Self-assembled particles for targeted delivery of immunomodulators to treat autoimmunity and cancer

Abstract

The present invention provides for delivery of therapeutic drug in a polymeric delivery system comprising a PEG-bl-PPS di-block polymer formed in a micelle, filomicelle, or polymersome structure, wherein the structure effectively binds and/or interacts through shape-based targeting with a targeted cell type.

Claims

1. A composition comprising a therapeutically effective amount of chloroquine or hydroxychloroquine to treat systemic lupus erythematosus, wherein the chloroquine or hydroxychloroquine in the composition is encapsulated and/or adsorbed on a polymeric delivery system, wherein the polymeric delivery system comprises a PEG-bl-PPS di-block polymer formed in a micelle, filomicelle, or polymersome structure, wherein the structure effectively binds through shape-based targeting of immune cell types to reduce an autoimmune response, wherein the immune cell types comprise B cells or plasmacytoid dendritic cells (pDCs).

2. The composition according to claim 1, wherein the shape-based targeting is effective in increasing efficacy of, and accelerating activity of, the chloroquine or hydroxychloroquine.

3. The composition according to claim 1, wherein the PEG-bl-PPS di-block copolymer comprises different degrees of polymerization, wherein “n” of a PEG.sub.n block is from about 15 to about 50, and wherein “n” of a PPS.sub.n block is from about 5 to about 50.

4. The composition according to claim 1, wherein the PEG-bl-PPS di-block copolymer is PEG.sub.45-bl-PPS.sub.20, PEG.sub.17-bl-PPS.sub.30, or PEG.sub.44-bl-PPS.sub.45.

5. The composition according to claim 1, wherein loading capacity of the PEG-bl-PPS di-block copolymer is about 40% to about 60%.

6. The composition according to claim 1, wherein the PEG-bl-PPS di-block copolymer is effective in decreasing MX1 gene expression.

7. The composition according to claim 1, wherein the composition is formulated in an administration form selected from the group consisting of oral, nasal, ophthalmic, topical, buccal, sublingual, rectal, vaginal, intravenous, intraperitoneal, subcutaneous, inhalation, intramuscular, and transdermal.

8. The composition according to claim 1, wherein the micelle, filomicelle, or polymersome structures are nanoparticles.

9. The composition according to claim 1, comprising a therapeutically effective amount of chloroquine.

10. A method of treating systemic lupus erythematosus, the method comprising: administering to a subject a composition comprising a therapeutic dose of chloroquine or hydroxychloroquine to treat systemic lupus erythematosus, wherein the chloroquine or hydroxychloroquine is encapsulated and/or adsorbed on a polymeric delivery system, wherein the polymeric delivery system comprises a PEG-bl-PPS di-block polymer formed in a micelle, filomicelle, or polymersome structure, wherein the structure effectively binds and/or interacts through shape-based targeting of immune cell types to reduce an autoimmune response, wherein the immune cell types comprise B cells or plasmacytoid dendritic cells (pDCs).

11. The method according to claim 10, wherein the shape-based targeting increases efficacy of, and accelerates activity of, the chloroquine or hydroxychloroquine.

12. The method according to claim 10, wherein the PEG-bl-PPS di-block copolymer comprises different degrees of polymerization, wherein “n” of a PEG.sub.n block is from about 15 to about 50, and wherein “n” of a PPS.sub.n block is from about 5 to about 50.

13. The method according to claim 12, wherein the PEG-bl-PPS di-block copolymer is PEG.sub.45-bl-PPS.sub.20; PEG.sub.17-bl-PPS.sub.30 orPEG.sub.44-bl-PPS.sub.45.

14. The method according to claim 10, wherein loading capacity of the PEG-bl-PPS di-block copolymer is about 40% to about 60%.

15. The method according to claim 10, wherein the PEG-bl-PPS di-block copolymer decreases MX1 gene expression.

16. The method according to claim 10, wherein the composition is formulated in an administration form selected from the group consisting of oral, nasal, ophthalmic drop, topical, buccal, sublingual, rectal, vaginal, intravenous, intraperitoneal, subcutaneous, inhalation, intramuscular, and transdermal.

17. The method according to claim 10, wherein the micelle, filomicelle, or polymersome structures are nanoparticles.

18. The method according to claim 10, wherein the composition comprises a therapeutically effective amount of chloroquine.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A and B; FIG. 1A shows a schematic of chloroquine loaded PEG-bl-PPS filomicelles (FMs) in SLE. Chloroquine loaded FMs target pDCs, prevent TLR7 and TLR9 activation and mitigate proinflammatory responses via type I interferon cytokine production, plasma cell differentiation, and immune complex formation (3). FIG. 1B shows a schematic of PEG-bl-PPS polymer shapes targeting tumor cells and immune cells for cancer immunotherapies.

(2) FIG. 2 shows the differences in the PEG-bl-PPS morphology determines the structure of shape including polymersomes, filomicelles, and micelles and retention of drug payload and uptake by specific immune cells.

(3) FIGS. 3A and B shows FEI Morgagni M268 100 kV transmission electron microscopy (TEM) images of chloroquine. FIG. 3A shows loaded and FIG. 3B shows unloaded filomicelle PEG-bl-PPS at 12 mg/mL. Samples were stained with 2% uranyl acetate.

(4) FIGS. 4A and 4B; FIG. 4A shows the absorbance of unloaded and chloroquine loaded PEG-bl-PPS filomicelles at chloroquine excitation wavelength of 334 nm using UV-Vis. Normalized to baseline (PEG PPS). FIG. 4B shows the average loading capacity of chloroquine loaded PEG-bl-PPS nanoparticles via thin-film hydration is 46.905±12.108 μg drug/mg polymer (n=3). Error bar represent standard deviation. Standard curve of chloroquine dissolved in DMSO used for loading capacity calculations. Y=0.037X+0.005094 where Y is absorbance and X is concentration. R2=0.9879.

(5) FIGS. 5A and 5B shows (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine perchlorate) DiD loaded nanoparticles cultured with frozen hPBMCs for 8, 24 and 48 hours in RPMI 1640 with 10% FBS and 20 ng/mL IL-3 at 37° C. and 5% CO.sub.2 (n=3). Error bar represents standard deviation. Graphs are normalized to hPBMCs without nanoparticles. FIG. 5A shows plasmacytoid dendritic cells (pDCs) and FIG. 5B shows B cells were analyzed for nanoparticle distribution via flow cytometry.

(6) FIG. 6 shows the average MX1 gene expression (n=2) of fresh hPBMCs using real-time PCR (RT-PCR) after pre-incubation with soluble chloroquine or chloroquine loaded nanoparticle for 1 hour and stimulation with TLR9 agonist, CpG, for 6 hours. MX1 is upregulated in SLE patients versus healthy patients. Error bars show standard deviation.

(7) FIGS. 7A &B: FIG. 7A shows the nanoparticle (NP) uptake of murine breast cancer 4T1 cells in vitro by flow cytometry after 24 hours (n=3). FIG. 7B shows the median fluorescent intensity of data is FIG. 7A. (FM=PEG 44 PPS 45, MC=PEG 45 PPS 20, PS=PEG 17 PPS 44)

(8) FIGS. 8A, 8B and 8C shows the DiD loaded nanoparticles injected intratumorally in 4-9 month old Balb/c female mice with 4T1 tumors (n=2). Error bar represents range. Tumors were removed, enzymatically dissociated and analyzed for nanoparticle distribution for FIG. 8A B cells, FIG. 8B macrophages and FIG. 8C myeloid derived suppressor cells (MDSCs) via flow cytometry.

(9) FIG. 9 shows results of filomicelle (PEG PPS)+CQ for inhibiting RNA stimulation of TLR7.

(10) FIG. 10 shows the loading capacity of the filomicelles (PEG PPS) with other immunosuppressives used in lupus.

DETAILED DESCRIPTION OF THE INVENTION

(11) Systemic lupus erythematosus (SLE) is an autoimmune disease where the body attacks its healthy tissue and organs via autoantibody and nuclear antigen immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) and B cells via toll-like receptors (TLRs)-7 and -9 leading to inflammatory type I interferon cytokine production, triggering further autoantibody production by self-reactive B cells and worsening disease severity as shown in FIG. 1. As described herein, the present invention inhibits such activation. Antimalarials, such as chloroquine (CQ), are FDA-approved treatments for SLE and have been prescribed for SLE patients since the 1950s due to their low cost, limited, mild side effects and safety for pregnant women. Antimarlarials work by causing conformational changes to nucleic acid immune complexes, masking the TLR-binding epitope and preventing the production of inflammatory cytokines such as interferon-alpha (IFNα).

(12) A major limitation of antimalarials is their delayed therapeutic efficacy, requiring up to 6 months of continuous treatment before the patient receives any benefits from the drug. Notably, CQ treatment requires months before achieving therapeutic efficacy. The present invention uses self-assembled di-block copolymer nanoparticles, poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS), to deliver CQ to pDCs and B cells via passive, shape-based targeting. CQ loaded PEG-bl-PPS nanoparticles of varying shapes can directly target immune cell types, such as pDCs and B cells, increasing the efficacy per dose and accelerating the activity of CQ versus systemic administration of the drug. The results of the present invention show that filamentous micelle polymers (FMs) loaded with CQ accelerates drug activity by directly delivering CQ to immune cells via passive, shape-based targeting.

(13) Notably, depending on the individual block lengths, these copolymers can be engineered to assemble into a variety of different nanostructures in aqueous solutions, including spherical micelles, vesicles (i.e., polymersomes), and filamentous wormlike micelles (i.e., filomicelles). The filomicelles of the present invention are prepared by the assembly of PEG and hydrophobic PPS blocks. Such PEG-bl-PPS, a diblock copolymer poly(ethylene glycol)-block-poly(propylene sulfide) and production of same is described in U.S. Patent Publication No. 2018/002287, the content of which are incorporated by reference herein for all purposes. According to the specification in this cited U.S. patent publication, the copolymers are PEG-bl-PPS assembled by a flash nanoprecipitation method that encapsulate hydrophobic molecules, hydrophilic molecules, bioactive protein therapeutics, or other target molecules in amphiphilic copolymer nanocarriers.

(14) The method of assembly of the FM include the following steps as described in the cited publication and briefly includes the steps of a flash precipitation method comprising;

(15) (i) providing an organic phase solution comprising an amphiphilic copolymer and a process solvent, wherein the amphiphilic copolymer is poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) and has a glass transition temperature below 0° C. and preferably from −40° to −0°;
(ii) providing an aqueous phase solution comprising an aqueous solvent, such as water;
(iii) mixing by multiple impingement, the organic phase solution, contain the chloroquine molecule and the aqueous phase solution to form a mixture; and
(iv) introducing the mixture into a reservoir to cause precipitation of the amphiphilic copolymer as a nanocarrier of the chloroquine. Importantly, the organic solution and block copolymer are impinged upon an aqueous solution under turbulent wherein a supersaturated solution is generated by the turbulent mixing and such supersaturation induces precipitation of the block copolymer for stabilization of monodisperse nanoparticles which are loaded the target molecule chloroquine.

(16) The synthesized, self-assembled PEG-bl-PPS di-block copolymers can include different degrees of polymerization, for example, a PEGS block wherein “n” ranges from about 15 to about 50, and wherein the “n” of the PPS.sub.n block can range from about 15 to about 50. For example, a block copolymer can form a micelle (MC) with a structure of about PEG.sub.45-bl-PPS.sub.20; a structure of about PEG.sub.17-bl-PPS.sub.30 will form a polymersome (PS) and a structure of about PEG.sub.44-bl-PPS.sub.45 will form a filomicelle (FM) as shown in FIG. 2.

(17) As discussed herein, the PEG-bl-PPS drug compounds disclosed herein can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders (e.g., reconstitutable lyophilized powder), micronized compositions, granules, elixirs, tinctures, suspensions, ointments, vapors, liposomal particles, nanoparticles, syrups and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, topical (e.g., dermal, epidermal, transdermal, ophthalmically such as ocular eyedrop), intranasally, subcutaneous, inhalation, intramuscular or transdermal (e.g., patch, microneedles) form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Again, the ordinarily skilled physician, veterinarian or clinician or a clinical pharmacist may readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

(18) The compounds of the present invention may be used in combination with other drugs or therapies having similar or complementary effects to those of the compounds disclosed herein. The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms to patients or regions of such patients in need of such therapy. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.

(19) As used herein, the term “composition,” “pharmaceutical composition,” or the like, is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

(20) The amount of the active ingredient(s) which will be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration and all of the other factors described above. The amount of the active ingredient(s) which will be combined with a carrier material to produce a single dosage form will generally be that amount of the active ingredient(s) which is the lowest dose effective to produce a therapeutic effect.

Experimentation

(21) Systemic lupus erythematosus (SLE) is an autoimmune disease that attacks healthy organs via circulating immune complexes. Complexes activate immune cells via toll-like receptors (TLRs) 7 and 9, driving inflammatory type I interferon secretion. Chloroquine (CQ) is an FDA-approved TLR antagonist that mitigates inflammation in SLE, but CQ treatment requires months before achieving therapeutic efficacy. The present invention overcomes such a delay by a polymeric delivery system.

(22) Nanoparticles (NPs) are materials with dimensions between 1 and 100 nanometers that have applications in medicine, consumer products and optics. Self-assembled NPs derived from poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS, as discussed above, form different geometries based on their ratio of hydrophilic PEG to hydrophobic PPS. The differences in structure influences shape, retention of drug payload and uptake by specific immune cells. The present invention shows that filamentous micelle polymers (FMs) (PEG.sub.44-bl-PPS.sub.45) loaded with CQ accelerates drug activity by directly delivering CQ to immune cells via passive, shape-based targeting. Further, human blood immune cells were pretreated with soluble CQ or CQ in FMs, the cells were stimulated with a TLR9 agonist, and type I interferon response was analyzed via MX1 gene expression using real-time RT-PCR. The results shown herein indicate that CQ-FMs decreased MX1 gene expression similar to soluble CQ, while CQ in control polymer PLGA spheres had no effect. These results show FMs can increase CQ efficacy per dose for SLE-relevant pathways.

(23) Filomicelle PEG-bl-PPS nanoparticles (FMs) obtained from Northwestern University were assembled via thin film hydration. PLGA NPs were assembled via double-emulsion and served as a NP control. Chloroquine (CQ) was loaded in PEG-bl-PPS. Chloroquine diphosphate was loaded in PLGA. Different CQ drugs were used based on loading methods and polymer properties. CQ loaded and unloaded FMs were stained with 2% uranyl acetate and imaged using the FEI Morgagni M268 100 kV transmission electron microscopy (TEM). Loaded FMs were hydrolyzed in dimethyl sulfoxide (DMSO) and measured using Mettler Toledo UV5 Nano. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Ficoll-Paque medium density gradient. To check for FM cell distribution and plasmacytoid dendritic cell (pDC), myeloid dendritic cell (mDC) and B cell frequencies in PBMCs, cells were incubated with DiD loaded FMs for 8, 24 and 48 hours in RPMI 1640 with 10% FBS and 20 ng/mL IL-3 and stained with fluorescent antibodies for the following markers: CD123 (PE), CD1c (APC/Cy7), CD19 (BV 421) and Zombie Aqua. Flow cytometry samples were acquired on a CyAn™ ADP High-Performance Flow Cytometer instrument using Summit Software. Data analysis was performed using FlowJo v10 10.1r7. Cell populations were determined using frequency of total events and cell distribution from median fluorescent intensity. Cells were treated for 1 hour with soluble drug or drug loaded nanoparticles. Cells were then stimulated with CpG Class A for 6 hours. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze downstream IFN-response gene MX1. Expression levels were normalized to β-actin control.

(24) Characterization of CQ loaded FMs showed an average loading capacity of 46.905 μg drug/mg polymer and average encapsulation efficiency of 49.5%. Transmission electron microscopy (TEM) images of loaded and unloaded FMs show no difference in morphology or structure, as shown in FIG. 3. Notably, in FIG. 10, it is shown that other lupus treating drugs can also be loaded into the filomicelles (PEG-PPS) such as AZA load at almost 400 ug/mg of mg of filomicelles and methylprednisolone at about 200 ug drug to mg of filomicelles.

(25) PEG-bl-PPS NPs target immune cells, pDCs and B cells, important in SLE pathogenesis and TLR-7 and -9 induced inflammatory responses. The differences in PEG-bl-PPS morphology, such as polymersomes, filomicelles, and micelles, is to deliver toll-like receptor (TLR)-7 and -9 antagonist drugs to immune cells, such as pDCs and B cells, important in SLE pathogenesis and severity. CQ-loaded FMs are an effective targeted drug delivery vehicle for suppressing TLR7 and activation in B cells and pDCs, the major pathways responsible for SLE pathogenesis and end-stage symptoms such as nephritis. FIG. 5 shows DiD loaded nanoparticles cultured with frozen hPBMCs for 8, 24 and 48 hours in RPMI 1640 with 10% FBS and 20 ng/mL IL-3 at 37° C. and 5% CO.sub.2 (n=3). Both plasmacytoid dendritic cells (pDCs) and B cells were analyzed for nanoparticle distribution via flow cytometry. Clearly the FM were more effective in the B cells.

(26) CQ loaded PEG-bl-PPS decreased MX1 expression similar to soluble condition. CQ loaded in PLGA had no effect on MX1 gene expression. It was found that CQ-loaded filamentous micelle PEG-bl-PPS nanoparticles (FMs) decreased MX1 gene expression in human peripheral blood mononuclear cells (hPBMCs) similar to soluble CQ. As shown in FIG. 6, CQ loaded in a polymer control, poly(lactic-co-glycolic acid) (PLGA) spheres, exhibited no effect on MX1 gene expression. MX1 is a type I interferon-stimulated gene whose activity is directly induced by IFNα, a critical driver of inflammatory damage in SLE. However, when human blood immune cells were pretreated with soluble CQ or CQ in FMs, stimulated cells with a TLR9 agonist, and analyzed type I interferon response via MX1 gene expression using real-time RT-PCR. The results shown in FIG. 6 illustrate that CQ-FMs decreased MX1 gene expression similar to soluble CQ, while CQ in control polymer PLGA spheres had no effect. These results show FMs can increase CQ efficacy per dose for SLE-relevant pathways. These results are also shown in FIG. 9 where the filomicelle (PEG PPS)+CQ is better at inhibiting single-stranded RNA stimulation of TLR7 compared to PLGA+CQ.

(27) In addition to the efficacy in SLE, the nanoparticle platform of the present invention was used to specifically target tumor cells and immune cells, such as myeloid derived suppressor cells (MDSCs), B cells and macrophages, important in supporting tumor growth by suppressing anti-tumor immunity as shown in FIG. 1B, which is a schematic of PEG-bl-PPS polymer shapes targeting tumor cells and immune cells for cancer immunotherapies. By directly targeting these cells, drug toxicity and severe side effects associated with current systemic cancer immunotherapies is reduced and the nanoparticle shape was used to deliver targeted immunotherapies to specific immunosuppressive cell types. The enclosed data show that PEG-bl-PPS polymer shapes, FMs, micelles (MCs), bicontinuous nanostructures (BCNs) and polymersomes (PSs), have different uptake patterns in murine stage IV metastatic breast cancer cell line, 4T1. MCs and FMs were taken up more than PSs and BCNs. Nanoparticles injected intratumorally in 4-9 month old Balb/c female mice with established 4T1 tumors showed that PSs were taken up B cells and MDSCs; whereas, MCs and FMs were taken up by macrophages and MDSCs.

(28) FIG. 7 shows the nanoparticle (NP) uptake of murine breast cancer 4T1 cells in vitro by flow cytometry after 24 hours (n=3). Notably, the MCs and FMs were taken by 4T1 significantly more than PSs and BCNs (two-way ANOVA p<0.0001) and uptake was significantly more in MCs compared to FMs (two-way ANOVA p<0.0001). The results in FIG. 8 shows DiD loaded nanoparticles injected intratumorally in 4-9 month old Balb/c female mice with 4T1 tumors (n=2). When the tumors were removed, enzymatically dissociated and analyzed for nanoparticle distribution for (A) B cells, (B) macrophages and (C) myeloid derived suppressor cells (MDSCs) via flow cytometry. As viewed in FIG. 8, different structures were more effective in the different types of cells, for example the polymersomes were more effective in delivery in B-cells, the micelles were more effective delivery in macrophages and again delivery by the polymersomes were the most effective delivery shape in the Tumor MDSC cancer cells.

(29) In conclusion, the results shown herein show a multi-functional approach to 1) reduce drug toxicity of systemic administration of clinically accepted drugs, 2) utilize nanoparticle shape to target different immune cells based on disease model 3) improve efficacy per drug dose.

REFERENCES

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