U.S. patent application number 16/869071 was filed with the patent office on 2020-08-20 for controlled release system for pulmonary delivery of surfactant protein d.
The applicant listed for this patent is B. G. NEGEV TECHNOLOGIES AND APPLICATIONS LTD CHILDREN'S HOSPITAL MEDICAL CENTER. Invention is credited to Shani ATTIAS, Giora ENDEN, Riki GOLDBART, Paul Scot KINGMA, Joseph KOST, Tamar TRAITEL, Jeffrey A. WHITSETT.
Application Number | 20200261547 16/869071 |
Document ID | 20200261547 / US20200261547 |
Family ID | 1000004808836 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
United States Patent
Application |
20200261547 |
Kind Code |
A1 |
KOST; Joseph ; et
al. |
August 20, 2020 |
CONTROLLED RELEASE SYSTEM FOR PULMONARY DELIVERY OF SURFACTANT
PROTEIN D
Abstract
A controlled release system including surfactant protein D (SPD)
and a carrier suitable for controlled release that can be
polylactic-co-glycolic acid (PLGA). The system can be in the form
of a nanoparticle. A pharmaceutical composition including the
system and a pharmaceutically acceptable carrier. Methods of
treatment of a disease, disorder or condition associated with a
decreased level of SPD in a subject, or for pulmonary delivery of
SPD, include administering the pharmaceutical composition to a
subject in need of such treatment.
Inventors: |
KOST; Joseph; (Omer, IL)
; TRAITEL; Tamar; (Beer Sheva, IL) ; GOLDBART;
Riki; (Lehavim, IL) ; ATTIAS; Shani; (Kiryat
Motzkin, IL) ; KINGMA; Paul Scot; (Cincinnati,
OH) ; WHITSETT; Jeffrey A.; (Cincinnati, OH) ;
ENDEN; Giora; (Tel-Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
B. G. NEGEV TECHNOLOGIES AND APPLICATIONS LTD
CHILDREN'S HOSPITAL MEDICAL CENTER |
Beer Sheva
Cincinnati |
OH |
IL
US |
|
|
Family ID: |
1000004808836 |
Appl. No.: |
16/869071 |
Filed: |
May 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15735005 |
Dec 8, 2017 |
10682396 |
|
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PCT/IL2016/050606 |
Jun 9, 2016 |
|
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16869071 |
|
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62172881 |
Jun 9, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 11/00 20180101;
A61K 9/0019 20130101; A61K 9/0073 20130101; A61K 9/0043 20130101;
A61K 38/395 20130101; A61K 9/146 20130101; A61K 38/1709 20130101;
A61K 9/5153 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 9/51 20060101 A61K009/51; A61K 9/14 20060101
A61K009/14; A61K 9/00 20060101 A61K009/00; A61P 11/00 20060101
A61P011/00 |
Claims
1. A controlled release system comprising a nanoparticle or a
microparticle, wherein said nanoparticle or microparticle comprises
surfactant protein D (SPD) as the active agent, and a polymeric
carrier suitable for controlled release of the SPD, wherein said
system does not comprise surfactant components other than the
SPD.
2. The system according to claim 1, wherein said polymer carrier is
selected from the group consisting of polylactic-co-glycolic acid
(PLGA), polylactic acid (PLA), polyglycolic acid (PGA), chitosan,
gelatin, polycaprolactone, and poly-alkyl-cyanoacrylates.
3. The system according to claim 2, wherein said polymer carrier is
polylactic-co-glycolic acid (PLGA).
4. The system according to claim 3, wherein a ratio of lactic acid
to glycolic acid in said PLGA is selected from the group consisting
of 50:50, 65:35, 70:30, 75:25, 82:18 and 85:15.
5. The system according to claim 4, wherein an average radius of
said nanoparticle is selected from the group consisting of between
about 20 and about 300, between about 50 and about 150 nm, and
about 100 nm.
6. The system according to claim 1, further comprising a
pharmaceutically acceptable carrier.
7. The system according to claim 1, wherein said system is an
aerosol.
8. The system according to claim 1, wherein the SPD is the sole
active agent in the nanoparticle or microparticle.
9. A method for treating a disease, disorder or condition selected
from the group consisting of chronic obstructive lung disease
(COPD), asthma, acute bronchitis, chronic bronchitis,
bronchopulmonary dysplasia, emphysema, infant respiratory distress
syndrome (IRDS), acute respiratory distress syndrome (ARDS), lung
infections, persistent pulmonary hypertension, lung hypoplasia,
cancer, cystic fibrosis, alveolar proteinosis, upper respiratory
inflammation, congenital SP-B deficiency, respiratory syncytial
virus (RSV), allergic rhinitis, influenza, and a disease, disorder
or condition associated with a decreased level of SPD in a subject
in need thereof, comprising administering the system according to
claim 1 to said subject.
10. A method for pulmonary delivery of SPD comprising administering
to a subject in need thereof the system according to claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/735,005, filed Dec. 8, 2017, which is the
U.S. National Phase under 35 U.S.C. 371 of PCT/IL2016/050606, filed
Jun. 9, 2016, which claims priority to U.S. Provisional Application
No. 62/172,881, the entire contents of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a controlled release system
for administration of surfactant protein D for treating lung
disease.
BACKGROUND OF THE INVENTION
[0003] Preterm infants, particularly those of very low birth weight
that were born between week 23 and week 28 of gestation, suffer
from a very high incidence of respiratory distress syndrome (RDS)
related to pulmonary immaturity and inability to make pulmonary
surfactant lipids and proteins. These infants are supported by the
use of oxygen, ventilators, and routine administration of
surfactant replacement. The surfactant replacement preparations
currently at use contain surfactant protein (SP) B and SPC but do
not contain SPA, SPD, or other innate host-defense proteins
(Ikegami et al. 2006; WO 90/11768). SPD is a hydrophilic surfactant
protein that decreases pulmonary inflammation and facilitates
normal surfactant lipid structure and recycling. Its use as a
surfactant replacement has been described (Ikegami et al. 2006; WO
07/056195; WO 02/17878; WO 00/023569; US 2011/0189104; U.S. Pat.
No. 7,266,403) but never implemented commercially, possibly due to
problems associated with consistent delivery of large oligomerized
proteins such as SPD. Furthermore, since repeated intubation or
instillation into the airway is challenging in small infants, a
sustained release vehicle that provides surfactant replacement,
including SPD, for long periods of time in lungs of preterm infants
would be highly desirable, but there is currently no system or
device for sustained release of SPD in the alveoli of infants or
adults.
SUMMARY OF THE INVENTION
[0004] According to one aspect, the present invention provides a
controlled release system comprising surfactant protein D (SPD) and
a carrier suitable for controlled release.
[0005] According to another aspect, the present invention provides
a nanoparticle comprising SPD and PLGA.
[0006] According to an additional aspect, the present invention
provides a pharmaceutical composition comprising the controlled
release system or nanoparticle according to the invention.
[0007] According to yet another aspect, the present invention
provides a method of treatment of a disease, disorder or condition
associated with a decreased level of SPD in a subject, comprising
administering the system or nanoparticle according to the invention
or a pharmaceutical composition thereof to said subject.
[0008] According to still another aspect, the present invention
provides a method for pulmonary delivery of SPD comprising
administering to a subject in need thereof the system or
nanoparticle according to the invention or a pharmaceutical
composition thereof.
BRIEF DESCRIPTION OF DRAWINGS
[0009] FIGS. 1A-1D show scanning Electron Microscopy (SEM) images
of poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) with
surfactant protein D (SPD). FIGS. 1A and 1B: PLGA 1A NPs with SPD;
FIGS. 1C and 1D: PLGA 7A NPs with SPD. FIGS. 1A and 1C are at
.times.10,000 magnification, and FIGS. 1B and 1D are at
.times.35,000 magnification.
[0010] FIG. 2 shows hydrodynamic radius of PLGA NPs with SPD in PBS
solution measured with Dynamic Light Scattering (DLS). From left to
right: PLGA-7A, PLGA-7A with SPD, PLGA-1A, PLGA-1A with SPD.
[0011] FIG. 3 shows size distribution of PLGA 7A NPs with SPD
measured with Dynamic Light Scattering (DLS) (n=3).
[0012] FIG. 4 shows Zeta potential of PLGA 1A and 7A NPs with and
without SPD in PBS solution. From left to right: PLGA 1A, PLGA 7A,
PLGA 7A with SPD.
[0013] FIG. 5 shows toxicity test of C57bl/6 mice by intra-tracheal
injection of PLGA 7A empty NPs at 10 and 20 .mu.g particles per
gram body weight, 2 or 4 weeks after the injection. Control group
was injected with 100 .mu.l sterile PBS and tested after 2 weeks.
Each group: Macrophages: (left black bars), neutrophils: (right
gray bars).
[0014] FIG. 6 shows SDS-PAGE analysis of SPD released from NPs
during one week. First four lanes from the left include different
concentrations of SPD (left to right: 25, 50, 100 and 200
.mu.g/ml), next two lanes include washes, and the following lanes
include daily samples from day 0 to day 7. The right-most lane is
sample taken at day 1 without .beta.-ME (normally added to each
sample to keep a denatured state). 40 kD marks the approximate
location of the SPD monomer (43 kDa), and 120 kD marks the
approximate location of the SPD trimer (129 kDa).
[0015] FIG. 7 shows biological activity of SPD protein that was
released from PLGA 1A NPs during one week using bacterial
aggregation assay. PBS: blue diamond, wash: red square, Day 0:
green triangle, Day 1: purple X, Day 2: blue asterisk, Day 3:
orange circle, Day 4: perpendicular bar, Day 5: brown line, Day 6:
green line, Day 7: purple diamond.
[0016] FIG. 8 shows short term toxicity test of C57bl/6 mice after
intra-tracheal injection with PLGA 7A NPs at 10 .mu.g particles per
g body weight (left bar). Control group was injected with PBS (n=4,
right bar). IL-6 concentration in Bronchiolar Lavage Fluid (BALF)
was detected using ELISA.
[0017] FIG. 9 shows SP-D concentration, measured by human SP-D
ELISA, in bronchial lung lavage supernatant from wild-type mice
after intra-tracheal injection of 5 (left bar), 10 (middle bar) or
20 (third bar from left) .mu.g/g body weight (gbw) of PLGA 7A NPs
with SP-D. Control group were untreated mice, (n=3) (right
bar).
[0018] FIG. 10 shows SP-D concentration measured by human SP-D
ELISA, in bronchial lung lavage supernatant from knock-out mice
after intra-tracheal injection of PLGA 7A NPs with SP-D at 20 .mu.g
per gram body weight (left bars), or of empty PLGA 7A NPs (middle
bars). Control group was untreated mice, (n=3) (right bar). Each
group: left bar (dark)--measured after 3 days, right bar
(light)--measured after 7 days.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In the present application, the inventors have found that
poly lactic-co-glycolic acid (PLGA) can form nanoparticles with
surfactant protein D (SPD). Importantly, it was found that the SPD
protein is released from the nanoparticles and maintains its
biological activity, and therefore a system comprising PLGA and SPD
can be used as a carrier in a delivery system for controlled
release of SPD in the lungs.
[0020] Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable and
biocompatible FDA-approved copolymer used as a vehicle for
delivery, including controlled delivery of drugs. PLGA is
synthesized by means of ring-opening co-polymerization of two
different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of
glycolic acid and lactic acid. Polymers can be synthesized as
either random or block copolymers thereby imparting additional
polymer properties. During polymerization, successive monomeric
units (of glycolic or lactic acid) are linked together in PLGA by
ester linkages, thus yielding linear, aliphatic polyester as a
product. Depending on the ratio of lactide to glycolide used for
the polymerization, different forms of PLGA can be obtained: these
are usually identified in regard to the molar ratio of the monomers
used (e.g. PLGA 75:25 identifies a copolymer whose composition is
75% lactic acid and 25% glycolic acid).
[0021] Additional carriers may be used in accordance with the
present invention instead of PLGA, including polymeric carriers
such as polylactic acid (PLA), polyglycolic acid (PGA), chitosan,
gelatin, polycaprolactone and poly-alkyl-cyanoacrylates (Kumari et
al, 2010). In some embodiments the carrier is biodegradable.
[0022] Examples 1-3 show that PLGA and SPD form complexes having
the size of nanoparticles. Example 5 further demonstrates that SPD
protein continues to be released from these nanoparticles for a
duration of at least a week, and Example 6 shows that the released
SPD is biologically active. Examples 9 and 10 show that SPD is
released from the SPD NPs of the invention after administration to
lungs of wild-type or knock-out mice.
[0023] Accordingly, the present invention provides a controlled
release system comprising SPD and a carrier suitable for controlled
release.
[0024] According to some embodiments, the carrier of the
system/nanoparticle is polymeric, i.e. comprising a polymer. In
some embodiments the carrier is PLGA.
[0025] The term "complex" as used herein refers to a molecular
entity formed by loose association involving two or more component
molecular entities (ionic or uncharged), or the corresponding
chemical species; and this term and the term "system" are used
herein interchangeably.
[0026] The ratio of lactic acid to glycolic acid in the PLGA
polymer of the system/nanoparticle may vary. The ratio which was
used in the experiments of the present invention is 50:50. However,
other ratios may also be suitable as indicated below. Accordingly,
in some embodiments, the ratio of lactic acid to glycolic acid in
said PLGA is 50:50, 65:35, 70:30, 75:25, 82:18 or 85:15, in
particular the ratio is 50:50.
[0027] The polymer may be at any of various inherent viscosities
between 0.05 and 7.0 dL/g, for example 0.05 to 1 dl/g or between
0.1 to 0.7 dl/g (depending on the molecular weight, molecular
weight distribution, and consistency of the polymer). The molecular
weight of the PLGA may be selected from between 1 to 200, between 3
to 150 or between 5 to 100 kDa. In the examples presented
hereinbelow, two types of PLGA polymers were used, with molecular
weights of 96 kDa and (inherent viscosity of 0.63 dl/g) and 5.6 kDa
(and inherent viscosity of 0.11 dl/g).
[0028] From Example 2 it can be seen that the radius of the
nanoparticles is about 100 nm. Accordingly, in some embodiments,
the system is a nanoparticle or a microparticle, in particular a
nanoparticle. In some embodiments, the average radius of said
nanoparticle is selected from between 20 and 300, between 50 and
150 nm, or about 100 nm.
[0029] The zeta potential affects the tendency of the particles to
aggregate; charged particles have a lesser tendency to aggregate
due to the repulsing forces of particles having the same charge,
while neutrally charged particles have a tendency to aggregate.
There is therefore an advantage to particles having a charge. The
SPD-loaded PLGA nanoparticles disclosed in herein have a negative
zeta potential of about -35 mV (Example 3).
[0030] In some embodiments the present invention provides a
nanoparticle comprising SPD and PLGA.
[0031] SPD was prepared as previously described (Ikegami et al.,
2006). In some embodiments, the SPD may be recombinant SPD. In some
embodiments, the SPD is a mammalian SPD, in particular human
SPD.
[0032] In some embodiments, the system does not contain surfactant
components other than SPD. For example, the system comprises SPD
but is void of SPA, SPB or SPC.
[0033] The system/nanoparticle of the present invention is prepared
by standard methods used in the art such as the double emulsion
evaporation technique, emulsification-solvent evaporation
technique, or the nanoprecipitation method also called the
interfacial deposition method (Danhier et al, 2012).
[0034] The term "controlled release" is used herein interchangeably
with the terms "prolonged-action", "repeat-action", "extended
release", and "sustained-release" and refers to the release of an
active agent at predetermined intervals or gradually, in such a
manner as to make the contained active agent available over an
extended period of time
[0035] In some embodiments, the system of the invention as
described hereinabove is for use in pulmonary delivery of SPD, i.e.
for use in delivery of SPD to the lungs.
[0036] The present invention also provides a pharmaceutical
composition comprising the system/complex/nanoparticle of the
invention as described hereinabove and a pharmaceutically
acceptable carrier.
[0037] Methodology and components for formulation of pharmaceutical
compositions are well known and can be found, for example, in
Remington's Pharmaceutical Sciences, Eighteenth Edition, A. R.
Gennaro, Ed., Mack Publishing Co. Easton Pa., 1990. Pharmaceutical
compositions for use in accordance with the present invention thus
may be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active agents into
preparations that can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0038] The pharmaceutical composition may be aerosolized by any
known method such as by a pneumatic, jet or ultrasonic nebulizer.
Methods and devices for aerosolization are described, for example,
in Labiris and Dolovich 2003, Br. J. Clin. Pharmacol. 56: 600-612,
and in Ibrahim et al., 2015, Medical Devices: Evidence and Research
8: 131-139.
[0039] In some embodiments, the pharmaceutical composition is in
the form of an aerosol, spray or mist.
[0040] The term "pharmaceutically acceptable carrier" refers to a
vehicle which delivers the active components to the intended target
and which will not cause harm to humans or other recipient
organisms. As used herein, "pharmaceutical" will be understood to
encompass both human and veterinary pharmaceuticals. Useful
carriers include, for example, water, acetone, ethanol, ethylene
glycol, propylene glycol, butane-1, 3-diol, isopropyl myristate,
isopropyl palmitate, mineral oil and polymers composed of chemical
substances like polyglycolic acid or polyhydroxybutyrate or natural
polymers like collagen, fibrin or polysaccharides like chitosan and
alginate. The carrier may be in any form appropriate to the mode of
delivery, for example, solutions, colloidal dispersions, emulsions
(oil-in-water or water-in-oil), suspensions, gels, sprays and the
like.
[0041] Several human lung diseases are characterized by decreased
levels of bronchoalveolar SPD and may therefore be treated by
administration of the system of the present invention.
[0042] The term "decreased levels of bronchoalveolar SPD" as used
herein refers to partial or complete deficiency of bronchoalveolar
SPD as compared with the level of bronchoalveolar SPD in normal
healthy individual(s). It has been shown for several diseases,
including acute respiratory distress syndrome (ARDS) and
respiratory syncytial virus (RSV) that the levels of SPD, even if
they are within the normal range, correlate with mortality rates or
with disease outcome. Accordingly, the term "decreased levels of
bronchoalveolar SPD" may be also used to further refer to a
decreased level of bronchoalveolar SPD which is still within the
normal range but is associated with poor prognosis of a
disease.
[0043] Non-limiting examples of individuals having decreased levels
of bronchoalveolar SPD, i.e. individuals in need for treatment,
include preterm infants, particularly those of very low birth
weight that were born between about week 23 and about week 28 of
gestation, and individuals with a mutated spd gene resulting in
reduced expression or altered activity.
[0044] Thus, in some embodiments, the system/nanoparticle or the
pharmaceutical composition of the invention as described
hereinabove is for use in the treatment of a disease, disorder or
condition associated with a decreased level of a SPD.
[0045] In some embodiments, the disease, disorder or condition is
selected from chronic obstructive lung disease (COPD), asthma,
acute bronchitis, chronic bronchitis, bronchopulmonary dysplasia,
emphysema, infant respiratory distress syndrome (IRDS), acute
respiratory distress syndrome (ARDS), lung infections, persistent
pulmonary hypertension, lung hypoplasia, cancer, cystic fibrosis,
alveolar proteinosis, upper respiratory inflammation, congenital
SP-B deficiency, respiratory syncytial virus (RSV), allergic
rhinitis, and/or influenza. In some embodiments, the disease,
disorder or condition is selected from IRDS, cystic fibrosis, and
emphysema.
[0046] The present invention further provides a method of treatment
of a disease, disorder or condition associated with a decreased
level of SPD in a person, comprising administering to said person a
therapeutically effective amount of the system of the invention as
described hereinabove or a pharmaceutical composition thereof.
[0047] The present invention also provides a method for pulmonary
delivery of SPD comprising administering to a subject in need
thereof a therapeutically effective amount of the system of the
invention as described hereinabove or a pharmaceutical composition
thereof.
[0048] As explained above, the controlled release feature of the
system allows for a lower frequency of administration thereby
causing less inconvenience to the patient.
[0049] The term "treating" or "treatment" as used herein includes
alleviating, abrogating, substantially inhibiting, slowing,
reducing or reversing the progression of a condition, substantially
ameliorating or reducing clinical symptoms of a condition,
substantially preventing the appearance of clinical symptoms of a
condition, or complete cure of the disease.
[0050] The controlled release system/nanoparticle of the invention
or the pharmaceutical composition comprising it can be administered
by various pulmonary delivery methods, for example, by inhalation,
nebulization, intratracheal administration such as by intratracheal
injection, or a nasal spray.
[0051] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a "therapeutically effective
amount" means an amount of an active ingredient effective to
prevent, alleviate or ameliorate symptoms of a disease or disorder
or prolong the survival of the subject being treated.
[0052] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0053] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations or a single administration of a slow release
composition, with course of treatment lasting from several days to
several weeks or until cure is effected or diminution of the
disease state is achieved.
[0054] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0055] The term "about" as used herein means that values of 10% or
less above or below the indicated values are also included.
[0056] The invention will now be illustrated in the following
non-limiting examples.
EXAMPLES
Materials and Methods
Materials
[0057] 50:50 poly lactic-co-glycolic acid (PLGA) 7A, MW 96 kDa with
an inherent viscosity of 0.63 dl/g and 50:50 PLGA 1A, MW 5.6 kDa
with an inherent viscosity of 0.11 dl/g were purchased from
Lakeshore Biomaterials. Polyvinyl alcohol (PVA, MW 85-124 kDa;
87-89% hydrolyzed, 9002-89-5), Ethyl acetate (34848) and Sodium
Hydroxide (1310-73-2) were obtained from Sigma-Aldrich Inc.
Surfactant protein D (SPD) protein was synthesized in Cincinnati
Children's Hospital Medical Center (CCHC) by methods as described
in Ikegami et al. 2006. Micro BCA.TM. protein assay (23235) was
purchased from ORNAT, Biochemicals & Laboratory Equipment Ltd.
Thiazolyl Blue Tetra-zolium Bromide (MTT) (M2128) was purchased
from Sigma-Aldrich Inc. Hydrochloric acid 32% (08460201), was
purchased from Bio-Lab. DMEM high glucose medium (01-055-1A), Fetal
Bovine Serum (FBS) (04-121-1A), Trypsin Ethylene diamine tetra
acetic acid (EDTA) (03-052-1B), L-glutamine (03-020-1B),
Penicillin-streptomycin (03-031-1B) were purchased from Biological
Industries Beit Haemek.
Fabrication of Nanoparticles
[0058] Two types of PLGA 50:50 with different molecular weights
(PLGA 7A-96 kDa and PLGA 1A-5.6 kDa) were used to prepare PLGA
nanoparticles (NPs) using a double emulsion evaporation technique
(w/o/w=water/oil/water). 100 mg PLGA were dissolved in 2 ml of
ethyl acetate solvent in a glass tube. 2.5 mg of surfactant protein
D (SPD) were dissolved in 100 .mu.L DDW and then added to the
polymer/solvent mixture and emulsified by sonication (Q-Sonica, 700
W, 20 KHz, 35% amplitude) for 10 s in an ice bath to form a primary
emulsion. The solution was then added at a high setting vortex to 4
ml of an external aqueous phase, containing 5% w/v PVA (polyvinyl
alcohol) as a stabilizer. The contents were sonicated 3 times for
10 s (35% amplitude). The resulting solution was then added drop
wise into 50 ml of a 0.5% w/v aqueous PVA solution under magnetic
stirring. The resulting nano-sized particles were stirred in
solution for 3 h to allow for ethyl acetate evaporation. The NPs
were centrifuged, washed 3 times with DDW, and finally resuspended
in 4 ml DDW and dried in a lyophilizer.
Dynamic Light Scattering (DLS)
[0059] Mean particle size and size distribution of the SPD-loaded
and empty PLGA 1A and 7A NPs were determined by dynamic light
scattering using CGS-3 (ALV, Langen, Germany). The laser power was
20 mW at the He--Ne laser line (632.8 nm). Correlograms were
calculated by ALV/LSE 5003 correlator, which were collected at
90.degree., during 10s, 20 times, at 25.degree. C. The samples were
diluted to an appropriate concentration using phosphate-buffered
saline (PBS) (PH=7.4). The PBS was previously filtered through a
0.22 .mu.m membrane filter (Millipore, USA) to avoid the presence
of any interfering particles. All measurements were carried out in
triplicates; hence, each value is the mean of three independent
readings within a batch and an error represents the standard
deviation of the mean particle size as an index of particle size
polydispersity.
Scanning Electron Microscopy
[0060] The surface morphology and shape of the nanoparticles were
investigated by scanning electron microscopy (HRSEM, JSM-7400F). To
this end, approximately one drop of nanoparticles suspension was
mounted on an aluminum stub and sputter coated for 30 s with a thin
layer of gold under vacuum and then observed with a scanning
electron microscope operating at 3 kV and 20.degree. C.
Zeta Potential
[0061] The surface charge of the NPs was determined by Zeta
potential. Samples from DLS were transferred to U-tube cuvette
(DTS1060C, Malvern) for subsequent zeta potential measurements
using Zetasizer (ZN-NanoSizer, Malvern, England). SPD-loaded and
empty PLGA 1A and 7A NPs were measured at automatic mode, at
25.degree. C., and the Smoluchowski model was used to calculate the
zeta potential. For each sample the zeta potential value was
presented as the average value of three runs.
In Vivo Toxicity Test
[0062] To determine if treatment with nanoparticles induces a
chronic inflammatory response by looking primarily at macrophage
and neutrophils in Bronchiolar Lavage Fluid (BALF), mice were
treated with either 10 .mu.g/g body weight (n=10), 20 .mu.g/g body
weight PLGA nanoparticles (n=4), or PBS for controls (n=5). 2 and 4
weeks after tracheal injection subsets of mice were lavaged and
some had lungs inflation fixed for histology. BALF was spun at
4000.times.g for 10 minutes and pelleted cells resuspended in 150
ul PBS. 140 ul of suspension was used for Cytospin, 5 minutes at
500 RPM. Slides were allowed to dry and H&E stain was performed
and cells were counted.
SPD Nanoparticle Elution Profile 7.5 mg of SPD-loaded NPs were
mixed with 150 ul PBS, spun for 5 minutes at 800 RPM to wash. Wash
process was repeated and fresh PBS placed on bead after second
wash. Tubes containing particles were rotated at room temp and
samples were collected daily for 1 week, stored at -20.degree. C.
and analyzed by silver stain gel according to standard
procedures.
Bacterial Aggregation Assay
[0063] The biological activity of SPD released from NPs was
analyzed using Bacterial Aggregation Assay (Hartshorn et al. 1998,
Am. J. Physiol.--Lung Cellular and Molecular Physiology
274(6):L958-L969). E. coli Y1088 cultures were spun down, media
removed, and washed in 1.times.TBS (Tris-buffered saline). Bacteria
was spun down again and resuspended in Hank's Balanced Salt
Solution (HBSS) such that the absorbance reading at A700 is 1.
Samples are loaded into Beckman DU 640 spectrophotometer cuvette
containing 5 mM CaCl.sub.2, E. coli, and SPD (5 ul of eluate per
elution day tested) and A700 readings are taken every 2.5 minutes
using a kinetics program. Control sample with PBS was also run for
comparison.
MTT Assay
[0064] MTT stock solution was prepared by dissolving 5 mg of MTT
powder in 1 mL of filter sterilized PBS. The cell medium was
replaced with 0.5 mL of fresh starvation medium and 50 .mu.L of MTT
solution was added to each well. Cells were incubated for 4 hours
in the incubator after which 1 mL of MTT solvent, dimethylsulfoxide
(DMSO), was added to each well. The cell plates were incubated over
night at room temperature in a sterile hood. Absorbance of each
well was read at wavelength of 570 nm and reference of blank
solution (MTT+DMEM medium+DMSO) was used. Cell viability was
calculated by equation 1:
Cell viability (%).times.Optical density (OD) of the treated
cells/OD of the non-treated cells.times.100% (1)
SP-D ELISA
[0065] In Human Surfactant Protein D ELISA, standards and samples
were incubated in microplate wells pre-coated with monoclonal
anti-human surfactant protein D antibody. 100 .mu.l of standard and
sample were added to the microplate wells and incubate for 2 hour
at room temperature on an orbital shaker at 300 rpm. After
incubation, the wells were washed 5 times. 100 .mu.l of Biotin
labelled monoclonal anti-human SP-D antibody was added into each
well and incubated with the captured SP-D for 60 minutes on an
orbital shaker at 300 rpm. After another washing (5 times), 100
.mu.l of Streptavidin-HRP Conjugate was added and shaken for 60
minutes. After incubation and the last washing step, the remaining
HRP conjugate is allowed to react with 100 .mu.l Substrate Solution
(TMB) that was added to each well. The microplate was incubated for
15 min after covering the plate with aluminum foil. The reaction
was stopped by addition of 100 .mu.l acidic solution and absorbance
of the resulting yellow product was measured at 450 nm wavelength.
The absorbance is proportional to the concentration of surfactant
protein D.
Example 1: Scanning Election Microscope
[0066] In order to investigate the physicochemical characterization
of nanoparticles prepared by the double emulsion method,
nanoparticles were observed by Scanning Electron Microscope. As
shown in FIGS. 1A-1D, the SPD-loaded PLGA NPs were spherical and
had a smooth surface without pores or cavities which could affect
the release of the encapsulated protein. The PLGA 1A NP size was
not homogeneous compared to PLGA 7A NPs. In both types of PLGA
there was no difference in shape between the NPs with protein and
the empty NPs
Example 2: Dynamcic Light Scattering (DLS)
[0067] The DLS method was used in order to determine the mean size
and size distribution of the NPs. DLS can calculate the
hydrodynamic diameter of a particle from Stokes-Einstein equation
by measuring the scattered light of the particles that are moving
in Brownian motion. PLGA-NPs 1A and 7A had similar average
hydrodynamic radius of approximately 100 nm with or without protein
(FIG. 2). The size distribution of nanoparticles is shown in FIG.
3. PLGA 1A with SPD showed two populations of particles on DLS and
the SEM results. One population demonstrated particles with
hydrodynamic radius of around 100 nm, similar to empty NPs, and an
additional population with radius of around 600 nm. For further
experiments or treatment the population including the larger
particles was removed by filtration before using the NPs.
Example 3: Zeta Potential
[0068] Zeta potential measures the surface charge of particles and
can give indication of the tendency of the particle suspension to
aggregate and sediment. FIG. 4 shows the Zeta potential of PLGA 1A
without SPD and PLGA 7A with and without SPD. The low values
indicate that the charge of the empty NPs is 0 and the particles
aggregate as can be observed from the SEM images.
[0069] NPs including SPD showed a highly negative Zeta potential
(-35 mV). The pI value of SPD is 5-8 according to the literature.
The large value of the zeta potential is predictive of high
colloidal stability.
Example 4: In Vivo Toxicity Text
[0070] FIG. 5 presents a toxicity test of C57bl/6 mice
intra-tracheal injection with PLGA 7A NPs at 10 or 20 .mu.g
particles per gram of body weight. The control group is a solution
with 100 .mu.l sterile PBS. In this test the number of macrophage
and neutrophils recovered was compared to the control group and
tested after 2 and 4 weeks.
[0071] As we can see in FIG. 5, the number of macrophage and
neutrophils was similar to the number in the control group
indicating that the PLGA NPs were not toxic to the mice.
Example 5: Surfactant Protein D (SPD) Nanoparticle Elution
Profile
[0072] Samples of surfactant protein-D (SPD) that were released
from PLGA NPs during one week were analyzed every day using silver
stain gel. As we can see in FIG. 6, SPD keeps being released from
nanoparticles for a duration of at least one week and the integrity
of the protein was also maintained during the encapsulation
process.
Example 6: Bacterial Aggregation Assay
[0073] To determine if the SPD released from nanoparticles is
biologically active a bacterial aggregation assay was performed.
The decreasing absorbance in FIG. 7 shows that SPD eluted from
nanoparticles is biologically active, with activity slowly
decreasing around day 2 and rapidly deteriorating by day 6. Though
activity decreases over time, nanoparticles do release biologically
active SPD protein for several days.
Example 7: In-Vitro Evaluation of PLGA NPs Toxicity
[0074] In order to investigate whether the NPs have toxic effect,
cell viability was assessed using the MTT assay. 0.5 ml of A549
Lung cells were seeded in a 24 well plate at a density of 30,000
cells/well in DMEM growth medium and incubated at 37.degree. C. and
5% CO.sub.2 for 24 hours to promote cell attachment. Following
incubation, the culture medium was removed, and the samples were
washed twice with 0.5 ml phosphate buffered saline (PBS). PLGA 7A
NPs with SP-D, as well as empty NPs for negative control, were
dissolved in DMEM starvation medium (containing 1% L-glutamine, 1%
penicillin-streptomycin, and 5% FBS) to final NPs concentration of
0.5 mg/ml or 1 mg/ml and incubated at 37.degree. C. for 24 hr.
Untreated A549 cells were used as the control. Following 24 hr
incubation, a standard MTT assay was performed according to
manufacturer's procedure. The samples were taken in triplicate.
[0075] Treatment with 0.5 mg/ml of PLGA 7A NPs showed around 80%
cell viability and treatment with 1 mg/ml of PLGA 7A NPs showed
cell viability values of about 70% (data not shown). The viability
of the cells with empty NPs was about 80-90%. Although the
concentration of NPs influences the cell viability, these high
values indicate that the NPs are not toxic to the cells for both
concentrations.
Example 8: Short Term Experiment--Acute Inflammatory Response
[0076] In order to determine whether NP administration induces an
acute inflammatory response, C57Bl6 mice were treated with 10 .mu.g
per gram body weight of PLGA 7A NPs (2.0 mg/ml in PBS, n=4) or PBS
for controls (n=4) given via tracheal injection. Six hours after
injection, mice were subjected to bronchiolar lavage using 1 ml of
PBS that was injected to the trachea. Bronchiolar Lavage Fluid
(BALF) was collected, spun at 4000.times.g for 10 minutes and the
remaining supernatant was tested for IL-6, a cytokine that drives
acute inflammatory response thus can be an inflammation marker,
using ELISA according to manufacture procedure.
[0077] The purpose of the In vivo toxicity test was to determine if
treatment with empty PLGA NPs induces inflammatory response in mice
before conducting in vivo experiments with SP-D loaded NPs. IL-6 is
a pleiotropic, .alpha.-helical, 22-28 kDa phosphorylated and
variably glycosylated cytokine that plays important roles in the
acute phase reaction, inflammation, hematopoiesis, bone metabolism,
and cancer progression. IL-6 drives the acute inflammatory response
and it is almost solely responsible for fever. It is important in
the transition from acute inflammation to either acquired immunity
or chronic inflammatory disease (C. Garbers, H. M. Hermanns, F.
Schaper, G. Muller-Newen, J. Grotzinger, S. Rose-John, J. Scheller,
Plasticity and cross-talk of interleukin 6-type cytokines, Cytokine
Growth Factor Rev. 23 (2012) 85-97). As can be seen from FIG. 8,
IL-6 concentration in the lungs of mice treated with NPs was 3.2
pg/ml, similar to the level in untreated mice (2.7 pg/ml),
indicating that no short term inflammation was observed as a result
from PLGA 7A NPs treatment on mice by using tracheal injection.
Example 9: In Vivo SPD Release Experiment in Wild-Type Mice
[0078] In order to determine whether SP-D will be released from the
NPs in the lungs, wild-type mice were injected intratracheally with
SP-D loaded PLGA 7A NPs dissolved in PBS (2 mg/ml) at two different
doses of 5 or 20 .mu.g NPs per gram of body weight, to assess which
dose will release the proper amount of SP-D. The control group was
untreated mice. After Two days, 1 ml of Bronchiolar Lavage Fluid
(BALF) was collected from the mice and centrifuge for 5 min at
10,000 RPM at temperature of 4.degree. C. The resulting supernatant
was assayed by human-SP-D ELISA, according to manufacture
procedure, for SP-D concentration determination.
[0079] As can be observed in FIG. 9, for 1 ml BALF that was
collected from the mice, negligible amount of SP-D was released
from 5 and 10 .mu.g/g body weight, while 239 ng of SP-D was
released from 20 .mu.g/g body weight. Since the ELISA is specific
for human SP-D, as expected no SP-D was detected in the untreated
mice control group. This finding emphasizes the fact that the SP-D
that was found by using the ELISA is SP-D that was released from
NPs only. The 239 ng SP-D that was released from 20 .mu.g/g body
weight of NPs fits to the preferred amount of SP-D needed in lungs
of mice (100-300 ng), thus this dose was applied for further
experiments with SP-D knock-out mice.
Example 10: In Vivo SPD Release Experiment in SPD Knock-Out
Mice
[0080] After determination of the appropriate dose based on the
wild-type mice experiment above, SP-D knock-out mice (disclosed in
WO 00/23569) were treated with 20 NPs per gram body weight of SP-D
loaded PLGA 7A NPs by an intra-tracheal injection. The control
groups were mice treated with empty PLGA 7A NPs or untreated. After
3 and 7 days, the amount of SP-D that was released from the NPs was
determined using SP-D ELISA.
[0081] As shown in FIG. 10, SP-D was released from NPs after 3 days
but after 7 days there was no SP-D in the lungs. Moreover, the SP-D
concentration extracted from the lungs fluids was lower than the
concentration in the wild-type mice experiment described above. One
explanation could be that for knock-out mice, more SP-D was
consumed than in the wild type mice, which have SP-D naturally, and
therefore the concentration was lower. It can be that changes in
the experiment such as larger dose of NPs to be delivered will
release appropriate amount of SP-D for one week.
Example 11: Decrease in Inflammation Following a Lung Injury Caused
by Infection or Hyperoxia
[0082] Lung injury is inflicted in wild-type or SPD knock-out mice
by intratracheal or intranasal administration of bacteria,
bacterial products such as LPS, virus or fungi; or by induction of
hyperoxia for example by exposing the mice to >95% O.sub.2 for
72 hrs. Injured mice are treated with an appropriate dose of PLGA
7A NPs complexed with human SPD by an intratracheal injection. The
control group is injured mice treated with empty PLGA 7A NPs. After
an appropriate time, for example when the control group exhibits
significant signs of injury, lung lavage is evaluated for human SPD
content by spinning 600 .mu.l for 5 min at 10,000 RPM at 4.degree.
C. and assaying by SPD ELISA, and the extent of inflammation is
assessed, for example by following neutrophilic alveolitis,
indicating the presence of an inflammatory response in the
alveoli.
[0083] To demonstrate treatment of emphysema and pulmonary
infections, clearance of bacteria, virus and fungi from the lung is
assessed in treated or un-treated SPD knock-out mice (for example
as taught in WO 00/23569).
REFERENCES
[0084] Danhier et al, J. Controlled Release, 2012, 161(2):505-522
[0085] Ikegami et al. 2006, Am. J. Respir. Crit. Care Med.
173:1342-1347 [0086] Kumari et al, Colloids and Surfaces 8:
Biointerfaces (2010) 75:1-18)
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