WO2016198121A1 - Means and methods for treating lung hypoplasia - Google Patents

Means and methods for treating lung hypoplasia Download PDF

Info

Publication number
WO2016198121A1
WO2016198121A1 PCT/EP2015/063214 EP2015063214W WO2016198121A1 WO 2016198121 A1 WO2016198121 A1 WO 2016198121A1 EP 2015063214 W EP2015063214 W EP 2015063214W WO 2016198121 A1 WO2016198121 A1 WO 2016198121A1
Authority
WO
WIPO (PCT)
Prior art keywords
vegf
nanodiamond particles
nanodiamond
lung
particles
Prior art date
Application number
PCT/EP2015/063214
Other languages
French (fr)
Inventor
Peter Carmeliet
Jan DEPREST
Stavros LOUKOGEORGAKIS
Noura AL-JUFFALI
Paolo De Coppi
Samuel JANES
Original Assignee
Vib Vzw
Katholieke Universiteit Leuven, K.U.Leuven R&D
Ucl Business Plc
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
Application filed by Vib Vzw, Katholieke Universiteit Leuven, K.U.Leuven R&D, Ucl Business Plc filed Critical Vib Vzw
Priority to PCT/EP2015/063214 priority Critical patent/WO2016198121A1/en
Publication of WO2016198121A1 publication Critical patent/WO2016198121A1/en

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
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/62—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 non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to the field of lung disorders.
  • the invention relates to compositions of nanodiamond particles conjugated to VEGF for the treatment of conditions suffering from lung hypoplasia and lung hypertension such as for example congenital diaphragmatic hernia.
  • CDH Congenital diaphragmatic hernia
  • pulmonary hypoplasia decreased airway number
  • pulmonary hypertension due to thickened blood vessels.
  • mortality exceeds 80%.
  • the lung pathology is caused mainly by intra-abdominal organs herniating into the chest and compressing the lungs thus preventing their growth. This condition is an ideal target for prenatal (in utero) treatment, as damage occurs before birth and cannot be treated postnatally.
  • VEGF vascular endothelial growth factor
  • VEGF vascular leakage in the lungs and oedema formation.
  • VEGF coupled to biocompatible nanoparticles induces dramatic lung growth and reverses defects in aveoli and arterioles when used in combination with tracheal occlusion in a pre-clinical model for CDH.
  • the observed effect is not present when unconjugated VEGF is administered in our model.
  • the ND-VEGF also correct the pulmonary hypertension which occurs in CDH.
  • Further advantages of delivering VEGF coupled to nanoparticles is that these particles enter the cell by endocytosis and are inert, not affecting normal cell function and not causing an immune response.
  • the ND-VEGF platform allows targeted and gradual release of VEGF in the lung, which mimics the spatially and temporally controlled release of VEGF during normal lung development.
  • Figure 1 effect of tracheal occlusion on fetal pulmonary uptake of nanodiamond particles following intra-tracheal in utero administration.
  • the left panel shows the uptake of fluorescent (AF488-ND) nanodiamond particles of fetal pneumocytes in conditions without tracheal occlusion (TO).
  • the right panel shows the enhanced uptake of the AF488-ND particles in combination with tracheal occlusion (TO).
  • Figure 2 localization of nanodiamonds following in utero administration to the fetal lungs with tracheal occlusion.
  • the left panel shows the uptake of free AF488.
  • the right panel depicts nanodiamond conjugated AF488.
  • Figure 3 chemical scheme depicting the conjugation of VEGF to nanodiamonds. The three steps are outlined in the materials and methods section.
  • FIG. 4 measurement of the lung to body weight ratio (LBWR).
  • the LBWR ratio is depicted as a %. It is apparent that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles to the pre-clinical CDH model have a similar LBWR as the healthy animal. The beneficial effect is absent in the presence of SU5416 (a potent and selective inhibitor of VEGF Receptor 2 [KDR/FLK-1].
  • SU5416 a potent and selective inhibitor of VEGF Receptor 2 [KDR/FLK-1].
  • Figure 5 measurement of the alveolar septal well thickness. It is apparent that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles to the pre-clinical model of CDH have a similar alveolar septal wall thickness as the healthy animal.
  • Figure 6 measurement of the alveolar internal diameter. It is striking that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles correct the alveolar internal diameter in the pre-clinical CDH model to the same alveolar diameter as in the healthy animal.
  • Figure 7 measurement of pulmonary arteriole medial thickness.
  • Administration of nanodiamond-VEGF (ND-VEGF + TO) particles restored the arteriole medial thickness to that observed in a healthy animal.
  • ND-VEGF + TO nanodiamond-VEGF
  • the invention provides nanodiamond particles which are chemically conjugated to VEGF.
  • the term 'chemically conjugated' is equivalent to 'chemically coupled' and means that a chemical bond (id est a covalent) bound is present between the nanodiamond particles and the VEGF protein.
  • the term "nanodiamonds” is known to the skilled person and a review describing the properties and applications of nanodiamonds is available from Mochalin VN et al (2012) Nature NanotechnologyVo ⁇ 7, p. 11-23.
  • nanodiamond particles chemically conjugated to VEGF are used for treating respiratory distress syndrome in a mammal.
  • Respiratory distress syndrome is a breathing disorder that affects newborns.
  • RDS rarely occurs in full-term infants. The disorder is more common in premature infants born about 6 weeks or more before their due dates.
  • RDS is more common in premature infants because their lungs are unable to make enough surfactant.
  • Surfactant is a liquid that coats the inside of the lungs. It helps keep them open so that infants can breathe in air once they're born. Without enough surfactant, the lungs collapse and the infant has to work harder to breathe. In some cases, the baby is unable to inhale enough oxygen to support the body's organs. Without proper treatment, the lack of oxygen leads to irreversible organ and brain damage.
  • nanodiamond particles chemically conjugated to VEGF are used for treating lung hypoplasia.
  • nanodiamond particles chemically conjugated to VEGF are used for treating disorders wherein lung hypoplasia occurs.
  • Pulmonary hypoplasia or lung hypoplasia is incomplete development of the lungs, resulting in an abnormally low number or size of bronchopulmonary segments or alveoli. Lung hypoplasia most often occurs secondary to other fetal abnormalities that interfere with normal development of the lungs, such as congenital diaphragmatic hernia (CDH).
  • Primary (idiopathic) pulmonary hypoplasia is rare and usually not associated with other maternal or fetal abnormalities. Incidence of pulmonary hypoplasia ranges from 9-11 per 10,000 live births and 14 per 10,000 births. Pulmonary hypoplasia is a common cause of neonatal death. It also is a common finding in stillbirths, although not regarded as a cause of these.
  • nanodiamond particles chemically conjugated to VEGF are used for treating lung hypertension.
  • Pulmonary hypertension or lung hypertension is an increase of blood pressure in the lung vasculature.
  • the disorder is characterized by a narrowing of blood vessels connected to and with the lungs, with resultant thickening and stiffening of affected blood vessels (fibrosis), and increased blood pressure in the lungs and impairment of blood flow.
  • fibrosis affected blood vessels
  • pulmonary hypertension often develops in patients with CDH.
  • nanodiamond particles chemically conjugated to VEGF are used for treating lung hypertension and lung hypoplasia.
  • nanodiamond particles chemically conjugated to VEGF are used for treating congenital diaphragmatic hernia.
  • the invention provides nanodiamond particles conjugated to VEGF wherein the chemical linker between the nanodiamond particles and VEGF comprises an amide bond and a reducible disulfide bond.
  • the invention provides a method for producing nanodiamond particles chemically linked to VEGF comprising the following steps: i) reacting amino- nanodiamond particles with sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate to generate ND-SPDP, ii) producing thiolated VEGF and iii) coupling the products obtained in steps i) and ii) to generate nanodiamond particles chemically linked to VEGF.
  • the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 2-14 days. In another particular embodiment the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 4-14 days. In yet another particular embodiment the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 6-14 days. In a certain embodiment, the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 2-3 days.
  • the present invention exemplifies the use of VEGF-A chemically coupled to nanodiamond particles, the present invention is not limited to the VEGF-A isoform.
  • VEGF-A multiple isoforms of VEGF-A exist as a result from alternative splicing.
  • VEGF121 isoform the VEGF145 isoform
  • VEGF189 the VEGF189
  • VEGF206 the rodent orthologs of these proteins contain one fewer amino acid.
  • the nanodiamond particles conjugated to VEGF are formulated with a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably).
  • the preferred administration of the nanodiamond particles conjugated to VEGF is intratracheal administration via the fetal trachea.
  • the intratracheal administration is followed by tracheal occlusion (TO).
  • nanodiamond particles conjugated to VEGF is intra- amniotic administration.
  • An amount effective to treat the disorders hereinbefore described depends on the usual factors such as the nature and severity of the disorders being treated and the weight of the mammal. However, a unit dose will normally contain 0.01 to 50 mg for example 0.01 to 10 mg, or 0.05 to 2 mg of the identified compound or a pharmaceutically acceptable salt thereof. It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.
  • Nanodiamond-VEGF particles improve lung growth in an experimental CDH model
  • Nanodiamonds coupled to Alexa Fluor demonstrated efficient nanodiamond (ND) uptake by fetal pulmonary epithelium that was enhanced by tracheal occlusion (TO), see Figure 1.
  • ND-FL were not found in any other fetal or maternal tissues, see Figure 2.
  • Nanodiamond particles coupled to VEGF (ND-VEGF) in combination with TO was associated with improved lung growth (LBWR: 5.5 ⁇ 0.3%), which was significantly greater than that observed in VEGF+TO (p ⁇ 0.01 ), raw ND+TO (p ⁇ 0.01 ), vehicle+TO (p ⁇ 0.01 ) and sham (p ⁇ 0.001 ), but was found similar to healthy controls, see Figure 4.
  • ND-VEGF+TO resulted in thinner alveolar septa (mean transection length/airspace: 19.6 ⁇ 1.8) see Figure 5, increased alveolar size (mean airspace chord length: 36.6 ⁇ 2.2) see Figure 6, and decreased muscularisation of pulmonary arterioles (medial thickness: 20.3 ⁇ 0.9%), see Figure 7, compared to other treatment groups (p ⁇ 0.01 vs. all), but were similar to healthy controls.
  • unconjugated VEGF+TO led to improvements in LBWR, airway and pulmonary vascular architecture, the effects did not exceed those seen in the vehicle+TO group.
  • Coadministration of SU5416 a VEGF Receptor-2 antagonist; Flk-1/KDR inhibitor
  • abrogated the beneficial effects of ND-VEGF but did not affect outcomes in any other of the treatment groups.
  • nanodiamonds were labeled with Alexa Fluor® 488 or 647 carboxylic acid succinimidyl ester.
  • the N- hydroxysuccinimide (NHS) ester is highly reactive towards primary amine groups and results in a stable amide bond (see scheme 1 ).
  • Alexa Fluor ® 488 or 647 succcinimidyl ester was reacted with ND-NH 2 particles at a weight ratio of 2:1 , in a 50:50 volume mix of DMSO and 0.1 M sodium bicarbonate buffer (pH 8). The reaction was maintained under stirring conditions for 3 hours, protected from light, and at room temperature. The solution was then washed 5 times with acetone and twice with ddH 2 0 via repeated centrifugation cycles (14.8K RPM, 99 minutes, 4°C). The resulting solution was subsequently transferred to a pre-weighed eppendorf tube and placed in a SAVANT DNA 120 speedvac concentrator (Thermo-Scientific, USA), to dry overnight. Alexa Fluor® conjugated nanodiamond yield was calculated by subtracting the weight of the empty eppendorf tube from the weight of the eppendorf tube containing ND-AF particles.
  • UV-Vis Ultraviolet-visible spectroscopy
  • UV-Vis spectroscopy measures the optical absorbance of samples in the visible and near visible (near-infrared, near-UV) regions, and is a reflectance of electronic transitions from the ground to the excited states(19). UV-Vis measurements were performed to ascertain fluorophore conjugation to the ND surface and to quantify the amount of cargo functionalized.
  • FITC conjugation was confirmed via a Jaz Oceanic Optics Spectrophotometer (USA), with the appearance of a 490 nm absorbance peak. Alexa Fluor® dye conjugation and the amount of dye conjugated to the ND surface was determined using a Varian Cary 300 Bio UVA is instrument and a Cary WinUV software package (Agilent, USA).
  • AF and ND-AF solutions were diluted 1 in 1000 in ddH 2 0 and their absorbance measured at 650 nm and at 494 nm for AF647 and 488 dyes, respectively.
  • ND-VEGF Nanodiamond Vascular Endothelial Growth Factor
  • ND-VEGF conjugation was a sequential process. ND-NH 2 particles were first reacted with the sulfhydryl-to-amine cross-linker sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate (Sulfo-LC-SPDP), which contains an amine-reactive NHS ester group and a thiol-reactive pyridyldithiol group.
  • Sulfo-LC-SPDP sulfhydryl-to-amine cross-linker sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate
  • the reaction was performed under stirring conditions, and at room temperature.
  • the resulting solution was then washed thrice with ddH 2 0 via repeated centrifugation cycles (14.8K RPM, 99 minutes, 4°C), and subsequently transferred to a pre-weighed eppendorf tube and placed in a SAVANT DNA 120 speedvac concentrator (Thermo-Scientific, USA), to dry overnight.
  • ND-SPDP yield was calculated by subtracting the weight of the empty eppendorf tube from the weight of the eppendorf tube containing ND-SPDP particles.
  • VEGF Thiolation 2-lminothiolane.HCL (Traut's reagent, Thermo-Scientific, USA) is a thiolation compound that reacts with primary amines to yield accessible sulfhydryl groups(27, 28).
  • Traut's reagent was reacted with lyophilized rat VEGF (in PBS) at a molar ratio of 4:1 in 0.1 M sodium bicarbonate buffer (pH 8), for 4 hours under strirring conditions, and at room temperature.
  • the resultant mixture was then filtered through illustra NAP-10 columns (GE Healthcare Life Sciences, UK) to permit solvent exchange of residual PBS with sodium bicarbonate buffer.
  • the BCA assay is a two-step reaction based on the reduction of cupric ions (Cu 2+ ) to cuprous ions (Cu + ) by protein, in alkaline medium (the biuret reaction). In the second step of the reaction, bicinchoninic acid reacts with the newly reduced cuprous ions (Cu + ), resulting in a blue to purple color shift that can be detected calorimetrically(29, 30).
  • BSA Bovine Serum Albumin
  • ND-SPDP functionalized particles were reacted with thiolated VEGF at a molar ratio of 1 :1.
  • the reaction was performed in 0.1 M sodium bicarbonate buffer (pH 8) overnight under stirring conditions, and at room temperature. The following day, the resulting product was washed thrice with ddH 2 0, via sequential ultracentrifugation cycles (45000 RPM, 90 mins, 4°C), using a Beckman Ultracentrifuge with a T100.2 rotor (Beckman Coulter, USA), and resuspended in 1 ml of ddH 2 0. 6.
  • the assay was used according to manufacturer protocols. Briefly, 100 ⁇ of ND-VEGF samples of various dilutions and VEGF standards (supplied with the kit) were pipetted into a 96-well plate, pre-coated with rat VEGF antibody, in duplicate and incubated at 4°C overnight on a horizontal orbital microplate shaker at 500 RPM.
  • the plate was washed 4 times with wash buffer, using an automated Denley Cellwash plate washer (Triad Scientific, USA) and 100 ⁇ of biotinylated VEGF detection antibody was added to each well and incubated for 1 hour, with gentle shaking at room temperature. Following which, the plate was washed 4 times and 100 ⁇ of horseradish peroxidase streptavidin was added to each well for 45 minutes, in order to bind to the biotinylated detection antibody. The plate was subsequently washed 4 times and 100 ⁇ of 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (chromogenic solution that develops proportionally to the amount of VEGF bound) was added to each well and incubated for 30 minutes, protected from light with gentle shaking.
  • TMB 3,3',5,5'-Tetramethylbenzidine

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to the field of lung disorders. In particular the invention relates to compositions of nanodiamond particles conjugated to VEGF for the treatment of conditions suffering from lung hypoplasia and lung hypertension such as for example congenital diaphragmatic hernia.

Description

Means and methods for treating lung hypopl
Field of the invention
The present invention relates to the field of lung disorders. In particular the invention relates to compositions of nanodiamond particles conjugated to VEGF for the treatment of conditions suffering from lung hypoplasia and lung hypertension such as for example congenital diaphragmatic hernia.
Introduction of the invention
Congenital diaphragmatic hernia (CDH) is a congenital malformation (birth defect) of the diaphragm and affects approximately 1 in 2000 newborns (300-400 new cases per year in the UK). CDH is a life-threatening pathology in infants, and a major cause of death due to two complications: pulmonary hypoplasia (decreased airway number) and pulmonary hypertension due to thickened blood vessels). In the more severe cases, mortality exceeds 80%. The lung pathology is caused mainly by intra-abdominal organs herniating into the chest and compressing the lungs thus preventing their growth. This condition is an ideal target for prenatal (in utero) treatment, as damage occurs before birth and cannot be treated postnatally. The only available treatment to date involves occlusion of the fetal trachea (at around 26-28 weeks gestational age), with encouraging initial results (the therapy is currently tested in a multicentre clinical trial; http://www.totaltrial.eu/). Although tracheal occlusion has been shown to be beneficial in this setting, it is likely that further therapies will be required in order to promote lung growth and improve outcomes. VEGF is described in the art as a pivotal growth factor for healthy lung development, and it has activity on both the airways (promotes pulmonary epithelial cell proliferation and maturation), as well as the pulmonary blood vessels (promotes angiogenesis, as well as blood vessel remodelling). Previously it was shown that the administration of recombinant VEGF is useful for the treatment of respiratory distress syndrome (see US7307060) due to an increased lung maturation and surfactant production. In our recent research we however observed that the acute administration of VEGF causes vascular leakage in the lungs and oedema formation. We therefore sought a more optimal administration of VEGF. In the present invention we identified an optimal treatment for CDH via the combination of the standard tracheal occlusion approach for CDH treatment and diamond nanoparticle-bound VEGF (nanodiamond-VEGF; ND-VEGF). In particular we show that the prenatal intra-pulmonary delivery of VEGF coupled to biocompatible nanoparticles induces dramatic lung growth and reverses defects in aveoli and arterioles when used in combination with tracheal occlusion in a pre-clinical model for CDH. Remarkably the observed effect is not present when unconjugated VEGF is administered in our model. Even more remarkable is that the ND-VEGF also correct the pulmonary hypertension which occurs in CDH. Further advantages of delivering VEGF coupled to nanoparticles is that these particles enter the cell by endocytosis and are inert, not affecting normal cell function and not causing an immune response. We believe that one of the key aspects of the invention is that the ND-VEGF platform allows targeted and gradual release of VEGF in the lung, which mimics the spatially and temporally controlled release of VEGF during normal lung development.
Figures
Figure 1 : effect of tracheal occlusion on fetal pulmonary uptake of nanodiamond particles following intra-tracheal in utero administration. The left panel shows the uptake of fluorescent (AF488-ND) nanodiamond particles of fetal pneumocytes in conditions without tracheal occlusion (TO). The right panel shows the enhanced uptake of the AF488-ND particles in combination with tracheal occlusion (TO).
Figure 2: localization of nanodiamonds following in utero administration to the fetal lungs with tracheal occlusion. The left panel shows the uptake of free AF488. The right panel depicts nanodiamond conjugated AF488. Figure 3: chemical scheme depicting the conjugation of VEGF to nanodiamonds. The three steps are outlined in the materials and methods section.
Figure 4: measurement of the lung to body weight ratio (LBWR). The LBWR ratio is depicted as a %. It is apparent that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles to the pre-clinical CDH model have a similar LBWR as the healthy animal. The beneficial effect is absent in the presence of SU5416 (a potent and selective inhibitor of VEGF Receptor 2 [KDR/FLK-1].
Figure 5: measurement of the alveolar septal well thickness. It is apparent that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles to the pre-clinical model of CDH have a similar alveolar septal wall thickness as the healthy animal. Figure 6: measurement of the alveolar internal diameter. It is striking that the administration of nanodiamond-VEGF (ND-VEGF + TO) particles correct the alveolar internal diameter in the pre-clinical CDH model to the same alveolar diameter as in the healthy animal.
Figure 7: measurement of pulmonary arteriole medial thickness. Administration of nanodiamond-VEGF (ND-VEGF + TO) particles restored the arteriole medial thickness to that observed in a healthy animal. Detailed description of the invention
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
In the present invention we have shown that prenatal intra-pulmonary delivery of VEGF with biocompatible nanoparticles induces dramatic lung growth and reverses structural alveolar and arterial abnormalities in an experimental animal model of congenital diaphragmatic hernia (CDH). While not limiting the invention to a particular mode of action of mechanism we believe that the lack of measurable effects of unconjugated VEGF suggests that gradual release - mimicking the spatial and temporal expression of VEGF in normal lung development - is a requirement for bioactivity in this experimental model.
Accordingly in a first embodiment the invention provides nanodiamond particles which are chemically conjugated to VEGF. The term 'chemically conjugated' is equivalent to 'chemically coupled' and means that a chemical bond (id est a covalent) bound is present between the nanodiamond particles and the VEGF protein. The term "nanodiamonds" is known to the skilled person and a review describing the properties and applications of nanodiamonds is available from Mochalin VN et al (2012) Nature NanotechnologyVo\ 7, p. 11-23.
In a particular embodiment nanodiamond particles chemically conjugated to VEGF are used for treating respiratory distress syndrome in a mammal.
Respiratory distress syndrome (RDS) is a breathing disorder that affects newborns. RDS rarely occurs in full-term infants. The disorder is more common in premature infants born about 6 weeks or more before their due dates. RDS is more common in premature infants because their lungs are unable to make enough surfactant. Surfactant is a liquid that coats the inside of the lungs. It helps keep them open so that infants can breathe in air once they're born. Without enough surfactant, the lungs collapse and the infant has to work harder to breathe. In some cases, the baby is unable to inhale enough oxygen to support the body's organs. Without proper treatment, the lack of oxygen leads to irreversible organ and brain damage.
In yet another embodiment nanodiamond particles chemically conjugated to VEGF are used for treating lung hypoplasia.
In yet another embodiment nanodiamond particles chemically conjugated to VEGF are used for treating disorders wherein lung hypoplasia occurs.
Pulmonary hypoplasia or lung hypoplasia is incomplete development of the lungs, resulting in an abnormally low number or size of bronchopulmonary segments or alveoli. Lung hypoplasia most often occurs secondary to other fetal abnormalities that interfere with normal development of the lungs, such as congenital diaphragmatic hernia (CDH). Primary (idiopathic) pulmonary hypoplasia is rare and usually not associated with other maternal or fetal abnormalities. Incidence of pulmonary hypoplasia ranges from 9-11 per 10,000 live births and 14 per 10,000 births. Pulmonary hypoplasia is a common cause of neonatal death. It also is a common finding in stillbirths, although not regarded as a cause of these.
In yet another embodiment nanodiamond particles chemically conjugated to VEGF are used for treating lung hypertension.
Pulmonary hypertension or lung hypertension is an increase of blood pressure in the lung vasculature. The disorder is characterized by a narrowing of blood vessels connected to and with the lungs, with resultant thickening and stiffening of affected blood vessels (fibrosis), and increased blood pressure in the lungs and impairment of blood flow. Like pulmonary hypoplasia, pulmonary hypertension often develops in patients with CDH. In yet another embodiment nanodiamond particles chemically conjugated to VEGF are used for treating lung hypertension and lung hypoplasia.
In yet another embodiment nanodiamond particles chemically conjugated to VEGF are used for treating congenital diaphragmatic hernia. In yet another embodiment the invention provides nanodiamond particles conjugated to VEGF wherein the chemical linker between the nanodiamond particles and VEGF comprises an amide bond and a reducible disulfide bond.
In yet another embodiment the invention provides a method for producing nanodiamond particles chemically linked to VEGF comprising the following steps: i) reacting amino- nanodiamond particles with sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate to generate ND-SPDP, ii) producing thiolated VEGF and iii) coupling the products obtained in steps i) and ii) to generate nanodiamond particles chemically linked to VEGF.
In a particular embodiment the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 2-14 days. In another particular embodiment the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 4-14 days. In yet another particular embodiment the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 6-14 days. In a certain embodiment, the nanodiamond conjugated VEGF particles of the invention release the VEGF part in a time period of between 2-3 days. Although the present invention exemplifies the use of VEGF-A chemically coupled to nanodiamond particles, the present invention is not limited to the VEGF-A isoform. The skilled person is aware that multiple isoforms of VEGF-A exist as a result from alternative splicing. For example in humans there also exists the VEGF121 isoform, the VEGF145 isoform, the VEGF189 and the VEGF206 isoform (the rodent orthologs of these proteins contain one fewer amino acid). Thus it is clear that all these isoforms of VEGF are within the scope of the invention.
In a specific embodiment the nanodiamond particles conjugated to VEGF are formulated with a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably). The preferred administration of the nanodiamond particles conjugated to VEGF is intratracheal administration via the fetal trachea. Preferably the intratracheal administration is followed by tracheal occlusion (TO).
Yet another way of administration of the nanodiamond particles conjugated to VEGF is intra- amniotic administration. An amount effective to treat the disorders hereinbefore described depends on the usual factors such as the nature and severity of the disorders being treated and the weight of the mammal. However, a unit dose will normally contain 0.01 to 50 mg for example 0.01 to 10 mg, or 0.05 to 2 mg of the identified compound or a pharmaceutically acceptable salt thereof. It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.
Examples
1. Nanodiamond-VEGF particles improve lung growth in an experimental CDH model
The administration of prenatal nanodiamonds did not have noticeable adverse feto-maternal effects. Nanodiamonds coupled to Alexa Fluor (ND-FL) demonstrated efficient nanodiamond (ND) uptake by fetal pulmonary epithelium that was enhanced by tracheal occlusion (TO), see Figure 1. ND-FL were not found in any other fetal or maternal tissues, see Figure 2. Nanodiamond particles coupled to VEGF (ND-VEGF) in combination with TO was associated with improved lung growth (LBWR: 5.5±0.3%), which was significantly greater than that observed in VEGF+TO (p<0.01 ), raw ND+TO (p<0.01 ), vehicle+TO (p<0.01 ) and sham (p<0.001 ), but was found similar to healthy controls, see Figure 4. Moreover, ND-VEGF+TO resulted in thinner alveolar septa (mean transection length/airspace: 19.6±1.8) see Figure 5, increased alveolar size (mean airspace chord length: 36.6±2.2) see Figure 6, and decreased muscularisation of pulmonary arterioles (medial thickness: 20.3±0.9%), see Figure 7, compared to other treatment groups (p<0.01 vs. all), but were similar to healthy controls. Although unconjugated VEGF+TO led to improvements in LBWR, airway and pulmonary vascular architecture, the effects did not exceed those seen in the vehicle+TO group. Coadministration of SU5416 (a VEGF Receptor-2 antagonist; Flk-1/KDR inhibitor) abrogated the beneficial effects of ND-VEGF, but did not affect outcomes in any other of the treatment groups.
We conclude that we have shown that prenatal intra-pulmonary delivery of VEGF with biocompatible nanoparticles induces dramatic lung growth and reverses structural arterial abnormalities via KDR/Flk-1 activation, when used in combination with TO in an experimental model of CDH. While not limiting the invention to a particular mode of action of mechanism we tend to believe that the lack of measurable effects of unconjugated VEGF suggests that gradual release - mimicking the spatial and temporal expression of VEGF in normal lung development - is a requirement for bioactivity in this experimental model.
Materials and methods 1. General procedures
CDH was induced in fetuses of pregnant Wistar rats by administration of nitrofen at E9 (term=E22). Maternal laparotomy and hysterotomy were performed at E19, and nanodiamonds (ND; 2-8nm carbon nanoparticles; 75μg mL in 50μΙ_ vehicle/saline) were administered into the fetal trachea followed by tracheal occlusion (TO). ND were either fluorescently-labelled (ND- FL), conjugated with recombinant VEGF164 (ND-VEGF; 2μg/mL VEGF164) or unlabelled/unconjugated (raw ND). Blinded assessment of lung-to-body weight ratio (LBWR), as well as airway and pulmonary vascular morphometric parameters was performed at E21.5 in CDH offspring. Comparisons were made between fetuses receiving ND-VEGF+TO, unconjugated VEGF+TO, raw ND+TO, vehicle+TO, and sham surgery. To examine the mechanism of action of VEGF in this setting, a potent and selective inhibitor of the VEGF- receptor-2 (KDR/Flk-1 ) was co-administered intra-tracheally to selected fetuses at the time of intervention (SU5416; 2mg/kg of fetal weight). Results are expressed as mean±SEM, and statistical analysis was performed using 1 -way AN OVA with Bonferroni post-hoc tests. 2. Nanodiamond Alexa Fluor® (ND-AF)
To be able to track the localization of nanodiamonds in cells and in animals, nanodiamonds were labeled with Alexa Fluor® 488 or 647 carboxylic acid succinimidyl ester. The N- hydroxysuccinimide (NHS) ester is highly reactive towards primary amine groups and results in a stable amide bond (see scheme 1 ).
Figure imgf000008_0001
Primary amine on nanodiamond Alexafluor dye NHS ester Alexafluor conjugated nanodiamond NHS Scheme 1 depicts the NHS ester reaction chemistry between amine-terminated nanodiamond and Alexa Fluor ® succinimidyl ester.
Alexa Fluor ® 488 or 647 succcinimidyl ester was reacted with ND-NH2 particles at a weight ratio of 2:1 , in a 50:50 volume mix of DMSO and 0.1 M sodium bicarbonate buffer (pH 8). The reaction was maintained under stirring conditions for 3 hours, protected from light, and at room temperature. The solution was then washed 5 times with acetone and twice with ddH20 via repeated centrifugation cycles (14.8K RPM, 99 minutes, 4°C). The resulting solution was subsequently transferred to a pre-weighed eppendorf tube and placed in a SAVANT DNA 120 speedvac concentrator (Thermo-Scientific, USA), to dry overnight. Alexa Fluor® conjugated nanodiamond yield was calculated by subtracting the weight of the empty eppendorf tube from the weight of the eppendorf tube containing ND-AF particles.
3. Ultraviolet-visible spectroscopy (UV-Vis)
UV-Vis spectroscopy measures the optical absorbance of samples in the visible and near visible (near-infrared, near-UV) regions, and is a reflectance of electronic transitions from the ground to the excited states(19). UV-Vis measurements were performed to ascertain fluorophore conjugation to the ND surface and to quantify the amount of cargo functionalized.
4. Nanodiamond-fluorophore conjugates characterization
FITC conjugation was confirmed via a Jaz Oceanic Optics Spectrophotometer (USA), with the appearance of a 490 nm absorbance peak. Alexa Fluor® dye conjugation and the amount of dye conjugated to the ND surface was determined using a Varian Cary 300 Bio UVA is instrument and a Cary WinUV software package (Agilent, USA).
In essence, AF and ND-AF solutions were diluted 1 in 1000 in ddH20 and their absorbance measured at 650 nm and at 494 nm for AF647 and 488 dyes, respectively. Dye concentration was determined using the Beer-Lambert equation: A=e.l.c, where A= absorbance (a.u.), ε= molar extinction coefficient (M"1cm"1), l= path length (cm), c= concentration (M); given a path length of 1 cm and molar extinction coefficients of 239,000 M"1cm"1 and 71000 M"1cm"1 for AF647 and 488, respectively. Concentrations were then multiplied by the dilution factor (i.e., 1000) and the degree of AF labeling on the ND surface was calculated using the following equation: (Concentration of dye on ND surface / Concentration of free dye) x 100% 5. Nanodiamond Vascular Endothelial Growth Factor (ND-VEGF) Coupling
ND-VEGF conjugation was a sequential process. ND-NH2 particles were first reacted with the sulfhydryl-to-amine cross-linker sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate (Sulfo-LC-SPDP), which contains an amine-reactive NHS ester group and a thiol-reactive pyridyldithiol group. The NHS ester arm of the linker reacts with amine terminated nanodiamond particles to form a stable amide bond, while the pyridyldithiol arm reacts with thiol-modified VEGF to yield reducible disulfide bonds(13-15) (see Figure 3). (a) Nanodiamond SPDP (ND-SPDP) Functionalization:
Sulfo-LC-SPDP (Thermoscientific, USA) was reacted with ND-NH2 particles overnight, at a molar excess of 377 in 0.1 M sodium bicarbonate buffer (pH 8).
The reaction was performed under stirring conditions, and at room temperature. The resulting solution was then washed thrice with ddH20 via repeated centrifugation cycles (14.8K RPM, 99 minutes, 4°C), and subsequently transferred to a pre-weighed eppendorf tube and placed in a SAVANT DNA 120 speedvac concentrator (Thermo-Scientific, USA), to dry overnight.
ND-SPDP yield was calculated by subtracting the weight of the empty eppendorf tube from the weight of the eppendorf tube containing ND-SPDP particles.
(b) VEGF Thiolation: 2-lminothiolane.HCL (Traut's reagent, Thermo-Scientific, USA) is a thiolation compound that reacts with primary amines to yield accessible sulfhydryl groups(27, 28).
Traut's reagent was reacted with lyophilized rat VEGF (in PBS) at a molar ratio of 4:1 in 0.1 M sodium bicarbonate buffer (pH 8), for 4 hours under strirring conditions, and at room temperature. The resultant mixture was then filtered through illustra NAP-10 columns (GE Healthcare Life Sciences, UK) to permit solvent exchange of residual PBS with sodium bicarbonate buffer.
(c) Bicinchoninic Acid (BCA) Protein Assay:
To quantify the yield of VEGF resulting from the thiolation reaction, a BCA assay (Thermo- Scientific, USA) was performed. The BCA assay is a two-step reaction based on the reduction of cupric ions (Cu2+) to cuprous ions (Cu+) by protein, in alkaline medium (the biuret reaction). In the second step of the reaction, bicinchoninic acid reacts with the newly reduced cuprous ions (Cu+), resulting in a blue to purple color shift that can be detected calorimetrically(29, 30). A set of standards made of Bovine Serum Albumin (BSA) protein were used in this assay and ranged, in concentration, from 31.25 μg ml to 2000 μg ml. In a 96-well plate, 20 μΙ of each standard and sample was added to 180 μΙ of BCA working reagent. The plate was then incubated at 37°C for 30 minutes and the absorbance measured at 550 nm, using a Titertek Multiscan MCC/340 plate reader (Labsystems, Finland). Absorbance values from samples were compared to a generated standard curve to determine sample protein concentrations.
(d) Nanodiamond VEGF (ND-VEGF) Coupling:
ND-SPDP functionalized particles were reacted with thiolated VEGF at a molar ratio of 1 :1. The reaction was performed in 0.1 M sodium bicarbonate buffer (pH 8) overnight under stirring conditions, and at room temperature. The following day, the resulting product was washed thrice with ddH20, via sequential ultracentrifugation cycles (45000 RPM, 90 mins, 4°C), using a Beckman Ultracentrifuge with a T100.2 rotor (Beckman Coulter, USA), and resuspended in 1 ml of ddH20. 6. Enzyme-Linked Immunosorbent Assay (ELISA) for VEGF Quantification
An ELISA assay was performed, using a rat VEGF ELISA kit (Abeam, UK), to determine the quantity of VEGF conjugated to the nanodiamond surface.
The assay was used according to manufacturer protocols. Briefly, 100 μΙ of ND-VEGF samples of various dilutions and VEGF standards (supplied with the kit) were pipetted into a 96-well plate, pre-coated with rat VEGF antibody, in duplicate and incubated at 4°C overnight on a horizontal orbital microplate shaker at 500 RPM.
The plate was washed 4 times with wash buffer, using an automated Denley Cellwash plate washer (Triad Scientific, USA) and 100 μΙ of biotinylated VEGF detection antibody was added to each well and incubated for 1 hour, with gentle shaking at room temperature. Following which, the plate was washed 4 times and 100 μΙ of horseradish peroxidase streptavidin was added to each well for 45 minutes, in order to bind to the biotinylated detection antibody. The plate was subsequently washed 4 times and 100 μΙ of 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (chromogenic solution that develops proportionally to the amount of VEGF bound) was added to each well and incubated for 30 minutes, protected from light with gentle shaking. 50μΙ of stop solution was then added to quench the substrate reaction and the plate absorbance was read at 450 nm, using a Titertek Multiscan MCC/340 plate reader (Labsystems, Finland). Absorbance values from samples were compared to a generated standard curve to ascertain the concentration of VEGF, grafted onto NDs.

Claims

Claims
1. Nanodiamond particles chemically coupled to VEGF.
2. Nanodiamond particles of claim 1 for treating lung hypertension.
3. Nanodiamond particles of claim 1 for treating lung hypoplasia.
4. Nanodiamond particles of claim 1 for treating lung hypoplasia and lung hypertension.
5. Nanodiamond particles of claim 1 for treating congenital diaphragmatic hernia.
6. Nanodiamond particles linked to VEGF wherein the chemical linker comprises an amide bond and a reducible disulfide bond.
7. Nanodiamond particles of claim 6 for treating lung hypertension.
8. Nanodiamond particles of claim 6 for treating lung hypoplasia.
9. Nanodiamond particles of claim 6 for treating lung hypoplasia and lung hypertension.
10. Nanodiamond particles of claim 6 for treating congenital diaphragmatic hernia.
11. A method of producing nanodiamond particles chemically linked to VEGF comprising the following steps: i) reacting amino-nanodiamond particles with Sulfosuccinimidyl 6- (3'-[2-pyridyldithio]-propionamido)hexanoate to generate ND-SPDP, ii) producing thiolated VEGF and iii) coupling the products obtained in steps i) and ii) to generate nanodiamond particles chemically linked to VEGF.
PCT/EP2015/063214 2015-06-12 2015-06-12 Means and methods for treating lung hypoplasia WO2016198121A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2015/063214 WO2016198121A1 (en) 2015-06-12 2015-06-12 Means and methods for treating lung hypoplasia

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2015/063214 WO2016198121A1 (en) 2015-06-12 2015-06-12 Means and methods for treating lung hypoplasia

Publications (1)

Publication Number Publication Date
WO2016198121A1 true WO2016198121A1 (en) 2016-12-15

Family

ID=53398090

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/063214 WO2016198121A1 (en) 2015-06-12 2015-06-12 Means and methods for treating lung hypoplasia

Country Status (1)

Country Link
WO (1) WO2016198121A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019006155A1 (en) 2017-06-28 2019-01-03 Children's Medical Center Corporation Promoting lung growth
EP3939612A4 (en) * 2019-03-13 2023-03-29 National Institute Of Advanced Industrial Science And Technology Optically heat-generating composite material, nanocluster, substance delivery carrier and pharmaceutical composition

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7307060B2 (en) 2001-04-24 2007-12-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Hypoxia inducible factor 2α for curing neonatal respiratory distress syndrome and as a target for the treatment of pulmonary hypertension

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7307060B2 (en) 2001-04-24 2007-12-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Hypoxia inducible factor 2α for curing neonatal respiratory distress syndrome and as a target for the treatment of pulmonary hypertension

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 2012, JOHN WILEY & SONS
DEBEER A ET AL: "Antenatal fetal VEGF therapy to promote pulmonary maturation in a preterm rabbit model", EARLY HUMAN DEVELOPMENT, SHANNON, IR, vol. 86, no. 2, 1 February 2010 (2010-02-01), pages 99 - 105, XP026939808, ISSN: 0378-3782, [retrieved on 20100223], DOI: 10.1016/J.EARLHUMDEV.2010.01.025 *
KLARITSCH P ET AL: "439: Intra-amniotic VEGF administration in fetal rats with nitrofen induced lung hypoplasia - A placebo controlled study", AMERICAN JOURNAL OF OBSTETRICS & GYNECOLOGY, MOSBY, ST LOUIS, MO, US, vol. 199, no. 6, 1 December 2008 (2008-12-01), pages S131, XP025985790, ISSN: 0002-9378, [retrieved on 20081201], DOI: 10.1016/J.AJOG.2008.09.468 *
MOCHALIN VN ET AL., NATURE NANOTECHNOLOGY, vol. 7, 2012, pages 11 - 23
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR PRESS
VADYM N. MOCHALIN ET AL: "The properties and applications of nanodiamonds", NATURE NANOTECHNOLOGY, vol. 7, no. 1, 18 December 2011 (2011-12-18), pages 11 - 23, XP055133599, ISSN: 1748-3387, DOI: 10.1038/nnano.2011.209 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019006155A1 (en) 2017-06-28 2019-01-03 Children's Medical Center Corporation Promoting lung growth
EP3939612A4 (en) * 2019-03-13 2023-03-29 National Institute Of Advanced Industrial Science And Technology Optically heat-generating composite material, nanocluster, substance delivery carrier and pharmaceutical composition

Similar Documents

Publication Publication Date Title
TWI633117B (en) Fibronectin based scaffold domain proteins that bind to myostatin
Yang et al. Non-invasive photobiomodulation treatment in an Alzheimer Disease-like transgenic rat model
Wang et al. WISP1 (CCN4) autoregulates its expression and nuclear trafficking of β-catenin during oxidant stress with limited effects upon neuronal autophagy
EA027380B1 (en) Tolerogenic nanocarriers for generating cd8+ regulatory t cells
Saric et al. Endomembrane tension and trafficking
CN108472380A (en) Control with increased NEP stability discharges CNP agonists
JP7056932B2 (en) Compounds for inducing histogenesis and their use
Jha et al. Dual effect of chitosan-based nanoparticles on the inhibition of β-amyloid peptide aggregation and disintegration of the preformed fibrils
WO2016198121A1 (en) Means and methods for treating lung hypoplasia
EP3724232A1 (en) Exosome packaging and targeted autophagy
Walter et al. Functionalisation of allergen-loaded microspheres with wheat germ agglutinin for targeting enterocytes
Li et al. Intracellular antibody delivery mediated by lipids, polymers, and inorganic nanomaterials for therapeutic applications
KR20140084020A (en) Nanoparticle Tumour Vaccines
Li et al. Nanoparticle-mediated simultaneous downregulation of placental Nrf2 and sFlt1 improves maternal and fetal outcomes in a preeclampsia mouse model
WO2016024627A1 (en) Method and kit for determining risk of preterm birth and/or birth of low-birth-weight baby
Janßen et al. A pivotal role of nonmuscle myosin II during microglial activation
JP2011526145A (en) Functional polypeptide
US20240016900A1 (en) Collagen peptide-based medicament compositions and uses thereof
AU2016246730B2 (en) Anti-Flt-1 antibodies in treating bronchopulmonary dysplasia
US20120083586A1 (en) Nmdar biomarkers for diagnosing and treating cerebral ischemia
AU2016310551A1 (en) Compounds for inducing tissue formation and uses thereof
US20180289836A1 (en) Complexes and methods of reducing inflammation
WO2010081975A1 (en) Polypeptides for specific targeting to otx2 target cells
EP4173626A1 (en) Pentagalloyl glucose (pgg) for use in treatment of pulmonary hypertension
Kang et al. Development of three-component conjugates: to get nano-globes with porous surfaces, high in vivo anti-osteoporosis activity and minimal side effects

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15729151

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15729151

Country of ref document: EP

Kind code of ref document: A1