[go: up one dir, main page]

EP4041227A1 - Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides - Google Patents

Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides

Info

Publication number
EP4041227A1
EP4041227A1 EP20886771.3A EP20886771A EP4041227A1 EP 4041227 A1 EP4041227 A1 EP 4041227A1 EP 20886771 A EP20886771 A EP 20886771A EP 4041227 A1 EP4041227 A1 EP 4041227A1
Authority
EP
European Patent Office
Prior art keywords
solid lipid
drug
lipid nanoparticle
nanoparticles
mct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20886771.3A
Other languages
German (de)
English (en)
Other versions
EP4041227A4 (fr
Inventor
Senay SANLIER
Güliz AK
Habibe YILMAZ
Ayse ÜNAL
Tugba KARAKAYALI
Özge SARI TURGUT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ege Universitesi
Original Assignee
Ege Universitesi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from TR2020/07578A external-priority patent/TR202007578A2/tr
Application filed by Ege Universitesi filed Critical Ege Universitesi
Publication of EP4041227A1 publication Critical patent/EP4041227A1/fr
Publication of EP4041227A4 publication Critical patent/EP4041227A4/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention is related to a MCT-1 (Monocarboxyl ate transporter 1) receptor targeted solid lipid nano drug delivery system that can cross the blood-brain barrier for use in the treatment of Glioblastoma (GBM).
  • MCT-1 Monocarboxyl ate transporter 1
  • GBM Glioblastoma
  • brain tumor is the most common type of cancer after leukemia in individuals aged 0 to 19.
  • brain tumors are the second most common cause of death after leukemia in individuals aged 1 to 19 years. Meningiomas take the first place among the brain tumors with a rate of 36.4%. This is followed by glioma with a rate of 27% and it constitutes 80% of malignant brain tumors.
  • GBM Grade IV astrocytoma glioblastoma
  • TMZ temozolomide
  • the prior art is to apply a radiotherapy treatment regimen with the FDA's recommendation carmustine (BCNU) in the treatment of recurrent glioblastoma.
  • BCNU carmustine
  • This application can increase the survival by only 2,5 months on average in glioblastoma treatment. Therefore, there is a need to develop new therapeutic approaches for glioblastoma, which covers a very high proportion of malignant brain tumors.
  • the American Cancer Society defines cancer treatment methods as chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted therapy, stem cell or bone marrow transplantation, and surgery.
  • Carmustine (BCNU) and temozolomide (TMZ) are drugs belonging to the group of anti cancer agents that have alkylating agent properties that slow the growth of cancer cells. It is an accepted view by scientists that inhibition of a single pathway in cancer treatment cannot give very successful results. Therefore, there is a need for treatment methods using innovative and combined drugs.
  • WO16026942A1 titled "Methods for enhancing permeability to blood-brain barrier and uses thereof', it is aimed to cross the blood brain barrier by using TMZ and VEGF combined drugs for the treatment of brain tumors.
  • drugs were given intravenously and intranasal administration was not used. Since the plasma half-life of the TMZ drug is 1,8 hours, the effectiveness of the drug decreases when the drug enters the systemic circulation.
  • repamycin-bound albumin nanoparticles were prepared and stated to be administered to patients after treatment with TMZ and irinotecan.
  • the technical problem that this system cannot solve is that since the drug is administered intravenously and the delivery system is not targeted, some of the drug is metabolized in the liver and eliminated from the body after it enters the systemic circulation. It takes time for the administered drug to reach the brain as it is distributed throughout the body, and the effectiveness of the drug decreases by the time the drug reaches the brain.
  • paclitaxel loaded albumin nanoparticles were prepared and a phase II study was carried out using carboplatin and TMZ. This study was used for advanced malignant melanoma. Its effectiveness in the treatment of glioblastoma has not been investigated.
  • TMZ was used in combination with SGT-53 gene therapy.
  • the drug systems used in these studies were given by intravenous administration. Therefore, with the addition of the drug to the systemic circulation, loss of the drug amount and side effects on the tissues in the systemic circulation can be seen.
  • solid lipid nanoparticles SPN containing carmustine (BCNU) and temozolomide (TMZ) are prepared and their surface is coated with polysorbate 80 to cross the blood brain barrier (BBB).
  • BBB blood brain barrier
  • the drug system is formulated for intranasal administration in order to reach the brain with a higher concentration of solid lipid nanoparticles loaded with the prepared dual drug system and in vitro and in vivo studies have been conducted.
  • the invention can also be administered intravenously. Since the permeability of the structure of the solid lipid nanoparticles used through cell membranes is higher compared to other carrier systems, intranasal application can be performed. In addition, by adding polysorbate 80 to the solid lipid nanoparticle structure, membrane permeability was further increased.
  • the target group of the invention is patients with glioblastoma, and by means of the invention, drug release and MCT-1 targeting are performed in a controlled manner, providing more benefits to the patient with low-dose drugs with increased therapeutic index of drugs in the treatment, and this allows both an increase in patient welfare and a decrease in cost.
  • MCT-1 protein plays a role as a carrier for monocarboxylic acids.
  • MCT-1 receptor targeting of the invention can also be used with receptors that accept the HBA-SA conjugate as a substrate and increase in tumor tissue.
  • the created system can be defined as targeted chemotherapy. Unlike the existing methods, more than one targeting strategy is used in the developed system. Clinical database searches have shown that there is no study involving a system in which TMZ and BCNU are used together and loaded into nanoparticles. Also, inhaler therapy applications of this nanoparticle system are not encountered. Many chemotherapeutic agents are administered as intravenous treatment, where the liquid substance is given directly through the vein, but inhaler therapy, where the drug is administered to the body by inhalation, helps the drug to reach the brain by passing the BBB, in other words, it allows the chemotherapeutic agent to reach the malignant tissue directly. This situation provides an advantage in treatment.
  • solid lipid nanoparticles passing through BBB Considering the success of solid lipid nanoparticles passing through BBB, toxicity and stability characteristics, it is seen as one of the most suitable systems for drug delivery to the brain.
  • the solid lipid nanoparticle by targeting the solid lipid nanoparticle to the MCT-1 receptor, it is aimed to pass drugs that normally cannot pass through the blood-brain barrier easily and to provide an effective treatment in the cancerous area without damaging healthy cells as much as possible.
  • a MCT-1 receptor targeted solid lipid nano drug carrier system that can cross the blood brain barrier is created for use in the treatment of glioblastoma.
  • Solid lipid nanoparticles are synthesized with polysorbate-80 surface coating to cross the blood brain barrier, and it is also present in healthy tissue in order for the system to deliver drugs more selectively for glioblastoma treatment, but the number of MCT-1 receptors in solid brain tumor tissue increases compared to healthy tissue.
  • SA Stearylamine
  • HBA b-Hydroxybutyrate conjugate
  • SA-HBA Stearylamine
  • SA-HBA b-Hydroxybutyrate conjugate
  • the system created with SA-HBA acts as a substrate for the MCT-1 receptor and thus interacts with the MCT-1 receptor increased in glioblastoma and the HBA-SA conjugate, enabling targeted therapy to be formed.
  • temozolomide and carmustine chemotherapy agents used in the prior art are combined and loaded into solid lipid nanoparticles to provide dual therapy.
  • the biocompatibility, binding to serum proteins and the amount of hemolysis of the invention are tested, and the effect of cytotoxicity and apoptosis is determined by cell culture studies. It is determined that the system is directed to the targeted tissue for its purpose by conducting single dose and repeated dose toxicity studies, biodistribution and pharmacokinetic trials and it provides therapeutic effect by controlled release in that region.
  • the invention differs from the prior art in that it allows the synthesized nanoparticle to cross the blood-brain barrier, its ability to accumulate only in the area where the system will act with MCT-1 targeting, allowing controlled release of drugs, and being administered intranasally.
  • targeted therapy is used with a drug delivery system and this treatment is administered intranasally.
  • the drugs are loaded into the solid lipid nanoparticle and these nanoparticles are increasingly targeted to the MCT-1 receptor, the passage through the blood-brain barriers is facilitated, and after the intranasal administration of the drug, approximately 49% of the drug has been observed to accumulate in the brain.
  • the drug is provided to reach the brain faster with intranasal administration.
  • Figure 1 FTIR spectrum of stearylamine and HBA-SA conjugate.
  • FIG. 1 FTIR spectrum of b-hydroxy butyrate (HBA) and HBA-SA conjugate.
  • Figure 4 SEM image of solid lipid nanoparticles.
  • Figure 5 A) FTIR spectrum of Polysorbate 80 B) Overlapped FTIR spectra of SLN, Cetyl Palmitate and HBA-SA conjugate.
  • Figure 7 A) Hydrodynamic diameter B) ZetaPotential result of solid lipid nanoparticles.
  • Figure 8 HPLC chromatogram of the supernatant of nanoparticles prepared at 0,1% Temozolomide concentrations.
  • Figure 9 HPLC chromatogram of the supernatant of nanoparticles prepared at 0,1% Carmustine concentration.
  • Figure 10 A) hydrodynamic diameter and B) zeta potential result of a SLN sample containing 0,1% drug
  • Figure 11 SEM image of a SLN sample containing at 0,1% drug concentration.
  • Figure 12 FTIR spectrum of nanoparticles containing a dual drug system.
  • Figure 13 Thermogram curve of nanoparticles containing a dual drug system.
  • Figure 14 Time dependent release curve of temozolomide and carmustine from nanoparticles.
  • Figure 15 Drug Release Graph in Solid Lipid Nanoparticles containing TMZ and BCNU made using Franz cell
  • Figure 16 Percentages of viability versus doses of solid lipid nanoparticles containing BCNU, solid lipid nanoparticles containing TMZ, and solid lipid nanoparticles containing BCNU-TMZ, applied to U87MG cells for 48 hours.
  • Figure 17 Percentages of viability versus doses of solid lipid nanoparticles containing BCNU, solid lipid nanoparticles containing TMZ, and solid lipid nanoparticles containing BCNU-TMZ, applied to U87MG cells for 72 hours.
  • Figure 18 A) Conjugate-bearing nanoparticle B) non-conjugate-bearing nanoparticle C) chromatograms for TMZ analysis showing uptake amounts of free drug samples into bEnd.3 cells.
  • Figure 19 IVIS image detected at the end of 3 hours in a solid lipid nanoparticle containing FITC loaded conjugate and nude mice given FITC only.
  • Figure 20 IVIS images of brain tissue extracted after sacrificing of mice applied with solid lipid nanoparticles containing FITC-labeled temozolomide and carmustine at the end of 3 hours
  • FIG. 21 Time dependent plasma concentration graph for TMZ Figure 22: Time dependent plasma concentration graph for BCNU Figure 23: 28-day Subacute Toxicity Weight Change Graph Figure 24: Cancer control group IVIS images.
  • Figure 25 IVIS images of the low-dose solid lipid nanoparticle group.
  • Figure 26 IVIS images of the high-dose solid lipid nanoparticle group.
  • Figure 27 IVIS images of free drug group.
  • Figure 28 Ex vivo staining images of the control group.
  • Figure 29 Ex vivo staining images of the free drug group.
  • Figure 30 Ex vivo staining images of the low dose nano group.
  • Figure 31 Ex vivo staining images of the high dose nano group.
  • Figure 32 A) Control group B) Free drug group C) Low dose nano group and D) Ki67 staining results of high dose nanoparticles.
  • SA Stearylamine
  • HBA b-Hydroxybutyrate
  • Stearylamine and b-hydroxybutyrate act as substrates for the MCT-1 receptor.
  • the monocarboxylate carrier MCT-1 mediates the transport of lactate, acetoacetate and pyruvate in addition to b-hydroxybutyrate.
  • b-hydroxybutyrate preferably lactate, acetoacetate and pyruvate can be used b- hydroxybutyrate, lactate, acetoacetate, and pyruvate can be referred to as ketone bodies transported with the MCT-1 receptor.
  • the substrate system for the MCT-1 receptor can be formed.
  • the main purpose of using stearylamine is to act as a spacer arm to create a better interaction of b-hydroxybutyrate substrate (or ketone bodies transported with the alternative MCT-1 receptor) with the increased MCT-1 receptors in cancer cells.
  • b-hydroxybutyrate 150 mg is dissolved in 5 mL of N, N-dimethyl formamide (DMF) in order to provide the conjugation of SA and HBA, which is available in the state of the art.
  • DMF N, N-dimethyl formamide
  • 40 mg of N-hydroxy succinimide (NHS) and 38 mg of 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) are dissolved in DMF, respectively, and added to the b-hydroxy butyrate solution to reach the final volume of 10 mL, and stirred at room temperature for 2 hours.
  • NHS N-hydroxy succinimide
  • EDC 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • the SA-HBA conjugate is precipitated by adding 50 mL of purified water to the reaction medium. Then, the conjugate is dialyzed against distilled water for 3 days in order to remove the contaminants in the medium. After dialysis, the dialysate is filtered by means of the por 3 nuche funnel. The conjugate remaining on the filter is dried in a 30°C oven for 3-5 days and stored in dry state at +4°C for the next reactions.
  • Cetyl palmitate one of the elements that constitutes the solid lipid nanoparticle, is used to form the lipid core.
  • mono-, di- and tri-glycerides such as glyceryl monostearate, soy lecithin, tripalmitine, docosanoic acid, stearic acid can be used to form the lipid core.
  • glyceryl monostearate glyceryl monostearate
  • soy lecithin soy lecithin
  • tripalmitine docosanoic acid
  • stearic acid can be used to form the lipid core.
  • 100 mg of cetyl palmitate is melted in a water bath at 65°C. Then, to this organic phase is added 5 mg of HBA-SA conjugate dissolved in 250 pL of chloroform. HBA- SA is added to solid lipid nanoparticle structure for MCT-1 targeting.
  • an aqueous solution of polysorbate 80 is prepared so that the final concentration is 2% (w/w) and the solution is heated in a water bath to a temperature of 65°C.
  • polysorbate 80 polysorbate 20 can also be used, but better encapsulation efficiency is obtained with polysorbate 80.
  • the aqueous phase is added to the lipid phase at the same temperature and homogenized for 2 minutes at 22000 rpm by means of a high speed blender (Ultraturrax, ISOLAB) to obtain a pre-emulsion. This pre-emulsion is continued to be homogenized for 1 minute in the sonic bath that reaches 65 °C.
  • the emulsion obtained at the end of the period is rapidly cooled to 4°C and then the temperature is increased to 25°C.
  • the mixture is centrifuged at 12000 rpm and the pellet is collected by separating the supernatant.
  • Solid lipid nanoparticles containing a dual drug system attached with b-hydroxybutyrate ligand and drug-free solid lipid nanoparticles (0,1%) are dispersed in PBS. Since PBS is a physiological phosphate buffer, it is used to create minimum irritation during drug administration to live animals. Nanoparticles are added to fetal bovine serum at a serum: nanoparticle volume ratio (v: v) of 10:90, 20:80, 40:60, 60:40, respectively, and a total volume of 600 pL.
  • the samples are left to incubate at 37°C for 2 hours at 160 rpm and after incubation the samples are centrifuged at 13000 rpm for 40 minutes. After centrifugation, protein was determined in the supernatant according to the Bradford method (Bradford, 1976) and the amount of protein bound to mg drug carrier and % binding values were calculated.
  • the damage that can be caused by solid lipid nanoparticles containing dual drugs on erythrocytes is determined by in vitro hemolysis studies and the methods applied by Mayer et al., 2009 and Yallapu et al. 2015 are used for the method. Accordingly, erythrocytes are separated from whole blood, washed with PBS and suspended in PBS to a final concentration of 2%. dual drug-containing and drug-free nanoparticles are dispersed in PBS at concentrations of 0,5; 0,1; 0,05; 0,01 mg nanoparticles/mL. The nanoparticle and erythrocyte suspension are mixed in a ratio of 1 : 1 (v.v) and incubated at 160 rpm for 2 hours at 37°C.
  • PBS is used as negative control and 1% Triton X-100 solution is used as positive control.
  • the samples are centrifuged at 1000 rpm and the cells are separated from the medium, and then the nanoparticles are removed by centrifugation at 13000 rpm for 40 minutes. Hemoglobin is determined at 540 nm in supernatants.
  • Stability studies of the formulations are carried out in accordance with the stability guide at 5 ⁇ 3°C, 25 ⁇ 2°C 60 ⁇ 5% relative humidity and at 40 ⁇ 2°C at 75 ⁇ 5% relative humidity.
  • the formulations are taken for stability study in colored glass vials and the stability of the nanoparticles is evaluated. The quantity of active substance was assayed by HPLC.
  • Cells are set to an initial density of 5xl0 3 cells/100 pL in 96-well plates and are incubated to adhere for 24 hours before sample groups (free TMZ, free BCNU, mixture of free TMZ and BCNU, SLN containing BCNU and TMZ, SLN containing BCNU and SLN containing TMZ) are added.
  • sample groups free TMZ, free BCNU, mixture of free TMZ and BCNU, SLN containing BCNU and TMZ, SLN containing BCNU and SLN containing TMZ
  • Dosage ranges of free temozolomide, free carmustine and carmustine- temozolomide combination are prepared as 1,563-100 pg/mL.
  • the dose range of SLN containing carmustine is 119-1900 pg/mL
  • the dose range of SLN containing Temozolomide is 124-3975 pg/mL
  • the dose range of SLN containing carmustine and temozolomide is 87,5-2800pg/mL.
  • the dose range of 400-1400 pg/mL of solid lipid nanoparticles containing BCNU and TMZ was more effective.
  • the media containing the drug is removed from the wells and 90 pL of FBS-free fresh media is added instead.
  • 10 pL of MTT (5 mg/mL in sterile PBS- 7.4) solution is added and incubated for 4 hours at 37°C.
  • formazan crystals formed by discarding the supernatant containing MTT are dissolved in 100 pL of DMSO.
  • Quantitative measurement is evaluated colorimetrically by measuring at 540 nm wavelength in a microplate reader (Polarstar Omega) at the end of 15 minutes. The results are compared by determining IC50 values of the drug groups on the cells.
  • Brain endothelial cells, bEnd.3 cells, expressing the MCT-1 receptor are plated into a 6-well plate at 76000 cells per well and incubated for 24 hours to adhere and grow. The conditions of 5% C0 2 and temperature of 37°C are provided for the growth of cells. At the end of the period, diluted free drugs and solid lipid nanoparticles carrying dual drug and solid lipid nanoparticles carrying dual drug without target molecule (conjugate) are added to the wells whose supernatant has been removed with 2 mL. Drug groups containing 154 pg of TMZ and 126 pg of BCNU are applied to the cells. Drug-free cells are considered a control. Cells are incubated at 5% CO2 and 37°C conditions.
  • the supernatant of the cells is removed after 2, 4, 6 and 16 hours for all three substances following drug administration. After washing with cold PBS four times, the cells removed with the cell scraper are centrifuged at 1200 rpm and the supernatant is removed. For cell lysis, 200 pL 0.5% (v/v) Triton X-100 and 200 pL water mixture is added to the cells. After 10 minutes of incubation at room temperature, it is centrifuged at 14000 rpm to extract the cell contents into the aqueous phase. Carmustine and temozolomide in the collected supernatants are determined by HPLC.
  • the apoptotic effects of solid lipid nanoparticles containing TMZ and BCNU, a mixture of free TMZ and BCNU and control groups on the U87MG cell line are determined by two separate methods (Annexin V and ApoDirect). Nanoparticles and free drug mixture are applied taking into account the IC 50 dose found in the cytotoxicity analysis. Accordingly, the nanoparticles containing BCNU and TMZ are used by being diluted at a concentration of 100, 300, 500 pg/mL, and the mixture of free BCNU and TMZ at a concentration of 2,5, 7,5, 12,5 pg/mL in EMEM medium, which is a cell growth medium. Drug-free cells are considered a control. Apoptosis analysis is performed with 2 different methods.
  • Annexin V Method The protocol of Annexin V-FITC method is as follows:
  • U87MG cells are expected to induce apoptosis for 48 hours.
  • the cells are resuspended with 400 pi of IX Binding Buffer.
  • U87MG cells are expected to induce apoptosis for 48 hours. 2. Cells are collected by centrifugation at 2000 rpm for 5 minutes.
  • DNA labeling solution is added on the cell pellets and incubated for 60 minutes at room temperature.
  • Biodistribution studies are carried out in 2 separate stages. While healthy CD-I mice are used in the first group study, in the second stage, studies are carried out with nude CD-I mice that have been created with glioblastoma model.
  • the prepared drug loaded SLNs are administered intranasally to CD-I mice.
  • mice are sacrificed and heart, liver, spleen, kidney, brain and blood samples are collected.
  • the received organs are homogenized. After homogenization processes, the biodistribution of the drug is determined by performing TMZ and BCNU analysis with HPLC method on all organs and blood samples.
  • FITC marked solid lipid nanoparticles are intranasally applied to nude mice carrying glioblastoma.
  • 3 nude mice are given FITC by only intranasal application.
  • samples of heart, liver, spleen, kidney, brain tissues and feces taken from mice placed in metabolism cages and sacrificed at the end of certain periods are homogenized.
  • the determination of drugs in homogenates is carried out by HPLC method.
  • drug analyzes are carried out by HPLC method on plasma samples obtained from blood taken into tubes with EDTA.
  • drug analysis is performed on urine samples collected in metabolism cages.
  • mice in each group (10 mice in each group; 5 males and 5 females) are administered intranasally for 4 weeks, in 3 replicates per week. The mice are weighed every week during the application and their situations are monitored. When the study is completed (at the end of 4 weeks), the mice are sacrificed and their blood is taken into lithium heparin tubes and blood parameters (such as ALB, ALT, GLU, TP, BUN) are examined on the Vetscan VS2 device using the Comphrehensive Diagnostic Profile rotor. Toxicological evaluation is made using all the data obtained.
  • blood parameters such as ALB, ALT, GLU, TP, BUN
  • mice placed in a stereotactic frame were given 5 pL and 2x105 U87MG cells suspended in nutrient medium (Geletneky et ah, 2010).
  • mice are anesthetized by applying 3% isofluorane anesthesia (60-80 seconds).
  • the mice are firstly placed in the chamber, and then in 1,5% isofluorane anesthesia by masking and fixed to the stereotactic frame.
  • a small amount of ophthalmic ointment is then applied to the mice's cornea to keep their eyes moist, and the effective antibiotic is administered subcutaneously to the mice for 2 weeks.
  • the operation area is first cleaned with alcohol and then povidone.
  • the operation area is exposed by incision with scissors and blunt-tipped tissue forceps.
  • the area to be operated is adjusted to be 1 mm anterior and 2 mm lateral of the bregma.In the skull, a hole is drilled by means of a 0.6 mm tip and a drill to create a hole with a lateral diameter of 2 mm. 5 pL of cell suspension is injected into 1-3 mm depth within 1 minute using a 10 pL Hamilton glass injector. The needle is moved very slowly as the tip is withdrawn. Haemostat (oxidized regenerated cellulose) is applied to the area where the cells are injected, and after the operation area is closed with 4.0 vicryl suture, the area is dressed with antibiotic cream.
  • Haemostat oxidized regenerated cellulose
  • Body temperatures of the treated mice are measured with a digital non-contact thermometer.
  • the mice taken for postoperative care are observed until they come out of anesthesia and liquid analgesic is added to their drinking water. From the first day of cell implantation, it is monitored for the purpose of following daily activities and general conditions. Regular dressings are made, body weight and body temperature are measured.
  • Tumor formation is analyzed at 7-10 days following cell implantation of tumor-induced mice with the orthotopic model.
  • Tumor size is examined using the luciferin substrate in an IVIS (Caliper Perkin Elmer) imaging device, utilizing the luciferase activity of U87MG tumor cells. Since the best image in IVIS imaging is obtained with the BIN 8, FI device setting, this setting is used throughout the studies.
  • Luciferin solution prepared in 200 pL 12 mg/mL water is given subcutaneously from the neck of the mice, and 5 minutes after the application, IVIS imaging is performed under isofluorane anesthesia. Tumor size is calculated with the formula 0.5x (Width)2xLength. Mice with tumor size over 200 mm3 are randomly grouped as 5-7 mice in each group. The details of the groups and the dose of intranasal therapy are as follows:
  • Control group 20 pL of PBS is applied 3 times a week.
  • Low-dose nanoparticles group Solid lipid nanoparticles prepared in PBS and at concentration of 300 pg/mL are applied 3 times a week at 20 pL.
  • High-dose nanoparticles group Solid lipid nanoparticles prepared in PBS and at concentration of 1000 pg/mL are applied 3 times a week at 20 pL.
  • Free drug group Free drug mixture containing 35,33 pg/mL BCNU and 30,53 pg/mL TMZ prepared in PBS is applied 3 times a week at 20 pL.
  • the cardiac blood of the mice sacrificed under anesthesia is first collected and then the brain tissues are collected for ex vivo studies.
  • analyzes are performed using the Comphrehensive Diagnostic Profile rotor in the Vetscan VS2 device.
  • Brain tissues are examined in gross after fixing in 10% formalin between 24-48. Serial sections are applied and the entire tissue is processed in an automatic tissue tracking device to create formalin-fixed paraffin-embedded tissue. 4 micron sections are taken on positively charged slides and stained with Hematoxylin eosin and Ki67 (Dako- MIB1) using Ventana automatic Stainer. All tissues are evaluated by a single neuropathologist under a light microscope, without the sample source known (blind). Slides were scanned with 3DHISTECH Pannoramic P250- Flash III Slide Scanner. Ki67 ratio is determined by "Cell- Quant" method in "Digital Quant Center” module in 3DHISTECH-CaseViewer.
  • Ki67 is an antibody that displays proliferating cells within the tumor. Ki67 ratio is known as a poor prognostic factor. Therefore, it is very important to determine this ratio in treatment groups.
  • the N-H stretching peaks belonging to the primary amine group in the structure of stearyl amine are seen at 3331 cm 1 and 3251 cm 1 .
  • the primary amine group is expected to be replaced by the secondary amine group.
  • the broad and single peak observed between 3325 cm 1 and 3215 cm 1 is the N-H stress peak belonging to the secondary amine group.
  • the peak seen at —1550 cm 1 in the literature data is the N-H bending peak of the secondary amine group, which is expected to be in the HBA-SA conjugate (Venishetty et ak, 2013).
  • N-H bending peak of the primary amine group is observed at 1606 cm-1 in the spectrum of stearyl amine.
  • C-C (sp3) asymmetric bending peak at 1462 cm 1 and C-H stretch peaks at 2916 and 2848 cm 1 belonging to the SA structure are observed.
  • the findings are compatible with the literature data (Varshosaz et ak, 2012).
  • FTIR spectrum of HBA is parallel to FTIR spectrum of poly beta- hydroxy butyrate in the study conducted by Ramezani (2015). It is the O-H stress peak of the flat peak carboxylic acid observed at 3400 cm 1 and 3100 cm 1 .
  • the C-H bending peak at 1462 cm 1 is found in spectra of cetyl palmitate, conjugate, polysorbate 80 components and SLN. According to the obtained results, it can be said that the starting components are included in the nanoparticle structure.
  • thermogram curve seen in Figure 6 no weight loss was observed in the structure up to approximately 200°C. It was found that the structure was completely degraded at approximately 360°C and the maximum degradation temperature was 362°C according to DTGA data.
  • Figure 7 it is known that the size of the aqueous form of nanoparticles can vary according to the dry form.
  • the hydrodynamic diameter of SLNs prepared under optimum conditions was found to be 220.9 ⁇ 35,73 nm and the zeta potential as -7,55 mV ( Figure 10.)
  • Lockman et al. (2004) it has been observed that negatively charged nanoparticles cross the blood-brain barrier faster at high concentrations and show more effect in this region.
  • BBB blood brain barrier
  • the retention time (rt) of the sample is 3.147.
  • the retention time (rt) of the sample is 4.434.
  • SLNs have a spherical/ellipsoidal shape and a smooth surface.
  • the hydrodynamic diameter of SLNs containing drugs at a concentration of 0,1% by weight was determined as 227 ⁇ 46,80 nm, and the dimensions of the dry form measured by SEM were approximately 170-180 nm. Similar to the obtained results, in the study conducted by Padya et al. (2018), it has been shown that SLNs differ from their dry dimensions measured by SEM and hydrodynamic diameter measured with zeta sizer in aqueous form. In the study, the size of SLNs loaded with olmesartan medoxomil was approximately 131 nm, while the hydrodynamic dimension was observed as 152.40 ⁇ 2.92 nm.
  • the FTIR spectrum of the nanoparticles loaded with temozolomide and carmustine is similar to the spectrum of non-drug loaded, empty nanoparticles and the peaks coincide with each other. It is thought that carbonyl stretch, C-H bending, C-0 stretching signals increase due to the structure of drugs.
  • weight loss was not observed up to approximately 200°C in the nanoparticle structure carrying dual drugs as in the empty nanoparticles. It was found that the structure was completely degraded at 360°C and the maximum degradation temperature was 367°C according to DTGA data. It is estimated that this value has shifted by 5°C compared to the empty nanoparticles and this is due to the drugs added to the structure. As with empty nanoparticles, it is believed that solid lipid nanoparticles containing drugs will retain their properties under operating conditions.
  • temozolomide was released at the rate of 9,78% and 18,4%, and carmustine at 9,23% and 28,1% in 1 and 5 hours, respectively. At the end of 25 hours, the release percentages are 19,82 for temozolomide and 30,22 for carmustine. It is observed that the release of drugs from solid lipid nanoparticles begins rapidly in the first 5 hours and continues in a controlled manner. As is known, nanoparticles can provide slow and controlled release of active ingredients.
  • Protein binding amounts and binding percentages are given in Table 2. As can be seen from the table, the amount of protein bound to the structures of the empty nanoparticles and the nanoparticles containing the drug mixed in the ratios of 10:90 and 20:80 was not found. In systems prepared to contain more serum, the percentage of bound protein is approximately the same for empty and drug-loaded nanoparticles and ranges from 21-28%. Table 2. The amount and percentage of nanoparticles binding to serum proteins.
  • Hemolysis testing was performed with positive control, negative control, SLN samples containing drug at a concentration of 0,5; 0,1; 0,05; 0,01 mg/mL, drug-free SLN samples at a concentration of 0,5; 0,1; 0,05; 0,01 mg/mL and it was found that nanoparticles at varying concentrations did not cause any hemolysis. It is an expected result that the used empty and drug-loaded nanoparticles, considering they are loaded, do not create hemolysis. When looking at all the protein binding and hemolysis results, it can be said that nanoparticles are biocompatible.
  • Cytotoxicities of BCNU-SLN, TMZ-SLN and BCNU-TMZ-SLN groups on U87MG cell are shown in Figures 16 and 17 for 48 and 72 hours, respectively. It was determined that more cytotoxicity occurred with increasing drug concentration of all drug groups. The highest lethal effect in the drug-containing solid lipid particle group was achieved in the solid lipid particle containing BCNU-TMZ.
  • IC50 values of the solid lipid nanoparticle containing dual drug in terms of carmustine in 48 and 72 hours were 36,23 mM ⁇ 4,65 and 40,16 mM ⁇ 3,23, respectively; IC50 values for temozolomide in 48 and 72 hours were calculated as 57,64 mM ⁇ 7,40 and 63,90 mM ⁇ 5,13, respectively.
  • bEnd.3 healthy brain cells and dual drug loaded nanoparticles have been found to have more than 90% viability in cells even at the highest drug doses. In the free drug combination, it was determined that the viability decreases to 74-82% at concentrations of 50 and 100 pg/mL, respectively.
  • nanoparticles may cause minimal damage to healthy endothelial cells, they may inhibit cell growth and show anticancer activity in glioblastoma cells and support the main goal of our project.
  • the intake percentage of bEnd.3 cells is 0,664%, 0,394% and 0,186% for conjugate loaded solid lipid nanoparticles containing dual drug, non-conjugate loaded solid lipid nanoparticles containing dual drug and free drug mixture, respectively.
  • the penetration of nanoparticles containing conjugates specifically to MCT-1 carrier is 1.68 times higher than non-conjugate-carrying nanoparticles and 3,57 times higher than free drug.
  • apoptosis rates of cells treated with free drug (TMZ+BCNU Combination) (2.5-7.5-12.5 pg/ml) and SLN (100-300-500 pg/ml) are 1%, 1.4% and 1.9%, respectively, in free drug, while they were determined as 1%, 1.7% and 2.3% as a result of SLN application.
  • TMZ+BCNU Combination free drug
  • SLN SLN
  • apoptosis rates of cells treated with free drug and nanoparticle bound drug were compared, it was observed that they increased in a concentration-dependent manner.
  • nanoparticle-bound drug it was determined that the apoptosis rates of cells increased 21.4 and 26.3 times with the increase in concentration compared to free drug. Accordingly, it is thought that the use of drug delivery systems in drug treatment methods will reduce the stress on the cell by administering drugs to the medium more slowly, leading to apoptosis, which is a more controlled form of cell death, and provides an effective treatment.
  • the apoptosis rates of cells treated with free drug (TMZ+BCNU Combination) and SLN were determined as 2.1%, 3.2%, 4.7% and 1.1%, 2.2% and 3.5%, respectively. According to this method, it was found that apoptosis rates are increased in increasing concentrations, but the apoptosis rates of cells treated with the free drug combination were higher than the nanoparticle-bound drug. As a result of the treatment with nanoparticle-bound drug, it was determined that the apoptosis rates of the cells increased with the increase in concentration compared to the free drug. Since both early and late apoptosis rates were shown in the Apodirect In Situ DNA fragmentation method, it was observed that the apoptosis rates of the cells treated with the free drug combination were higher than the nanoparticle-bound drug.
  • Table 4. Zero time-measurement results of nanoparticles.
  • Table 5. 3rd Month measurement results of nanoparticles.
  • Biodistribution results The biodistribution results are shown in table 9. While creating the table values, the average of the data of 3 mice belonging to each period and organ was calculated. When the table data was examined, the drug could not be determined by the method applied in spleen, heart and lung samples. It can be said that there is no accumulation of solid lipid nanoparticles containing drugs in these organs. However, accumulation is seen when drug analysis is performed in the brain and kidney.
  • a two-stage treatment protocol was created to determine the cancer treatment potential of solid lipid nanoparticles containing dual drugs. Initially, solid lipid nanoparticles prepared in PBS and at a concentration of 300 pg/mL were administered intranasally to 5 nude mice with cancer, 3 times a week, and survival was monitored and tumor sizes were calculated by IVIS imaging at certain intervals. In Table 15, nude mouse tumor sizes given low dose solid lipid nanoparticles are shown. In Figure 25, IVIS images are given. Table 15. Low-dose solid lipid nanoparticle group.
  • the free drug containing the mixture of drugs trapped in the solid lipid nanoparticle at the specified concentration 35,33 pg/mL BCNU and 30,53 pg/mL TMZ was administered to 7 nude mice with cancer by intranasal administration and tumor sizes and survival were monitored.
  • Cardiac blood was drawn from mice that completed or failed to complete the treatment process, and whole blood analyzes were performed on a biochemistry analyzer (VetScan VS2) using a rotor (Comphrehensive Diagnostic Profile), and the results are given in Table 18.
  • Biochemistry results of healthy control, cancer control groups, free drug group and solid lipid nanoparticle group were evaluated statistically using an independent sample t test.
  • Tumor/tissue ratio in the brain in nude mice The values found for tumor/tissue ratio; Independent sample t test analysis results of the difference between free and control, low dose nano and control, high dose nano and control, free and low dose nano, free and high dose nano and low dose and high dose nano are shown in Table 20.
  • the difference between other groups is not significant (p>0.05).
  • Ki67 ratio in samples stained with Ki67 (Dako-MIBl) using hemotoxylin eosin and Ventana automatic staining device was determined by "Cell-Quant” method in "Digital Quant Center” module in 3DHISTECH-CaseViewer and the findings are given in Table 21. Table 21. Ki67 ratio
  • FIG. 28 Images selected from staining samples in ex-vivo studies are shown in Figures 28, 29, 30 and 31. Selected images of Ki67 staining index are shown in Figure 32.
  • Glioblastoma are tumors that consist of astrocytic cells, have high mitotic activity and a parallel high Ki67 staining index, and often show signs of necrosis. They are characterized by the formation of a large mass in a short time because they have a very fast growth potential.
  • the most important factors in evaluating the treatment response in glioblastoma are reduction in tumor size and decrease in Ki67 staining index. Since the presence of necrosis may reflect both the characteristics of the tumor and the response to treatment, it is not appropriate to use it as a treatment response criterion.
  • the tumors created in our study are phenotypically identical to glioblastoma.
  • Ki67 staining index which shows the growth rates of tumors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une nanoparticule lipidique solide, qui est un système d'administration ciblée de médicament qui peut traverser la barrière hémato-encéphalique pour une utilisation dans le traitement du glioblastome. Selon l'invention, on prépare des nanoparticules lipidiques solides (NLS) contenant de la carmustine (BCNU) et du témozolomide (TMZ), et la surface de ces nanoparticules est revêtue de polysorbate 80 pour traverser la barrière hémato-encéphalique (BHE). Le système de médicament est formulé pour une administration intranasale afin d'atteindre le cerveau avec une concentration plus élevée de nanoparticules lipidiques solides chargées avec le système de double médicament préparé.
EP20886771.3A 2019-11-11 2020-11-09 Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides Pending EP4041227A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
TR201917444 2019-11-11
TR2020/07578A TR202007578A2 (tr) 2019-11-11 2020-05-14 Gli̇oblastoma tedavi̇si̇ne yöneli̇k kati li̇pi̇d taşiyicilar i̇le nanoteknoloji̇k yeni̇ bi̇r yaklaşim
PCT/TR2020/051062 WO2021096464A1 (fr) 2019-11-11 2020-11-09 Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides

Publications (2)

Publication Number Publication Date
EP4041227A1 true EP4041227A1 (fr) 2022-08-17
EP4041227A4 EP4041227A4 (fr) 2023-08-02

Family

ID=75912135

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20886771.3A Pending EP4041227A4 (fr) 2019-11-11 2020-11-09 Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides

Country Status (2)

Country Link
EP (1) EP4041227A4 (fr)
WO (1) WO2021096464A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114588277B (zh) * 2022-04-07 2023-04-25 江苏省苏北人民医院 装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法
KR20250073220A (ko) * 2022-10-06 2025-05-27 미레카 메디슨즈 게엠베하 신경망막 약물 전달을 위한 접합체

Also Published As

Publication number Publication date
WO2021096464A1 (fr) 2021-05-20
EP4041227A4 (fr) 2023-08-02

Similar Documents

Publication Publication Date Title
Zhang et al. Disruption of dual homeostasis by a metal-organic framework nanoreactor for ferroptosis-based immunotherapy of tumor
Ak et al. Brain-targeted, drug-loaded solid lipid nanoparticles against glioblastoma cells in culture
Tang et al. A simple self-assembly nanomicelle based on brain tumor-targeting peptide-mediated siRNA delivery for glioma immunotherapy via intranasal administration
Sekerdag et al. A potential non-invasive glioblastoma treatment: Nose-to-brain delivery of farnesylthiosalicylic acid incorporated hybrid nanoparticles
Guo et al. TRAIL and doxorubicin combination enhances anti-glioblastoma effect based on passive tumor targeting of liposomes
Wang et al. Precise engineering of prodrug cocktails into single polymeric nanoparticles for combination cancer therapy: extended and sequentially controllable drug release
CN109953972A (zh) 基于巨噬细胞膜包被的乳腺癌靶向纳米粒及其制备方法
Zhou et al. Shape regulated anticancer activities and systematic toxicities of drug nanocrystals in vivo
EA011612B1 (ru) Состав с иринотеканом
Yuan et al. Regulating tumor-associated macrophage polarization by cyclodextrin-modified PLGA nanoparticles loaded with R848 for treating colon cancer
Huang et al. Development of curcumin-loaded galactosylated chitosan-coated nanoparticles for targeted delivery of hepatocellular carcinoma
Chen et al. Response of pH-sensitive doxorubicin nanoparticles on complex tumor microenvironments by tailoring multiple physicochemical properties
CN1785430A (zh) 磁热疗用纳米磁粉－抗人肝癌单抗HAb18靶向药物
Wang et al. Self-assembly of maltose-albumin nanoparticles for efficient targeting delivery and therapy in liver cancer
TW202108127A (zh) 可改變構型及帶電特性之酸鹼敏感聚合物與細胞核導向奈米粒針對多種癌症遞送抗癌藥物和基因治療之技術平台
EP4041227A1 (fr) Nouvelle approche nanotechnologique pour le traitement du glioblastome avec des supports lipidiques solides
US12128041B2 (en) Nanoparticles for delivering drug, whose surface is modified with peptide for targeting brain cancer, preparation method thereof and use thereof
US20200078471A1 (en) Renal clearable drug delivering nanoparticles and methods of use therefor
Qin et al. Hybrid cell membranes camouflaged copper-loaded nano-prodrug for tumor angiogenesis inhibition and cell cuproptosis
Guo et al. Tumor-derived nanovesicles enhance cancer synergistic chemo-immunotherapy by promoting cGAS/STING pathway activation and immunogenetic cell death
Xie et al. Ultrasound-triggered topical oxygen delivery enhances synergistic sonodynamic and antibody therapies against hypoxic gastric cancer
Tian et al. Platelets camouflaged nanovehicle improved bladder cancer immunotherapy by triggering pyroptosis
Li et al. pH/redox dual-responsive drug delivery system with on-demand RGD exposure for photochemotherapy of tumors
Zhang et al. Biomimetic cancer cell membrane engineered lipid nanoparticles for enhanced chemotherapy of homologous malignant tumor
US9326938B2 (en) Oligomer-contained nanoparticle complex release system

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220510

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Free format text: PREVIOUS MAIN CLASS: A61K0031418800

Ipc: A61K0047140000

A4 Supplementary search report drawn up and despatched

Effective date: 20230704

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 35/00 20060101ALI20230629BHEP

Ipc: A61K 31/17 20060101ALI20230629BHEP

Ipc: A61K 31/4188 20060101ALI20230629BHEP

Ipc: A61K 9/51 20060101ALI20230629BHEP

Ipc: A61K 47/14 20170101AFI20230629BHEP