U.S. patent application number 15/456004 was filed with the patent office on 2017-06-29 for dry powder fibrin sealant.
This patent application is currently assigned to Quadrant Drug Delivery Limited. The applicant listed for this patent is Mallinckrodt Pharma IP Trading D.A.C.. Invention is credited to Jos Grimbergen, Jaap Koopman, Nicola Whitfield.
Application Number | 20170182135 15/456004 |
Document ID | / |
Family ID | 42240815 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170182135 |
Kind Code |
A1 |
Whitfield; Nicola ; et
al. |
June 29, 2017 |
DRY POWDER FIBRIN SEALANT
Abstract
The invention provides a composition comprising a mixture of
first microparticles that comprise fibrinogen and trehalose, and
second microparticles that comprise thrombin and trehalose. The
invention further provides methods for treating wounds by
administering the novel microparticle composition.
Inventors: |
Whitfield; Nicola;
(Nottingham, GB) ; Koopman; Jaap; (Leiden, NL)
; Grimbergen; Jos; (Leiden, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mallinckrodt Pharma IP Trading D.A.C. |
Hazelwood |
MO |
US |
|
|
Assignee: |
Quadrant Drug Delivery
Limited
Nottingham
GB
|
Family ID: |
42240815 |
Appl. No.: |
15/456004 |
Filed: |
March 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12636718 |
Dec 12, 2009 |
|
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15456004 |
|
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61122063 |
Dec 12, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/4833 20130101;
A61P 17/02 20180101; A61K 9/5084 20130101; A61K 41/17 20200101;
A61K 9/1623 20130101; A61K 38/363 20130101; A61K 9/0014 20130101;
C12Y 304/21005 20130101; A61K 9/4858 20130101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 9/00 20060101 A61K009/00; A61K 41/00 20060101
A61K041/00; A61K 9/50 20060101 A61K009/50; A61K 38/36 20060101
A61K038/36; A61K 9/48 20060101 A61K009/48 |
Claims
1. A dry powder fibrin sealant composition, comprising: a mixture
of first microparticles that comprise 0.5 to 20% w/w fibrinogen and
trehalose, and second microparticles that comprise 10 to 20,000
IU/g thrombin and trehalose, wherein trehalose is the only
stabilizing sugar in the first microparticles and second
microparticles.
2. The composition of claim 1, wherein the composition is
sterilizable by gamma irradiation.
3. The composition of claim 2, wherein the concentration of
trehalose in the first microparticles is sufficient to preserve at
least 70% of fibrinogen activity after exposure of the first
microparticles to a 25 kGy dose of gamma irradiation delivered at a
rate of 8 kGy/hour.
4. The composition of claim 3, wherein fibrinogen activity is
measured by ELISA.
5. The composition of claim 2, wherein the first microparticles
have a bioactivity of at least 10 mg fibrinogen per 100 mg
microparticles after exposure to gamma irradiation.
6. The composition of claim 2, wherein the second microparticles
have a bioactivity of at least 70 units thrombin per 100 mg
microparticles after exposure to gamma irradiation.
7. The composition of claim 5, wherein the bioactivity is measured
by ELISA.
8. The composition of claim 6, wherein the bioactivity is measured
by chromogenic assays.
9. The composition of claim 1, wherein the mixture of first
microparticles and second microparticles has a clot strength of at
least 50 g after exposure to gamma irradiation.
10. The composition of claim 1, wherein the concentration of
trehalose in the first microparticles and second microparticles is
sufficient to preserve at least 70% of expected clot strength after
exposure of the mixture of microparticles to a 25 kGy dose of gamma
irradiation delivered at a rate of 8 kGy/hour.
11. The composition of claim 1, wherein the microparticles are
spray-dried microparticles.
12. The composition of claim 11, wherein the composition has a
residual moisture content no greater than 5% (w/w).
13. The composition of claim 12, wherein the residual content is no
great than 3% (w/w).
14. The composition of claim 1, wherein said first and second
microparticles are mixed in a weight ratio of 1:1.
15. A method for preparing a dry powder fibrin sealant composition
comprising a mixture of first microparticles that comprise 0.5 to
20% w/w fibrinogen and trehalose and second microparticles that
comprise 10 to 20,000 IU/g thrombin and trehalose, wherein
trehalose is the only stabilizing sugar in the first microparticles
and second microparticles, the method comprising: (i) spray-drying
a first aqueous solution comprising fibrinogen and trehalose to
produce said first microparticles, wherein said first aqueous
solution does not include a second stabilizing sugar; (ii)
spray-drying a second aqueous solution comprising thrombin and
trehalose to produce said second microparticles, wherein said
second aqueous solution does not include a second stabilizing
sugar; and (iii) mixing the first and second microparticles.
16. The method of claim 15, wherein the first microparticles have
at least 70% of fibrinogen activity after exposure of the first
microparticles to a 25 kGy dose of gamma irradiation delivered at a
rate of 8 kGy/hour.
17. The method of claim 15, wherein the mixture of the first and
second microparticles preserves at least 70% of expected clot
strength after exposure of the mixture of microparticles to a 25
kGy dose of gamma irradiation delivered at a rate of 8
kGy/hour.
18. The method of claim 15, wherein each of the first and second
aqueous solutions is spray-dried with an outlet temperature of at
least about 80.degree. C.
19. The method of claim 15, wherein the dry powder fibrin sealant
composition has a residual moisture content no greater than 5%
(w/w).
20. The method of claim 15, wherein step (iii) comprises mixing the
first and second microparticles in a weight ratio of 1:1.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application claims the benefit of U.S. application Ser.
No. 12/636,718 filed Dec. 12, 2009 which claims priority to U.S.
provisional application Ser. No. 61/122,063, filed Dec. 12, 2008,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a dry powder fibrin sealant.
BACKGROUND OF THE INVENTION
[0003] WO97/44015 describes a dry powder fibrin sealant based on
micro-particles of fibrinogen and thrombin. This has been
demonstrated to be an easy-to-use, stable and efficacious topical
haemostat. The product can be used immediately, without
reconstitution. On contact with aqueous fluid such as blood, the
exposed active thrombin immediately converts the exposed fibrinogen
into insoluble fibrin polymers.
SUMMARY OF THE INVENTION
[0004] The novel fibrin sealant is a blend of spray-dried
fibrinogen and thrombin, each of which has been individually
co-spray dried with an excipient. A number of excipients have been
used in the fibrin sealant formulation to stabilise the active
ingredients fibrinogen and thrombin and the physical stability
evaluated. In addition, the fibrin sealant formulation has been
exposed to electron beam/gamma irradiation or heat sterilisation in
order to terminally sterilize the product. The results of the
evaluation indicate that trehalose is the most effective excipient
in terms of protein protection during stability storage and
electron beam exposure. The superior stabilization afforded by the
trehalose-based formulations may be attributed to the higher glass
transition temperature of trehalose compared to other excipients
such as sucrose.
[0005] The influence of different parameters on the efficacy of the
product was determined in pig liver biopsy models and pig liver
resection models. The efficacy of the fibrin sealant powder to stop
severely bleeding injuries, with blood loss of >10 ml/min, was
enhanced by the opportunity to apply pressure directly after
administration of the product. Fibrin sealant powders with a
fibrinogen content of at least 4% w/w and a thrombin content of at
least 139 IU/g were shown to be effective in stopping severe
bleeding. The optimum fibrinogen and thrombin content was 7.5% w/w
and .about.400 IU/g, respectively. Fibrinogen and thrombin from 3
different suppliers all performed equally well, demonstrating the
robustness of the product. Terminal sterilization of the product
using electron beam or gamma irradiation of up to 15 or 25 kGy had
no effect on the efficacy of the product and is considered to
reduce the risk of bacterial contamination before use. In summary,
the invention provides a fibrin sealant product that demonstrates
high efficacy at low fibrinogen levels in severely bleeding wounds
and can be terminally sterilized using standard irradiation
methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a plot of the particle size distribution of
spray-dried thrombin:trehalose according to the invention.
[0007] FIG. 2 is a plot of the particle size distribution of
spray-dried fibrinogen:trehalose according to the invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0008] Respective fibrinogen-containing and thrombin-containing
soluble microparticles can be formulated together, in stable, dry
form. This formulation can be subsequently activated, as desired,
to give a fibrin sealant that is useful in wound therapy and
surgical repair. It can meet the primary objectives of achieving
good flow properties, enhanced, effective delivery to the active
site, and dissolution only at the site, not in the delivery
system.
[0009] The content of fibrinogen in the microparticles containing
it may be about 0.1 to 50% w/w, preferably about 0.5 to 20 w/w. The
content of thrombin in the microparticles containing it may be
about 10 to 20,000 IU/g, preferably about 25 to 1,000 IU/g.
[0010] Microparticles comprising fibrinogen or thrombin may be
prepared by the procedures described in WO92/18164, WO96/09814 and
WO96/18388. These spray-drying and associated particle manipulation
processes enable the production of soluble protein microcapsules
with defined size distribution, e.g. of up to 50 .mu.m in diameter.
For example, as described in those documents, the microparticles
may be produced reproducibly, e.g. with 90% or more (by volume) up
to 30 .mu.m, e.g. 10 to 20 .mu.m, in size.
[0011] Microparticles of the invention are preferably prepared by
spray-drying. Typically, a 2-fluid nozzle is used which utilises
compressed air during the atomisation process; this results in the
production of hollow microparticles. The maximum particle size
(X50) of microparticles that can be manufactured using this
atomisation system on the Niro Mobile Minor spray dryer is
.about.30 .mu.m. Preferred X50 values for the microparticles of the
invention are between 5 and 50 microns, most preferably between 10
and 20 microns.
[0012] Microparticles of the invention may be prepared by
spray-drying a solution of the active component with trehalose
alone. An alternative procedure comprises co-spray-drying, in which
fibrinogen or thrombin and another wall-forming material are
formulated and spray-dried, to give microparticles in which the
active component is incorporated in the wall of the particle. The
product is preferably amorphous or in the form of a glass, as
measured by a suitable technique such as FTIR or DSC., with a glass
transition temperature of at least 50 Celsius, most preferably at
least 80 Celsius.
[0013] The fibrinogen or thrombin may be full-length or any active
fragment thereof. Fragments are known; see Coller et al, J. Clin.
Invest. 89:546-555 (1992). Fibrinogen raw material may be a frozen
solution, although, lyophilised powder which requires
reconstitution prior to spray-drying may be used.
[0014] Suitable other proteins may be naturally occurring or
recombinant. They may act as "wall-forming materials", as described
in W09.sup.2/.sub.18164, where various examples are given. A
preferred material is HSA (human serum albumin). For example,
fibrinogen is spray-dried alone or in the presence of varying
amounts of excipients such as HSA (e.g. fibrinogen: HSA ratios of
1:1, 1:3, 3:1) and trehalose. Other suitable substitutes for HSA
include surfactants, such as Tween 20, Tween 80, Poloxamer 407 or
Poloxamer 188.
[0015] Calcium ion, e.g. as calcium chloride, may be incorporated
in the thrombin feedstock. Alternatively, calcium chloride may be
added to the microcapsules after processing.
[0016] Microparticles of the invention may be sterilised, if
necessary or desired. Sterile processing, electron beam
irradiation, Y-irradiation and ethylene oxide are examples of
suitable techniques.
[0017] Although the components of the microcapsules in a fibrin
sealant of the invention are preferably water-soluble, and the
microparticles are preferably obtained by spray-drying a suitable
solution, the microparticles that are obtainable may be
free-flowing, discrete and substantially anhydrous, with a residual
moisture content preferably no greater than 5% w/w, most preferably
no greater than 3% w/w. This means that the compounds of fibrin
sealant in accordance with this invention are not activated until
they are wetted, e.g. by coming into contact with liquid at a wound
site. The active components may therefore be delivered as a dry
mixture, although separate application of the different
microparticles is also envisaged.
[0018] A dry powder fibrin sealant product may be of particular
value where application to a large surface area is required. This
includes surgery and repair of traumatic injuries to various organs
such as the liver and spleen. A further advantageous application is
in skin grafting for burns patients, and specifically where skin
epidermal sheets are cultured in vitro and then transferred to the
wound site. The use of fibrin sealant in the latter indication may
be particularly effective in patients with extensive burns,
providing a biocompatible anchorage for skin grafts. It may also be
suitable in the treatment of topical ulcers.
[0019] The following Examples illustrate the invention.
Example 1
[0020] Spray-dried fibrinogen microparticles were prepared by
dissolving 73.8 g human fibrinogen in 1650 mL water containing
275.1 g trehalose dihydrate. The resultant solution was spray-dried
on a Niro Mobile Minor spray dryer using the following operating
parameters:
[0021] Inlet temperature: 160.degree. C.
[0022] Atomisation type: 2--Fluid Nozzle
[0023] Liquid insert: 0.5 mm
[0024] Atomisation pressure: 0.5 bar
[0025] Feed rate: 18 g/minute
[0026] The spray-dried powder had a particle size (X50, geometric
diameter) of 18.4 .mu.m and a fibrinogen content of 152 mg/g. The
moisture content (Karl-Fischer) was 2%.
[0027] Spray-dried thrombin microparticles were prepared by
dissolving 751,230 IU human thrombin in 1653 mL water containing
11.5 g calcium chloride dihydrate and 507.3 g trehalose dihydrate.
The resultant solution was spray-dried on a Niro Mobile Minor spray
dryer using the following operating parameters:
[0028] Inlet temperature: 160.degree. C.
[0029] Atomisation type: 2--Fluid Nozzle
[0030] Liquid insert: 0.5 mm
[0031] Atomisation pressure: 0.5 bar
[0032] Feed rate: 18 g/minute
[0033] The spray-dried powder had a particle size (X50, geometric
diameter) of 12.5 .mu.m and a thrombin content of 977 IU/g. The
moisture content (Karl-Fischer) was 3%.
[0034] The two spray-dried powders were blended in a 1:1% w/w ratio
using a drum mixer at 18 rpm for 15 minutes. The resultant blend
had a particle size of 15.5 .mu.m, and a fibrinogen content of 69.1
mg/g.
[0035] The respective particle size distributions are shown in
FIGS. 1 and 2. FIG. 1 shows the cumulative distribution as
follows:
TABLE-US-00001 x.sub.0/.mu.m Q.sub.3/% 1.80 6.71 2.20 8.45 2.60
10.02 3.00 11.48 3.60 13.58 4.40 16.36 5.20 19.25 6.20 23.15 7.40
28.33 8.60 33.97 10.00 40.87 12.00 50.65 15.00 64.07 18.00 75.17
21.00 83.61 25.00 91.27 30.00 96.53 36.00 99.11 42.00 99.85 50.00
100.00 60.00 100.00 72.00 100.00 86.00 100.00 102.00 100.00 122.00
100.00 146.00 100.00 174.00 100.00 206.00 100.00 246.00 100.00
294.00 100.00 350.00 100.00
TABLE-US-00002 Evaluation: WINDOX 5.1.2.0, HRLD Product: Fibrocaps
Revalidation: Density: 1.00 g/cm.sup.3, shape factor: 1.00
Reference measurement: Disp. Meth: Set up for Fibrocaps R/M 08-20
11:42:05 C.sub.opt = 1.56% Contamination: 0.00%
TABLE-US-00003 Trigger condition: Fibrocaps User parameters: Time
base: 200.00 ms Batch Number: PV Thrombin Start: c.opt >=0.2%
Formulation: EM/08/126 Valid: always Name: aks Stop: 2.000 sc.opt
<=0.2% or Run Number: Run 1 10000 s real time
[0036] FIG. 2 shows the cumulative distribution as follows:
TABLE-US-00004 x.sub.0/.mu.m Q.sub.3/% 1.80 2.68 2.20 3.58 2.60
4.53 3.00 5.52 3.60 7.06 4.40 9.19 5.20 11.39 6.20 14.20 7.40 17.63
8.60 21.10 10.00 25.15 12.00 30.88 15.00 39.17 18.00 46.85 21.00
53.70 25.00 61.53 30.00 69.48 36.00 76.91 42.00 82.47 50.00 87.80
60.00 92.11 72.00 95.07 86.00 96.87 102.00 97.94 122.00 98.72
146.00 99.34 174.00 99.81 206.00 100.00 246.00 100.00 294.00 100.00
350.00 100.00
TABLE-US-00005 Evaluation: WINDOX 5.1.2.0, HRLD Product: Fibrocaps
Revalidation: Density: 1.00 g/cm.sup.3, shape factor: 1.00
Reference measurement: Disp. Meth: Set up for Fibrocaps R/M 08-29
13:35:41 C.sub.opt = 7.30% Contamination: 0.00%
TABLE-US-00006 Trigger condition: Fibrocaps User parameters: Time
base: 200.00 ms P1: SD Fibrinogen: trehalose clinical Start: c.opt
>=0.2% P2: EM/08/129 Valid: always P3: TR Stop: 2.000 sc.opt
<=0.2% or P4: run 1 10000 s real time
Example 2
[0037] Four batches of microparticles were produced, using the
following formulations and a Mini spray dryer. [0038] 200 mg/ml
trehalose--200 units/ml thrombin [0039] 200 mg/ml sucrose--200
units/ml thrombin--1% HSA w-v [0040] 200 mg/ml trehalose--40
mg/fibrinogen [0041] 200 mg/ml sucrose--40 mg/ml fibrinogen.
[0042] The spray-drying parameters were selected so as to produce
particles in the region of 10 .mu.m.
[0043] Thrombin Formulation: [0044] Inlet temperature: 130.degree.
C. [0045] Outlet temperature: -80.degree. C. [0046] Atomisation
Airflow: 5/-min [0047] Drying Airflow: 5/-sec [0048] Feed Rate: 5.0
g-min
[0049] Fibrinogen Formulation: [0050] Inlet temperature:
130.degree. C. [0051] Outlet temperature: -83.degree. C. [0052]
Atomisation Airflow: 15/-min [0053] Drying Airflow: 51-sec [0054]
Feed Rate: 3.0 g-min
[0055] Each of the microcapsules batches was aliquoted into clear
10 ml glass vials both as separate components and as
excipient-matched blends. A stability study at 4.degree. C. was
conducted over four weeks. Four timepoints were selected; initial,
1 week, 2 weeks and 4 weeks, and the following assays were employed
to compare the effect of the different excipients on the stability
and bioactivity retention.
[0056] Fibrinogen Analysis used a polyconal antibody to human
fibrinogen as a capture antibody and a second peroxidise-labelled
antibody to human fibrinogen is used for detection in a chromogenic
assay.
[0057] Thrombin Analysis was based on a commercial substrate which
is sensitive to thrombin and gives a colour change which can be
measured. The initial rate of change in absorbance is proportional
to thrombin concentration.
[0058] Particle Size was measured using a L5230 Laser Sizer in
conjunction with Medium Chain Trigylceride oil to determine the
particle size of the spray dried material.
[0059] Thermogravimetric Analysis was carried out to assess
moisture content.
[0060] Flow time was the time taken for a microcapsule blend to
pass through a funnel of a pre-determined size was used as a
comparative measure of flowability between batches.
[0061] Angle of Repose indicates the flowability of a powder and
was measured by the calculation of the angle created upon the flow
of a powder through a funnel and subsequent accumulation on a flat
surface.
[0062] Packed and tap density were measured using the Jolting
Volume Meter and the values used in Carrs Compressibility Index (%
CCI).
[0063] Clot Strength utilises the formulation of a clot from a
blend in a plastic syringe. A bead is suspended in the syringe
prior to clot formation and the weight required to pull the bead
through the clot is recorded.
[0064] Adhesive Strength: blends are applied to a piece of rat skin
via a 10 ml glass pipette fitted with a compressed air supply. The
weight required to separate two pieces of the tissue bonded
together by a blend is used as a measurement of adhesive strength.
(This assay is based on a Gottlob skin test method--Gesting and
Lerner: Autologous fibrinogen for tissue adhesion haemostatis
(1983)). Additional assays were performed at the four week
timepoint; SDS PAGE to assess the effects of spray-drying on the
structural integrity of the bioactives and a BCA assay for total
protein determination. Scanning electron micrographs (SEM) were
also obtained for each of the individual formulations.
[0065] Results demonstrated no significant changes over the
stability period for either formulation, but the data do suggest a
greater retention of activity generated for the trehalose
formulation when compared with the sucrose formulation. The clot
strength values also indicate an increased activity retention with
the trehalose formulation. The addition of NSA to the
trehalose-thrombin formulation showed no significant differences in
bioactivity retention compared to the trehalose-thrombin
formulation. The flow properties were retained over the stability
period which is reflected in the consistent adhesive strength
values.
[0066] SDS PAGE data demonstrated the retention of structural
integrity post-30 spray-drying.
[0067] Scanning electron micrographs revealed similar morphology
for all formulations.
[0068] Dry heat viral inactivation step was conducted for 72 hours
at 80.degree. C.
[0069] The individual fibrinogen and thrombin components were
assessed using ELISA and chromogenic assays respectively and the
blends were analysed using the clot strength assay. The results are
documented in Table 1.
TABLE-US-00007 TABLE 1 Dry Heat Sterilisation Bioactivity -
Concentration per 100 mg Clot Strength Sample spray-dried Product
(g) Trehalose - Thrombin 101.6 units (97%) * microcapsules
Trehalose - Fibrinogen 14.17 mg (103%) * microcapsules Trehalose -
Active blend * 64.8 g Sucrose - Thrombin 93.8 units (89%) *
microcapsules Sucrose - Fibrinogen 11.04 mg (80.5) * microcapsules
Sucrose - Active blend * 62.7 g Theoretical thrombin concentration
= 105 units - 100 mg spray-dried product Theoretical fibrinogen
concentration = 13.7 mg - 100 mg spray-dried product % Retention is
shown in brackets Expected clot strength = ~70 g
[0070] The bioanalytical results indicate the excipient trehalose
allows a greater retention of the active during the dry heat
step.
[0071] Gamma-irradiation employed 25 KG at a rate of 8 KG-hour.
Samples were exposed to these irradiation conditions both as
separate components and as a 15 blend.
[0072] The components were assessed using ELISA and chromogenic
assays respectively and the blends examined via the clot strength
assay. The results are documented in Table 2.
TABLE-US-00008 TABLE 2 Gamma Irradiation Sterilisation Bioactivity
- Concentration per 100 mg Clot Strength Sample spray-dried Product
(g) Trehalose - Thrombin 71 units (71%) * microcapsules Trehalose
Fibrinogen 10.3 mg (73%) * microcapsules Trehalose - Active blend *
59.5 g Sucrose - Thrombin 71 units (71%) microcapsules Sucrose -
Fibrinogen 7.7 mg (55.5 * microcapsules Sucrose - Active blend *
36.9 g % Retention is shown in brackets.
[0073] The conditions investigated in the terminal sterilisation
study (72 hours at 80.degree. C.) suggest that trehalose offers a
higher level of protection to the protein, as reflected by the
activity retention. This observation may also suggest a trehalose
formulation may be capable of room temperature storage. Gamma
irradiation of the sucrose formulation resulted in a 50% drop in
fibrinogen activity. The trehalose formulation was found to have a
significantly higher fibrinogen activity retention (70%) as
measured by the ELISA and clot strength assays.
* * * * *