U.S. patent application number 16/551505 was filed with the patent office on 2020-02-20 for cellular-based method for determining the biological activity of defibrotide.
The applicant listed for this patent is GENTIUM S.R.L.. Invention is credited to Terenzio IGNONI, Vijay KUMAR, Claudio VERGA.
Application Number | 20200057051 16/551505 |
Document ID | / |
Family ID | 52000693 |
Filed Date | 2020-02-20 |
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United States Patent
Application |
20200057051 |
Kind Code |
A1 |
IGNONI; Terenzio ; et
al. |
February 20, 2020 |
CELLULAR-BASED METHOD FOR DETERMINING THE BIOLOGICAL ACTIVITY OF
DEFIBROTIDE
Abstract
The present invention relates to cell-based methods for
determining the biological activity of defibrotide. In particular,
the invention provides a method for assessing the potency of
defibrotide by assessing the viability of mammalian cells in the
presence of at least one cytotoxic agent and one or more
concentrations of defibrotide. Such methods are particularly useful
for standardizing pharmaceutical compositions comprising
defibrotide.
Inventors: |
IGNONI; Terenzio; (San Fermo
Della Battaglia (CO), IT) ; KUMAR; Vijay; (Casnate
(CO), IT) ; VERGA; Claudio; (Bregnano (CO),
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENTIUM S.R.L. |
Villa Guardia (CO) |
|
IT |
|
|
Family ID: |
52000693 |
Appl. No.: |
16/551505 |
Filed: |
August 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15529814 |
May 25, 2017 |
10393731 |
|
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PCT/EP2015/077355 |
Nov 23, 2015 |
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16551505 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5014 20130101;
G01N 33/5064 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2014 |
EP |
14195277.0 |
Claims
1-15. (canceled)
16. A pharmaceutical composition comprising defibrotide at a
standardized potency ratio between 0.9 and 1.2 compared with a
reference batch of defibrotide, wherein at least 10 .mu.g/mL of the
defibrotide protects epithelial cells from damage in vitro, and
wherein the potency of the defibrotide is assessed by a method
comprising: growing mammalian cells in culture; incubating the
cells with a solution containing at least one cytotoxic agent and
at least one concentration of defibrotide from a sample batch;
measuring the viability of the cells after an incubation period;
comparing the cell viability to the cell viability for a reference
batch of defibrotide; and calculating the potency of the sample
batch of defibrotide based on the comparison.
17. The pharmaceutical composition of claim 16, wherein the cells
are incubated with the cytotoxic agent and at least four different
concentrations of defibrotide from the sample batch, and wherein
the cell viability is determined for each of the concentrations to
create a dose-response curve.
18. The pharmaceutical composition of claim 16, wherein the cell
viability measured for the sample batch of defibrotide is compared
to a calibration curve obtained from cell viability measurements
with the reference batch of defibrotide.
19. The pharmaceutical composition of claim 16, wherein the
mammalian cells are human endothelial cells, human epithelial
cells, human liver sinusoidal endothelial cells, or human
microvascular endothelial cells.
20. The pharmaceutical composition of claim 16, wherein the
mammalian cells are present at a density of about 5.times.10.sup.4
cells/ml to about 5.times.10.sup.5 cells/ml.
21. The pharmaceutical composition of claim 16, wherein the
cytotoxic agent is fludarabine,
9-beta-D-arabinofuranosyl-2-fluoroadenine (F-Ara-A), or
doxorubicin.
22. The pharmaceutical composition of claim 21, wherein fludarabine
or F-Ara-A is present in the solution at a concentration of about
10 .mu.g/ml to about 50 .mu.g/ml or wherein doxorubicin is present
in the solution at a concentration of about 0.1 .mu.g/ml to about
10 .mu.g/ml.
23. The pharmaceutical composition of claim 16, wherein the at
least one concentration of defibrotide from the sample batch is in
the range of about 1 .mu.g/ml to about 100 .mu.g/ml.
24. The pharmaceutical composition of claim 16, wherein the
incubation period is at least 24 hours, at least 48 hours, or at
least 72 hours.
25. The pharmaceutical composition of claim 16, wherein measuring
cell viability comprises performing a colorimetric assay based on
the reduction of tetrazolium dyes.
26. The pharmaceutical composition of claim 25, wherein the
tetrazolium dye is
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-te-
trazolium.
27. The pharmaceutical composition of claim 16, wherein measuring
cell viability comprises measuring the absorbance of the solution
following incubation with the cells.
28. The pharmaceutical composition of claim 16, wherein the sample
batch of defibrotide is extracted from bovine tissue or porcine
tissue.
29. A pharmaceutical composition comprising defibrotide at a
standardized potency ratio between 0.9 and 1.2 compared with a
reference batch of defibrotide, wherein at least 10 .mu.g/mL of the
defibrotide protects epithelial cells from damage in vitro, and
wherein the potency of the defibrotide is assessed by a method
comprising: growing mammalian cells in culture in a multi-well
microtiter plate; incubating the cells with a solution containing
at least one cytotoxic agent and at least four different
concentrations of defibrotide from a sample batch; collecting dose
response data points simultaneously; measuring the viability of the
cells after an incubation period; comparing the cell viability to
the cell viability for a reference batch of defibrotide; and
calculating the potency of the sample batch of defibrotide based on
the comparison.
30. The pharmaceutical composition of claim 29, wherein the cells
are incubated with a solution containing defibrotide at a
concentration between 1.25 to 80 .mu.g/mL.
31. The pharmaceutical composition of claim 29, wherein the cell
viability is determined for each of the concentrations to create a
dose-response curve.
32. The pharmaceutical composition of claim 29, wherein the
cytotoxic agent fludarabine or F-Ara-A is present in the solution
at a concentration of about 10 .mu.g/ml.
33. The pharmaceutical composition of claim 29, wherein the
multi-well microtiter plate is a 96-well plate.
34. The pharmaceutical composition of claim 29, wherein at least 3
replicates of incubating the cells with a solution containing at
least one cytotoxic agent and at least four different
concentrations of defibrotide from the sample batch is performed.
Description
BACKGROUND OF THE INVENTION
[0001] Medicinal substances should be produced at a constant
specific activity level so that they can be delivered safely. For
example, assays for biological molecules such as heparin have
variability from batch to batch in terms of chain length, molecular
weight, composition, degree of sulphation, etc. Other substances
that are extracted from natural substances also need to be
standardized. See for example, U.S. Pat. No. 7,575,886. One such
substance is defibrotide. Defibrotide is a heterogeneous mixture of
single-stranded polynucleotides of varying lengths that is
extracted from mammalian organs.
[0002] There are assays available to evaluate the biological
activity of defibrotide, including the fibrin plate test and the
thromboelastographic recording of the euglobulin lysis time (Prino
G. et al., Indagini preliminari sull'attivitfibrinolitica,
nell'animale e nell'uomo, di una nuova sostanza presente in diversi
organi animali, Simposio Internazionale: La ricerca scientifica
nell'industria farmaceutica in Italia, Rome, 2-4 Oct. 1975-11
Farmaco, Ed. Prat.) (1969),24,552-561), the plasmin method (U.S.
Pat. No. 7,338,777), and the euglobulin method (WO2013/190582).
Although these methods are useful pharmaceutical manufacturing
tools, all these methods, which are based on the pro-fibrinolytic
properties of defibrotide, involve an assessment of defibrotide's
activity on isolated proteins or enzymes.
[0003] Thus, there is a need in the art for novel methods to
determine the biological activity of defibrotide in a cellular
context that provides an accurate and reliable process for
assessing the potency, e.g., by comparison with a reference
defibrotide standard preparation, of new batches of defibrotide
regardless of the manufacturing process used.
SUMMARY OF THE INVENTION
[0004] The present invention is based, in part, on the discovery
that defibrotide protects mammalian cells from cytotoxicity induced
by particular cytotoxic agents in a dose-dependent manner. The
inventors have taken advantage of this cell protection effect and
developed a cell-based method for assessing the potency of
defibrotide batches and defined a measurement unit to facilitate
effective and safe administration. Such methods allow for, inter
alia, quality control during the defibrotide manufacturing process,
standardization of defibrotide batches produced by different
methods or sources, and consistent dosing of patients with
defibrotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1. Viability of HMEC cells incubated with fludarabine
in the presence or absence of varying concentrations of defibrotide
as measured by MTT assay. HMEC-1 were incubated with fludarabine
(F-Ara) at 10 .mu.g/ml in the presence or absence of varying
concentrations of defibrotide (DF) (1 .mu.g/m1-100 mg/ml) for 72 hr
and the viability of the cells was measured with the MTT assay.
Student t-test: .sctn.p<0.01, F-ara 10 .mu.g/ml vs. control
(ctr); *p<0.05, cells treated with DF at 1 .mu.g/ml vs. F-ara 10
.mu.g/ml; **p<0.001, DF at 10 and 100 .mu.g/ml vs. F-ara 10
.mu./ml.
[0006] FIG. 2. Viability of SK-HEP-1 cells incubated with
doxorubicin in the presence or absence of varying concentrations of
defibrotide as measured by CCK-8 assay. SK-HEP-1 cells were
incubated with doxorubicin (Dox) at 0.1 .mu.g/ml in the presence or
absence of varying concentrations of defibrotide (DF) (1
.mu.g/ml-100 .mu.g/ml) for 72 hr and the viability of the cells was
measured with the CCK-8 assay. Student t-test: .sctn.p<0.01, Dox
0.1 .mu.g/ml vs. control (ctr); *p<0.01, cells treated with DF
at 50 or 100 .mu.g/ml vs. Dox 0.1 .mu.g/ml.
[0007] FIG. 3. Viability of HMEC-1 cells incubated with fludarabine
in the presence or absence of varying concentrations of
defibrotide, AC or ACTG as measured by MTT assay. HMEC-1 cells were
incubated with fludarabine (F-Ara) at 50 .mu.g/ml in the presence
or absence of varying concentrations of random synthetic
Adenine-Cytosine (AC) oligonucleotides of about 16 kDa (1-500
.mu.g/ml) (FIG. 3A), random synthetic
Adenine-Cytosine-Guanine-Thymine (ACGT) oligonucleotides of about
17 kDa (12.5-50 .mu.g/ml) (FIG. 3B), or defibrotide (5-100
.mu.g/ml) (FIG. 3C) for 72 hr and the viability of the cells was
measured with the MTT assay. Student t-test: *p<0.01, F-ara 50
.mu.g/ml vs. control (Ctr), **p<0.01 F-ara 50 .mu.g/ml vs.
defibrotide. There was no significant protection by either of the
synthetic oligonucleotides.
[0008] FIG. 4. Viability of SK-HEP-1 cells incubated with
fludarabine in the presence or absence of varying concentrations of
ACTG, tpA, or glutathione as measured by CCK-8 assay. SK-HEP-1
cells were incubated with fludarabine (F-Ara) at 10 .mu.g/ml in the
absence or presence, (A-G), of varying concentrations of random
synthetic Adenine-Cytosine-Guanine-Thymine (ACGT) oligonucleotides
of about 17 kDa (1.25-80 .mu.g/ml), tPA (10-320 IU/ml), or
glutathione (1.25-80 .mu.g/ml) for 72 hr and the viability of the
cells was measured with the CCK-8 assay. Student t-test:
*p<0.01, F-Ara 10 .mu.g/ml vs. control (Ctr). There was no
significant protection from F-Ara-induced cytoxicity by ACGT, tPA,
or glutathione.
[0009] FIG. 5. Comparison of the dose response curves of standard
defibrotide versus acid-stressed (FIG. 5A) and basic-stressed (FIG.
5B) defibrotide samples in the cell protection assay. The raw
absorbance data was processed using the PLA2 statistical analysis
program (4-parameter logistic function analysis). Absorbance of the
cell viability indicator dye (CCK-8) is plotted on the Y-axis as
"response." Dose is plotted on the X-axis and is a 2-fold dilution
series of defibrotide in the assay (1.25-80 .mu.g/ml); STD
represents the reference standard defibrotide. The lower traces in
each panel, which correspond to the stressed samples, indicate a
reduced potency. Using this statistical analysis program, both
stressed samples failed to meet the statistical criteria of
acceptance.
[0010] FIG. 6. Viability of SK-HEP-1 cells incubated with
fludarabine in the presence or absence of varying concentrations of
defibrotide as measured by CCK-8 assay. SK-HEP-1 cells were
incubated with fludarabine (F-Ara) at 10 .mu.g/ml in the absence or
presence of varying concentrations of defibrotide (DF) (1
.mu.g/ml-100 .mu.g/ml) for 72 hr and the viability of the cells was
measured with the CCK-8 assay. Student t-test: p<0.01, F-Ara 10
.mu.g/ml vs. control (Ctr) and for cells treated with DF at >1
.mu.g/ml vs. F-Ara 10 .mu.g/ml.
[0011] FIG. 7. Assessment of the potency ratio between a
standardized Reference defibrotide sample (standard) and a sample
of defibrotide of unknown biological activity. SK-HEP-1 cells were
exposed to 6 serial dilutions (1:2) of standard and sample
defibrotide to give concentration of 80, 40, 20, 10, 5, 2.5 and
1.25 .mu.g/ml in the presence of fludarabine (F-Ara) (10 .mu.g/ml).
Each concentration of the standard and the sample consisted of 4
replicates. After 72 hr incubation at 37.degree. C., the viability
of the cells was measured with the CCK-8 assay. The absorbance
measurements were submitted to statistical analysis for sample
potency determination (4-parameter logistic analysis).
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides a reliable method for
determining the biological activity of defibrotide based on the
ability of defibrotide to protect living cells from the effects of
certain cytotoxic agents. This cell protection effect is important
for defibrotide's use as a medicinal product. This method allows
for the standardization of the activity for defibrotide that is
obtained by different methods or sources. The method also allows
for the establishment and assignment of a unit of measurement to
facilitate effective and safe administration of defibrotide.
[0013] In one embodiment of the invention, the method of assessing
the potency of a sample batch of defibrotide comprises (i) growing
mammalian cells in culture, (ii) incubating the cells with a
solution containing at least one cytotoxic agent and at least one
concentration of defibrotide from the sample batch, (iii)
determining the viability of the cells after an incubation period,
and (iv) calculating the potency of the sample batch of defibrotide
based on the cell viability measurement. In some embodiments, the
method further comprises comparing the cell viability for the
sample batch of defibrotide to the cell viability for a reference
batch of defibrotide, and calculating the potency of the sample
batch of defibrotide based on the comparison.
[0014] For the purpose of the present invention the term "potency"
refers to the measure of the biological activity of defibrotide, in
particular based on the measure of the ability of defibrotide to
protect living cells from the effects of cytotoxic agents.
Defibrotide (Merck Index, 1996, no. 2915; CAS number 83712-60-1) is
a substance of natural origin. It is the sodium salt of low
molecular weight polydeoxyribonucleotides which are obtained by
extraction from animal organs. Defibrotide is known to have a
molecular weight (MW) between 14 and 19 kDa, but specific
measurement techniques show defibrotide to have an average
molecular weight (MW) around 16.1 kDa.+-.2.0 kDa if determined by
SEC-HPLC technique; a MW around 17.6 kDa.+-.1.0 kDa if determined
by PAGE technique; and a MW around 16.7 kDa.+-.1.6 kDa if
determined by Multi-Angle Laser Light Scattering technique.
"Analysis of Aggregates and Particles in Protein Pharmaceuticals"
H. Mahler and W. Jiskoot (eds.), 2012 John Wiley & Sons, Inc.
Defibrotide has numerous therapeutic applications, including use as
an anti-thrombotic agent (U.S. Pat. No. 3,829,567), treatment of
peripheral arteriopathies, treatment of acute renal insufficiency
(U.S. Pat. No. 4,694,134), and treatment of acute myocardial
ischaemia (U.S. Pat. No. 4,693,995). More recently, defibrotide has
been used for the treatment and prevention of sinusoidal
obstruction syndrome/venous occlusive disease (EU clinical trial
EudraCT:2004-000592-33, US clinical trial 2005-01
(ClinicalTrials.gov identifier: NCT00358501). Other uses of
defibrotide are described in the following patents and patent
applications, each of which is hereby incorporated by reference in
its entirety: U.S. Pat. Nos. 3,770,720; 3,829,567; 3,899,481;
4,693,134; 4,693,995; 4,938,873; 4,985,552; 5,081,109; 5,116,617;
5,223,609; 5,646,127; 5,646,268; 5,977,083; 6,046,172; 6,699,985;
6,767,554; 7,338,777; 8,551,967; 8,771,663, US Patent Publication
Nos. 20080194506; 20090131362; 20110092576; 20130231470;
20140005256, U.S. patent application Ser. No. 14/323,918; and WO
2013/190582.
[0015] The methods described herein can be used to assess the
potency of defibrotide batches manufactured by different methods or
extracted from different animal organs. For instance, in some
embodiments, the defibrotide sample batch is extracted from bovine
tissue, such as bovine lung, intestine, or mucous membranes. In
other embodiments, the defibrotide sample batch is extracted from
porcine tissue, such as porcine lung, intestine, or mucous
membranes. Defibrotide sample batches may also be extracted from
other organs from other animal species, including sheep and
horses.
[0016] In certain embodiments, the defibrotide sample batches to be
evaluated for potency by the methods described herein are
manufactured by a process such as that described in U.S. Pat. Nos.
4,985,552 and 5,223,609, both of which are hereby incorporated by
reference in their entireties. In particular, the defibrotide
obtained with this process is a polydeoxyribonucleotide
corresponding to the following formula of random sequence: [0017]
P.sub.1-5,(dAp).sub.12-24,(dGp).sub.10-20,(dTp).sub.13-26,(dCp).sub.10-20
[0018] wherein:
[0019] P=phosphoric radical
[0020] dAp=deoxyadenylic monomer
[0021] dGp=deoxyguanylic monomer
[0022] dTp=deoxythymidylic monomer
[0023] dCp=deoxycytidylic monomer.
[0024] The defibrotide sample batches may have one or more or all
of the following chemico-physical properties:
electrophoresis=homogeneous anodic mobility; extinction
coefficient, E.sub.1 cm.sup.1% at 260.+-.1 nm=220.+-.10; extinction
ratio, E.sub.230/E.sub.260=0.45.+-.0.04; coefficient of molar
extinction (referred to phosphorus), .epsilon.(P)=7750.+-.500;
rotary power [.alpha.]D.sup.20.degree.=53.degree..+-.6; reversible
hyperchromicity, indicated as % in native DNA, h=15.+-.5; and a
purine:pyrimidine ratio of 0.95.+-.0.5.
[0025] In certain embodiments, the defibrotide sample batches to be
evaluated for potency by the methods of the invention may have been
subjected to a physiochemical stress or suspected of being exposed
to a physiochemical stress, such as high temperature, extreme pH,
hydrogen peroxide, etc. Thus, the methods of the invention can also
be used to assess the potency of defibrotide batches or
compositions comprising defibrotide that have been stored at
sub-optimum conditions or for extended periods of time. In certain
embodiments, the methods can be used to monitor the stability of
defibrotide batches or compositions comprising defibrotide, for
example, to predict shelf-life.
[0026] The methods of the invention comprise growing mammalian
cells in culture. In certain embodiments, the mammalian cells are
human cells. In some embodiments, the human cells are human
epithelial cells. In other embodiments, the human cells are human
endothelial cells. In one particular embodiment, the human
endothelial cells are human liver sinusoidal endothelial cells,
such as SK-HEP-1 cells. In another particular embodiment, the human
endothelial cells are human microvascular endothelial cells, such
as HMEC-1 cells. In another particular embodiment, the epithelial
cells are keratinocytes (e.g. HaCaT cells) or alveolar epithelial
cells (e.g. A549 cells). Mammalian cells can be obtained from
recognized depositories, such as the American Type Culture
Collection (ATCC) as well as other sources.
[0027] Suitable growth media for growing mammalian cells in culture
are well known in the art and are disclosed for instance in
"Culture of Animal Cells: A Manual of Basic Technique and
Specialized Applications" R. I. Freshney, 2010, Wiley-Blackwell.
The optimal medium for each type of cells can be obtained from
specialised suppliers of the cells (e.g.: ATCC-LGC, MI, Italy; CDC,
Atlanta, Ga., USA). In certain embodiments, the mammalian cells are
grown in Eagle's Minimum Essential Medium (EMEM) supplemented with
10% (v/v) fetal bovine serum (FBS), 100 units/mL Penicillin, 100
.mu.g/mL Streptomycin, and 2.5 .mu.g/mL Amphotericin B. In other
embodiments, the mammalian cells are grown in RPMI 1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL
Penicillin, 100 .mu.g/mL Streptomycin, and 2.5 .mu.g/mL
Amphotericin B. In certain embodiments, the growth media may
contain L-glutamine (e.g. 2 mM), hydrocortisone (e.g. 10 .mu.g/ml),
and epidermal growth factor (e.g. 10 .mu.g/ml).
[0028] The density of the mammalian cells may be from about
5.times.10.sup.4 cells/ml to about 5.times.10.sup.5 cells/ml, from
about 2.5.times.10.sup.4 cells/ml to about 2.5.times.10.sup.5
cells/ml, or from about 5.times.10.sup.4 cells/ml to about
2.times.10.sup.5 cells/ml. In order to obtain optimal assay
response, the cell density may, in certain embodiments, be
optimised taking into account the nature of the cytotoxic agent and
the type of cell used for the assay.
[0029] For example, in embodiments in which human endothelial cells
are used for the assay, particularly suitable cell densities range
from about 2.5.times.10.sup.4 cells/ml to about 2.times.10.sup.5
cells/ml, preferably about 5.times.10.sup.4 cells/ml. These cell
densities are particularly suitable for assays in which doxorubicin
or fludarabine is the cytotoxic agent.
[0030] In another aspect of the invention, the methods comprise
incubating the mammalian cells in culture with a solution
comprising a cytotoxic agent and at least one concentration of
defibrotide from the sample batch under evaluation. As used herein,
the term "cytotoxic agent" refers to a compound that has a toxic
effect on a cell, such as inducing cell necrosis, inhibiting cell
growth or cell division, or inducing cell apoptosis. The
cytotoxicity of compounds can result from various properties,
including, but not limited to, anti-metabolite properties,
alkylating properties, nucleic acid intercalating properties, or
apoptotic properties. An anti-metabolite property is the ability of
the compound, or its metabolites, to interfere with the proper
synthesis of biomolecules, such as DNA and RNA. Examples of
compounds having an anti-metabolite property include nucleobase
analogs (e.g. purine and pyrmidine analogs), nucleoside and
nucleotide analogs, and antifolate compounds. Exemplary nucleobase
and nucleoside analogs that have cytotoxic effects include, but are
not limited to, azathioprine, thiopurines (e.g. thioguanine,
mercaptopurine), fludarabine, pentostatin, 5-fluorouracil,
6-azauracil, clofarabine, nelarabine, cladribine, cytarabine,
floxuridine, capecitabine, gemcitabine, azacitidine, and
decitabine. Examples of antifolates include methotrexate,
aminopterin, pemetrexed, pralatrexate, and raltitrexed.
[0031] An alkylating property is the ability of the compound, or
its metabolites, to transfer alkyl groups to biomolecules or form
covalent bonds with reactive groups within biomolecules (e.g.
amino, carboxyl, sulfhydryl, and phosphate groups), which can
inactivate or interfere with their biological function. Many
alkylating agents can cross-link DNA strands impairing DNA
replication, which can lead to the induction of apoptosis. Examples
of alkylating agents include nitrogen mustards (e.g.
mechlorethamine, cyclophosphamide, melphalan, chlorambucil,
ifosfamide and busulfan), nitrosoureas (e.g.
N-Nitroso-N-methylurea, carmustine, lomustine, and semustine,
fotemustine and streptozotocin), tetrazines (e.g. dacarbazine,
mitozolomide and temozolomide), aziridines (e.g. thiotepa,
mytomycin and diaziquone), and cisplatins (e.g. cisplatin,
carboplatin and oxaliplatin).
[0032] A nucleic acid intercalating property is the ability of the
compound, or its metabolites, to insert into the DNA double helix,
which can cause mutations, or intercalate within regions of helical
structures of RNA. Examples of intercalating agents include
ethidium bromide, mitomycin, actinomycin, plicamycin,
anthracyclines (e.g. doxorubicin, daunorubicin, epirubicin,
idarubicin, valrubicin, and mitoxantrone), thalidomide and
bleomicins.
[0033] An apoptotic property is the ability of the compound, or its
metabolites, to induce programmed cell death. One particular class
of compounds that can induce apoptosis is anti-microtubule agents,
which interfere with mitosis and result in cell cycle arrest,
thereby inducing apoptosis. Anti-microtubule agents include vinca
alkaloids, such as vincristine, vinblastine, vinorelbine,
vindesine, and vinflunine, and taxanes, such as paclitaxel and
docetaxel.
[0034] Topoisomerase inhibitors also are cytotoxic by virtue of
their ability to prevent DNA replication and transcription and/or
by causing DNA strand breaks, thereby inducing apoptosis.
Topoisomerase inhibitors include, but are not limited to,
irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone and
teniposide.
[0035] The cytotoxic agent used in the methods of the invention is
generally a synthetic, semi-synthetic, or natural chemical
compound. The compound may have one or more of the properties
described above. The cytotoxic agent can be any of the compounds
described herein or a metabolite thereof. In some embodiments, the
cytotoxic agent may be selected from fludarabine, cytarabine,
5-fluorouracil, methotrexate, busulfan, melphalan, cisplatin,
ethidium bromide, doxorubicin, anthracyclines, thalidomide, or
combinations thereof. In certain embodiments, the cytotoxic agent
used in the methods of the invention is fludarabine or its active
metabolite, 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-Ara-A). In
other embodiments, the cytotoxic agent used in the methods of the
invention is doxorubicin.
[0036] Alternative cytotoxic agents commonly known to the person
skilled in the art are equally suitable for use in the methods of
the present invention. For example, in some embodiments, the
cytotoxic agents slow or arrest cell cycle progression, and/or
induce apoptosis of cells. Such types of cytotoxic agents include
Staurosporine, Bendamustine, Carmustine, Imatinib and salts thereof
(marketed as Gleevec), Ara-C, Gemtuzumab (such as Gemtuzumab
ozogamicin, marketed as Mylotarg), Azacitidine (marketed as
Vidaza), Decitabine (marketed as Dacogen), Vorinostat (marketed as
Zolinza), and Thapsigargin, H.sub.2O.sub.2, and Phorbol Myristate
Acetate. See also the NIOSH list of Antineoplastic and Other
Hazardous Drugs in Healthcare Settings 2012, HHS, Publication No.
2012-150
(http://www.edc.gov/niosh/docs/2012-150/pdfs/2012-150.pdf).
[0037] The concentration of the cytotoxic agent used in the methods
of the invention will vary depending on the particular cytotoxic
agent and mammalian cell type being used. In embodiments in which
fludarabine or F-Ara-A is the cytotoxic agent, the agent is present
in the growth medium at a final concentration from about 10
.mu.g/ml to about 50 .mu.g/ml. In other embodiments in which
doxorubicin is the cytotoxic agent, the agent is present in the
growth medium at a final concentration from about 0.1 .mu.g/ml to
about 10 .mu.g/ml.
[0038] The cytotoxic agents may be used singly or in combination of
2, 3, 4, 5, 6, or more agents. In certain embodiments, the potency
of a single sample batch of defibrotide may be assessed by
evaluating independently its cell protection effect for two
different cytotoxic agents. By way of example, a first potency
value of the defibrotide sample batch may be obtained by performing
the method with a first cytotoxic agent (e.g. fludarabine) and a
second potency value may be obtained by performing the method with
a second cytotoxic agent (e.g. doxorubicin). An overall potency of
the defibrotide sample batch may be determined by a mathematical
comparison of the first and second potency values, for example by
averaging the two value or calculating a ratio of the two
values.
[0039] In certain embodiments, a particular set of culture
conditions may be used to induce cytotoxicity of the mammalian
cells rather than employing a specific cytotoxic agent or agents.
For instance, the methods may comprise exposing mammalian cells to
an apoptosis-inducing culture medium in the presence of at least
one concentration of defibrotide, determining the viability of the
cells after an incubation period, and calculating the potency of
the defibrotide based on the cell viability measurement. An
apoptosis-inducing culture medium can include medium having an
acidic pH (e.g. pH of about 2 to about 6 or about 4.5 to about 6.5)
or a basic pH (e.g. pH of about 7.5 to about 10 or about 8 to about
9.5). Apoptosis-inducing culture medium also includes medium that
does not contain essential growth factors (e.g. fibroblast growth
factor, epidermal growth factor, platelet-derived growth factor) as
withdrawal of growth factors is recognized as an inducer of
apoptosis. As used herein, "apoptosis-inducing medium" can also
refer to medium at a particular temperature range (e.g. greater
than 37.degree. C.) or oxygen concentration range (less than 5%
oxygen) that induces apoptosis. The apoptosis-inducing culture
medium or conditions can be readily adjusted by a person of
ordinary skill in the art for the particular mammalian cell type
being employed in the methods. U.V. or other types of radiation can
also be used to induce apoptosis. In some embodiments, the
incubation solution comprises at least one concentration of
defibrotide from the sample batch under evaluation in addition to
the cytotoxic agent. The concentration of defibrotide from the
sample batch (e.g. final concentration in cell-containing medium)
can be in the range from about 1 .mu.g/ml to about 1 mg/ml, from
about 1 mg/ml to about 100 .mu.g/ml, from about 1.25 .mu.g/ml to
about 80 .mu.g/ml, or from about 5 .mu.g/ml to about 50
.mu.g/ml.
[0040] In certain embodiments, multiple concentrations of the
defibrotide from the sample batch are tested. For instance, in one
embodiment, at least two different concentrations of defibrotide
from the sample batch are separately tested. In another embodiment,
at least three different concentrations of defibrotide from the
sample batch are separately tested. In a particular embodiment, at
least four different concentrations of defibrotide from the sample
batch are separately tested. The multiple concentrations of
defibrotide from the sample batch are preferably within the ranges
disclosed above. In some embodiments, the multiple concentrations
of the defibrotide from the sample batch are prepared by successive
1:2 dilutions of a stock solution.
[0041] In some embodiments, the method further comprises testing a
reference defibrotide batch simultaneously with the defibrotide
sample batch. The reference defibrotide batch is typically tested
at various known concentrations of defibrotide. Multiple
concentrations of the reference defibrotide batch may, in some
embodiments, be tested. As with the multiple concentrations of the
defibrotide sample batch, the multiple concentrations of the
defibrotide reference batch can be prepared by serial dilution of a
stock solution in accordance with a predetermined dilution factor.
The concentrations of the defibrotide from the reference batch are
preferably in the same concentration range as the concentrations
from the defibrotide from the sample batch. For example, the
concentrations of defibrotide from the reference batch (e.g. final
concentration in cell-containing medium) can be from about 1
.mu.g/ml to about 1 mg/ml, from about 1 .mu.g/ml to about 100
.mu.g/ml, from about 1.25 .mu.g/ml to about 80 .mu.g/ml, or from
about 5 .mu.g/ml to about 50 .mu.g/ml.
[0042] In some embodiments of the method, at least 4 concentrations
of the defibrotide sample batch and the defibrotide reference batch
are prepared with at least 3 replicates for each concentration of
the sample batch and reference batch.
[0043] In certain embodiments, the methods comprise a positive
control of cytotoxicity. For instance, the mammalian cells are
incubated with the cytotoxic agent alone (i.e. without any
defibrotide) under the same conditions.
[0044] In some embodiments, the methods comprise a negative control
of cytotoxicity. For example, in one embodiment, the mammalian
cells are incubated in a solution without any defibrotide or
cytotoxic agent under the same conditions. Such solutions may
contain the cell growth medium and optionally any vehicle or
solvent.
[0045] In one particular embodiment, the incubation of the
mammalian cells with the cytotoxic agent with and without
defibrotide (reference and sample batches, positive and negative
controls) is conducted in a multi-well microtiter plate (e.g.
96-well). The subsequent determination of cell viability may also
be performed in the microtiter plate. In some related embodiments,
the wells of the microtiter plate are coated with a cell attachment
matrix, such as poly-D-lysine.
[0046] The incubation period to obtain an acceptable assay response
can be optimised, in relation to the cytotoxic agent and type of
cell used in the method. One of skill in the art can adjust these
parameters based on the common general knowledge.
[0047] In certain embodiments of the methods, the cells may be
incubated with the cytotoxic agent and defibrotide from the sample
and/or reference batches for a period ranging from about 12 to
about 120 hours, from about 24 to about 96 hours, from about 48 to
about 72 hours, or from about 48 to about 96 hours. In one
embodiment, the incubation period is at least about 24 hours. In
another embodiment, the incubation period is at least about 48
hours. In still another embodiment, the incubation period is at
least about 72 hours.
[0048] Suitable incubation conditions for specific mammalian cell
types can be found in general laboratory manuals, such as "Culture
of Animal Cells: A Manual of Basic Technique and Specialized
Applications" R. I. Freshney, 2010, Wiley-Blackwell. The set points
for temperature and % CO.sub.2 during the incubation period are
other variables that can be adjusted to optimize the assay
response. According to one embodiment of the present invention, the
mammalian cells are incubated at a temperature ranging from between
about 35.degree. C. to about 39.degree. C. In another embodiment,
the mammalian cells are incubated at a temperature ranging from
between about 36.degree. C. and about 38.degree. C. According to a
further aspect of the present invention, the mammalian cells are
incubated at a CO.sub.2 concentration ranging from about 0 to about
10% (v/v) to maintain an optimal pH of the medium for cell growth.
In another embodiment, the CO.sub.2 concentration may be from about
1 to about 5%.
[0049] In another aspect of the methods of the invention, the
viability of the mammalian cells is determined following incubation
with the cytotoxic agent and defibrotide from a sample batch.
Multiple techniques are available to assess cellular viability
(see, e.g., Assay Guidance Manual, NCBI, 2013, G. Sitta Sittampalam
et al. Eds., available on the Internet at
http://www.ncbi.nlm.nih.gov/books/NBK53196/; Stoddart M J., Cell
viability assays: introduction; Methods Mol Biol. 2011; 740:1-6 and
Riss et al., ASSAY and Drug Development Technologies, Vol. 2(1):
51-62, 2004, both of which are hereby incorporated by reference in
their entireties), and any specific techniques described herein are
illustrative only. In some embodiments, cell viability is assessed
by using commercially available kits, such as the Cell Counting Kit
8 (Dojindo Molecular Technology Inc.; Sigma-Aldrich) and those
available from Life Technologies (see
http://www.lifetechnologies.com/us/enthome/references/molecular-probes-th-
e-handbook/assays-for-cell-viability-proliferation-and-function.html)
and Thermo Scientific (see
http://www.piercenet.com/product/alamarblue-cell-viability-assay-reagent)-
.
[0050] Some suitable methods for determining cell viability that
can be used with the methods of the invention include methods of
assessing membrane integrity, assays measuring reduction or
oxidation, methods that measure cellular ATP content, mitochondrial
activity assays, and caspase assays. Methods of assessing membrane
integrity (e.g. cytolysis or membrane leakage assays) include vital
dye exclusion methods, such as those utilizing trypan blue,
propidium iodide, erythrosin B or 7-Aminoactinomycin D, lactose
dehydrogenase assays, and assays for protease biomarkers. Such
methods generally entail measuring the presence of intracellular
enzymes in the extracellular milieu (e.g. lactose dehydrogenase) or
the presence of membrane impermeable dyes intracellularly as
indications of compromised cell membranes.
[0051] Redox-based assays are typically colorimetric or
fluorimetric methods in which certain classes of compounds
(dyes/stains) change color or fluorescence as a result of
biochemical reactions carried out by living cells. One example of
these types of assays include the MTT assay in which cellular
oxidoreductase enzymes reduce the tetrazolium dye MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its
insoluble formazan, which has a purple color. Other closely related
tetrazolium dyes can be used in similar assays to measure cellular
viability. Thus, in certain embodiments of the methods of the
invention, cell viability is determined by performing a
colorimetric assay based on the reduction of tetrazolium dyes.
Suitable tetrazolium dyes include
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT), 3
-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulf-
ophenyl)-2H-tetrazolium (MTS), and water soluble tetrazolium salts,
such as WST-1 and WST-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-t-
etrazolium). Such techniques are well known in the art and are
described, for instance, in Mosmann, "Rapid colorimetric assay for
cellular growth and survival: application to proliferation and
cytotoxicity assays," J Immunol Methods. 1983 Dec. 16; 65: 55-63,
which is hereby incorporated by reference in its entirety. A
similar redox-based assay for determining cell viability utilizes
the fluorescent dye, resazurin (7-Hydroxy-3H-phenoxazin-3-one
10-oxide). Resazurin is reduced to highly red fluorescent resorufin
in live cells and thus cell viability can be determined by
measuring the increase in fluorescence in the presence of the
dye.
[0052] Cell viability can also be assessed by measuring changes in
intracellular processes, such as changes in intracellular free
radicals (e.g. reactive oxygen species, nitric oxide), free ion
concentration (e.g. Ca.sup.2+, Mg.sup.2+, Zn.sup.2+), and membrane
potential. Fluorescence indicators to monitor and quantitate such
changes are commercially available from various sources, such as
the fluorescent-based reagents available from Life Technologies and
Promega. One such assay involves the use of calcein AM, which is a
cell permeable dye that is a substrate for cellular esterases.
Enzymatic activity in live cells converts calcein AM to a
fluorescent product thereby allowing the determination of the
number of live cells by increases in fluorescence. Quantitation of
adenosine triphosphate (ATP) content has also been used as a marker
of cell viability. Cellular ATP content can be measured by the
amount of light produced through reaction with the luciferase
enzyme using, for example, a luminometer.
[0053] In some embodiments, cell viability may be measured
manually, for example by counting living cells with the aid of
suitable microscopes or by means of suitable equipment evaluating
the absorbance/fluorescence change, selected from a
spectrophotometer, a spectrofluorimeter, a flow cytometer or a
combination thereof. Other techniques for assessing cell viability
are known to those of skill in the art and may be adopted for use
with the methods of the present invention,
[0054] Depending on the assay used to assess cell viability, the
cell viability measurement may be a change in the absorbance or
fluorescence of the cell-containing solution or medium, a
percentage or number of living cells, or a percentage or number of
dead cells. In some embodiments, a change in absorbance or
fluorescence can be converted into a percentage or number of living
cells or dead cells. For instance, in embodiments in which a change
in color or fluorescence occurs as a result of a dye or stain
permeating a compromised cell membrane (e.g. trypan blue,
erythrosin B or propidium iodide), an increase in absorbance or
fluorescence at a particular wavelength indicates an increase in
the number of dead cells. In other embodiments in which a change in
color or fluorescence occurs as a result of a cellular reaction
(e.g. MTT assay, calcein AM assay), the number of live cells
correlates with an increase in absorbance or fluorescence at a
particular wavelength. Thus, in certain embodiments of the methods
of the invention, cell viability is determined by measuring the
absorbance or fluorescence of the solution containing the mammalian
cells following incubation with the cytotoxic agent and
defibrotide. In one embodiment, the cell viability (e.g. absorbance
or fluorescence) for each concentration of defibrotide from the
sample batch (e.g. absorbance/fluorescence of each well of the
microtiter plate containing different concentrations of defibrotide
from the sample batch) is measured and plotted against the
corresponding concentration of defibrotide to create a
dose-response curve. In some embodiments, the dose-response curve
is a sigmoidal curve (i.e. "S-shaped"). The range of defibrotide
concentrations that are tested may be expanded or additional number
of concentrations added to obtain a sigmoidal dose-response
curve.
[0055] The absorbance or fluorescence readings or other cell
viability measurements (e.g. number or percentage of live cells;
e.g. number or percentage of dead cells) for each of the samples
(e.g. the positive and negative controls, defibrotide reference
batch and defibrotide sample batch), known as raw data, can be
processed and subject to further statistical analysis. Dedicated
software can be employed for statistical analysis, such as that
especially designed for bioassay evaluation like, for example, PLA
2 (Stegmann Systems GmbH, Germany) or, alternatively, a commercial
off-the shelf spreadsheet customised for the statistical evaluation
of biological assay data.
[0056] In certain embodiments, the methods of the invention
comprise comparing the cell viability measured for samples
containing the sample batch of defibrotide to the cell viability
measured for a reference batch of defibrotide. In some embodiments,
the cell viability measurements for the reference batch of
defibrotide were obtained prior to the analysis of the sample batch
of defibrotide. Such prior measurements for reference batches of
defibrotide can be stored in a reference database or computer
readable storage medium. In certain embodiments the cell viability
measurements for the reference batch represent an average of cell
viability measurements obtained from a population of defibrotide
reference batches. Thus, potency of the defibrotide sample batch
can be calculated based on the cell viability measurements for the
sample batch by comparison to a standard calibration curve obtained
from prior analysis of a defibrotide reference batch or population
of defibrotide batches. In other embodiments, the cell viability
measurements for the reference batch of defibrotide are acquired at
the same time as the cell viability measurements for the sample
batch of defibrotide. For example, a series of concentrations for
the defibrotide sample batch are run in parallel with a series of
concentrations for the defibrotide reference batch.
[0057] The reference batch of defibrotide is preferably a
standardized defibrotide batch having a known biological activity
(e.g. pro-fibrinolytic activity, cell protection activity). For
instance, in one particular embodiment, the reference batch of
defibrotide has a cell protection activity of between 630-905
Units/mg. The standardized defibrotide batch can have one or more
of the following characteristics: an average molecular weight of
between about 14 to about 19 kDa as measured by SEC-HPLC, an
extinction coefficient (.epsilon.1%) of about 207-233, an
extinction ratio (Emin/Emax) of about 0.41-0.49, a purine to
pyrimidine ratio greater than about 0.80 (e.g. about 0.80 to about
1.50), coefficient of molar extinction (.epsilon.(P), referred to
phosphorus) of about 7200 to about 8400, rotary power
([.alpha.]D.sup.20.degree.) of about 45.degree. to about
60.degree., and reversible hyperchromicity, indicated as % in
native DNA (h) of about 8 to about 22. In some embodiments, the
reference batch of defibrotide is a batch of defibrotide
manufactured under GMP conditions for clinical use. In other
embodiments, the reference batch of defibrotide is a commercial
batch of defibrotide available from Gentium (Villa Guardia,
Italy).
[0058] In some embodiments, cell viability measurements for
multiple concentrations of defibrotide from the reference batch are
acquired to create a calibration curve. In one embodiment, creation
of the calibration curve comprises the acquisition of the
absorbance data relating to the samples at known increasing
concentrations of defibrotide from the reference batch and the
statistical processing of those data to obtain the calibration
curve, which represents the correlation between the increase in
cell viability in the presence of a cytotoxic agent and the dose of
defibrotide. In certain embodiments, the cell viability measured
for the sample batch of defibrotide is compared to a calibration
curve obtained from cell viability measurements with a reference
batch of defibrotide to determine the potency of the sample batch
of defibrotide.
[0059] In some embodiments, a dose-response curve obtained from
cell viability measurements from the defibrotide sample batch is
compared to the calibration curve obtained from cell viability
measurements with a reference batch of defibrotide. In such
embodiments, the dose-response curve and the calibration curve may
be sigmoidal curves (see FIG. 7, for example). The difference
between the two curves is a function of the difference in
biological activity between the defibrotide sample batch and the
defibrotide reference batch. This difference is the potency of the
sample batch of defibrotide compared to the reference batch. In one
embodiment, a four-parameter logistic function model (4-PL,
European Pharmacopoeia, section 5.3.2) is used to determine the
difference between the dose-response curve for the defibrotide
sample batch and the calibration curve for the defibrotide
reference batch to calculate the potency of the sample batch of
defibrotide. In another embodiment, a five-parameter logistic
function model (5-PL, R. A. Dudley et al., "Guidelines for
immunoassay data processing," Clin. Chem., 1985, 31: 1264-1271) is
used to determine the difference between the dose-response curve
for the defibrotide sample batch and the calibration curve for the
defibrotide reference batch to calculate the potency of the sample
batch of defibrotide.
[0060] In some embodiments, the data obtained from the cell
viability measurements of samples with the defibrotide sample batch
can be evaluated for additional statistical parameters to ensure
the data are valid. For example, the data can be required to
satisfy certain statistical criteria, such as those mandated by
regulatory agencies. Such tests may include tests for linearity,
parallelism, and linear regression at the significance level of,
for example 0.05, such that, F.sub.non-linearity<F.sub.critical,
F.sub.non-parallclism<F.sub.critical, and
F.sub.Regression>F.sub.critical, respectively as detailed in for
example, European Pharmacopoeia, section 5.3.2, 2014 and United
States Pharmacopeia Chapter (1034) Analysis of Biological Assays,
2014.
[0061] The potency of the defibrotide sample batch calculated from
statistical methods above can be expressed as a percent of the
defibrotide reference batch, protection activity units per weight
of defibrotide, or other units that may or may not be arbitrary. In
some embodiments, a defibrotide protection unit is the
concentration of defibrotide that mediates half-maximal
cell-protection of SK-HEP-1 cells in the presence of 10 .mu.g/ml
fludarabine under the given assay conditions. In one embodiment,
the potency of the defibrotide sample batch is expressed as a
potency ratio relative to the potency of the reference batch of
defibrotide. In certain embodiments, the potency ratio for the
defibrotide sample batch is calculated using the following
formula:
Potency Ratio=C.sub.ref/C.sub.samp
[0062] Where, C is the concentrations of reference (ref) and sample
(samp) defibrotide materials required to achieve the same
effect.
[0063] The methods of assessing potency of a defibrotide sample
batch as described herein can be used in the preparation of
pharmaceutical compositions comprising defibrotide to adjust the
quantity of defibrotide included in the compositions to ensure the
compositions comprise accurate and consistent dosages. Thus, the
methods of the invention can be used during the manufacturing
process to assess the potency of different batches of defibrotide
prepared at different locations, by different methods, or from
different sources.
[0064] The methods of the invention can also be used to monitor the
stability of defibrotide batches or pharmaceutical compositions
comprising defibrotide over time. For instance, the potency of a
batch or composition can be determined by the methods described
herein periodically over time (e.g., monthly, biannually, annually)
or following exposure to extreme conditions to monitor the activity
of the defibrotide and identify batches or compositions that have
deteriorated or degraded.
[0065] These and other aspects of the invention will be better
illustrated in the following examples, which are not, however, to
be regarded as limiting the invention.
[0066] All patent and non-patent documents referenced throughout
this disclosure are incorporated by reference herein in their
entirety for all purposes.
EXAMPLES
[0067] Materials and Methods
[0068] The following materials were used in the Examples given here
below.
[0069] Apparatus
[0070] A Victor 3 microplate reader equipped with different
emission and absorption filters (Perkin Elmer, Milan, Italy),
operated by Wallac software (Perkin Elmer, Milan, Italy).
[0071] Single and multi-channel pipettes with continuous volume
adjustment equipped with sterile tips for molecular biology
(Gilson, Milan, Italy.
[0072] Cell incubator with temperature and CO.sub.2 control (Thermo
Fischer Scientific, Milan, Italy.
[0073] Laminar flow hoods for tissue culture model HERAcell-150
(Thermo Fischer Scientific, Milan, Italy).
[0074] Analytical balance AX 26 DR (Mettler, Milan, Italy).
[0075] pH meter model 780 (Metrohom Italia, Milan, Italy).
[0076] Cell culture flasks, 25 and 75 cm.sup.2, vent cap (Coming
Incorporated, NY, USA).
[0077] Sterile, clear 96 well poly-D-lysine coated or uncoated
(Sigma Aldrich, Milan, Italy).
[0078] Neubauer counting chamber and optical microscope (Carl
Zeiss, Milan, Italy). Vacuum medium filter sterilization unit
(Sigma Aldrich, Milan, Italy?).
[0079] Computer Programs
[0080] Microsoft Excel 2003. (Microsoft Corporation, Redmond,
Wash., USA)
[0081] PLA 2 (Stegmann Systems GmbH, Germany)
[0082] Cells
[0083] Human Microvascular Endothelial Cell line (HMEC-1, CDC,
Atlanta, Ga., USA)
[0084] Liver sinusoidal endothelial cell line (SK-HEP-1, ATCC,
Manassas, Va., USA)
[0085] Reagents and Chemicals
[0086] Defibrotide (Gentium, Italy)
[0087] 9-beta-D-arabinofuranosyl-2-fluoroadenine, analytical grade
(Sigma Aldrich, Milan, Italy), referred to as fludarabine or F-ara
in the Figures and Examples below
[0088] Doxorubicin, analytical grade (Sigma Aldrich, Milan,
Italy)
[0089] Amphotericin B (Sigma-Aldrich, Milan, Italy)
[0090] Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Milan, Italy)
[0091] Gelatin, 2% in water, tissue culture grade (Sigma-Aldrich,
Milan, Italy)
[0092] Dulbecco's phosphate buffered saline (D-PBS) (Sigma-Aldrich,
Milan, Italy)
[0093] Ethanol Absolute (Sigma-Aldrich, Milan, Italy)
[0094] Fetal Bovine Serum (FBS) (Sigma-Aldrich, Milan, Italy)
[0095] Penicillin-Streptomycin 100.times. (Sigma-Aldrich, Milan,
Italy)
[0096] MTT (Sigma-Aldrich, Milan, Italy)
[0097] CCK-8 (Sigma Aldrich, Milan, Italy)
[0098] Trypan Blue (Sigma-Aldrich, Milan, Italy)
[0099] Eagle's Minimum Essential Medium (EMEM) ATCC Number: 30-2003
(ATCC Manassas, Va., USA)
[0100] Oligonucleotide (ACGT).sub.n of about 17 Kda (Sigma Genosys,
Milan, Italy)
[0101] Oligonucleotide (AC).sub.n of about 17 KDa (Sigma Genosys,
Milan, Italy)
[0102] Glutathione (Sigma-Aldrich, Milan, Italy)
[0103] Human tissue plasminogen activator (tPA) (Sigma-Aldrich,
Milan, Italy)
[0104] Molecular biology grade water (Sigma-Aldrich, Milan,
Italy)
[0105] Preparation of Cell Growth Medium for SK-HEP-1
[0106] Cell growth medium for SK-HEP-1 was Eagle's Minimum
Essential Medium (EMEM) supplemented with 10% (v/v) of foetal
bovine serum (FBS), 1.times. Penicillin-Streptomycin and 1.times.
Amphotericin B. From 500 ml of EMEM medium 65 ml was removed and 50
ml of FBS, 5 ml of a 100.times. concentrate of
Penicillin-Streptomycin stock and 10 ml 50.times. concentrate of
Amphotericin B stock was added. The medium was filter sterilised
using a medium filter sterilisation unit.
[0107] Preparation of Cell Growth Medium for HMEC-1
[0108] Cell growth medium for HMEC-1 was RPMI 1640 medium
supplemented with 10% (v/v) FBS, 1.times. Penicillin-Streptomycin
and lx Amphotericin B. From 500 ml of RPMI 1640 medium 65 ml was
removed and 50 ml of FBS, 5 ml of a 100.times. concentrate of
Penicillin-Streptomycin stock and 10 ml 50.times. concentrate of
Amphotericin B stock, 2 mM L-glutamine, 10 .mu.g/ml hydrocortisone
was added. The medium was filter sterilised using a medium filter
sterilisation unit and sterile epidermal growth factor was added to
a concentration of 10 .mu.g/ml.
[0109] SK-HEP-1 Cultivation and Preparation
[0110] The human liver sinusoidal endothelial cell line SK-HEP-1
was obtained from the American Type Culture Collection (ATCC) and
cultured in complete EMEM medium in a humidified cell incubator
containing 5% CO.sub.2 at 37.degree. C. using gelatin-coated tissue
culture flasks. The cells were sub-cultured by trypsin mediated
detachment every 2-3 days following the instructions provided by
the ATCC. Cells were serially transferred into culture flasks when
the culture was 80-90% confluent and used for the protection assay
between passages +3 to +10. That is, 3 to 10 passages beyond the
characterised passage number of the cells received from the
ATCC.
[0111] A suspension of SK-HEP-1 for use in the cell protection
assay was prepared and counted. Briefly the cells were washed with
D-PBS, and detached using 1 ml of trypsin solution and resuspended
in complete medium to a cell concentration of 10.sup.5,
2.times.10.sup.5 or 4.times.10.sup.5 cells/ml. Cells were counted
using a Neubauer counting chamber in the presence of trypan blue to
assess the percent viability of the cultures. The cell culture used
in the cell protection assay had a viability of >90%.
[0112] HMEC-1 Cultivation and Preparation
[0113] The human microvascular endothelial cell line (HMEC-1) was
obtained from the Centers for Disease Control and Prevention (CDC)
and cultured in complete RPMI 1640 medium in a humidified cell
incubator containing 5% CO.sub.2 at 37.degree. C. The cells were
sub-cultured by trypsin mediated detachment every 2-3 days and
serially transferred into culture flasks when the culture was
80-90% confluence and used for the protection assay between
passages +3 to +10.
[0114] A suspension of HMEC-1 for use in the cell protection assay
was prepared and counted. Briefly the cells were washed with D-PBS,
and detached using 1 ml of trypsin solution and resuspended in
complete medium to a cell concentration of 10.sup.5,
2.times.10.sup.5 or 4.times.10.sup.5 cells/ml. Cells were counted
using a Neubauer counting chamber in the presence of trypan blue to
assess the percent viability of the cultures. The cell culture used
in the cell protection assay had a viability of >90%.
[0115] Preparation of Stock Solutions
[0116] 1. Fludarabine
[0117] A 10 mg vial of Fludarabine was dissolved in 1 ml of DMSO to
give a solution of 10 mg/ml and stored at 4.degree. C. The stock
solution was diluted 1:1 with complete growth medium to give the
working stock solution of 5 mg/ml.
[0118] 2. Defibrotide
[0119] Defibrotide stock solution was prepared on the day of use.
Approximately 100 mg of defibrotide drug substance was accurately
weighed into a 50 ml sterile tube and dissolved in 20 ml of D-PBS
to give a solution of 5 mg/ml. This solution was diluted 1:10 with
complete growth medium to give the working stock solution of 0.5
mg/ml used to produce the concentration dilution series.
[0120] 3. Tissue Plasminogen Activator (tPA)
[0121] The content of two 10 .mu.g vials of t-PA (about 400,000
IU/mg per vial) were dissolved in 2 ml of D-PBS to give a solution
of 4000 IU/ml and stored at -80.degree. C.
[0122] 4. ACGT Oligonucleotide,
[0123] A 1 mg vial of ACGT oligonucleotide was dissolved in 2 ml of
D-PBS to give a solution of 0.5 mg/ml and stored at 4.degree.
C.
[0124] 5. Glutathione
[0125] 100 mg of glutathione were dissolved in 20 ml of PBS? and
diluted 1:10 with complete growth medium to a final concentration
of 0.5 mg/ml.
[0126] 6. Doxorubicin
[0127] A 10 mg vial of doxorubicin was dissolved in 10 ml DMSO and
stored at -80.degree. C. A working stock was prepared by dilution
in complete medium to 200 .mu.g /ml.
[0128] Plate Depositions
[0129] Fifty .mu.l of the cell suspensions, or medium alone for
blanks, prepared at the concentrations described above was placed
in wells of a poly-D-lysine coated 96-well microtiter plate. The
plates were placed in the cell incubator for 3 hr after which 50
.mu.l of the challenge solution was added to cell-containing wells.
Three or four replicate wells were used for each experimental
condition. For example, the preparation of solutions containing
fludarabine in the absence or presence of defibrotide is given in
Table I. Following addition of the solution to the wells, the
plates were returned to the incubator and cell viability was
assessed after 24, 48 or 72 hr. For a background measurement, 100
.mu.l of complete medium alone was included in 3 to 4 replicate
wells.
TABLE-US-00001 TABLE 1 Preparation of fludarabine and defibrotide
solutions Fludarabine Defibrotide Sample Defibrotide (*) Medium
Stock Stock type (.mu.g/ml) (.mu.l) (.mu.l) (.mu.l) Negative 0 3486
14 0 Control Defibrotide 1.25 3468.5 14 17.5 Sample 1:1 Defibrotide
2.5 3451 14 35 Sample 1:2 Defibrotide 5 3416 14 70 Sample 1:4
Defibrotide 10 3346 14 140 Sample 1:8 Defibrotide 20 3206 14 280
Sample 1:16 Defibrotide 40 2926 14 560 Sample 1:32 Defibrotide 80
2366 14 1120 Sample 1:64 Blank 0 3500 0 0 (*) final concentration
in each well following addition of 50 .mu.L of cell suspension and
50 .mu.L of the indicated solution
[0130] Cell Viability Using MTT Assay
[0131] After the specified period of incubation, cell viability in
each well was measured using the MTT assay. The MTT assay is based
on the cleavage of tetrazolium salts by mitochondrial dehydrogenase
in viable cells leading to the production of an insoluble formazan
dye. MTT dye, 10 .mu.l of a 2 mg/ml solution in D-PBS, was added to
each well and then the plates were incubated for 3 hours. Plates
were then centrifuged and each well aspirated. The dye was
solubilised with 200 .mu.l of a mixture DMSO/Ethanol (1:1) and
absorbance in the wells was read at 570-590 nm on a microplate
reader. A blank well containing only media and cytotoxic drug
(fludarabine or doxorubicin) was also run as a control in all
experiments.
[0132] Cell Viability Using CCK-8 Assay
[0133] After the specified period of incubation, cell viability in
each well was measured using the CCK-8 cell counting kit (Sigma
Aldrich, Milan, Italy) following the manufacturer's instructions.
The assay is based on the reduction by dehydrogenase activities of
viable cells of the water soluble tetrazolium salt
WST-(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)--
2H-tetrazolium). The reduced formazan dye is soluble in tissue
culture media. The amount of the formazan is directly proportional
to the number of viable cells. The detection sensitivity of CCK-8
is higher than the other tetrazolium salts such as MTT. Unlike MTT,
no solubilisation step is required and thus the assay can be
measured continuously.
[0134] Briefly, after the specified incubation time, 10 .mu.l of
the supplied reagent was added to each well of the microtitre plate
and the plate returned to the incubator. After 3 hours, the
absorbance was measured at 450 nm with a background correction at
590 nm. Absorbance of the medium blank was subtracted from the test
samples.
Example 1
[0135] The present example shows the magnitude of the protection
effect of defibrotide against fludarabine-induced cytotoxicity of
HMEC-1 cells at physiologically relevant concentrations of
fludarabine and defibrotide.
[0136] HMEC-1 cells were cultivated according to the above
mentioned procedure. A cell density of 500,000 cells/ml was used
for the assay. The cytotoxic agent was fludarabine at a
concentration of 10 .mu.g/ml. Defibrotide was added to the
microplate well at a concentration of 100, 10 or 1 .mu.g/ml. Four
replicates of each condition were performed. The viability of the
HMEC cells was assessed after 72 hours with the MTT assay described
above. Defibrotide protected the cells from fludarabine-induced
cytotoxicity in a dose-dependent manner with a protection effect of
more than 50% observed with 100 .mu.g/ml of defibrotide (FIG.
1).
Example 2
[0137] The present example shows the magnitude of the protection
effect of defibrotide against doxorubicin-induced cytotoxicity of
SK-HEP-1 cells at physiologically relevant concentrations of
doxorubicin and defibrotide.
[0138] SK-HEP-1 cells were grown according to the above mentioned
procedure. A cell density of about 50,000 cells/ml was used for the
experiment. The cytotoxic agent was doxorubicin at a concentration
of 0.1 .mu.g/ml. Defibrotide was added to the microplate well at a
concentration of 100, 50, 20, 10, 5 or 1 .mu.g/ml. Three replicates
of each condition were performed. The viability was assessed after
72 hours with the CCK-8 assay kit as described above. At
concentrations of 50 .mu.g/ml or greater, defibrotide significantly
protected SK-HEP-1 cells from doxorubicin-induced cytotoxicity.
(FIG. 2).
Example 3
[0139] The present example compares the protective effective
against fludarabine-induced cytotoxicity of defibrotide and
synthetic oligonucleotides having similar average length and base
composition to defibrotide.
[0140] HMEC-1 cells were cultivated according to the above
mentioned procedure. A cell density of about 500,000 cells/ml was
used for the experiment. The cytotoxic agent was fludarabine at a
concentration of 50 .mu.g/ml. Synthetic oligonucleotides
(Adenine-Cytosine (AC) of about 16 kDa or
Adenine-Cytosine-Guanine-Thymine (ACGT) of about 17 kDa) or
defibrotide were added to each well at varying concentrations.
Specifically, the AC oligonucleotides were added to each well at a
concentration of 1, 10, 100 or 500, whereas the ACGT
oligonucleotides were added to each well at a concentration of
12.5, 25 or 50 .mu.g/ml. Defibrotide was added to each well at a
concentration of 5, 25, 50, or 100 .mu.g/ml. Each treatment
condition was performed in triplicate. The viability was assessed
with the MTT assay after 24, 48 and 72 hours.
[0141] As shown in FIGS. 3A and 3B, neither the AC oligonucleotides
nor the ACGT oligonucleotides had any protective effect against
fludarabine-induced cytotoxicity of HMEC-1 cells after 72 hours of
incubation. In contrast, defibrotide exhibited a dose-dependent
protection of the cells from fludarabine-induced cytotoxicity (FIG.
3C).
Example 4
[0142] The experiments described in this example tested the ability
of a synthetic ACGT oligonucleotide, tPA, and glutathione to
protect SK-HEP-1 cells from fludarabine-induced toxicity.
[0143] SK-HEP-1 cells were grown and expanded according to the
above mentioned procedure. A cell density of about 50,000 cells/ml
was used for the experiment. The cytotoxic agent was fludarabine at
a concentration of 10 .mu.g/ml. A random synthetic oligonucleotide
(ACGT), tPA, or glutathione was added to each well at varying
concentrations. Specifically, the ACGT oligonucleotide or
glutathione was added to each well at a concentration of 1.25, 2.5,
5, 10, 20, 40, or 80 .mu.g/ml, whereas tPA was added at a
concentration of 10, 20, 40, 80, 160, or 320 IU/ml. Each treatment
condition was performed in triplicate. The viability was assessed
with the CCK-8 assay kit after 72 hours incubation. No protection
of the SK-HEP-1 cells from fludarabine-induced cytotoxicity was
observed with any of the three compounds (FIG. 4).
Example 5
[0144] The present example evaluates the protective effective
against fludarabine-induced cytotoxicity of defibrotide which has
been modified as a result of physicochemical stress.
[0145] Defibrotide samples were stressed by submitting a standard
sample of defibrotide to either 1) an Acidic Stress or 2) a Basic
Stress. The Acidic Stress entailed incubating the standard
defibrotide sample in a phosphate buffer having a pH of about 3 at
about 80.degree. C. for 18 hours. The Basic stress involved
incubating the standard defibrotide sample in a phosphate buffer
having a pH of about 12 at about 80.degree. C. for 18 hours. After
the incubation period, the solutions were brought to neutrality
with phosphoric acid or sodium hydroxide.
[0146] SK-HEP-1 cells were grown according to the above mentioned
procedure. A cell density of about 50,000 cells/ml was used for the
experiment. The cytotoxic agent was fludarabine at a concentration
of 10 .mu.g/ml. Standard defibrotide (unmodified), defibrotide
submitted to acid stress, or defibrotide submitted to basic stress
were added to each well at a concentration of 80, 40, 20, 10, 5,
2.5, or 1.25 .mu.g/ml. Each treatment condition was performed in
triplicate.
[0147] The viability of the cells was assessed with the CCK-8 assay
kit after 72 hours incubation. Dose-response curves were
constructed for unmodified defibrotide, acid-stressed defibrotide,
and basic-stressed defibrotide. A comparison of the dose-response
curves is shown in FIG. 5. Both the acid-stressed and
basic-stressed defibrotide samples were less potent than unmodified
defibrotide in protecting SK-HEP-1 cells from fludarabine-induced
cytotoxicity (FIG. 5).
Example 6
[0148] The present example shows the magnitude of the protection
effect of defibrotide against fludarabine-induced cytotoxicity of
SK-HEP-1 cells at physiologically relevant concentrations of
defibrotide and fludarabine.
[0149] SK-HEP-1 cells were grown and expanded according to the
above mentioned procedure. The cells at a cell density of about
50,000 cells/ml were plated in a poly-D-lysine coated 96-well
microplate. The cytotoxic agent was fludarabine at a concentration
of 10 .mu.g/ml. Defibrotide was added to each well at a
concentration of 100, 50, 40, 20, 10, 5 or 1 .mu.g/ml. Each
treatment condition was run in 4 replicate wells. The viability was
assessed after 72 hours of incubation with the CCK-8 assay kit.
Defibrotide produced a dose-dependent cell protection effect from
fludarabine-induced cytotoxicity with greater than 80% of the cells
surviving with concentrations of defibrotide of 40 .mu.g/ml or
greater (FIG. 6).
Example 7
[0150] The present example shows the application of the cell-based
protection assay for the assessment of the potency of a defibrotide
sample of unknown biological activity.
[0151] SK-HEP-1 cells were grown and expanded according to the
above mentioned procedure. The cells were plated at a cell density
of about 50,000 cells/ml in poly-D-lysine-coated microplates. The
cytotoxic agent was fludarabine at a concentration of 10 .mu.g/ml.
The defibrotide reference standard and the defibrotide test sample
were added to separate wells at a concentration of 80, 40, 20, 10,
5, 2.5, or 1.25 .mu.g/ml. Four replicate wells were run for each
treatment condition. The viability of the cells was assessed with
the CCK-8 assay kit after 72 hours of incubation.
[0152] The absorbances measured for the defibrotide reference
standard samples and the defibrotide test samples were submitted to
a 4-parameter logistic function analysis. That is, the
dose-response of the reference and sample defibrotide curves can be
described by a 4-parameter logistic function:
.upsilon. = .delta. + .alpha. - .beta. 1 + e - .beta. ( x - .gamma.
) ##EQU00001##
[0153] Where, .upsilon. is the response, .alpha. is the upper
asymptote, .delta. is the lower asymptote, .beta. is the
slope-factor, and .gamma. is the horizontal location of the sample
curve on the x axis. The potency of the defibrotide test sample was
determined by calculating a potency ratio against the defibrotide
reference standard. The potency ratio for the defibrotide test
sample was 1.157 (FIG. 7).
Example 8
[0154] The present example evaluates the precision of the
defibrotide potency determinations of the cell-based protection
assay. The potency of the same defibrotide test sample was
measured, against a defibrotide reference standard, in repeated
assays by different analysts, using different batches of qualified
medium, cell batches and pipetting devices.
[0155] SK-HEP-1 cells were grown and expanded according to the
above mentioned procedure. The cells were plated at a cell density
of about 50,000 cells/ml in poly-D-lysine-coated microplates. The
cytotoxic agent was fludarabine at a concentration of 10 .mu.g/ml.
The defibrotide reference standard and the defibrotide test sample
were added to separate wells at the same series of concentrations
as described in Example 7. Four replicate wells were run for each
experimental condition. The viability of the cells was assessed
with the CCK-8 assay kit after 72 hours of incubation.
[0156] The absorbances measured for each well in each assay run
were subject to a 4-parameter logistic function analysis and
potency ratio against the defibrotide reference standard was
calculated as described in Example 7. The potency ratio of the same
defibrotide test sample in each assay run is shown in Table 2. From
the potency measurements by different analysts under the variable
conditions shown in Table 2, the assay has a high a precision (%
relative standard deviation of 7.8) and a low bias of <3%.
TABLE-US-00002 TABLE 2 Precision of the assay is demonstrated by
analysing the same test sample, against the defibrotide standard,
in different assay runs, on different days and cells of a different
passage number. Cell passage Pipetting Measured Assay run Day
Analyst number device set Potency 1 1 1 1 #1 0.901 2 2 1 1 #2 0.998
3 2 1 2 #1 0.937 4 2 1 1 #1 1.087 5 2 1 2 #2 1.011 6 3 2 1 #1 1.057
7 3 2 2 #1 1.068 8 3 1 2 #1 0.944 9 4 2 1 #1 0.971 10 4 2 2 #1 1.11
11 5 2 1 #2 1.135 12 5 2 2 #2 1.13
Example 9
[0157] The present example shows a comparison of the potency as
determined by the cell-based protection assay for three different
batches of defibrotide and a defibrotide reference standard.
[0158] SK-HEP-1 cells were grown and expanded according to the
above mentioned procedure. The cells were plated at a cell density
of about 50,000 cells/ml in poly-D-lysine-coated microplates. The
cytotoxic agent was fludarabine at a concentration of 10 .mu.g/ml.
The defibrotide reference standard and the different defibrotide
test samples from three separate batches were added to separate
wells at a concentration of 80, 40, 20, 10, 5, 2.5, or 1.25
.mu.g/ml. Four replicate wells were run for each experimental
condition. The viability of the cells was assessed with the CCK-8
assay kit after 72 hours of incubation.
[0159] The absorbances measured for each well were submitted to a
4-parameter logistic function analysis and the potency ratio for
each batch of defibrotide was calculated as described in Example 7.
The potency ratio relative to the defibrotide standard, of each of
the defibrotide test samples from the three batches is shown in
Table 3.
TABLE-US-00003 TABLE 3 Potency ratios for three different batches
of defibrotide analysed against a defibrotide reference standard.
Linearity, 95% Regression and Confidence Defibrotide batch
Parallelism tests Interval Potency 1080030021 Passed 0.823-0.989
0.902 1080060117 Passed 0.697-0.889 0.787 1080010016 Passed
1.116-1.725 1.387
[0160] It is understood that the disclosed invention is not limited
to the particular methodology, protocols and materials described as
these can vary. It is also understood that the terminology used
herein is for the purposes of describing particular embodiments
only and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0161] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Example 10
[0162] It would be useful to add experimental data directly
comparing the method of the present invention with that of D3.
* * * * *
References