solution instead of water and buffer. Zeta potential was immediately assessed using a Malvern Nano-ZS (Malvern Panalytical).
Fluorexcent Intensity Characterization
Colloid suspensions were prepared as described above. A 100 μL sample of each formulation was pipetted into a 96 well plate. Fluorescence was measured using a Tecan Infinite Pro 200 plate reader.
Characterization by Transmission Electron Microscopy (TEM)
Colloidal formulations (5 μL) were deposited onto freshly glow-discharged 400 mesh carbon coated copper TEM grids (Ted Pella, Inc.) and allowed to adhere for 5 min. Excess liquid was removed, followed by a quick wash with 5 μL water. Grids were then imaged on a LEO 912B Energy Filtered TEM operating at 120 kV.
Assessment of Colloid-Bound Transferrin
Colloid formulations were prepared as described above with transferrin-Alexa Fluor 488 conjugate as the stabilizer. At predetermined time points during incubation at 37° C., formulations were centrifuged at 16,000×g for 1 h, followed by withdrawal of 100 μL, of supernatant for measurement. The amount of transferrin remaining in the supernatant was measured using fluorimetry (λex=488 nm, kem=530 nm). The fraction of transferrin bound to the colloid was calculated with Equation 1.
Assessment of Drug Release
Drug release was measured by centrifuging to pellet the colloids, and then by quantifying the drug in the supernatant. Colloid formulations were centrifuged at 16,000×g for 1 h, followed by withdrawal of 100 μL for quantification. Another 100 μL was withdrawn for DLS analysis to confirm that the colloids had completely settled out of solution. Non-centrifuged colloid suspensions were used as controls.
Membrane Permeability Assay
Supported artificial lipid membranes were prepared according to the manufacturer instructions (EMD Millipore cat #MATRNPS50). Briefly, a 1% (w/w) solution of egg lecithin in dodecane was prepared by sonicating until the solution was no longer cloudy. A 5 μL aliquot of this phospholipid solution was dropped by pipette into each well of the mesh plate, which wet the membrane and caused it to become translucent. Receiver solution was prepared as 1% (v/v) DMSO and 10% (v/v) FBS in PBS and 340 μL was pipetted into each well of the receiver plate. Lapatinib and fulvestrant solutions ranging from 0 to 5 μM for constructing the standard curve were prepared in identical media and pipetted into separate wells of the receiver plate. Colloids were prepared in PBS containing 10% (v/v) FBS as described above, and 150 μL was pipetted into the mesh plate wells corresponding to the receiver solution in the receiver plate. The mesh plate and receiver plate were gently mated, resulting in the receiver solution wetting the bottom of the phospholipid membrane. A plate cover was added, and the assembly edges sealed with parafilm to prevent evaporation. The sealed assembly was then incubated at 37° C. for 6 h. Finally, the stack was carefully disassembled and a 200 μL sample of receiver and standard curve solutions was withdrawn for drug quantification as described below. Each donor and receiver well were tested with a pH strip to verify integrity of the membrane. A separate test with Trypan blue verified that the membranes were impermeable to this colloidal dye.
Drug Concentration Quantification
Drug concentration in samples from drug release and membrane permeability assays was quantified by high pressure liquid chromatography coupled with tandem mass spectrometry (HPLC-MS-MS). Protein was precipitated from samples in serum containing media by spiking with 10 μL of formic acid and adding acetonitrile to a final volume of 1 mL. The precipitated samples were then centrifuged at 16,000×g for 5 min to pellet the proteins. The drug-containing supernatant was then diluted in methanol such that the final drug concentration was less than 100 ng mL−1. During the final dilution, internal standards (nilotinib for lapatinib and norethindrone for fulvestrant) were added to a final concentration of 25 ng/mL each. Standard curves were prepared in a similar way, by diluting 100 mM solutions of drug in DMSO with methanol to final concentrations of 100, 75, 50, 25, 10, 5, 2.5, and 1 ng mL−1, each with 25 ng mL−1 of internal standard.
Cell Culture
MDA-MB-231-H2N cells were a generous gift from Dr. Robert Kerbel (Sunnybrook Research Institute, Toronto, ON, Canada). Cells were maintained in a humidified incubator at 37° C. with 5% atmospheric CO2. Cells were grown in 75 cm2 tissue culture flasks with 10 mL RPMI 1640 supplemented with 10% FBS, 10 UI/mL penicillin, and 10 μg mL−1 streptomycin. Cells were passaged twice per week with typical subculture ratio of 1:16.
Cell Viability Experiments
After passaging, cell suspensions were diluted into fresh media and 200 μL was pipetted into each well of a 96 well plate. Two thousand cells per well were plated and allowed to adhere overnight. Then, the media was withdrawn and replaced with treatment formulations (prepared as described above). Cells were incubated during the experiment in a humidified incubator at 37° C. with 5% atmospheric CO2. For treatments lasting less than 3 d, the treatment solutions were removed after the prescribed time, cells were washed with fresh media, and 200 μL of fresh media was added. Cell viability was assessed 3 days after commencing treatment using the PrestoBlue™ viability assay according to the manufacturer's protocol. Cell viability was reported as a percentage of the vehicle (DMSO with no drug or excipient) control.
Confocal Imaging of Treated Cells
Cells were seeded at approximately 2×105 cells per well in 8-chamber tissue culture treated glass cover slips and allowed to adhere overnight. Treatments were prepared as described above and 300 μL applied to each well. Cells were incubated during the experiment in a humidified incubator at 37° C. with 5% (v/v) atmospheric CO2. The treatment solutions were removed after the prescribed time, cells washed with fresh media, and 300 μL of fresh media added. For fixed cells, 4% (w/w) aqueous paraformaldehyde was applied for 10 min, followed by staining (when appropriate), and finally blank PBS. For live cell conditions, cells were washed with fresh media, stained, and imaged under Hank's balanced salt solution. Cells were imaged on an Olympus FV1000 inverted confocal microscope using a 1.42 N.A. 60× oil immersion lens (Olympus PLAPON 60XO). Laser and detector settings were held constant between different treatment conditions.
Flow Cytometry
Cells were seeded at approximately 2×105 cells per well in 24-well plates and allowed to adhere overnight. Treatments were prepared as described above and 500 μL was applied to each well. Cells were incubated during the experiment in a humidified incubator at 37° C. with 5% (v/v) atmospheric CO2. For treatments lasting less than 3 h, the treatment solutions were removed after the prescribed time, cells were washed with fresh media, and 500 μL of fresh media was added. Three hours after treatment initiation, cells were washed three times with media, detached using 500 μL of accutase solution, spiked with 500 μL of media, then centrifuged at 400×g to pellet the cells. Cells were then resuspended in cold flow buffer (PBS supplemented with 2% (v/v) FBS and 2 mM EDTA) and kept on ice until measurement. The flow buffer was supplemented with 7-AAD at 2 μg mL−1 as a vital stain, except for blank controls and in the endosome escape assay where 7-AAD had already been added. Cell fluorescence was quantified using a BD Accuri™ C6 flow cytometer with excitation wavelength of 488 nm and emission filters of 585/40 nm (BODIPY colloid dye) and >670 nm (7-AAD). Data were analyzed using the BD Accuri™ C6 Plus software and reported as the fluorescence of the live cell fraction (gated using scattering and 7-AAD) averaged between three biological replicates.
Results
This study was initiated by identifying colloid-forming drugs that could respond to acidic conditions by measuring the critical aggregation concentration (CAC) of several aggregators as a function of pH (Table 1). Critical aggregation concentrations were calculated by plotting the scattering intensity (indicative of colloid number and size) from dynamic light scattering (DLS) versus concentration (Table 2,
A stable colloidal formulation of lapatinib was developed due to its potency and robust response to acidic pH. Although lapatinib forms colloidal aggregates on its own, they are only transiently stable and precipitate within a few hours (
The response of the stable colloids to acidic conditions was investigated. Colloids were formulated in PBS and subsequently acidified, mimicking the pH within the endo-lysosomal pathway. To determine whether the lapatinib-fulvestrant co-colloids changed as a function of pH, colloid size and scattering intensity were measured by DLS (
The endocytosis of the stable, acid-responsive colloidal drug aggregates was investigated. To measure uptake, a hydrophobically-modified BODIPY dye, which is fluorescent only when incorporated within the colloid, 22 was incorporated. These colloids appeared as punctate structures within MDA-MB-231-H2N cells (
The in vitro toxicity of acid-responsive, stable lapatinib/fulvestrant colloids was then examined against lapatinib-sensitive, HER2-overexpressing MDA-MB-231-H2N cells. The transferrin-stabilized lapatinib/fulvestrant colloids were substantially more toxic than any other formulation, including PLAC-PEG stabilized lapatinib/fulvestrant colloids that were minimally endocytosed (
To determine the mechanism of the increased toxicity of endocytosed lapatinib-containing colloids versus non-endocytosed colloids, the ability of colloidal lapatinib to disrupt the endosomes was investigated using a fluorescence dequenching assay.29, 30 Incorporating the membrane impermeant nuclear stain, 7-aminoactinomycin D (7-AAD), into the colloids allowed measurement of its fluorescence in MDA-MB-231-H2N cells. The fluorescence of 7-AAD intensifies on DNA binding. Therefore, 7-AAD staining should only be observed in the nucleus following both colloid endocytosis and subsequent endosomal escape. Lapatinib/fulvestrant-transferrin colloids resulted in higher nuclear 7-AAD fluorescence than fulvestrant-transferrin colloids and PLAC-PEG stabilized formulations (
An alternative explanation to endosome disruption is simple diffusion of lapatinib from the endosomes, which was probed with a membrane permeability assay. The transmembrane diffusion of lapatinib was reduced when the colloids were acidified (
Notwithstanding the importance of transferrin for cellular uptake, its stability in serum was investigated since displacement of the macromolecular coronas by serum proteins can affect circulating nanoparticles.27 To test whether serum proteins could displace the transferrin stabilizer, colloidal drug aggregates stabilized with fluorophore-labelled transferrin were formulated and then pelleted by centrifugation. Quantifying the fraction of transferrin remaining in the supernatant, it was found that most of the transferrin was displaced after 6 hours of incubation in 10% serum (
Discussion
This study demonstrates that it is possible to trigger the release of weakly basic colloid-forming drugs from stable colloidal drug aggregates by acidification of the medium. By exploiting the local acidity in the endo-lysosomal pathway, drug release and its consequent cytotoxicity was triggered, thereby overcoming a key challenge of colloidal stability, and hence inactivity, after cell uptake. Thus, whereas previous research has shown that colloidal aggregation can cause false negative hits in cytotoxicity assays,11, 12 this effect can be overcome by protonation of the colloidal drug and subsequent en-dosome disruption.
Interestingly, it was found that the amount of lapatinib released was less than its CAC at a comparable pH. Without wishing to be bound by theory, this behaviour may be attributed to the fulvestrant, which remains in the colloidal state and acts as a sink for lapatinib. This hypothesis is supported by studies on the phase behaviour of co-colloids that show a reduction in effective CAC when multiple drugs co-exist in the colloid.31-33 This physical interaction between lapatinib and fulvestrant suggests that they mix to form co-colloids and may explain how fulvestrant stabilized lapatinib against precipitation.
It was found that endocytosis of the acid-responsive lapatinib-fulvestrant colloids greatly enhanced their cytotoxicity due to increased drug transport from the endosomes into the cytosol. This endosomal escape could occur by one of two mechanisms: either the increased concentration of free lapatinib enhances the amount of lapatinib diffusion across the endosomal membrane or the weakly basic lapatinib disrupts the integrity of the endosomes through osmotic pressure effects.29, 34, 35 As lapatinib is slow to cross membranes under acidic conditions, the enhanced diffusion mechanism is unlikely. Transport of ionizable drugs, such as protonated (cationic) lapatinib at reduced pH, diffuse more slowly through the lipid membranes.36, 37 This prediction was supported by the estimate of free, uncharged lapatinib as a function of pH. With respect to the proton-sponge mechanism, the 7-AAD fluorescence quenching assay showed that acid-responsive colloidal drug aggregates can disrupt endosomes, leading to drug leakage into the cytosol and ultimately enhancing drug cytotoxicity. This type of endosomal escape strategy has been exploited to deliver therapeutics that are unable to escape the endosomes themselves.35, 38, 39 This approach has been adapted here and has been surprisingly found to effectively deliver a new type of cargo: colloidal drug aggregates. Furthermore, the approach employed here successfully avoided the use of acid-responsive but pharmacologically inert excipients by using a drug that is naturally acid-responsive. Such pharmacologically active drug colloids can be used instead of the traditionally used inert carriers to deliver proteins or nucleic acids.
Notwithstanding these results, colloidal drug aggregates are admittedly in dynamic equilibrium with free drug, and a small amount of that free drug may diffuse across lipid membranes. As a result, cells that are highly sensitive to lapatinib may be killed even in the absence of colloid endocytosis. Another consequence of this behaviour is that drug diffusion out of the endo-lysosomal pathway could slowly occur simultaneously with the proton sponge effect.
The present study demonstrates that endosomal escape of lapatinib is enhanced by the acidic microenvironment. Importantly, endosomal escape in live cells was shown to occur through a membrane disruption mechanism. Furthermore, it was shown that only colloidal drugs that are inherently acid-responsive can have their release triggered by acidic conditions whereas those that are unresponsive to acid cannot.
Conclusions
Recent attempts to exploit colloidal drug aggregates have been, hindered, until now, by the inability to control drug release. The present study showed acid-triggered release from stable colloidal drug aggregates, endosomal disruption, and enhanced cytotoxicity. The selective, stimulus-responsive release of drugs from colloidal aggregates was demonstrated. This provides demonstration of controlled release of a therapeutic molecule from a colloid that is inactive until its target is reached.
Methods
Materials
Fulvestrant was purchased from Selleck Chemicals. Clotrimazole, IgG from human serum, and RPMI 1640 cell culture media were purchased from Sigma-Aldrich. Trastuzumab was obtained from Roche. CholEsteryl BODIPY 542/563 C11, Hoechst 33342, and wheat germ agglutinin Alexa Fluor 647 conjugate were purchased from Thermo Fisher scientific. SKOV3 cells were purchased from ATCC. Fetal bovine serum was purchase from Wisent Bio Products.
Colloid Formulation and Characterization by Dynamic Light Scattering
Colloidal drug aggregates were formulated as described as described in Example 1 using clotrimazole or fulvestrant in place of lapatinib. In particular, colloids of fulvestrant (non-responsive) or clotrimazole (acid-responsive) were formulated at 50 μM and stabilized with 3 μM trastuzumab at pH 7 followed by acidification of pH 5. These formulations were assessed by dynamic light scattering as described in Example 1.
Cell Culture and Confocal Microscopy
Cell culture was carried out as described in Example 1. SKOV3 cells were seeded at 10,000 per well in 16-well glass chamber slides. Fluorescent clotrimazole colloids were prepared as described above, with the addition of 500 nM CholEsteryl BODIPY 542/563 C11. Trastuzumab or IgG were added to a final concentration of 3.5 μM, the formulations were incubated for 10 minutes, and then FBS was added to a final concentration of 5% (v/v). The cell media was replaced with the colloid formulations and incubated for 3 h. The formulations were removed and the cells were fixed with 4% paraformaldehyde. Following fixation, wheat germ agglutininin Alefa Fluor 647 conjugate was added to stain the cell membranes according to the manufacturer protocol and counterstained with Hoechst. Cells were then imaged on an Olympus FV1000 confocal microscope at 60× magnification.
Results
As shown in
Discussion
These results demonstrated that the colloid-forming compound clotrimazole is acid-responsive, and that colloids of clotrimazole can target cancer cells by coating the colloids with targeting antibodies.
Methods
Materials
A sorafenib analogue containing a pendant imidazole functional group on the N-methylpyridine-2-carboximide was synthesized to specifications by Enamine. Phosphate buffered saline was purchased from Wisent Bio Products. Hydrochloric acid was purchased from VWR. Cell lines were obtained as a generous gift from R. Kerbel (MDA-MB-231-H2N) or from ATCC (BT474, SKBR3, SKOV3).
Colloid Formulation and CAC Measurement
Colloids were formulated as described in Example 1. Briefly, solutions of sorafenib analogue in DMSO were prepared at various concentrations and then diluted 100-fold with PBS at different concentrations. 100 μL of each sample was analyzed by DLS and the CAC's calculated as described in Example 1.
Cell Culture and IC50 Determination
Cell culture and experiments were carried out as described in Example 1. IC50's were calculated using GraphPad Prism 7 from cell viabilities measured after treating with difference concentrations of drug for 3 days.
Results
This study also included an investigation of the cytotoxicity of sorafenib in comparison to the cytotoxicity of the sorafenib analogue. As shown in
Discussion
These experiments demonstrate that an aggregator (in this case a drug) that does not respond to acid can be made responsive by modification with an ionizable functional group, while retaining the pharmacologic activity of the aggregator.
Methods
Materials
Fulvestrant was purchased from MedChemExpress. Dimethyl sulfoxide (DMSO), tert-butyldimethylsilyl chloride (TBSCl), (diacetoxyiodi)benzene, ammonium carbamate, N-Boc-glycine, N-Boc-valine, N,N-diisopropyl-N-ethylamine (DIPEA), tetra-N-butyl ammonium fluoride (TBAF) solution, tetrahydrofuran (THF), trifluoroacetic acid (TFA) bromoacetyl bromide, dimethylamine hydrochloride, and morpholine were purchased from Sigma-Aldrich. Dimethylformamide (DMF) was purchased from Alfa Aesar. Ethyl acetate, tert-butyl methyl ether (MTBE), hexanes, methanol, dichloromethane (DCM), ethanol, and glacial acetic acid were purchased from Caledon. Imidazole was purchased from Bio Basic. HCTU was purchased from AnaSpec. Silica (SilicaFlash P60™) was purchased from SiliCycle.
Synthesis of Ionizable Fulvestrant Analogues Modified with Tertiary Amines
Fulvestrant (species 1 in
3,17β-Bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphinyl) nonyl]estra-1,3,5-(10)-triene (2): 3,4-Dihydro-2H-pyran (248 μL, 2.72 mmol) and trifluoroacetic acid (12.6 μL, 165 μmop were added under stirring to a solution of fulvestrant (500 mg, 824 μmol) in dichloromethane (8 mL). The reaction was stirred at room temperature for 3 d, then extracted (1× sat. NaHCO3, 1× brine), dried (MgSO4), and filtered. The solvent was removed under reduced pressure, yielding a clear viscous oil which was used without further purification. Rf=0.25 (1:1 hexanes:ethyl acetate).
3,17β-Bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentyl sulphonimidoyl)nonyl] estra-1,3,5-(10)-triene (3): Finely ground ammonium carbamate (85.2 mg, 1.09 mmol) and (diacetoxyiodo)benzene (221 mg, 685 μmop were added to a stirred solution of crude 2 (192 mg, ≤248 μmot) in methanol (5 mL). After a few minutes, gas (presumably CO2) was evolved and a yellow color developed. After 4 h, the reaction was diluted in ethyl acetate and extracted (2×PBS, 1×brine), dried (MgSO4), and filtered. The solvent was removed and the product purified by column chromatography (silica, 1:1 hexanes:ethyl acetate) to give 3 as a clear oil (57.88 mg, 73.26 μmol 30% cumulative yield). Rf=0.2 (1:1 hexanes:ethyl acetate); MS (ESI+) m/z [M+H]+ calculated for C42H65F5NO5S: 790.4511, found: 790.4503.
N-(2-Bromoacetyl)-(3,17β-bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene) (4): 3 (57.9 mg, 73.3 μmop was dissolved in dichloromethane (2 mL) and N,N-diisopropylethylamine (63.8 μL, 366 μmol) added. The solution was cooled on ice, and then a solution of bromoacetyl bromide (12.8 μL, 147 μmop in dichloromethane (2 mL) added under agitation. The now amber-coloured solution was allowed to warm to room temperature and stirred for 30 minutes. Then, 100 μL of saturated Na2CO3 was added and the solvent removed under air. The residue was then dispersed in ethyl acetate and extracted (3×PBS, 1× brine), dried (MgSO4), filtered, and evaporated under nitrogen. The crude product was purified by column chromatography (silica, 4:1 hexanes:ethyl acetate) to give 4 as a slightly yellow oil (40.5 mg, 44.1 μmol, 60%). Rf=0.4 (4:1 hexanes:ethyl acetate), 0.85 (1:1 hexanes:ethyl acetate); 1H NMR (500 MHz, CDCl3) δ 7.17-7.10 (m, 1H), 6.63 (dd, J=8.4, 2.8 Hz, 1H), 6.54 (d, J=3.1 Hz, 1H), 5.05 (dt, J=5.2, 2.7 Hz, 1H), 4.69-4.59 (m, 1H), 4.08-3.96 (m, 2H), 3.96-3.84 (m, 3H), 3.90-3.87 (m, 2H), 3.79-3.69 (m, 2H), 3.66-3.42 (m, 6H), 3.40-3.27 (m, 3H), 2.90-2.82 (m, 1H), 2.70 (dd, J=16.7, 4.8 Hz, 1H), 2.38-1.08 (m, 27H), 1.08-0.85 (m, 6H), 0.83-0.76 (m, 4H); MS (ESI+) m/z [M+Na]+ calculated for C44H65BrF5NNaO6S: 932.3528, found: 932.3529.
N-(2-Dimethylaminoacetyl)-(3,17β-bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene) (5): A solution of dimethylamine hydrochloride (285 mg) and DIPEA (70 μL, 400 μmol) in DCM (1.5 mL) was added to a solution of 4 (97 mg, 100 μmol) in 1 mL DCM. The reaction was agitated for 2.5 h, diluted with 10 mL ethyl acetate, and extracted (2× water, 1× brine). The extract was dried (MgSO4) and the solvent removed under nitrogen. The resulting amber oil was used without further purification.
N-(1H-Imidazol-1-ylacetyl)-(3,17β-bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene) (6): A solution of imidazole (16 mg, 0.23 mmol) in dichloromethane (1 mL) was added to 4 (18 mg, 20 μmol), agitated for 3 h, and then the solvent removed under nitrogen. The residue was then resuspended in ethyl acetate and extracted (2× water, 1× brine), dried (MgSO4), and the solvent removed under nitrogen followed by vacuum. The resulting oil (18 mg, 20 μmol, 99%) was used without further purification. MS (ESI+) m/z [M+H]+ calculated for C47H69F5N3O6S: 898.4822, found: 898.4820 ([M+H−THP]+ and [M+H−2THP]+ also observed).
N-(Morpholin-4-ylacetyl)-(3,17β-bis((tetrahydro-2H-pyran-2-yl)oxy)-7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene) (7): A solution of morpholine (26 μL, 300 μmol) and DIPEA (17.42 μL, 100 μmol) in DCM (1.5 mL) was added to a solution of 4 (97 mg, 100 μmol) in 1 mL DCM. The reaction was agitated for 2.5 h, diluted with 10 mL ethyl acetate, and extracted (2×water, 1×brine). The extract was dried (MgSO4) and the solvent removed under nitrogen. The resulting amber oil was used without further purification.
N-(2-Dimethylaminoacetyl)-(7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene-3,17β-diol) (8): See method for 9, but using 5 as in place of 6. 1H NMR (500 MHz, DMSO-d6) δ 9.21-8.80 (s, 1H), 7.07-7.00 (d, 1H), 6.50 (dd, J=8.4, 2.7 Hz, 1H), 6.41 (s, J=2.6 Hz, 1H), 4.54-4.41 (m, 1H), 3.66-3.47 (m, 7H), 2.62-2.57 (m, 1H), 2.54 (s, 6H), 2.45-2.39 (m, 1H), 2.34-1.10 (m, 42H), 0.93-0.87 (m, 1H), 0.66 (s, 3H); 13C NMR (126 MHz, DMSO) δ 154.96, 135.97, 129.61, 126.63, 115.75, 112.85, 80.10, 61.72, 50.72, 49.40, 46.01, 43.81, 42.96, 41.74, 37.79, 36.78, 34.15, 32.76, 29.86, 29.37, 29.02, 28.72, 28.42, 27.81, 27.56, 27.41, 27.06, 25.81, 25.11, 22.28, 21.42, 13.55, 11.30; MS (ESI+) m/z [M+H]+ calculated for C36H56F5N2O4S: 707.3875, found: 707.3876.
N-(1H-Imidazol-1-ylacetyl)-(7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene-3,17β-diol) (9): A mixture of trifluoroacetic acid (20 μL) and methanol (480 μL) was added to a solution of crude 6 (13 mg, 15 μmol) in methanol (500 μL). The mixture was agitated overnight, diluted with ethyl acetate and extracted (1× sat. Na2CO3, 1× brine), dried (MgSO4), filtered, and evaporated under nitrogen. The crude product (7.9 mg) was then purified by column chromatography (silica, ethyl acetate followed by ethanol), dried under nitrogen, redissolved in acetonitrile, and filtered (0.45 μm PTFE) to yield an amber-tinted film (1.6 mg, 2.2 μmol, 15%) after drying under nitrogen. Rf=0.1-0.3 (acetone), 0-0.2 (ethyl acetate), 0.9 (ethanol); 1H NMR (500 MHz, DMSO-d6) δ 8.97 (d, J=6.6 Hz, 1H), 7.64 (s, 1H), 7.09 (s, 1H), 7.04 (d, J=8.5 Hz, 1H), 6.89 (s, 1H), 6.50 (dd, J=8.4, 2.6 Hz, 1H), 6.41 (d, J=2.6 Hz, 1H), 4.77 (s, 2H), 4.48 (s, 1H), 3.65-3.43 (m, 6H), 2.46-0.78 (m, 33H), 0.66 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 174.99, 154.94, 135.99, 129.62, 126.64, 115.75, 112.85, 103.02, 80.09, 62.93, 51.18, 50.65, 49.31, 45.99, 42.96, 41.74, 37.78, 36.77, 34.15, 33.65, 32.75, 29.87, 29.36, 29.02, 28.72, 28.42, 27.79, 27.56, 27.41, 27.06, 25.10, 24.48, 22.28, 22.09, 21.43, 21.05, 18.39, 13.95, 13.53, 11.30; MS (ESI+) m/z [M+H]+ calculated for C37H53F5N3O4S: 730.3671, found: 730.3676.
N-(Morpholin-4-ylacetyl)-(7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl] estra-1,3,5-(10)-triene-3,17β-diol) (10): See method for 9, but using 7 in place of 6. Rf=0.1 (ethyl acetate), 0.8 (acetone), 0.9 (ethanol); 1H NMR (500 MHz, DMSO-d6) δ 8.97 (s, 1H), 7.04 (d, J=8.5 Hz, 1H), 6.50 (dd, J=8.4, 2.7 Hz, 1H), 6.41 (d, J=2.6 Hz, 1H), 4.47 (d, J=4.9 Hz, 1H), 3.66-3.40 (m, 10H), 3.04 (s, 2H), 2.60 (d, J=16.6 Hz, 1H), 2.48-2.43 (m, 4H), 2.30-0.74 (m, 32H), 0.66 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 177.68, 154.94, 135.98, 129.62, 126.63, 115.75, 112.84, 80.09, 66.15, 63.27, 52.66, 50.57, 49.24, 45.99, 42.95, 41.74, 37.78, 36.76, 35.77, 34.14, 32.74, 31.29, 31.27, 30.76, 29.86, 29.59, 29.35, 29.00, 28.67, 28.39, 28.01, 27.84, 27.68, 27.54, 27.38, 27.06, 26.60, 25.10, 22.27, 22.09, 21.46, 13.95, 13.59, 11.30; MS (ESI+) m/z [M+H]+ calculated for C38H58F5N2O5S: 749.3981, found: 749.3978.
Synthesis of Amino Acid-Modified Fulvestrant
Fulvestrant was modified by addition of a glycyl or valyl group at the sulfinyl to yield 15a and 15b, respectively, as shown in
N-(2-aminoacetyl)-(7-α-[9-(4,4,5,5,5,-pentafluoropentylsulphonimidoyl)nonyl]estra-1,3,5-(10)-triene-3,17β-diol) (9): 1H NMR (500 MHz, DMSO-d6) δ 7.04 (d, J=8.5 Hz, 1H), 6.50 (dd, J=8.5, 2.6 Hz, 1H), 6.41 (d, J=2.6 Hz, 1H), 4.48 (s, 1H), 3.66-3.42 (m, 5H), 3.23 (s, 2H), 2.75 (dd, J=16.6, 5.2 Hz, 1H), 2.63-2.55 (m, 1H), 2.47-2.34 (m, 2H), 2.25 (dd, J=12.1, 4.2 Hz, 1H), 2.21-2.11 (m, 1H), 2.05-1.58 (m, 7H), 1.58-1.13 (m, 22H), 0.97-0.79 (m, 1H), 0.66 (s, 3H); MS (ESI+) m/z [M+H]+ calculated for C34H52F5N2O4S: 679.3562, found: 679.3559.
Colloid Formulation and Determination of CAC
Colloids were formulated as described in Example 1. Briefly, solutions of fulvestrant analogue in DMSO were prepared at various concentrations and then diluted 100-fold with PBS at different concentrations. 100 μL of each sample was analyzed by DLS and the CAC's calculated as described in Example 1.
Results
Discussion
As in Example 3, this study demonstrates that an aggregator that does not respond to acid can be made acid-responsive by modification with an ionizable functional group. Furthermore, these results show that the pKa of the aggregator, and thus the pH at which it becomes ionized, can be tuned by adjusting the nature of the attached ionizable functional group.
Methods
Materials
Siramesine hydrochloride was purchased from MedChemExpress. Bovine serum albumin (BSA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS) was purchased from Wisent. Hydrochloric acid was purchased from VWR.
Colloid Formulation and CAC Measurement
Colloids were formulated as described in Example 1. Briefly, solutions of siramesine in DMSO were prepared at various concentrations and then diluted 100-fold with pH-adjusted PBS containing stabilizer (where applicable). 100 μL of each sample was analyzed by DLS and the CACs calculated as described in Example 1.
High Concentration Colloid Formulation
Siramesine was dissolved in DMSO at a concentration of 50 mg/mL, or in ethanol at a concentration of 10 mg/mL. 15 μL (DMSO) or 75 μL (ethanol) of this solution was diluted with 1.5 mL of water containing 5 mg/mL of BSA. The resulting colloids were flash frozen and lyophilized to yield a white cake. The lyophilized colloids were reconstituted by adding 150 μL of PBS and mixing with a pipette. The reconstituted colloids were measuring by DLS after diluting 10 μL with 90 μL of PBS.
Results
Discussion
These results demonstrated that the colloid-forming compound siramesine is acid-responsive, and that colloids of siramesine can be formulated at high concentrations.
Methods
Materials
Sorafenib was purchased from MedChemExpress. Bovine serum albumin (BSA) and RPMI 1640 cell culture media were purchased from Sigma-Aldrich. Attenuated diphtheria toxin (aDT) was obtained from Professor Roman Melnyk's (The Hospital for Sick Children, Toronto, ON, Canada). SKOV3 cells were purchased from ATCC. Fetal bovine serum was purchase from Wisent Bio Products. PrestoBlue™ cell viability reagent was purchased from Thermo Fisher Scientific.
Colloid Formulation and Characterization by Dynamic Light Scattering
Colloidal drug aggregates were formulated as described as described in Example 1 using sorafenib in place of lapatinib. In particular, colloids of sorafenib were formulated at 50 μM or 200 μM and stabilized with 10 μg/mL or 100 μg/mL diphtheria toxin in phosphate buffered saline. These formulations were assessed by dynamic light scattering as described in Example 1.
Cell Culture
SK-OV-3 cells were maintained in a humidified incubator at 37° C. with 5% atmospheric CO2. Cells were grown in 75 cm2 tissue culture flasks with 10 mL RPMI 1640 supplemented with 10% FBS, 10 UI/mL penicillin, and 10 μg mL−1 streptomycin. Cells were passaged twice per week with typical subculture ratio of 1:5.
Cell Viability Experiments
After passaging, cell suspensions were diluted into fresh media and 100 μL was pipetted into each well of a 96 well plate. Five thousand cells per well were plated and allowed to adhere overnight. Then, the media was withdrawn and replaced with treatment formulations (prepared as described above) or control treatments. Cells were treated with either cell culture medium, bovine serum albumin (BSA; 100 μg/mL), aDT (100 μg/mL), BSA-stabilized sorafenib colloids (100 μg/mL BSA; 200 μM sorafenib) or aDT-stabilized sorafenib colloids (100 μg/mL aDT; 200 μM sorafenib). All treatments were prepared in complete culture media. Cells were incubated during the experiment in a humidified incubator at 37° C. with 5% atmospheric CO2. For treatments lasting less than 3 d, the treatment solutions were removed after the prescribed time, cells were washed with fresh media, and 200 μL of fresh media was added. Cell viability was assessed 3 days after commencing treatment using the PrestoBlue™ viability assay according to the manufacturer's protocol. Cell viability was reported as a percentage of the blank (cell culture medium) control.
Results
Discussion
These results demonstrated that colloidal drug aggregates can be stabilized by an acid-responsive protein stabilizer/excipient (e.g., aDT), and this coating enhances cytotoxicity against a cancer cell line.
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All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
Number | Date | Country | Kind |
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1908716.2 | Jun 2019 | GB | national |
This invention was made with government support under grant no. GM071630 awarded by The National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/CA2020/050845 | 6/18/2020 | WO |