WO2019071031A1 - Administration exosomale d'anthocyanidines et de curcumine - Google Patents

Administration exosomale d'anthocyanidines et de curcumine Download PDF

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WO2019071031A1
WO2019071031A1 PCT/US2018/054450 US2018054450W WO2019071031A1 WO 2019071031 A1 WO2019071031 A1 WO 2019071031A1 US 2018054450 W US2018054450 W US 2018054450W WO 2019071031 A1 WO2019071031 A1 WO 2019071031A1
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exosomes
drug
anthos
composition
milk
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Ramesh Gupta
Farrukh Aqil
Jeyaprakash JEYABALAN
Radha MUNAGALA
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University of Louisville Research Foundation ULRF
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger

Definitions

  • the present invention generally relates to microvesicle compositions and methods for providing enhanced bioavailability and efficacy, particularly for treating cancers and more particularly for treating ovarian cancer and cervical cancer.
  • certain embodiments of the presently disclosed subject matter relate to microvesicle compositions including microvesicles encapsulating anthocyanidins and microvesicle encapsulating curcumin and methods for using these compositions in the treatment of specific cancers.
  • Ovarian cancer is the most lethal gynecological malignancy in women in the United States. Five to 10 percent of ovarian cancers diagnosed are hereditary. Front-line treatment for this lethal disease is cytoreductive surgery followed by intraperitoneal (i.p.) and/or intravenous (i.v.) platinum chemotherapy. Within 5 years after initial treatment, the disease recurs in 60-70% of patients and often exhibits cross-resistance to many structurally related or unrelated drugs. In addition, ⁇ 25% of ovarian cancers are "innately" resistant to platinum and respond poorly to initial chemotherapy. For the treatment of ovarian carcinomas, therefore, cisplatin resistance presents a very serious problem.
  • paclitaxel has been shown to be one of the most effective agents in such patients with relapsed platinum-refractory disease. Many of these drug combinations have severe toxic side effects and fail to increase the survival rates of patients with drug resistant tumors. Therefore, there is an urgent need to develop effective treatment strategies for the management of platinum-resistant ovarian cancers.
  • Therapeutic dosing with non-toxic plant bioactives in combination with chemotherapeutic drugs to treat the ovarian cancer and overcome drug resistance is a viable strategy.
  • berry bioactives exhibit poor oral bioavailability and stability, thus limiting their effectiveness to full potential for clinical applications. Identifying effective oral delivery systems that can achieve high oral bioavailability and are safe when used for prolonged periods are needed to overcome the limitations of Anthos for clinical translatability.
  • CUR curcumin
  • CU R is a chemopreventive agent which exists as a native mixture of curcumin (75%), demethoxycurcumin (18-20%), and bisdemethoxycurcumin (5-7%).
  • hydrophobic nature of CUR results in low water solubility and rapid intestinal/hepatic metabolism limits its oral bioavailability, impeding clinical development of CU R as potential therapeutic agent.
  • Exosomes are extracellular microvesicles or endogenous nanoparticles secreted by a wide variety of cells having a particle size of 30 nm - 100 nm and carry a cargo of proteins, lipids, RNA, and DNA. Their properties of shuttling in-and-out of the cells suggest that these particles can be exploited as a nano drug carrier.
  • M ilk exosomes, in particula r are potential carriers for drug-delivery because of their nano size, a scalable source, biocompatibility, ability of exosomes to stabilize drugs, lack of toxicity, tumor targetability, among other factors. Due to their known stability in acidic environment, milk exosomes may provide a desirable oral drug delivery carrier, with wide preventive and therapeutic applications, including for carrying anthocyanidins and curcumin. Summary of the Present Invention
  • the present development is a method for delivering anthocyanidins and for delivering curcumin using exosomes, particularly for the treatment of cancers and more particularly for the treatment of ovarian cancer and cervical cancer.
  • ExoCU R exosomal-CUR
  • Figure 1 is a graph showing the antiproliferative activity of the chemotherapeutic drug, Cisplatin against drug-sensitive (A2780) and drug-resistant (A2780/CP70, OVCA432 and OVCA433) human ovarian cancer cells by MTT assay;
  • Figure 2 is a graph showing the antiproliferative activity of the native mixture of Anthos from bilberry (B) against drug-sensitive (A2780) and drug-resistant (A2780/CP70, OVCA432 and OVCA433) human ovarian cancer cells by MTT assay;
  • Figure 3 is a graph showing the antiproliferative activity of Anthos-loaded milk exosomes vs free Anthos against human drug-resistant ovarian cancer OVCA433 cells by MTT assay, wherein exosomal protein concentration was maintained constant (50 ⁇ g/ml), and each data point represents an average of 3 to 4 replicates ⁇ SD, and wherein the Students' t-test was conducted to calculate the statistical significance;
  • Figure 4 is a graph showing the antiproliferative activity of Anthos (6 and 30 mg/kg b. wt.) and exosomal formulations of Anthos (6 mg/kg b. wt., 60 mg/kg Exo protein) given by oral gavage and compared with vehicle control against ovarian ca ncer A2780 tumor xenograft;
  • Figure 5 is a graph showing the cell viability of drug-resistant human ovarian cancer (OVCA433) cells treated with Cisplatin alone or in combination with 75 ⁇ Anthos;
  • Figure 6 is a bar graph showing the effect of Anthos and PAC on the PgP expression wherein cells were treated with the indicated concentrations of PAC, Anthos and a combination thereof for 24 h before the cells were collected and proteins were separated on SDS-PAGE and probed for the p-glyoprotein;
  • Figure 7 is a graph showing the effect of the exosomal formulations on ovarian cancer (A2780) tumor xenograft for tumors that grew to about 80 mm 3 and then the animals were treated with vehicle, PAC alone, ExoPAC (4 mg/kg), ExoAnthos (6 mg/kg) and combinations of the ExoPAC and ExoAnthos by oral gavage on alternate days;
  • Figure 8 is a graph showing the particle-size determination of milk-derived exosomes and ExoCU R by Zetasizer and atomic force microscopy (AFM );
  • Figure 9 is a graph showing the cell viability of H 1299 cells treated with CU R, freshly prepared ExoCU R, and ExoCU R that was stored at -80C for six months;
  • Figure 10 is a set of bar graphs showing the tissue distribution of CU R in rats treated with ExoCU R or free CU R, wherein the tissues examined were from the liver, lung and brain;
  • Figure 11 is a set of bar graphs showing the anti-proliferative activity of ExoCU R compared to curcumin and isolated milk exosomes with no drug loading for three different cell lines;
  • Figure 12 is a graph demonstrating the antitumor activity of CUR and ExoCU R against human cervical cancer (CaSki) xenografts in nude mice.
  • the presently-disclosed subject matter is based, at least in part, on the discovery that an exosomal formulation of anthocyanidins (Anthos) elicits potent therapeutic activity against both drug- sensitive and drug-resistant human ovarian cancer cells, and also synergistically enhance therapeutic activity when used in combination with cisplatin.
  • an exosomal formulation of anthocyanidins elicits potent therapeutic activity against both drug- sensitive and drug-resistant human ovarian cancer cells, and also synergistically enhance therapeutic activity when used in combination with cisplatin.
  • ExoAnthos Exosomal anthocyanidins
  • ExoPAC Exosomal Paclitaxel
  • Anthos is isolated from 36% anthocyanin- enriched bilberry extract ( Indena, Seattle, WA) using a recently developed solvent-solvent extraction procedure (Patent # 8,987,481 Bl).
  • the Anthos thus isolated has over 85% purity, and can be further purified by C18 Bond-Pak column to ⁇ 95% purity as determined by U PLC.
  • the final Anthos mixture contains delphinidin ( Dp), cyanidin (Cy), malvidin (Mv), peonidin ( Pe) and petunidin (Pt) in the proportion of 33:28: 16:16:7.
  • the molecular weight of the Anthos mixture represents the average molecular weight of these five individual anthocyanidins calculated by multiplying its content with respective molecular weights.
  • Authentic Anthos standards were purchased from Chromadex (Irvine, CA).
  • Primary P-glycoprotein antibody and secondary antibodies were purchased from cell signaling technology (Danvers, MA). All other chemicals received were of analytical grade.
  • Exosomes are isolated from the mature bovine milk by differential centrifugation.
  • the isolated exosomes are characterized by analysis of size and surface protein markers (including CD63 and CD81).
  • the size distribution of the isolated exosomes is measured by NanoSight and Zetasizer, and confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM).
  • SEM scanning electron microscopy
  • AFM atomic force microscopy
  • the proteins markers are characterized by western blot.
  • the exosomes are suspended in PBS and stored at -80°C until use.
  • Drug loading is achieved by dissolving the drug to be loaded in ethanol or 1: 1 mixture of ethanol and acetonitrile and then mixing with the milk exosomes. Free drug is removed by a low-speed centrifugation (8000 x g, 5 min) and the drug-loaded exosomes are collected by either Amicon ultra filtration using 500 kDa filter (for ExoAnthos), or by ultracentrifugation (135,000 x g, 90 min). Drug load is determined by analysis of the drug and protein in the formulation.
  • Human ovarian cancer cells (A2780, A2780/CP70, OVCA432, and OVCA433) were obtained and maintained in RPM I 1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 m M L-glutamine, and 0.2 units/ml insulin (Sigma-Aldrich, St. Louis, MO). Cells were cultured in an atmosphere of 95% humidity and 5% CO2 at 37°C. As is known in the art, these drug-resistant ovarian cancer cell lines overexpress p-glycoproteins (PgP) and show >100-fold resistance to chemotherapeutic drug cisplatin compared A2780.
  • PgP p-glycoproteins
  • Antiproliferative activity of the Anthos, PAC, cisplatin and combination of the drugs and their exosomal formulations is assessed against ovarian cancer cells by MTT assay. All the cell lines are plated (5000 cells per well) and antiproliferative activity is determined after 72 h. [0035] As shown in Figures 1 and 2 and in Table 1, the antiproliferative activity of Anthos against drug sensitive and cisplatin-resistant ovarian cancer cells was studied and the results compared to the efficacy of cisplatin.
  • the cisplatin-resistant (A2780/CP70, OVCA432 and OVCA433) cell overexpress PgP and show 100-fold resistance to cisplatin compared to the drug- sensitive (A2780) ovarian cancer cell lines.
  • Anthos resulted in dose-dependent inhibition of ovarian cancer cells, as shown in Figure 2.
  • the IC 5 o concentration, as calculated by Ca lcuSyn Software, for cisplatin compared to Anthos is shown for the four cell lines in Table 1.
  • Anthos demonstrated an IC50 concentration of nearly 25 ⁇ against the sensitive A2780 cells; however, it was almost 10-fold higher for the drug resistant OVCA432 cells. This is the first demonstration showing the ability of Anthos to inhibit growth of drug-resistant ovarian cancer cells.
  • the Anthos treatment demonstrated an inhibition of about 63% with compared with vehicle control (575 ⁇ 217 vs. 1535 ⁇ 340).
  • the sub-optimal concentration of Anthos (6 mg/kg) was included in the evaluation to demonstrate the synergistic effect of the exosomes with the Anthos (comparison being the ExoAnthos composition).
  • the ExoAnthos demonstrate greater efficacy with respect to ovarian cancer because the Anthos is protected in the exosomal formulation and remain viable after tolerating Gl conditions to elicit biological response.
  • ExoAnthos are capable of inhibiting ovarian cancer.
  • Paclitaxel is known in the art for treating drug-resistant recurrent ovarian cancers as they often retain sensitivity to PAC.
  • drug-resistant OVCA432 cells were treated with the Anthos and PAC, alone and in combination, and whole cell lysates were analyzed by Western blot.
  • the ovarian cancer OVCA432 cells are treated with the Anthos and PAC, individually and in combination at different concentrations. After 24 h cells are collected and whole cell lysates are prepared in RI PA buffer. Protein concentration is determined using the BCA method.
  • mice were (1) treated with a combination of ExoAnthos and ExoPAC containing 6 mg/kg Anthos a nd 60 mg/kg Exo three times a week (on alternate days), or (2) treated with an exosomal formulation of PAC (4 mg PAC/kg b. wt. and 60 mg Exo/kg b.
  • ExoAnthos (6 mg/kg Anthos and 60 mg/kg Exo) and ExoPac (4 mg PAC/kg b. wt. and 60 mg Exo/kg b. wt.) three times a week.
  • ExoPAC shows only modest but insignificant tumor inhibition.
  • berry Anthos are highly effective against ovarian cancer and that the milk exosomes serve as an excellent nano carrier to enhance the drug's oral bioavailability for management of ovarian cancer.
  • CU R is one of the most investigated plant bioactives for benefits against various diseases; however medicinal benefits of this compound are limited because of its low oral bioavailability.
  • poor aqueous solubility of CUR remains a major barrier in its bioavailability and clinical efficacy.
  • Efforts taken to increase its solubility and bioavailability include encapsulation of CUR in liposomes, polymeric nanoparticles, biodegradable microspheres, phospholipid mixtures and hydrogels.
  • none of these delivery systems has advanced to translational setting due to inherent toxicity, high costs and/or non-scalability.
  • the present invention demonstrates that milk-derived exosomes can overcome the bioavailability issues of CU R.
  • Exosomal formulation of CUR qualifies as ideal nanoformulation due to physical attributes such as average size of about 100 nm with a PDI of 0.19 ⁇ 0.02.
  • Sufficiently high drug load ( ⁇ 20-25%) can be achieved by simple mixing of CU R in presence of solvents concentration 10% which preserves the quality attributes of the exosomes.
  • loading of CU R on to exosomes presumably occurs due to the interactions between highly lipophilic CUR and hydrophobic domains on the surface exosomal proteins, however, we cannot rule out if part of the loading is "into" the exosome lumen and lipid membrane.
  • ExoCU R formulations stored at -80°C are stable for at least 6 months with unaltered drug load and particle size. Moreover, the storage of ExoCU R has no effects on its antiproliferative potency.
  • CU R can be delivered effectively using milk-derived exosomes.
  • CU R when mixed with exosomes in the presence of 10% ethanol: acetonitrile (1:1), provided a drug load of 18- 24%, and the formulation was stable for 6 months as determined by particle size analysis, drug load and anti-proliferative activity.
  • the uptake of exosomes by cancer cells involved caveolae/clathrin- mediated endocytosis.
  • Oral administration of exosomal CU R (ExoCU R) in Sprague-Dawley rats demonstrated 3-5 times higher levels in various organs versus free agent.
  • ExoCU R showed enhanced antiproliferative activity against multiple cancers including, breast, lung and cervical.
  • ExoCUR showed significantly higher anti-inflammatory activity measured as N F- ⁇ activation in lung and breast cancer cells.
  • nude mice bearing the cervical CaSki tumor xenograft were treated with ExoCU R by oral gavage, CU R diet, exosomes alone and PBS as controls. While CUR via dietary route failed to elicit any effect, exosomes had a modest (25-30%) tumor growth inhibition. However, ExoCU R showed significant inhibition (61%; p ⁇ 0.01) of the tumor xenograft. No gross or systemic toxicity was observed in the rats administered with the exosomes or ExoCU R. These results suggest that exosomes can be developed as potential nano-carriers for delivering CUR which otherwise has encountered significant tissue bioavailability issues in the past.
  • exosomes were isolated from raw bovine milk using differential centrifugation performed at 13,000 x g, 100,000 x g and 135,000 x g for 30, 60 and 90 min, respectively, to isolate the exosomes.
  • the exosome pellet was suspended in PBS and stored at -80 °C.
  • the size distribution was measured by Zetasizer and atomic force microscopy (AFM).
  • the buoyant density of exosomes and the ExoCU R was determined using 10-60% (w/v) sucrose density-gradient medium and centrifuged at 135,000 x g in a swing bucket rotor (SW 41Ti, Beckman Coulter Inc.) at 4 °C for 8 h.
  • CU R was loaded to the exosomes by mixing a solution of CU R in ethanol: acetonitrile (1: 1) with the exosomes at room temperature (22 °C). The concentration of organic solvent was kept less than 10%. A low speed centrifugation at 10,000 x g for 10 min was done to remove the unbound CU R, and the CU R-loaded exosomes were collected by ultracentrifugation. The pellet was suspended in PBS and stored at -80 °C until use. For drug load analysis, ExoCUR (50 ⁇ ) was mixed with 950 ⁇ of acetonitrile and centrifuged at 10,000 x g for 10 min to separate precipitated proteins.
  • CU R was analyzed in the supernatant by UPLC as described below. Protein pellet was dissolved in water and exosomal proteins were analyzed by BCA method. Exo and ExoCU R formulations were prepared and stored at -80°C prior to testing in cell culture and for animal experiments. To ensure that formulations were stable, storage stability at -80°C was tested. ExoCUR was stored in PBS at -80 °C and analyzed for CU R load after 3 and 6 months using UPLC. Stability of the exosomal formulation was determined by analyzing the drug load, size of the exosomes and antiproliferative activity at different time intervals.
  • the efficacy of the ExoCU R was assessed using human lung cancer H 1299 and A549 cells, cervical cancer (HeLa) and breast cancer (M DA-M B-231 and T47D) cells.
  • the cells were maintained in DM EM glutamax medium (A549 and H1299 cells), RPM I ( HeLa, CaSki and T47D cells) and L15 (M DA- M B-231 cells) containing 10% fetal bovine serum at 37 °C in 5% C02 and humidified air.
  • lung cancer H1299 cells were plated in 8-well chamber slide and treated with different endocytosis inhibitors for 2 h prior to incu bation with PKH-67-labelled exosomes (50 ⁇ g exosomal proteins/m L) for 4 h.
  • Green fluorescent cell linker (PKH-67) was used to label the milk exosomes as per manufacturer's instructions.
  • the inhibitors of caveolae (colchicine and genistein), clathrin (sucrose and phenothiazine), microtubule (actin) (cytochalasin D), golgi bodies (berfeldin A), and microtubule/macropinocytosis (nocodazole) were used (20). Actin and nuclei were stained with Alexa Fluor 555 Phalloidin and DAPI, respectively. To determine the metabolic inhibition, cells were treated with the metabolic inhibitor, sodium azide or incubated at 4 °C in the presence of PKH-67- labelled exosomes.
  • H 1299 cells were fixed and incubated with endocytosis markers caveolin-1, clathrin, early endosome markers EEA1, late endosome Rab 7 and recycling endosome Rabl l (Cell Signaling, Danvers, MA) primary antibody for lh followed by anti-rabbit alexa fluor 546 (Thermo Fisher Scientific, Waltham, MA). CellLight ® Lysosomes- RFP reagent was used to label lysosomes (red). Nuclei were visualized by staining with DAPI. Images were taken at 20x - 40x magnification on a confocal microscope.
  • the extracted CU R was analyzed by U PLC. Antiproliferative activity was determined against lung, cervical and breast cancer cells. Cells were plated at an initial density of 3 x 10 3 (lung cancer) and 5 x 10 3 (cervical and breast cancer) cells per well and treated with CU R or ExoCU R (0.25 - 50 ⁇ ) and incubated for 72 h. Exosome concentration was maintained constant at 50 ⁇ g/m L. Inhibition of cell proliferation was analyzed by MTT assay as described. DNA binding of N F- ⁇ was measured in the vehicle, CUR and ExoCU R treated lung and breast cancer cell by EMSA.
  • CU R - 20 mg/kg b. wt. and Exo - 80 mg/kg b. wt. An additional group of animals was provided diet supplemented with CU R (400 ppm) that is equivalent to 80 mg/kg b. wt. based on average 4 g diet consumption.
  • Tumor dimensions (length, width and height) of the tumors were measu red with a digital calipers. The animals were euthanized after 7-8 weeks of the treatment by CO2 asphyxiation.
  • Exosomes were isolated by differential centrifugation from bovine milk and characterized for structure morphology, buoyant density, particle size, and presence of exosomal markers.
  • the average size of isolated exosomes as determined by zetasizer was 84 ⁇ 7 nm and had polydispersity index (PDI) of 0.19 ⁇ 0.02 as shown in Figure 8.
  • the size and morphology of exosomes was confirmed by AFM and it was found that the size was somewhat smaller (60- 80 nm) than reported from the Zetasizer, which could have been due to shrinkage during drying.
  • CU R was loaded onto the exosomes in the presence of ethanol and acetonitrile (1:1; 10%) and resulted in a drug load of 18-24%. Unbound CU R was removed by slow-speed centrifugation and ExoCUR was harvested by ultracentrifugation. Size analysis of ExoCU R revealed that there was slight but insignificant increase in the size (93 ⁇ 6 nm) after CU R loading with a PDI of 0.21 ⁇ 0.04, as shown in Figure 8. The stability of CU R in exosomal formulation upon long-term storage was determined by solvent extraction and U PLC analysis. As shown in Figure 9, there was essentially no change in the CU R levels after 6 months of storage at - 80°C.
  • Exosomal formulation of CU R showed almost unaltered particle size (104 ⁇ 11 nm) after 6 months of storage.
  • the antiproliferative efficacy of the ExoCU R against A549 lung cancer cells after storing for six months remained similar to the efficacy determined with the fresh formulation.
  • the caveolae inhibitor, colchicine and clathrin inhibitor, phenothiazine resulted in 53% and 48% decrease in uptake of exosomes, respectively; microtubule inhibitor, nocodazole and actin inhibitor, cytochalasin D caused 32% and 21 % inhibition of exosome internalization by cells, respectively.
  • I nvolvement of calveolin-1 and clathrin proteins was confirmed by observation of colocalization with PH K-67-labeled exosomes in lung cancer cells.
  • CU R tissue distribution of CU R in the lung, liver and brain was also examined.
  • CU R is a well-established anti-inflammatory agent. ExoCUR inhibited both constitutive and TN Fa-induced N F- ⁇ levels in lung and breast cancer cells, while free CUR was essentially ineffective. Similar effects were observed against LPS-induced NF- ⁇ activation. A modest anti-inflammatory effect of the exosomes alone was also evident against the constitutive and TN Fa-induced N F- ⁇ levels. These protective effects are presumably exerted by the endogenous cargo of immune factors, miRNAs and proteins in the exosomes.
  • ExoCU R Anti-proliferative activity of ExoCU R was determined against several human cancer cell line. As shown in Figure 11, compared with free CU R, a n increased anti-proliferative efficacy of ExoCU R with all cancer types examined was observed. The growth inhibition increased from 23% to 66% against H 1299 lung cancer cells and from 34% to 61% against A549 lung cancer cells with free CU R and ExoCU R at 1.56 ⁇ concentration each. ExoCUR showed even greater inhibition of cell growth against cervical and breast cancer cells - 70-80% compared to 10-35% with CU R. M ilk exosomes per se exhibited significant growth inhibitory effects ranging from 15-45%, different cancer cell types.
  • ExoCU R CU R - 20 mg/kg b. wt. and Exo - 80 mg/kg b. wt.
  • An additional group was provided diet supplemented with CU R at 400 ppm.
  • Data represent average of 8-10 animals ⁇ SE.
  • Statistical analysis was done using student's t-test. Dietary CUR showed no effect on the growth of tumor xenograft. However, a significantly higher inhibition of tumor growth was observed with ExoCU R (61%; p ⁇ 0.01) compared to Exo alone (21%).
  • wild-type SD rats were treated with vehicle, exosomes alone, CU R and ExoCU R. Specifica lly, wild-type rats were treated with PBS, CUR (2.5 mg/kg b. wt.) or two doses ExoCU R (1.25 and 2.5 mg CU R/kg b. wt., each containing 25 mg Exo proteins/kg b. wt.) daily by oral gavage. After 14 days, animals were euthanized by CO2 asphyxiation. Blood was collected at the time of euthanasia, and hematological and various biochemical parameters were analyzed. Data represent average ⁇ SD of four animals. No differences were observed when statistical analysis was done using student's t-test.
  • Exosomal formulation of CU R was prepared with the goal of improving its tissue bioavailability. This nanoformulation overcomes limitations of low oral bioavailability of CUR, and reduces its effective dose. Exosomal formulation resulted in increased tissue accumulation of CU R presumably due to higher uptake, prolonged circulation, and protection from rapid hepatic degradation. ExoCUR demonstrated enhanced anti-proliferative, anti-inflammatory and anti-tumor activities compared to free CU R. Furthermore, the milk-derived exosomes and ExoCU R showed lack of any cross-species reaction. Thus, exosomes may offer an effective nanodelivery method for CU R and other plant bioactives many of which otherwise face oral bioavailability issues.
  • microvesicles are defined as particles that are in the form of small assemblies of lipid particles, are about 30 to 1000 nm in size, and are not only secreted by many types of in vitro cell cultures and in vivo cells, but are commonly found in vivo in body fluids, such as blood, urine and malignant ascites.
  • microvesicles include, but are not limited to, particles such as exosomes, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes, and oncosomes.
  • Microvesicles can be formed by a variety of processes, including the release of apoptotic bodies, the budding of microvesicles directly from the cytoplasmic membrane of a cell, and exocytosis from multivesicular bodies.
  • exosomes are commonly formed by their secretion from the endosomal membrane compartments of cells as a consequence of the fusion of multivesicular bodies with the plasma membrane.
  • the multivesicular bodies (MVBs) are formed by inward budding from the endosomal membrane and subsequent pinching off of small vesicles into the luminal space. The internal vesicles present in the MVBs are then released into the extracellular fluid as so-called exosomes.
  • microvesicle As is known in the art, as part of the formation and release of microvesicles, unwanted molecules are eliminated from cells. However, cytosolic and plasma membrane proteins are also incorporated during these processes into the microvesicles, resulting in microvesicles having particle size properties, lipid bilayer functional properties, and other unique functional properties that allow the microvesicles to potentially function as effective nanoparticle carriers of therapeutic agents.
  • microvesicle is used interchangeably herein with the terms “nanoparticle,” “liposome,” “exosome,” “exosome-like particle,” “nano-vector” and grammatical variations of each of the foregoing.
  • milk is used herein to describe the opaque liquid that contains proteins, fats, lactose, and various vitamins and minerals and that is produced by the mammary glands of mature female mammals including, but not limited to, after the mammals have given birth to provide nourishment for their young.
  • milk is further inclusive of colostrum.
  • colostrum is used herein to describe the liquid that is secreted by the mammary glands of mammals at the time of parturition and that is rich in antibodies and minerals.
  • the compositions of the presently-disclosed subject matter are comprised of colostrum-derived microvesicles.
  • the phrases “milk-derived” microvesicles or “colostrum-derived” microvesicles is used interchangeably herein with the phrases “milk microvesicles” or “colostrum microvesicles,” respectively to refer to microvesicles that have been isolated from milk or colostrum.
  • the phrase “milk-derived” can be used interchangeably with the phrase “isolated from milk” to describe a microvesicle of the presently-disclosed subject matter that is useful for encapsulating therapeutic agents.
  • the isolation of microvesicles is achieved by centrifuging raw (i.e., unpasteurized milk or colostrum) at high speeds to isolate the microvesicles.
  • the microvesicles of the presently-disclosed subject matter are isolated in a manner that allows for the isolation of clinical-grade microvesicles.
  • a method of isolating a microvesicles is further provided that includes the steps of: obtaining an amount of milk (e.g., raw milk or colostrum).
  • the series of sequential centrifugations comprises a first centrifugation at 20,000 x g at 4°C for 30 min, a second centrifugation at 100,000 x g at 4°C for 60 min, and a third centrifugation at 120,000 x g at 4°C for 90 min.
  • the isolated microvesicles can then be stored at a concentration of about 5 mg/ml to about 10 mg/ml as such a concentration has been found to prevent coagulation and allow the isolated microvesicles to effectively be used for the encapsulation of one or more therapeutic agents.
  • the isolated microvesicles are passed through a 0.22 ⁇ filter to remove any coagulated particles as well as microorganisms, such as bacteria.
  • microvesicle curcumin refers to a microvesicle whose lipid bilayer encapsulates or surrounds an effective amount of curcumin.
  • the encapsulation of various therapeutic agents within microvesicles can be achieved by mixing the one or more of the phytochemical agents or chemotherapeutic agents with isolated microvesicles in a suitable solvent, such as ethanol.
  • the microvesicle/therapeutic agent mixture is then subjected to a low-speed centrifugation (e.g., 10,000 x g) to remove any unbound therapeutic agent and one or more high-speed centrifugation centrifugations to isolate the microvesicles encapsulating the therapeutic agents.
  • a low-speed centrifugation e.g., 10,000 x g
  • the term "therapeutic agent” is used to refer to an agent that is capable of “treating” a disease, as defined herein below.
  • the therapeutic agent can comprise an anthocyanidin.
  • the phytochemical agent is a bilberry anthocyanidin mixture, which includes, in certain embodiments, a mixture of five anthocyanidins isolated from anthocyanin-enriched bilberry extract following acid hydrolysis and purification by solvent-solvent extraction.
  • the mixture of five anthocyanidins is a mixture of delphinidin, cyanidin, malvidin, peonidin, and petunidin.
  • the therapeutic agent that is encapsulated within the exosome is a chemotherapeutic agent.
  • chemotherapeutic agents that can be used in accordance with the presently disclosed subject matter include, but are not limited to, platinum coordination compounds such as cisplatin, carboplatin or oxalyplatin; taxane compounds, such as paclitaxel or docetaxel; topoisomerase I inhibitors such as camptothecin compounds for example irinotecan or topotecan; topoisomerase II inhibitors such as anti-tumor podophyllotoxin derivatives for example etoposide or teniposide; antitumor vinca alkaloids for example vinblastine, vincristine or vinorelbine; anti-tumor nucleoside derivatives for example 5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such as nitrogen mustard
  • the chemotherapeutic agent that is encapsulated by an exosome in accordance with the presently- disclosed subject matter is paclitaxel, which can be encapsulated in an exosome separate from the exosome encapsulating the anthocyanidin.
  • compositions including the milk-derived exosomes are further provided.
  • a pharmaceutical composition is provided that comprises a milk-derived microvesicle composition disclosed herein and a pharmaceutical vehicle, carrier, or excipient.
  • the pharmaceutical composition is pharmaceutically-acceptable in humans.
  • the pharmaceutical composition can be formulated as a therapeutic composition for delivery to a subject.
  • a pharmaceutical composition as described herein preferably comprises a composition that includes pharmaceutical carrier such as aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • pharmaceutical carrier such as aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient
  • aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.
  • the pharmaceutical compositions used can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents
  • formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried or room temperature (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.
  • solid formulations of the compositions for oral administration can contain suitable carriers or excipients, such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid.
  • suitable carriers or excipients such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid.
  • Disintegrators include, but are not limited to, microcrystalline cellulose, corn starch, sodium starch glycolate, and alginic acid.
  • Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose.
  • Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.
  • the solid formulations can be uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained/extended action over a longer period of time.
  • glyceryl monostearate or glyceryl distearate can be employed to provide a sustained-/extended-release formulation. Numerous techniques for formulating sustained release preparations are known to those of ordinary skill in the art and can be used in accordance with the present invention.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional techniques with pharmaceutically-acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g. lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethy
  • compositions can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • the compositions can take the form of capsules, tablets or lozenges formulated in conventional manner.
  • compositions can also be prepared by conventional methods for inhalation into the lungs of the subject to be treated or for intranasal administration into the nose and sinus cavities of a subject to be treated.
  • the compositions can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the desired compound and a suitable powder base such as lactose or starch.
  • compositions can also be formulated as a preparation for implantation or injection.
  • the compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • compositions can further be formulated as topical semi-sold ointment or cream formulations can contain a concentration of the presently-described microvesicle compositions in a carrier such as a pharmaceutical cream base.
  • a carrier such as a pharmaceutical cream base.
  • formulations for topical use include drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various supports and vehicles.
  • the optimal percentage of the therapeutic agent in each pharmaceutical formulation varies according to the formulation itself and the therapeutic effect desired in the specific pathologies and correlated therapeutic.
  • such ointment or cream formulations can be used for trans-dermal delivery of the pharmaceutical compositions described herein or for delivery to organs such as vagina or cervix in women.
  • Injectable formulations of the compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol), and the like.
  • water soluble versions of the compositions can be administered by the drip method, whereby a formulation including a pharmaceutical composition of the presently-disclosed subject matter and a physiologically-acceptable excipient is infused.
  • Physiologically-acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the compounds
  • a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.
  • a suitable insoluble form of the composition can be prepared and administered as a suspension in an aqueous base or a pharmaceutically-acceptable oil base, such as an ester of a long chain fatty acid, (e.g., ethyl oleate).
  • the microvesicle compositions of the presently-disclosed subject matter can also be formulated as rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the microvesicle compositions can also be formulated as a depot preparation by combining the compositions with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a cancer that include administration of a microvesicle composition described herein.
  • the cancer is ovarian cancer.
  • a therapeutic composition as disclosed herein e.g., a milk-derived microvesicle encapsulating a therapeutic agent
  • conventional methods of extrapolating human dosage based on doses administered to a murine animal model can be carried out using the conversion factor for converting the mouse dosage to human dosage:
  • Dose Human per kg Dose Mouse per kg x 1/12.
  • Doses can also be given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions.
  • body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species.
  • Suitable methods for administering a therapeutic composition in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and/or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, dermally (e.g., topical application), intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper- velocity injection/bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site.
  • the therapeutic compositions are administered orally, intravenously, intranasally, or intraperitoneally to thereby treat a disease or disorder.
  • the compositions of the presently-disclosed subject matter typically not only include an effective amount of a therapeutic agent, but are typically administered in an amount effective to achieve the desired response.
  • the term "effective amount” is used herein to refer to an amount of the therapeutic composition (e.g., a microvesicle encapsulating a therapeutic agent, and a pharmaceutically vehicle, carrier, or excipient) sufficient to produce a measurable biological response (e.g., a decrease in inflammation).
  • a measurable biological response e.g., a decrease in inflammation.
  • Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and/or application.
  • the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and the dose is escalated in the absence of dose- limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.
  • treatment relate to any treatment of a condition of interest (e.g., an inflammatory disorder or a cancer), including but not limited to prophylactic treatment and therapeutic treatment.
  • a condition of interest e.g., an inflammatory disorder or a cancer
  • the terms “treatment” or “treating” include, but are not limited to: preventing a condition of interest or the development of a condition of interest; inhibiting the progression of a condition of interest; arresting or preventing the further development of a condition of interest; reducing the severity of a condition of interest; ameliorating or relieving symptoms associated with a condition of interest; and causing a regression of a condition of interest or one or more of the symptoms associated with a condition of interest.
  • the term "subject” includes both human and animal subjects.
  • veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter.
  • the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos.
  • Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses.
  • carnivores such as cats and dogs
  • swine including pigs, hogs, and wild boars
  • ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels
  • horses are also provided.
  • domesticated fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including, but not limited to, domesticated swine, ruminants, ungulates, horses (including
  • the term "about”, when referring to a value or to an amount of mass, weight, time, volume, concentration, or percentage can encompass variations of, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments to ⁇ 0.1%, from the specified amount, as such variations are appropriate in the disclosed application.
  • ranges can be expressed as from “about” one particular value, and/or to "about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value "10" is disclosed, then “about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • an optionally variant portion means that the portion is variant or non-variant.

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Abstract

La présente invention concerne une méthode d'administration d'anthocyanidines ou de curcumine à l'aide d'exosomes (ExoAnthos ou ExoCUR). L'administration des anthocyanidines ci-décrite a trait à la prise en charge du cancer de l'ovaire ou du cancer du col de l'utérus. L'ExoAnthos déclenche une puissante activité thérapeutique contre les cellules cancéreuses ovariennes humaines tant sensibles aux médicaments que résistantes aux médicaments, et améliore de manière synergique l'activité thérapeutique lorsqu'il est utilisé en association avec le cisplatine. En outre, l'invention concerne l'association d'ExoAnthos et d'une formulation exosomale de Paclitaxel (ExoPAC) pour le traitement de cellules cancéreuses ovariennes résistantes au cisplatine sensibilisées par réduction des niveaux d'expression de PgP. L'ExoCUR déclenche des niveaux de CUR augmentés dans les tissus du cerveau, du foie et du poumon, et présente une efficacité biologique augmentée sous forme d'activités antiproliférative, anti-inflammatoire et antitumorale sans effets secondaires indésirables.
PCT/US2018/054450 2017-10-04 2018-10-04 Administration exosomale d'anthocyanidines et de curcumine Ceased WO2019071031A1 (fr)

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JP2022160101A (ja) * 2021-04-06 2022-10-19 株式会社ファンケル 乳由来エクソソーム様ベシクル含有皮膚外用剤、コラーゲン産生促進剤およびエラスチン産生促進剤
CN116672458A (zh) * 2023-01-18 2023-09-01 中国农业科学院油料作物研究所 一种芝麻叶外泌体纳米颗粒及其制备方法和在活性成分递送中的应用
CN121015626A (zh) * 2025-07-17 2025-11-28 哈尔滨商业大学 一种基于花青素包覆的纳米粒子的制备方法及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022160101A (ja) * 2021-04-06 2022-10-19 株式会社ファンケル 乳由来エクソソーム様ベシクル含有皮膚外用剤、コラーゲン産生促進剤およびエラスチン産生促進剤
JP7814847B2 (ja) 2021-04-06 2026-02-17 株式会社ファンケル 乳由来エクソソーム様ベシクル含有皮膚外用剤、コラーゲン産生促進剤およびエラスチン産生促進剤
CN116672458A (zh) * 2023-01-18 2023-09-01 中国农业科学院油料作物研究所 一种芝麻叶外泌体纳米颗粒及其制备方法和在活性成分递送中的应用
CN121015626A (zh) * 2025-07-17 2025-11-28 哈尔滨商业大学 一种基于花青素包覆的纳米粒子的制备方法及其应用

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