U.S. patent application number 17/410485 was filed with the patent office on 2022-02-10 for method of treating a mammal, including human, against cancer using methionine and asparagine depletion.
The applicant listed for this patent is ERYTECH PHARMA. Invention is credited to Karine AGUERA, Willy BERLIER, Fabien GAY, Yann GODFRIN.
Application Number | 20220040272 17/410485 |
Document ID | / |
Family ID | 1000005925611 |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220040272 |
Kind Code |
A1 |
AGUERA; Karine ; et
al. |
February 10, 2022 |
METHOD OF TREATING A MAMMAL, INCLUDING HUMAN, AGAINST CANCER USING
METHIONINE AND ASPARAGINE DEPLETION
Abstract
The invention is related to a new method for treating liquid and
solid cancers, in a mammal, including human, wherein methioninase
is administered before asparaginase. The invention also encompasses
the use of a dietary methionine deprivation, possibly combined with
methioninase administration, in advance of asparaginase treatment.
Methioninase and asparaginase may be used in particular under free
form, pegylated form or encapsulated into erythrocytes.
Inventors: |
AGUERA; Karine; (LYON,
FR) ; BERLIER; Willy; (LYON, FR) ; GAY;
Fabien; (LYON, FR) ; GODFRIN; Yann; (LYON,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ERYTECH PHARMA |
LYON |
|
FR |
|
|
Family ID: |
1000005925611 |
Appl. No.: |
17/410485 |
Filed: |
August 24, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16067398 |
Jun 29, 2018 |
11141468 |
|
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PCT/EP2017/050006 |
Jan 2, 2017 |
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17410485 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/18 20130101;
A61K 38/54 20130101; A61P 35/00 20180101; C12Y 404/01011 20130101;
A61K 38/50 20130101; A61K 38/51 20130101; A61K 31/675 20130101;
C12Y 305/01001 20130101; A61K 9/0019 20130101; A61K 9/10
20130101 |
International
Class: |
A61K 38/54 20060101
A61K038/54; A61K 38/50 20060101 A61K038/50; A61K 38/51 20060101
A61K038/51; A61K 9/10 20060101 A61K009/10; A61K 35/18 20060101
A61K035/18; A61K 9/00 20060101 A61K009/00; A61K 31/675 20060101
A61K031/675; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 31, 2015 |
EP |
15307197.2 |
Claims
1-25. (canceled)
26. A pharmaceutical composition or kit for use in treating cancer
in a mammal comprising an asparagine-reducing means and a
methionine-reducing means, wherein the methionine-reducing means is
administered before the asparagine-reducing means and wherein there
is a delay between the administration of the methionine-reducing
means and the asparagine-reducing means.
27. The composition or kit of claim 26, wherein when the
methionine-reducing means comprises or consists essentially of a
methioninase and/or when the asparagine-reducing means comprises or
consists essentially of an asparaginase, the methioninase and/or
the asparaginase are under free form, pegylated form or
encapsulated inside erythrocytes.
28. The composition or kit of claim 27, wherein the methioninase is
under free form or is pegylated and the delay between the end of
the methioninase administration and the initiation of the
asparaginase administration is between about 1 h and about 7 days,
between about 3 h and about 6 days, or between about 1 day and
about 5 days.
29. The composition or kit of claim 27, wherein the methioninase is
encapsulated into erythrocytes and the delay between the end of
methioninase administration and the initiation of the asparaginase
administration is between about 1 h and about 30 days, between
about 1 day and about 20 days, or between about 1 day and about 10
days.
30. The composition or kit claim 29, wherein the methioninase is
administered once or more in an amount of between about 100 and
about 100,000 IU, between about 500 and about 50,000 IU, or between
about 500 and about 5,000 IU; and wherein the asparaginase is
administered once or more in an amount of between about 500 and
about 100,000 IU, between about 1,000 and about 50,000 IU, or
between about 5,000 and about 30,000 IU.
31. The composition or kit of claim 30, wherein (a) the
methioninase is under free form or pegylated form and the delay
between the methioninase and the asparaginase administration is
between about 1 h and about 7 days, between about 3 h and about 6
days, or between about 1 day and about 5 days; or (b) the
methioninase is encapsulated inside erythrocytes and the delay
between the end of the methioninase administration and the
initiation of the asparaginase administration is between about 1 h
and about 30 days, between about 1 day and about 20 days, or
between about 1 day and about 10 days; wherein the methioninase is
administered once or more in an amount of between about 100 and
about 100,000 IU, between about 500 and about 50,000 IU, or between
about 500 and about 5,000 IU; and wherein the asparaginase is
administered once or more in an amount of between about 500 and
about 100,000 IU, between about 1,000 and about 50,000 IU, or
between about 5,000 and about 30,000 IU.
32. The composition or kit of claim 31, further comprising PLP or a
PLP precursor for simultaneous, separate or sequential
administration with the methioninase.
33. The composition or kit of claim 32, comprising methioninase
encapsulated inside erythrocytes and a non-phosphate precursor of
PLP for separate or sequential administration.
34. The composition or kit of claim 31, wherein the methioninase
encapsulated inside erythrocytes is administered at least once or
twice before the asparaginase encapsulated inside erythrocytes is
administered, and each methioninase administration is followed by
administration of a solution of non-phosphate precursor of PLP
before asparaginase is administered.
35. The composition or kit of claim 26, wherein the
methionine-reducing means comprises or consists essentially of a
methionine-restricted diet.
36. The composition or kit of claim 26, wherein the
asparagine-reducing means comprises or consists essentially of an
asparagine-restricted diet.
37. A method for treating a cancer in a mammal in need thereof, the
method comprising administering to the mammal in need thereof, a
composition comprising a methionine-reducing means, delaying for a
period of time, and then administering a composition comprising an
asparagine-reducing means, thereby treating the cancer.
38. The method of claim 37, wherein the methionine-reducing means
comprises or consists essentially of a methioninase and the
asparagine-reducing means comprises or consists essentially of an
asparaginase.
39. The method of claim 37, wherein the delay comprises between
about 1 h and about 30 days.
40. The method of claim 37, wherein the methionine-reducing means
comprises or consists essentially of a methionine-restricted
diet.
41. The method of claim 37, wherein the asparagine-reducing means
comprises or consists essentially of an asparagine-restricted
diet.
42. The method of claim 38, wherein the methioninase is
encapsulated inside erythrocytes and is administered at least once
or twice before the asparaginase, which is also encapsulated inside
erythrocytes, and each methioninase administration is accompanied
by administration of PLP or a precursor of PLP before the
asparaginase is administered.
43. The method of claim 38, wherein a non-phosphate precursor of
PLP is administered once or more after each administration of
methioninase.
44. The method of claim 43, wherein the non-phosphate precursor of
PLP is administered once a day, or twice or more per day, during
the time of methioninase administration.
45. The method of claim 37, wherein the cancer is leukemia or
gastric cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/067,398, having a filing date of Jun. 29, 2018, which is a
371 application of International Patent Application
PCT/EP2017/050006, filed Jan. 2, 2017, which claims the benefit of
EP application EP 15307197.2, filed Dec. 31, 2015, all of said
applications incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to a new method,
particularly enzymatic method of treating a mammal, including
human, against cancer, and to novel uses of asparaginase and
methioninase in the treatment of cancer. Enzymatic therapies are
intended to starve tumours and help in particular manage
cancers.
BACKGROUND OF THE INVENTION
[0003] Asparaginase hydrolyses and depletes asparagine, an amino
acid essential for the production of the proteins necessary for
cell life. Now, in contrast to normal cells, certain cancerous
lymphoblastic cells do not have the capacity to produce their
asparagine themselves and are dependent on extra-cellular sources
for the synthesis of their proteins. The enzyme may thus be used to
treat Leukemias (liquid or blood cancers). L-asparaginase has thus
been used in chemotherapy combination for the treatment of Acute
Lymphoblastic Leukemia (ALL) for the last thirty years. ERY-ASP
consists of red blood cell-encapsulated L-asparaginase.
Encapsulation enables L-asparaginase to destroy asparagine inside
the red blood cell, preventing allergic reactions and reducing
other adverse events (WO 2006/016247, incorporated herein by
reference).
[0004] Methionine-.gamma.-lyase (MGL; EC number 4.4.1.11; CAS
number 42616-25-1), also designated as methioninase, is a
pyridoxal-dependent enzyme involved in the metabolism of
L-methionine (Met), an essential, sulfur-containing proteinogenic
amino acid. Met requirement in cancers has been purposed in the
1970s: studies revealed that substitution of Met by its precursor
homocysteine in culture medium has no impact on normal cells such
as fibroblasts but leads to a slow growing rate of several
transformed or malignant cells. In PC-3 prostate cancer cells,
anti-tumor effects of Met starvation were also reinforced by using
a Met analogue which dramatically slowed the proliferation of
cancer cells both in vitro and in vivo and forced cells to enter
apoptosis. Complementary studies revealed that exogenous Met
restriction in Met-dependent cancer cells blocks cell division in
the late S or G2 phase of the cell cycle. As Met restriction
appeared to be effective for cancer treatment, therapeutic approach
using MGL enzyme from several sources was investigated for Met
depletion in the tumor microenvironment. The aim was to develop a
new therapeutic solution based on MGL encapsulated into
erythrocytes for systemic depletion of Met in patients harbouring
Met-dependent cancers (WO 2015/121348, incorporated herein by
reference).
SUMMARY OF THE INVENTION
[0005] There is still a need for new or additional therapeutic
solutions in cancer treatment.
[0006] The effect of drug combination is inherently unpredictable.
There is often a propensity for one drug to partially or completely
inhibit the effects of the other. In vitro studies were carried out
to assess cytotoxic effects of the enzymes constituting ERY-ASP and
ERY-MET, L-asparaginase and MGL, alone or in combination, on a
selected human leukemia cell line (HL-60). For each drug
separately, the concentration that gives a 50% inhibition of cell
viability (IC50) was previously determined. Then, assays were
performed to evaluate the benefits of treatment combination when
some delay, e.g. 72 hours were added between the additions of
L-asparaginase and MGL (IC50 dose for each enzyme), whatever the
order of combination.
[0007] The present invention is based on the surprising observation
that cell mortality could be increased with an addition of MGL at
IC50 dose followed by L-asparaginase at IC50 dose 3 days later. The
reverse design of enzyme addition did not permit to obtain such
increase of cell mortality in vitro in a liquid tumor model, say a
leukemia model. This remarkable effect has been confirmed in a
solid tumor, say gastric tumor, wherein an increase of cell
mortality in vitro and tumor volume regression in vivo were
observed. Without willing to be bound by theory, it can be
hypothesized that methionine deprivation induced by MGL activity
could make the cells more responsive to L-asparaginase and that
there is probably a link with the role of each enzyme involved in
the cell cycle regulation. This finding opens the way to treatment
regimens comprising sequential methionine deprivation or
methioninase treatment and asparagine deprivation or asparaginase
treatment. As it will be evident from the following disclosure, the
invention may encompass diet and/or drug administration that
induces the beneficial effect on cancer. Thus the invention may
combine diet and drug administration, in any combination wherein
methionine deprivation or methioninase treatment precedes
asparagine deprivation or asparaginase treatment. As methioninase
is also known as having a cysteinase activity, and asparaginase as
having a glutaminase activity, it cannot be excluded that a
cysteinase activity, respectively a glutaminase activity may be
involved in the mode of action of methioninase, respectively
asparaginase.
[0008] An object of the invention is a method for treating cancer
in a mammal in need thereof, the method comprising depriving the
mammal for methionine, then depriving the mammal for asparagine.
What is searched for is to reduce the amount of methionine and
asparagine available to the cancer cells. As it will be apparent
from the foregoing, methionine deprivation may be performed through
dietary methionine deprivation and/or methioninase administration,
whereas asparagine deprivation may preferably be performed using
asparaginase administration.
[0009] By deprivation, it is meant a sufficient reduction of
methionine or asparagine to produce beneficial effects in treating
cancer, the cancer cells being deprived for sufficient amount of
the amino acid.
[0010] By enzyme treatment, it is meant that the enzyme will
degrade the concerned amino acid and possibly induce other
beneficial effects such as inhibition of protein or amino acid
synthesis or any mechanism that leads to lack of sufficient amount
of the amino acid to the cancer cell.
[0011] An object of the present invention is a pharmaceutical
composition for use in treating cancer in a mammal comprising
asparaginase and methioninase for at least one sequential
administration with methioninase being administered before
asparaginase. As asparaginase and methioninase are to be
administered separately and sequentially, the composition may be
qualified of set or kit comprising separate formulations thereof or
of compositions to be used in accordance with order and frequence
of the invention.
[0012] In the context of the invention under its different aspects
or objects, at least one sequential administration means that the
same mammal may be treated sequentially more than once during a
treatment therapy or phase. However, one or several methioninase
administration(s) may be performed before one or several
asparaginase administration(s).
[0013] Another object of the present invention is the use of
asparaginase and methioninase for the preparation of a
pharmaceutical composition or pharmaceutical compositions or a kit
or set of pharmaceutical compositions (one containing methioninase,
another one containing asparaginase), wherein the composition(s) or
the kit is for use in treating cancer in a mammal with at least one
sequential administration with methioninase being administered
before asparaginase.
[0014] Other objects of the invention are: [0015] a pharmaceutical
composition comprising asparaginase for use in treating cancer in a
mammal, wherein the composition is to be administered to a mammal
that has been administered methioninase; [0016] a pharmaceutical
composition comprising asparaginase for use in treating cancer in a
mammal, wherein the composition is to be administered to a mammal
that has been subjected to methionine deprivation diet, i.e. has
been administered a methionine deprived food, therapeutic or not;
by therapeutic food in the meaning of this invention, it is meant a
food administered in medical environment and/or subjected to
marketing authorization by Regulatory Authority, especially a
liquid food, that may be or not administered by infusion; [0017] a
pharmaceutical composition comprising methioninase for use in
treating cancer in a mammal, wherein the composition is to be
administered to a mammal that will be further administered
asparaginase; [0018] a food composition or diet, therapeutic or
not, comprising no methionine or substantially no methionine for
use in depriving a mammal for methionine, before treating the
mammal with asparaginase.
[0019] Other objects of the invention are: [0020] the use of
asparaginase for the preparation of a pharmaceutical composition
for use in treating cancer in a mammal, wherein the composition is
to be administered to a mammal that has been administered
methioninase; [0021] the use of asparaginase for the preparation of
a pharmaceutical composition for use in treating cancer in a
mammal, wherein the composition is to be administered to a mammal
that has been subjected to methionine deprivation diet, i.e. has
been administered a methionine deprived food, therapeutic or not;
[0022] the use of methioninase for the preparation of a
pharmaceutical composition for use in treating cancer in a mammal,
wherein the composition is to be administered to a mammal that will
be further administered asparaginase.
[0023] Still another object of the invention is a kit comprising a
pharmaceutical composition containing methioninase or a therapeutic
food or diet for methionine deprivation, and a pharmaceutical
composition containing asparaginase, the compositions being
separately packaged. The compositions are for sequential
administration with methioninase or food/diet being administered
before asparaginase. The kit may further contain a leaflet
indicating that the compositions are for sequential administration
with methioninase or food/diet being administered before
asparaginase.
[0024] Still another object of the invention is a method of
treatment of cancer in a mammal comprising administering to a
mammal first an efficient amount of methioninase and second an
efficient amount of asparaginase.
[0025] Still another object of the invention is a method of
treatment of cancer in a mammal comprising administering to a
mammal first a food or diet, therapeutic or not, to deprive
methionine, and second an efficient amount of asparaginase.
[0026] Still another object of the invention is a method of
treatment of cancer in a mammal having a low methionine
bioavailable level, or having been subjected to a food or diet,
therapeutic or not, having deprived methionine, the method
comprising administering to the mammal an efficient amount of
asparaginase.
[0027] In these different objects, methioninase administration and
methionine diet deprivation may be combined.
[0028] The invention may be beneficial to any cancer, including
liquid, i.e. haematological cancers, and solid cancers.
[0029] A specific object of the invention is the application of
this invention to the treatment of cancers auxotrophic to
asparagine and/or methionine.
[0030] A specific object of the invention is the application of
this invention to the treatment of cancers not auxotrophic to
asparagine and/or methionine.
[0031] The invention may apply to any mammal and especially human,
companion animals such as dogs and cats and sport animals such as
horses.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0032] The person skilled in the art may understand from the
present disclosure that the duration of treatment with diet or one
of the drugs, and the delay between methionine deprivation and
asparaginase treatment, may vary depending on the treatment, on the
patient response and importantly on the half-life of the drug or
diet effect. There may be a difference depending on the dosage form
used in the invention, for example a free enzyme, a pegylated
enzyme and erythrocytes encapsulating the enzyme, or else enzyme
bound to microcapsules (e.g. made of PLA or PLGA) or liposomes or
encapsulated in these structures.
[0033] In a preferred embodiment of these different objects, the
delay between the end of methioninase administration and the
initiation of asparaginase administration is between about 1 h and
about 7 days, in particular between about 3 h and about 6 days,
preferably between about 1 day and about 5 days. Preferably, in
this embodiment, methioninase is under free form or pegylated form,
and asparaginase may be under any of the forms described
herein.
[0034] In another embodiment, the delay between the end of
methioninase administration and the initiation of asparaginase
administration is between about 1 h and about 30 days, in
particular between about 1 day and about 20 days, preferably
between about 1 day and about 10 days. Preferably, in this
embodiment, methioninase is encapsulated, preferably into
erythrocytes, and asparaginase may be under any of the forms
described herein.
[0035] In still another embodiment, the delay between the end of
methionine restriction and the initiation of asparaginase
administration is between about 1 h and about 7 days, in particular
between about 1 h and about 3 days, preferably between about 1 h
and about 1 day. Asparaginase may be under any of the forms
described herein.
[0036] Compositions Comprising Enzyme Under Free Form or Under
Pegylated Form, and the Like:
[0037] These compositions can be administered to a mammal using
standard techniques. Techniques and formulations generally may be
found in Remington's Pharmaceutical Sciences, 18.sup.th ed., Mack
Publishing Co., Easton, Pa., 1990 (hereby incorporated by
reference).
[0038] Pharmaceutically acceptable carriers and/or excipients can
also be incorporated into a pharmaceutical composition according to
the invention to facilitate administration of the particular
methioninase or asparaginase. Examples of carriers suitable for use
in the practice of the invention include calcium carbonate, calcium
phosphate, various sugars such as lactose, glucose, or sucrose, or
types of starch, cellulose derivatives, gelatin, vegetable oils,
polyethylene glycols, and physiologically compatible solvents.
Examples of physiologically compatible solvents include sterile
solutions of water for injection (WFI), saline solution and
dextrose.
[0039] Pharmaceutical compositions according to the invention can
be administered by different routes, including intravenous,
intraperitoneal, subcutaneous, intramuscular, oral, topical
(transdermal), or transmucosal administration. For systemic
administration, oral administration is preferred. For oral
administration, for example, the compounds can be formulated into
conventional oral dosage forms such as capsules, tablets, and
liquid preparations such as syrups, elixirs, and concentrated
drops.
[0040] Alternatively, injection (parenteral administration) may be
used, e.g. intramuscular, intravenous, intraperitoneal, and
subcutaneous injection. For injection, pharmaceutical compositions
are formulated in liquid solutions, preferably in physiologically
compatible buffers or solutions, such as saline solution, Hank's
solution, or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. For example, lyophilized forms of the methioninase or
asparaginase can be used.
[0041] Systemic administration can also be accomplished by
transmucosal or transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are well
known in the art, and include, for example, for transmucosal
administration, bile salts, and fusidic acid derivatives. In
addition, detergents may be used to facilitate permeation.
Transmucosal administration, for example, may be through nasal
sprays, inhalers (for pulmonary delivery), rectal suppositories, or
vaginal suppositories. For topical administration, compounds can be
formulated into ointments, salves, gels, or creams, as is well
known in the art.
[0042] The invention encompasses also the use of implanted devices
or applied on the mammal to deliver the enzyme, for instance
through infusion or another route. In a special embodiment, the
device comprises two chambers or vials, one containing
methioninase, the other containing asparaginase. The device has,
for each chamber or vial, a tube and the like for delivering the
enzyme into the blood circulation, an electronic or electrical
valve or pump, or an actuated piston, that is controlled by an
electronic circuit and a suitable software. The electronic circuit
and its software controls the delivery of methioninase first,
during a predetermined period of time, preferably at a certain
debit rate, a delay period, and then the delivery of asparaginase,
during a predetermined period of time, preferably at a certain
debit rate.
[0043] Compositions Comprising Erythrocytes (Red Blood Cells or
RBCs) Encapsulating the enzyme:
[0044] In an embodiment, asparaginase is encapsulated inside
erythrocytes and the composition comprises a suspension of these
erythrocytes in a pharmaceutically acceptable carrier or
vehicle.
[0045] In an embodiment, methioninase is encapsulated inside
erythrocytes and the composition comprises a suspension of these
erythrocytes in a pharmaceutically acceptable carrier or
vehicle.
[0046] In an embodiment, asparaginase is in free form or under a
pegylated form (PEG-asparaginase), in a pharmaceutically acceptable
carrier or vehicle.
[0047] In an embodiment, methioninase is in free form or under a
pegylated form (PEG-methioninase), in a pharmaceutically acceptable
carrier or vehicle.
[0048] In an embodiment, methioninase is administered in an amount
of between about 100 and about 100 000 IU, in particular between
about 500 and about 50 000 IU, preferably between about 500 and
about 5000 IU.
[0049] In an embodiment, asparaginase is administered once in an
amount of between about 500 and about 100 000 IU, in particular
between about 1000 and about 50 000 IU, preferably between about
5000 and about 30 000 IU.
[0050] In an embodiment, the composition is for use for two or more
sequential administrations, especially 2 or 3.
[0051] In an embodiment, asparaginase and methioninase are used
sequentially in accordance with the invention, and these enzymes
are both encapsulated into erythrocytes.
[0052] In an embodiment, asparaginase and methioninase are used
sequentially in accordance with the invention, with asparaginase
encapsulated into erythrocytes and methioninase in free form or
under a pegylated form.
[0053] In an embodiment, asparaginase and methioninase are used
sequentially in accordance with the invention, with methioninase
encapsulated into erythrocytes and asparaginase in free form or
under a pegylated form.
[0054] "Encapsulated" means that the enzyme is contained inside the
erythrocytes. It is possible however that some minor amount of
enzyme is retained within the erythrocyte wall.
[0055] Dietary Methionine Restriction:
[0056] Dietary methionine restriction has been proposed either in
association with cystemustine therapy in melanoma and glioma (E.
Thivat et al., Anticancer Research 2009, 29: 5235-5240) or with
FOLFOX as first line therapy of metastatic colorectal cancer (X.
Durando et al., Oncology 2010, 78: 205-209). Methionine restriction
or deprivation diet is a food regimen or feeding the mammal with a
food composition during a sufficient time to induce a full or
substantial decrease or elimination of free methionine in the
mammal.
[0057] The food may preferably be a liquid food that is
administered through parenteral route, especially infusion.
[0058] Also, methionine deprivation using methioninase aims at
inducing a full or substantial decrease or elimination of free
methionine in the mammal. Typically, this diet is performed in
order to decrease the methionine level of 30 to 100%, typically
from 30 to 60% with respect to the mean level in the mammal.
Reference may be done to the works by Thivat 2009 and Durando
2010.
[0059] Administration of the food may be done during one day or
more, for example from one day to seven days.
[0060] In an embodiment, the food is combined to methioninase
treatment, for example the food is administered during the whole or
part duration of treatment with methioninase.
[0061] Methioninase
[0062] Methioninase is further called, inter alia, L-methioninase,
Methionine Gamma Lyase MGL; this compound is receiving number EC
4.4.1.11 and CAS number 42616-25-1. In order to be aware of the
methioninase sources which may be used according to the invention,
mention may notably be made to the publication El Sayed A, Applied
Microbiol. Biotechnol. (2010) 86: 445-467.
[0063] A recombinant methioninase may be produced in the
Escherichia coli bacterium from a gene coding for the enzyme, for
example from the Pseudomonas putida bacterium. The thereby obtained
enzyme called rMETase may be used under free form or under a
modified form, e.g. pegylated form (PEG-rMETase). See X. Sun et al.
Cancer Research 2003, 63: 8377-8383. It may also be encapsulated
into erythrocytes, the composition or suspension advantageously
containing an amount of erythrocytes and an amount of encapsulated
methioninase that is sufficient to deliver to the patient the dose
of asparaginase that has been decided.
[0064] The person skilled in the art may refer to WO 2015/121348
for compositions and methods of use.
[0065] The composition of methioninase may further comprise the
cofactor of the enzyme, i.e. PLP, and/or a precursor thereof, which
may be a non-phosphate precursor, such as a non-phosphate form of
vitamin B6, and/or a phosphate precursor such as pyridoxine
phosphate (PNP).
[0066] Vitamin B6 exists in different forms, either phosphate or
non-phosphate. Pyridoxine phosphate (PNP), pyridoxal phosphate
(PLP) and pyridoxamine phosphate (PMP) are the phosphate forms
thereof. The corresponding non-phosphate forms are pyridoxine (PN),
pyridoxal (PL), and pyridoxamine (PM). The non-phosphate forms of
vitamin B6 may cross the erythrocyte membrane, which the phosphate
forms can only cross with difficulty. According to the predominant
route, pyridoxine (PN) is transformed inside the erythrocytes into
PNP under the effect of PN-kinase, PNP is then transformed into PLP
under the effect of PNP-oxidase. The PLP may then be transformed
into pyridoxal (PL) under the effect of PLP-phosphatase and the PL
may leave the erythrocytes. It is easily understood that the
provided precursor is able to undergo transformations in the
erythrocytes during the preparation method or during the storage of
the composition.
[0067] By a non-phosphate form of vitamin B6, will be meant here
one of the three "vitamers" of vitamin B6 or a mixture of two or
three vitamers: PL, PN and PM. The PN form is preferred. They may
also be in the form of a salt.
[0068] The composition may comprise PLP encapsulated in
erythrocytes. The PLP may be provided during the encapsulation
procedure or be totally or partly obtained in the erythrocytes from
its precursor. The PLP either present or formed may be associated
with the enzyme. The composition may therefore comprise the
corresponding holoenzyme, for example methioninase-PLP. Under these
conditions, the half-life of the active enzyme, as observed for
example with the duration of the plasma depletion of its substrate,
is considerably increased. The composition according to the
invention notably gives the possibility of preserving enzymatic
activity beyond 24 hours after administration, notably at or beyond
1, 5, 10 or 15 days.
[0069] In an embodiment, the composition of methioninase therefore
comprises pyridoxal phosphate (PLP) and/or a non-phosphate form of
vitamin B6 and/or a phosphate precursor, pyridoxine phosphate (PNP)
and/or pyridoxamine phosphate (PMP).
[0070] According to a feature, PNP and/or PMP is encapsulated
inside the erythrocytes within the composition. This precursor may
be co-encapsulated with the enzyme or be totally or partly obtained
in the erythrocytes from its own precursor.
[0071] The composition notably comprises from about 0.05 to about
600, notably from about 0.5 to about 100, preferably from about 5
to about 50 .mu.moles of PLP and/or PNP and/or PMP, encapsulated
per liter (L) of red blood cells (erythrocytes).
[0072] According to a feature, the composition comprises
erythrocytes encapsulating the PLP enzyme and PLP and further a
non-phosphate PLP precursor, encapsulated in the erythrocytes,
present inside the erythrocytes or present inside and outside the
erythrocytes. This non-phosphate precursor may be PN, PL or PM,
preferably PN, or a mixture of two or three of these compounds. The
non-phosphate precursor may be present inside and/or outside the
erythrocytes. The presence of this non-phosphate precursor gives
the possibility of reaching a remarkably higher intra-erythrocyte
PLP level than in the absence of this non-phosphate precursor.
[0073] In an embodiment, the composition comprises erythrocytes
encapsulating the methioninase and in addition PLP and one of its
phosphate precursors, PNP, PLP and/or PMP. This same composition
may further comprise advantageously a non-phosphate precursor,
notably PN, as this has just been described.
[0074] The composition or suspension advantageously contains an
amount of erythrocytes and an amount of encapsulated methioninase
that is sufficient to deliver to the patient the dose of
methioninase that has been decided.
[0075] The composition may thus further comprise PLP or a PLP
precursor for simultaneous, separate or sequential administration
with the methioninase. In an embodiment, the composition comprises
methioninase encapsulated inside erythrocytes and a non-phosphate
precursor of PLP for separate or sequential administration.
[0076] According to an embodiment, the composition comprises (i) a
formulation of erythrocytes and a pharmaceutically acceptable
vehicle, the erythrocytes encapsulating methioninase, and (2i) a
formulation of vitamin B6 in a non-phosphate form, preferably PN,
and a pharmaceutically acceptable vehicle. These formulations are
for simultaneous, separate or sequential administration, and
dedicated to methionine depletion according to the invention. The
method of use presented thereafter will detail the best modes of
administration. The composition may notably be in the form of a set
or kit, comprising separately these formulations. According to an
embodiment, the pharmaceutically acceptable vehicle in the
formulation of erythrocytes is a preservation solution for
erythrocytes, i.e. a solution in which the erythrocytes
encapsulating an active ingredient are suspended in their suitable
form for being stored while awaiting their injection. A
preservation solution preferably comprises at least one agent
promoting preservation of the erythrocytes, notably selected from
glucose, dextrose, adenine and mannitol. Possibly, the preservation
solution contains inorganic phosphate allowing inhibition of the
intra-erythrocyte PLP-phosphatase enzyme.
[0077] In an embodiment, methioninase encapsulated inside
erythrocytes is to be administered at least once, preferably at
least twice before asparaginase encapsulated inside erythrocytes is
administered, and each methioninase administration is to be
followed by administration of a solution of non-phosphate precursor
of PLP before asparaginase is administered.
[0078] MGL activity is expressed in IU which corresponds to the
amount of MGL required to liberate one micromole of ammonia per
minute under the following conditions.
[0079] In the presence of its cofactor PLP, MGL hydrolyzes
L-methionine into alpha-ketobutyric acid, forming one molecule of
ammonium per molecule of L-methionine:
L-methionine+H.sub.2O.fwdarw.methanthiol+NH.sub.4.sup.++alpha-ketobutyric
acid
[0080] The dosage of MGL activity is performed at 37.degree. C.,
pH=8.6, in presence of 0.26 .mu.g/mL of MGL, 20 nM of PLP and 25 mM
of L-methionine, a commercially available test may be used (e.g.
NH.sub.3 kit, Roche diagnostics).
[0081] The method consists in measuring the kinetics of ammonium
production between 5 min and 10 min of the reaction, when maximum
activity (Vmax) of MGL is reached. The measurement of ammonium
production is obtained by measuring the variation of optical
density at 340 nm due to the oxidation of NADPH to NADP.sup.+ by
the glutamate deshydrogenase (GLDH) in the presence of ammonium and
alpha-ketoglutaric acid, as follows:
Alpha-ketoglutaric acid+NH4.sup.++NADPH.fwdarw.L-glutamic
acid+NADP.sup.++H.sub.2O.
[0082] Asparaginase
[0083] Asparaginase itself is designated by the CAS number:
9015-68-3. Its usual name is asparaginase; other common names for
it are: colaspase, L-asparaginase and L-asparagine
aminohydrolase.
[0084] The term asparaginase in the sense of the present invention
covers asparaginase of any origin, it can in particular be of
natural or recombinant origin, and any derivative incorporating
asparaginase, such as for example a pegylated or PEG form
(PEG-asparaginase), or a fragment retaining the activity of
L-asparaginase. It also covers asparaginase whatever its bacterial
origin. Thus, the asparaginase may be of the E. coli type, in
particular E. coli HAP-A-1-3, of the Erwinia chrysanthemi type or
of the Wolinella succinogenes type. "Type" is understood to mean
that it can be obtained from a culture of the bacterium in question
or that it can be recombinant, in other words a form of
asparaginase of that bacterium obtained by genetic engineering. In
a preferred implementation mode, it is of the E. coli HAP-A-1-3
type.
[0085] Commercial products are available and usable herein: 5000 U
Medac, 10000 U Medac.RTM., Oncaspar.RTM.. The product is under
powder form, to be solubilized before use in an injectable liquid
or water. Excipients may be present, such as sodium
dihydrogenphosphate 1H.sub.2O, sodium monohydrogenphosphate
7H.sub.2O and/or sodium chloride.
[0086] The term asparaginase also covers asparaginase-like
substances which in the sense of the invention are bacterial
enzymes having an L-asparagine aminohydrolase activity. By way of
example, Acinetobacter Glutaminase Asparaginase (AGA) may be
cited.
[0087] According to an embodiment of the invention, asparaginase is
encapsulated into erythrocytes and the composition or suspension
advantageously contains an amount of erythrocytes and an amount of
encapsulated asparaginase that is sufficient to deliver to the
patient the dose of asparaginase that has been decided.
[0088] One IU asparaginase is defined as usual as the quantity of
enzyme required to liberate 1 .mu.mol ammonia per minute at pH 7.3
and 37.degree. C. from L-asparagine, the quantity of L-asparaginase
being in excess.
[0089] Encapsulation into Erythrocytes
[0090] According to an embodiment, the composition of methioninase
and/or the composition of asparaginase comprises erythrocytes
encapsulating the enzyme and a pharmaceutically acceptable vehicle.
Preferably, the erythrocytes are issued from a mammal of the same
species than the treated subject. When the mammal is a human, the
erythrocytes are preferably of human origin. In an embodiment, the
erythrocytes come from the patient itself.
[0091] According to an embodiment, the pharmaceutically acceptable
vehicle is a preservation solution for erythrocytes, i.e. a
solution in which the erythrocytes encapsulating the enzyme are
suspended in their suitable form for being stored while awaiting
their injection.
[0092] A preservation solution preferably comprises at least one
agent promoting preservation of the erythrocytes, notably selected
from glucose, dextrose, adenine and mannitol.
[0093] The preservation solution may be an aqueous solution
comprising NaCl, adenine and at least one compound from among
glucose, dextrose and mannitol.
[0094] The preservation solution may comprise NaCl, adenine and
dextrose, preferably an AS3 medium.
[0095] The preservation solution may comprise NaCl, adenine,
glucose and mannitol, preferably a SAG-Mannitol or ADsol
medium.
[0096] In particular, the composition or suspension, in a
preservation solution, is characterized by an extracellular
hemoglobin level maintained at a level equal to or less than 0.5,
in particular 0.3, notably 0.2, preferably 0.15, even better 0.1
g/dl at 72 h and preservation at a temperature comprised between 2
and 8.degree. C.
[0097] In particular, the composition or suspension, in a
preservation solution, is characterized by an extracellular
hemoglobin level maintained at a level equal to or less than 0.5,
in particular 0.3, notably 0.2, preferably 0.15, even better 0.1
g/dl for a period comprised between 24 h and 20 days, notably
between 24 and 72 h and preservation at a temperature comprised
between 2 and 8.degree. C.
[0098] The extracellular hemoglobin level is advantageously
measured by the manual reference method described in G. B. Blakney
and A. J. Dinwoodie, Clin. Biochem. 8, 96-102, 1975. Automatic
devices also exist which allows this measurement to be made with a
sensitivity which is specific to them.
[0099] In particular, the composition or suspension, in a
preservation solution, is characterized by a hemolysis rate
maintained at equal to or less than 2, notably 1.5, preferably 1%
at 72 h and preservation at a temperature comprised between 2 and
8.degree. C.
[0100] In particular, the composition or suspension, in a
preservation solution, is characterized by a hemolysis rate
maintained at equal to or less than 2, notably 1.5, preferably 1%
for a period comprised between 24 h and 20 days, notably between 24
and 72 h and at a temperature comprised between 2 and 8.degree.
C.
[0101] Methods of Encapsulation
[0102] Encapsulating the enzymes into erythrocytes may be performed
using an erythrocyte suspension that is put into contact with a
hypotonic liquid medium resulting in the opening of pores in the
erythrocyte membrane. There exist three alternatives in the
lysis-resealing technique, which are hypotonic dialysis, hypotonic
preswelling and hypotonic dilution, all based on the difference in
osmotic pressure between the inside and the outside of the
erythrocytes. Hypotonic dialysis is preferred.
[0103] The suspension of erythrocytes encapsulating the enzyme is
notably able to be obtained with the following method:
[0104] 1--suspending a pellet of erythrocytes in an isotonic
solution at a hematocrit level equal to or greater than 65%,
cooling between +1 and +8.degree. C.,
[0105] 2--a lysis procedure, at a temperature maintained between +1
and +8.degree. C., comprising the passing of the suspension of
erythrocytes at a hematocrit level equal or greater than 65% and of
a cooled hypotonic lysis solution between +1 and +8.degree. C.,
into a dialysis device, such as a coil or a dialysis cartridge (the
cartridge is preferred);
[0106] 3--an encapsulation procedure by adding, preferably
gradually, the enzyme to be encapsulated (notably in a solution
made up beforehand) into the suspension before or during lysis, at
a temperature maintained between +1 and +8.degree. C.; and
[0107] 4--a resealing procedure conducted in the presence of an
isotonic or hypertonic, advantageously hypertonic solution, at a
higher temperature, notably comprised between +30 and +42.degree.
C.
[0108] In a preferred alternative, use may be done of the method
described in WO-A-2006/016247 (EP 1 773 452; which is incorporated
herein by reference.):
[0109] 1--suspending a pellet of erythrocytes in an isotonic
solution at a hematocrit level equal to or greater than 65%,
cooling between +1 and +8.degree. C.,
[0110] 2--measuring osmotic fragility from a sample of erythrocytes
from this same pellet,
[0111] 3--a lysis procedure, at a temperature maintained between +1
and +8.degree. C., comprising the passing of the suspension of
erythrocytes at a hematocrit level equal to or greater than 65% and
of a hypotonic lysis solution cooled between +1 and +8.degree. C.,
into a dialysis device, such as a coil or a dialysis cartridge (the
cartridge is preferred); the lysis parameters being adjusted
according to the osmotic fragility measured earlier; notably,
depending on the measured osmotic fragility, the flow of the
erythrocyte suspension passing into the dialysis device is adjusted
or the osmolarity of the lysis solution is adjusted; and
[0112] 4--a procedure for encapsulation by adding, preferably
gradually, the enzyme to be encapsulated (notably in a solution
made beforehand) in the suspension before and during lysis, at a
temperature maintained between +1 and +8.degree. C.; and
[0113] 5--a resealing procedure conducted in the presence of an
isotonic or hypertonic, advantageously hypertonic solution, at a
higher temperature, notably comprised between +30 and +42.degree.
C.
[0114] Notably, for dialysis, the pellet of erythrocytes is
suspended in an isotonic solution with a high hematocrit level,
equal to or greater than 65%, and preferably equal to or greater
than 70%, and this suspension is cooled between +1 and +8.degree.
C., preferably between +2 and +6.degree. C., typically around
+4.degree. C. According to a particular method, the hematocrit
level is comprised between 65 and 80%, preferably between 70 and
80%.
[0115] When it is measured, the osmotic fragility is advantageously
measured on erythrocytes just before the lysis step, in the
presence or in the absence, preferably in the presence of the
enzyme to be encapsulated. The erythrocytes or the suspension
containing them are advantageously at a temperature close to, or
identical with the temperature selected for lysis. According to
another advantageous feature of the invention, the conducted
measurement of the osmotic fragility is rapidly utilized, i.e. the
lysis procedure is carried out in a short time after taking the
sample. Preferably, this lapse of time between the sampling and
beginning of lysis is less than or equal to 30 minutes, still
better less than or equal to 25 and even to 20 minutes.
[0116] As regards to how to conduct the lysis-resealing procedure
with measurement and taking into account of the osmotic fragility,
one skilled in the art may refer for more details to
WO-A-2006/016247. This document is incorporated herein by
reference.
[0117] An improvement of this encapsulation technique was described
in WO 2014/180897, to which one skilled in the art may refer and
which is incorporated herein by reference. Thus, according to an
embodiment, the erythrocytes encapsulating the enzyme, are obtained
by a method comprising the encapsulation of the active ingredient
inside erythrocytes by lysis-resealing, the obtaining of a
suspension or of a pellet comprising erythrocytes incorporating the
enzyme and a solution with an osmolality greater than or equal to
280 mOsmol/kg, in particular between about 280 and about 380
mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg,
the incubation of the pellet or of the suspension as such or after
adding an incubation solution, at an osmolality greater than or
equal to 280 mOsmol/kg, in particular between about 280 and about
380 mOsmol/kg, preferably between about 290 and about 330
mOsmol/kg. Incubation is notably carried out for a period greater
than or equal to 30 minutes, in particular greater than or equal to
1 h. It is then proceeded with removal of the liquid medium of the
incubated solution and the erythrocytes obtained are suspended in a
solution allowing injection of the suspension into a patient,
preferably a preservation solution allowing injection of the
suspension into a patient. The indicated osmolality is that of the
solution in which the erythrocytes are suspended or in a pellet at
the relevant moment.
[0118] By stabilized erythrocyte suspension , is notably meant a
suspension having an extracellular hemoglobin content which remains
less than or equal to 0.2 g/dl until its use in humans, the latter
may intervene notably from 1 to 72 hours after producing the
erythrocyte batch incorporating the active ingredient.
[0119] By ready-to-use stabilized erythrocyte suspension , is meant
the stabilized suspension in a solution allowing injection into a
patient, notably in a preservation solution. Its hematocrit is
generally equal to or greater than 35%, 40% or 45%.
[0120] By erythrocyte pellet , is meant a concentrate or
concentration of erythrocytes collected after separating the
erythrocytes of the liquid medium in which they were suspended
previously. The separation may be ensured by filtration or by
centrifugation. Centrifugation is the means generally used for such
a separation. A pellet comprises a certain proportion of liquid
medium. Generally, the pellet has a hematocrit comprised between 70
and 85%.
[0121] By incubation solution , is meant the solution in which the
erythrocytes encapsulating an active ingredient are present during
the incubation step. The incubation may be accomplished over a
large range of hematocrits, notably between 10 and 85% of
hematocrit.
[0122] By fragile erythrocytes , are meant the erythrocytes
stemming from the incorporation procedure which may, once suspended
in a preservation solution, be lyzed when the suspension is
preserved between 2 and 8.degree. C., notably after 1 to 72 h.
[0123] By initial hematocrit , is meant the hematocrit before cell
loss due to lysis of the fragile erythrocytes during
incubation.
[0124] The method may notably comprise the following steps:
[0125] (a) encapsulation of the enzyme inside erythrocytes,
comprising the putting of the erythrocytes into contact with a
hypotonic medium (allowing opening of pores in the membrane of the
erythrocytes), the contacting with the active ingredient (for
allowing it to enter the erythrocytes), the resealing of the
erythrocytes, notably by means of an isotonic or hypertonic medium,
advantageously hypertonic,
[0126] (b) obtaining or preparing a suspension or pellet comprising
erythrocytes incorporating the enzyme and a solution with an
osmolality greater than or equal to 280 mOsmol/kg, in particular
between about 280 and about 380 mOsmol/kg, preferably between about
290 and about 330 mOsmol/kg,
[0127] (c) incubating the pellet or the suspension of step (b) as
such or after adding an incubation solution, at an osmolality
greater than or equal to 280 mOsmol/kg, in particular between about
280 and about 380 mOsmol/kg, preferably between about 290 and about
330 mOsmol/kg, for a period greater than or equal to 30 minutes,
notably greater than or equal to 1 h,
[0128] (d) removing the liquid medium of the incubated suspension
of step (c),
[0129] (e) suspending the erythrocytes obtained under (d) into a
solution allowing injection of the suspension into a patient,
preferably a preservation solution allowing injection of the
suspension into a patient.
[0130] According to a first method, the step following the
encapsulation by lysis-resealing, notably step (b), includes at
least 1 washing cycle, preferably 2 or 3 washing cycles, by
dilution of the obtained suspension or pellet in the
lysis-resealing step or step (a) in a solution, at an osmolality
greater than equal to 280 mOsmol/kg, in particular between about
280 and about 380 mOsmol/kg, preferably between about 290 and about
330 mOsmol/kg, and then obtaining a pellet of erythrocytes or a
suspension. This pellet or this suspension comprises erythrocytes
incorporating the enzyme and a solution with an osmolality greater
than or equal to 280 mOsmol/kg, in particular between about 280 and
about 380 mOsmol/kg, preferably between about 290 and about 330
mOsmol/kg. The following steps, e.g. (c), (d) and (e) are then
applied.
[0131] According to a second method, in the lysis-resealing step or
step (a), resealing of the erythrocytes by means of an isotonic or
hypertonic medium produces the suspension of erythrocytes which may
then be subject to incubation, e.g. the suspension of step (b), in
a solution with an osmolality greater than or equal to 280
mOsmol/kg, in particular between about 280 and about 380 mOsmol/kg,
preferably between about 290 and about 330 mOsmol/kg. In other
words, the lysis-resealing step or step (a) includes a step for
resealing the erythrocytes wherein the suspended erythrocytes
encapsulating the enzyme are mixed with an isotonic or hypertonic
resealing solution, advantageously hypertonic, producing a
suspension of erythrocytes with an osmolality greater than or equal
to 280 mOsmol/kg, in particular between about 280 and about 380
mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg. In
this method, the incubation step or step (c) comprises incubation
of the suspension stemming from the resealing. The incubation is
carried out for a period greater than or equal to 30 minutes,
notably greater than or equal to 1 h. The following steps, e.g. (d)
and (e) are then applied.
[0132] The steps following the lysis-resealing, e.g. (b) to (e),
are conducted under conditions resulting in the lysis of fragile
erythrocytes, or of a majority of them, notably more than 50, 60,
70, 80 or 90%, or more. To do this, it is possible to act on the
incubation period, the incubation temperature and on the osmolality
of the solution in which the erythrocytes are suspended. The higher
the osmolality, the longer the incubation time may be. Thus the
lower the osmolality, the shorter may be the incubation in order to
obtain the same effect. Also, the higher the temperature, the
shorter the incubation time may be, and vice versa. One or several
washing cycles will then allow removal of cell debris and
extracellular hemoglobin, as well as the extracellular enzyme.
[0133] According to the invention, a washing cycle comprises the
dilution of the suspension or pellet of erythrocytes, and then the
separation between the erythrocytes and the washing solution.
Preferably, a washing step comprises preferably 2 or 3
dilution-separation cycles. The separation may be achieved by any
suitable means, such as filtration and centrifugation.
Centrifugation is preferred.
[0134] Incubation is not limited by the hematocrit of the
suspension. In this way, a suspension having an initial hematocrit
generally comprised between 10 and 85%, notably between 40 and 80%
may be incubated. This is rather referred to as a pellet from 70%
and as a suspension below this value.
[0135] The removal step or step (d) aims at removing the liquid
portion of the suspension or of the incubated pellet, in order to
notably remove cell debris and the extracellular hemoglobin, as
well as consequently the extracellular enzyme.
[0136] According to a first method for the removal step or step
(d), separation, notably centrifugation is carried out, this being
notably applicable to a suspension. This separation may be followed
by one or several, for example 2 or 3, washing cycles, by dilution
in an isotonic solution, and then separation, notably by
centrifugation.
[0137] According to a second method for the removal step or step
(d), dilution before separation notably centrifugation is carried
out, this being applicable to a suspension or to a pellet. The
dilution may notably be carried out with an isotonic washing
solution or with a preservation solution.
[0138] The final step or step (e) consists of preparing the final
suspension such that it may be administered to the patient, without
any other treatment.
[0139] According to a first method for this step, a dilution of the
erythrocyte pellet from the removal step or step (d) is carried out
with the injection solution, notably the preservation solution.
[0140] According to a second method for this step, one or several
cycles for washing the erythrocyte pellet stemming from the removal
step or step (d) is carried out with the injection solution,
notably the preservation solution, by dilution followed by
separation. After washing, the erythrocytes are re-suspended in the
injection solution, notably the preservation solution.
[0141] The method of the invention may further comprise one,
several or the totality of the following features: [0142] the
incubation step or step (c) is carried out at a temperature
comprised between about 2 and about 39.degree. C., over sufficient
time for ensuring lysis of fragile erythrocytes; [0143] the
incubation step or step (c) is carried out at a low temperature,
notably comprised between about 2 and about 10.degree. C., in
particular between about 2 and about 8.degree. C., and lasts for
about 1 h to about 72 h, notably from about 6 h to about 48 h,
preferably from about 19 h to about 30 h; [0144] the incubation
step or step (c) is conducted at a higher temperature comprised
between about 20 and about 39.degree. C., notably at room
temperature (25.degree. C..+-.5.degree. C.) and lasts for about 30
min to about 10 h, notably from about 1 h to about 6 h, preferably
from about 2 h to about 4 h; it is possible to operate at an even
higher temperature than room temperature, but this may have a
negative impact on the cell yield, P50 and/or the 2,3-DPG content;
[0145] in the incubation step or step (c), the suspension is at an
initial hematocrit comprised between 10 and 85%, notably between 40
and 80%; a pellet from separation, having for example a hematocrit
between 70 and about 85%, or a diluted pellet having a hematocrit
comprised between about 40 and 70% may be incubated; [0146] the
incubation step comprises stirring of the suspension; [0147] the
incubation step does not comprise any stirring; [0148] as a
solution for washing and/or incubation, a metered aqueous NaCl
solution is used for obtaining the desired osmolality; as an
example, a solution may thus comprise 0.9% of NaCl; this solution
may also comprise, notably in addition to NaCl, glucose, notably
glucose monohydrate, monosodium phosphate dihydrate, disodium
phosphate dodecahydrate; as an example, a composition comprises:
0.9% of NaCl, 0.2% of glucose monohydrate, 0.034% of monosodium
phosphate dihydrate, 0.2% of disodium phosphate dodecahydrate;
[0149] the washing in the final step or step (e) is carried out
with the preservation solution; [0150] the osmolality of the
solution (liquid portion) in the ready-to-use suspension or which
may be injected into the patient is comprised between about 280 and
about 380 mOsmol/kg, preferably between about 290 and about 330
mOsmol/kg; [0151] the hematocrit of the ready-to-use suspension or
which may be injected into the patient is equal to or greater than
35%, 40% or 45%; [0152] all the steps for washing, incubation are
carried out with the preservation solution; [0153] the washing
solution of step (b) and/or the washing solution of step (e) and
the preservation solution are of the same composition and comprise
compound(s) promoting preservation of the erythrocytes; [0154] the
preservation solution (and the washing solution(s) or the
incubation solutions if necessary) is an aqueous solution
comprising NaCl, adenine and at least one compound from among
glucose, dextrose and mannitol; [0155] the preservation solution
(and the washing or incubation solution(s) if necessary) comprises
NaCl, adenine and dextrose, preferably an AS3 medium; [0156] the
preservation solution (and the washing or incubation solution(s),
if necessary) comprise NaCl, adenine, glucose and mannitol,
preferably a SAG-Mannitol or ADsol medium.
[0157] The methods according to the invention notably comprise the
following step:
[0158] (a) encapsulating the enzyme inside erythrocytes, comprising
the contacting with a hypotonic medium allowing opening of pores in
the membrane of the erythrocytes, the contacting with the enzyme in
order to allow its entry into the erythrocytes, the resealing of
the erythrocytes by means of an isotonic or hypertonic medium. It
should be noted that the enzyme may be present in the suspension of
erythrocytes before the lysis of the latter, or further be added
during lysis or after lysis, but always before resealing. In an
embodiment of this step (a), the method comprises the following
sub-steps:
[0159] (a1) having a suspension of erythrocytes at a hematocrit
equal to or greater than 60 or 65%,
[0160] (a2) measuring the osmotic fragility of the erythrocytes in
this suspension,
[0161] (a3) a procedure for lysis and internalization of the active
ingredient(s), comprising the passing of the erythrocyte suspension
into a dialysis device, notably a dialysis cartridge, counter to a
lysis solution, adjusting the flow of the erythrocyte suspension or
adjusting the flow rate of the lysis solution or adjusting the
osmolarity of the lysis solution, depending on the osmotic
fragility measured under (a2),
[0162] (a4) a procedure for resealing the erythrocytes.
[0163] Methods of Use
[0164] In a first aspect, the invention is a method for treating
cancer in a mammal in need thereof, the method comprising depriving
the mammal for methionine, then depriving the mammal for
asparagine, especially through administering asparaginase in
sufficient amount. What is searched for is to reduce the amount of
methionine and asparagine available to the cancer cells. Methionine
deprivation may be performed as mentioned above through dietary
methionine deprivation and/or methioninase administration.
[0165] In a second aspect, the invention is a method for treating
cancer in a mammal in need thereof, the method comprising
administering, especially injecting, to the mammal in need thereof,
a composition comprising methioninase and then a composition
containing asparaginase.
[0166] Sequential administration, delay between methionine
deprivation and/or methioninase administration, and asparaginase
administration, dosages, repeated administrations and forms of
pharmaceutical compositions (free form, pegylated form and/or
suspension of erythrocytes (RBCs) encapsulating the enzyme) has
been detailed above and apply to the methods of use.
[0167] In an embodiment, methioninase (e.g. under free form,
pegylated form or encapsulated) is administered once or more.
[0168] In another embodiment, free or pegylated methioninase is
administered more than once before asparaginase administration, for
example two or more (e.g. 3, 4, 5) doses of methioninase are
administered to the mammal, typically at different days, e.g.
daily.
[0169] In an embodiment, an effective amount of the cofactor of
methioninase is administered to the patient. It may be administered
before, at the same time or after the administration of
methioninase. In an embodiment it is present in the same
composition than methioninase. In another embodiment, it is
administered in a separate composition.
[0170] In an embodiment, administration of methioninase
encapsulated into erythrocytes is performed, and cofactor may be
encapsulated as well or the cofactor may be in free form in a
solution. In a preferred embodiment, the cofactor is in solution in
a pharmaceutically acceptable vehicle and is a non-phosphate form
of vitamin B6, preferably PN. This solution of non-phosphate form
of vitamin B6 may be administered by injection or oral route, or
via any other route. In an embodiment, the solution is administered
once or more after each injection of encapsulated methioninase, for
example between 1 and 10 hours after. Preferably, the solution is
administered advantageously once a day, or else twice or more per
day, during the time of methioninase treatment or duration of
methioninase activity in blood circulation (depending on the
half-life thereof). With methioninase encapsulated inside
erythrocytes, the cofactor in solution may be administered at least
once a day during between 10 and 30 days.
[0171] In an embodiment, asparaginase under free form, pegylated
form or encapsulated is administered once or more.
[0172] In another embodiment, free or pegylated asparaginase is
administered more than once, for example two or more (e.g. 3, 4, 5)
doses of asparaginase are administered to the mammal, typically at
different days, e.g. daily.
[0173] In an embodiment, the methioninase and/or asparaginase is
under powder form and the method of use comprises the
solubilization thereof in a pharmaceutically acceptable solution or
liquid before administering to the mammal.
[0174] In an embodiment, use is made of a device as described
above. Thus the method of cancer treatment comprises the
implantation or placing on the mammal, especially human, a device
as described. The implantation or placing may comprise the
connection of the tubes to a blood vessel or to a catheter and the
like that is already in place. The method may then comprise
starting the device for its sequential delivery according to a
programming of its software in accordance with the method of the
invention.
[0175] Advantageously, the suspension of erythrocytes encapsulating
methioninase or asparaginase in preservation solution is ready to
use, and preferably may have a low extracellular haemoglobin level,
conforming in particular to FDA recommendations.
[0176] In a first embodiment, the injection is given to a mammal,
especially a human patient of a suspension of RBCs encapsulating
the active ingredient prepared between 1 and 72 h, in particular
between 10 and 72 h before injection. The haematocrit of this
suspension is 40% or higher. It is contained in a preservation
solution. The extracellular haemoglobin level is 0.5 or lower, in
particular 0.3 or lower, more particularly 0.2 or lower, preferably
0.15 or lower, further preferably 0.1 g/dl or lower, and/or the
haemolysis rate is 2 or lower, in particular 1.5 or lower,
preferably 1% or lower. The suspension is not subjected to washing
or similar before injection.
[0177] In another embodiment, this method comprises the steps of
providing packed red blood cells, placing it in suspension in
physiological buffer at a haematocrit of 60 or 65% or higher,
encapsulating the active ingredient in these RBCs using lysis and
resealing procedure, incubating the RBCs obtained, washing the
latter and collecting a final suspension of RBCs. The haematocrit
of the suspension is 40% or higher. It is contained in a
preservation solution. This suspension is stored at a temperature
between 2 and 8.degree. C. This final suspension is injected in the
mammal, especially a human patient between 1 h and 72 h preferably
between 24 and 72 h after preparation of the suspension. The
extracellular haemoglobin level of this suspension is 0.5 or lower,
in particular 0.3 or lower, more particularly 0.2 or lower,
preferably 0.15 or lower, further preferably 0.1 g/dl or lower
and/or its haemolysis rate is 2 or lower, in particular 1.5, or
lower preferably 1% or lower. The suspension is not subjected to
washing or similar before injection.
[0178] Compositions, kits and methods aim at treating liquid
(hematologival) and solid tumors auxotrophic for asparagine and/or
methioninase. As example leukemia (acute myeloid leukemia, acute
promyelocytic leukemia) and gastric cancer (carcinoma stage IV,
adenocarcinoma) may be cited.
[0179] The invention will now be described in further detail using
the following non-limiting embodiments.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0180] FIG. 1 is a graph showing % cell viability under different
conditions of treatment.
[0181] FIGS. 2 and 3 are graphs showing % cell viability under
different conditions of treatment.
[0182] FIG. 4 is a graph showing individual tumor volume with
median in function of time.
EXAMPLE 1
I. Abbreviations
[0183] CCK-8: Cell counting kit-8
DPBS: Dulbecco's Phosphate-Buffered Saline
IMDM: Iscove's Modified Dulbecco's
[0184] MGL: Methionine-.gamma.-lyase v/v: Volume to volume
II. Operating Conditions
II.1 Test Item
II.1.1. L-Asparaginase
Description: Medac.RTM. (Germany), E. Coli L-asparaginase 10 000
IU
[0185] One concentration of L-asparaginase (2.53 IU/mL) was
prepared by serial dilutions in Dulbecco Phosphate Buffered Saline
(DPBS) 1.times.. Concentration of L-asparaginase was diluted
11-fold to obtain final concentration of 0.23 IU/mL (IC50).
II.1.2. Methionine-.gamma.-Lyase (MGL)
[0186] Description: P. Putida methionine-.gamma.-lyase (MGL)
produced in E Coli. One concentration of MGL (2.09 IU/mL) was
prepared by serial dilutions in Dulbecco Phosphate Buffered Saline
(DPBS) 1.times.. Concentration of MGL was diluted 11-fold to obtain
final concentration of 0.19 IU/mL (IC50).
II.2 Cell Lines
II.2.1. Description
[0187] Name: HL-60 cell line Description: Human promyelocytic
leukemia cell line (suspension) Supplier and reference number:
ATCC, CCL-240
II.2.2. Culture Conditions
[0188] Cells were cultivated in a IMDM with L-glutamine medium and
supplemented with 20% (v/v) of foetal bovine serum, 100 IU/mL of
penicillin and 100 .mu.g/mL of streptomycin. Subculturing was
performed according to PO-CELL-002 and PO-CELL-005.
II.2.3. Colorimetric Kit
Name: Cell Counting Kit-8 (CCK-8)
[0189] Supplier and reference number: Fluke 96992 Principle: the
CCK-8 reagent contains a highly water-soluble tetrazolium salt
WST-8. WST-8 is reduced by dehydrogenases in cells to give a yellow
colored product (formazan) which is soluble in the tissue culture
medium. The amount of the formazan dye generated by the activity of
dehydrogenases in cells is directly proportional to the number of
living cells.
[0190] The colorimetric test was performed according to
PO-CELL-004.
III. Cytotoxicity Assay
III.1 Method
[0191] Fifteen thousand cells in 100 .mu.L/well were dispensed in
five 96-well flat bottom plates. In addition, 2 wells were filled
with culture medium for blank control on each plate. All empty
wells were filled with culture medium in order to minimize
evaporation and condensation. On day 0 (D0), 10 .mu.L of IC50
concentrations of L-asparaginase or MGL was added to the
corresponding wells. Controls (blank wells and control plate)
received 10 .mu.L of DPBS 1.times.. On day 3 (D3), medium was
removed from wells and replaced by fresh medium and 10 .mu.L of
DPBS 1.times. or 10 .mu.L of IC50 concentrations of L-asparaginase
(for cells previously incubated with MGL) or MGL (for cells
previously incubated with L-asparaginase) added to the
corresponding wells. Controls (blank and positive control) received
10 .mu.L of DPBS 1.times.. Then, plates were incubated for 3 more
days in the incubator. At the end of the incubation period (D6), 10
.mu.L of CCK-8 solution were added to each well according to
PO-CELL-004 and plates incubated for 2 hours in the incubator.
Optical density (OD) was then determined at 450 nm using a
microplate reader.
III.2 Internal Controls
[0192] Controls were performed in duplicate.
III.2.1. Blank Wells
[0193] Slight spontaneous absorbance around 460 nm occurs in
culture medium with CCK-8. This background absorbance depends on
the culture medium, pH, incubation time and length of exposure to
light. Therefore blank wells were performed containing 100 .mu.L of
culture medium and 10 .mu.L of L-asparaginase or MGL diluent, DPBS
1.times.. The average absorbance of these control wells was
subtracted to the others wells containing cells.
III.2.2. Viability Control (Positive Control)
[0194] As positive control for the HL-60 cell line (100% cell
viability), cells were cultivated in the culture medium (100 .mu.L)
without L-asparaginase nor MGL, but with 10 .mu.L of the diluent
(DPBS 1.times.).
III.3 Determination of Cell Viability
[0195] Culture medium without cells constituted blank controls (OD
Blank). Cells without L-asparaginase nor MGL constituted positive
controls (viability control).
[0196] Percentage of living cells was calculated as shown
below:
O .times. D L - aspa + MGL * - OD Blank OD viability - control ** -
O .times. D Blank .times. 100 ##EQU00001##
*: cells with L-asparaginase and MGL treatment **: cells without
L-asparaginase and MGL treatment [0197] Calculations were
automatically performed via the Gen 5 software that pilots the
microplate reader. The mean optical density (OD) of the 2 blank
wells was automatically subtracted from all optical densities.
Calculations of cell viability were realized for sequential
treatment.
IV. Results
IV.1 Internal Control
[0198] Internal controls were acceptable when it was not specified
in raw data.
IV.2 IC50 Calculations with L-Asparaginase or MGL Alone
[0199] Percentages of cell viability with drug alone (MGL or
L-asparaginase) were controlled in each experiment of drugs
combination
IV.2.1. Sequential Addition of L-Asparaginase and MGL
[0200] The experiment with sequential treatment of L-asparaginase
and MGL was done once with duplicate data. All quality controls
(blank and positive control) were accepted in experiments. Details
of % of cell viability calculations and graphical representation
are presented below in table 1 and FIG. 1.
TABLE-US-00001 TABLE 1 % of cell viability for controls and enzyme
association % cell viability at D6 Mean SD Cells alone 100 25 Cells
+ IC50 L-aspa D0 34 0 Cells + IC50 MGL D0 27 8 Cells + IC50 L-aspa
D0 + IC50 32 15 MGL D3 Cells + IC50 MGL D0 + IC50 L- 8 2 aspa
D3
[0201] Results indicated that enzyme association with MGL added at
IC50 dose before the addition of L-asparaginase at IC50 dose (in
red on FIG. 1) permitted to reduce cell viability of: [0202] 76%
compared to IC50 L-asparaginase (IC50 control for L-asparaginase),
[0203] 70% compared to MGL (IC50 control for MGL), [0204] 75%
compared to enzyme association with L-asparaginase added in first
at IC50 dose.
[0205] Yet, the reverse order of enzyme association did not give
such results, with no benefits of the association on cell viability
compared to enzymes alone (controls).
V. Conclusion
[0206] Sequential enzyme association demonstrated that cell
mortality could be increased with an addition of MGL at IC50 dose
followed 3 days later by the addition of L-asparaginase at IC50
dose. Yet, the reverse design of enzyme addition did not permit to
obtain such results.
[0207] We can hypothesize that Met deprivation induced by MGL
enzyme activity makes HL-60 leukemia cells more sensitive to
L-asparaginase activity. Moreover, the roles of L-asparaginase and
MGL have to be discussed considering their known respective effect.
Indeed, L-asparaginase is known to trigger apoptosis in leukaemia
cells (Ueno et al., 1997), therefore, it could probably plays a
role of cytotoxic agent. MGL being known for blocking cell division
in S or G2 phase of the cell cycle probably acts more as a
cytostatic agent.
EXAMPLE 2: METHOD FOR ENCAPSULATION OF L-ASPARAGINASE IN MURINE
ERYTHROCYTES
[0208] The L-asparaginase (Medac.RTM. E. Coli L-asparaginase) is
encapsulated in murine erythrocytes (OF1 mice) by the method of
hypotonic dialysis in a dialysis bag. The blood is centrifuged
beforehand to remove the plasma, and then washed three times with
0.9% NaCl. The haematocrit is adjusted to 70% in the presence of
the asparaginase, added to a final concentration of 400 IU/ml of
erythrocytes or red blood cells (RBC) before starting the dialysis.
The dialysis lasts 50 minutes at 4.degree. C. against a lysis
buffer of low osmolarity. The murine erythrocytes are then resealed
through the addition of a high osmolarity solution and incubating
30 minutes at 37.degree. C. After two washings with 0.9% NaCl and
one washing with Sag-mannitol supplemented with bovine serum
albumin BSA (6%), the erythrocytes are adjusted to haematocrit 50%.
The erythrocytes encapsulating the L-asparaginase are called L-Aspa
RBC. The encapsulation generates L-Aspa RBC at a concentration of
40 IU of asparaginase/ml of RC at 50% haematocrit.
[0209] During the encapsulation procedure, the whole blood, the
washed RBC, the RBC mixed with the L-asparaginase (before dialysis)
and the RBC loaded with L-asparaginase (after dialysis) are tested
for: [0210] haematocrit (Ht) [0211] average corpuscular volume
(ACV) [0212] average corpuscular haemoglobin concentration (ACHC)
[0213] total haemoglobin concentration and [0214] cell count.
[0215] Aliquots of the cell suspensions are withdrawn before and
after the hypotonic dialysis for measurement of the L-asparaginase
enzyme activity. The estimation of the L-asparaginase was performed
according to the protocol published in: Orsonneau et al., Ann Biol
Clin, 62: 568-572.
EXAMPLE 3: ENCAPSULATION OF L-ASPARAGINASE IN HUMAN
ERYTHROCYTES
[0216] The method described in WO-A-2006/016247 is used to produce
a batch of erythrocytes encapsulating L-asparaginase. In accordance
with the teaching of WO-A-2006/016247, the osmotic fragility is
considered and the lysis parameters are adjusted accordingly (flow
rate of the erythrocyte suspension in the dialysis cartridge is
adjusted). The method is further performed in conformity with the
physician prescription, which takes into account the weight of the
patient and the dose of L-asparaginase to be administered. The
specifications of the end product are as follows: [0217] mean
corpuscular volume (MCV): 70-95 fL [0218] mean corpuscular
haemoglobin concentration (MCHC): 23-35 g/dL [0219] extracellular
haemoglobin.ltoreq.0.2 g/dL of suspension [0220] osmotic
fragility.ltoreq.6 g/L of NaCl [0221] mean corpuscular
L-asparaginase concentration: 78-146 IU/mL [0222] extracellular
L-asparaginase.ltoreq.2% of the total enzyme activity.
[0223] The suspension of erythrocytes so obtained is called
GRASPA.RTM. and is mentioned in the literature.
EXAMPLE 4. METHOD FOR OBTAINING AND CHARACTERIZING METHIONINE GAMMA
LYASE (MGL)
[0224] Production of the strain and isolation of a hyper-producing
clone: the natural sequence of MGL of Pseudomonas putida (GenBank:
D88554.1) was optimized by modifying rare codons (in order to adapt
the sequence stemming from P. putida to the production strain
Escherichia coli). Other changes have been made to improve the
context of translation initiation. Finally, silent mutations were
performed to remove three elements that are part of a putative
bacterial promoter in the coding sequence (box -35, box -10 and a
binding site of a transcription factor in position 56). The
production strain E. coli HMS174 (DE3) was transformed with the
expression vector pGTPc502_MGL (promoter T7) containing the
optimized sequence and a producing clone was selected. The
producing clone is pre-cultivated in a GY medium+0.5%
glucose+kanamycin for 6-8 h (pre-culture 1) and 16 h (pre-culture
2) at 37.degree. C.
[0225] Fermentation: the production is then achieved in a fermenter
with GY medium, with stirring, controlled pressure and pH from the
pre-culture 2 at an optical density of 0.02. The growth phase (at
37.degree. C.) takes place until an optical density of 10 is
obtained and the expression induction is achieved at 28.degree. C.
by adding 1 mM IPTG into the culture medium. the cell sediment is
harvested 20 h after induction in two phases: the cell broth is
concentrated 5-10 times after passing over a 500 kDa hollow fiber
and then cell pellet is recovered by centrifugation at
15900.times.g and then stored at -20.degree. C.
[0226] Purification: The cell pellet is thawed and suspended in
lysis buffer (7v/w). Lysis is performed at 10.degree. C. in three
steps by high pressure homogenization (one step at 1000 bars, and
then two steps at 600 bars). The cell lysate then undergoes
clarification at 10.degree. C. by adding 0.2% PEI and
centrifugation at 15900.times.g. The soluble fraction is then
sterilized by 0.2 .mu.m before precipitation with ammonium sulfate
(60% saturation) at 6.degree. C., over 20 h. Two crystallization
steps are carried out on the re-solubilized sediment using
solubilization buffer, the first crystallization step is realized
by addition of PEG-6000 at 10% (final concentration) and ammonium
sulfate at 10% saturation, and the second crystallization is then
performed by addition of PEG-6000 at 12% final concentration and
0.2M NaCl (final concentration) at 30.degree. C. The pellets
containing the MGL protein are harvested at each stage after
centrifugation at 15900.times.g. The pellet containing the MGL
protein is re-suspended in a solubilization buffer and passed over
a 0.45 .mu.m filter before being subject to two anion exchange
chromatographies (DEAE sepharose FF). The purified protein is then
subject to a polishing step and passed over a Q membrane
chromatography capsule for removing the different contaminants
(endotoxins, HCP host cell protein, residual DNA). Finally, the
purified MGL protein is concentrated at 40 mg/ml and diafiltered in
formulation buffer using a 10 kDa cut-off tangential flow
filtration cassette. Substance is then aliquoted at .about.50 mg of
protein per vial, eventually freeze-dried under controlled pressure
and temperature, and stored at .about.80.degree. C.
[0227] Characterization: The specific activity of the enzyme is
determined by measuring the produced NH.sub.3 as described in WO
2015/121348. The purity is determined by SDS-PAGE. The PLP level
after being taken up in water was evaluated according to the method
described in WO 2015/121348. The osmolarity is measured with an
osmometer (Micro-Osmometer Loser Type 15).
[0228] The following table 2 summarizes the main characteristics of
one produced batch of MGL:
TABLE-US-00002 MGL of P. putida Freeze-dried (amount per tube: 49.2
mg). Formulation Characteristics after being taken up in 625 pL of
water: 78.7 mg/ml, ~622 .mu.M of PLP, 50 mM of Na phosphate, pH
7.2, Osmolarity 300 mOsmol/kg. Specific activity 13.2 IU/mg Purity
>98%
[0229] Discussion of the production method. The method for
purifying the MGL described in in WO 2015/121348 is established on
the basis of the method detailed in patent EP 0 978 560 B1 and of
the associated publication (Takakura et al., Appl Microbiol
Biotechnol 2006). This selection is explained by the simplicity and
the robustness of the crystallization step which is described as
being particularly practical and easily adaptable to large scale
productions according to the authors. This step is based on the use
of PEG6000 and of ammonium sulfate after heating the MGL solution
obtained after the lysis/clarification and removal of impurities by
adding PEG6000/ammonium sulfate steps. The other notable point of
this step is the possibility of rapidly obtaining a high purity
level during the step for removing the impurities by achieving
centrifugation following the treatment of the MGL solution with
PEG6000. The impurities are again found in the centrifugation
pellet, the MGL being in majority found in solution in the
supernatant. Because of this purity, the passing of the MGL
solution in a single chromatography step over an anion exchanger
column (DEAE), associated with a purification step by gel
filtration on a sephacryl S200 HR column, gives the possibility of
obtaining a purified protein.
[0230] Upon setting into place the patented method for small scale
tests, it appeared that the obtained results were not able to be
reproduced. According to patent EP 0 978 560 B1, at the end of the
step for removing the impurities (treatment with PEG6000/ammonium
sulfate and centrifugation), the MGL enzyme is in majority found in
the soluble fraction, centrifugation causing removal of the
impurities in the pellet. During small scale tests conducted
according to the described method in EP 0 978 560 B1, the MGL
protein is again in majority found (.about.80%) in the
centrifugation pellet. The table 3 below lists the percentage of
MGL evaluated by densitometry on SDS-PAGE gel in soluble
fractions.
TABLE-US-00003 MGL percentage in Purification the soluble fraction
Average Test no. 1 11% 17% Test no. 2 23%
[0231] This unexpected result therefore led to optimization of the
patented method by: 1) operating from the centrifugation pellet
containing MGL, 2) carrying out two successive crystallization
steps for improving the removal of the impurities after loading on
a DEAE column, 3) optimizing chromatography on a DEAE column.
[0232] For this last step, it is found that the DEAE sepharose FF
resin is finally not a sufficiently strong exchanger in the tested
buffer and pH conditions. After different additional optimization
tests, the selection was finally directed to 1) replacement of the
phosphate buffer used in the initial method with Tris buffer pH 7.6
for improving the robustness of the method and 2) carrying out a
second passage on DEAE in order to substantially improve the
endotoxin level and the protein purity without any loss of MGL (0.8
EU/mg according to Takakura et al., 2006 versus 0.57 EU/mg for the
modified method).
[0233] Finally, in order to obtain a method compatible with the
requirements for large scale GMP production, a polishing step on a
membrane Q was added in order to reduce the residual endotoxins and
HCP levels. This final step of polishing avoids the implementation
of the S200 gel filtration chromatography which is a difficult step
to be used in production processes at an industrial scale (cost and
duration of the chromatography).
[0234] Product obtained is summarized in the following table 4
using the two methods.
TABLE-US-00004 Patent Method of EP 978 560 B1 the application
Amount of Yield Amount of Yield Step enzyme (g) (%) enzyme (g) (%)
Solubilised pellet 125 100 70 100 before DEAE Concentrated
solution.sup.$ 80 64 46 65 .sup.$post sephacryl S-200 HR (EP 978
560) or post Membrane Q (method of the invention).
EXAMPLE 5. CO-ENCAPSULATION OF MGL AND PLP IN MURINE
ERYTHROCYTES
[0235] Whole blood of CD1 mice (Charles River) is centrifuged at
1000.times.g, for 10 min, at 4.degree. C. in order to remove the
plasma and buffy coat. The RCs are washed three times with 0.9%
NaCl (v:v). The freeze-dried MGL is re-suspended in water at a
concentration of 78.7 mg/ml and added to the erythrocyte suspension
in order to obtain a final suspension with a hematocrit of 70%,
containing different concentrations of MGL and of the PLP. The
suspension was then loaded on a hemodialyzer at a flow rate of 120
ml/h and dialyzed against a hypotonic solution at a flow rate of 15
ml/min as a counter-current. The suspension was then resealed with
a hypertonic solution and then incubated for 30 min at 37.degree.
C. After three washes in 0.9% NaCl, 0.2% glucose, the suspension
was taken up in a preservation solution SAG-Mannitol supplemented
with 6% BSA. The obtained products are characterized at D0 (within
the 2 h following their preparation) and at D1 (i.e. after
.about.18 h-24 h of preservation at 2-8.degree. C.). The
hematologic characteristics are obtained with a veterinary
automaton (Sysmex, PocH-100iV).
[0236] Results:
[0237] In the different studies mentioned hereafter, the MGL
activity in the finished products is assayed with the method
described in example 5 against an external calibration range of MGL
in aqueous solution. These results, combined with explanatory
studies, show that MGL activity in the finished products increases
with the amount of enzyme introduced into the method and that it is
easily possible to encapsulate up to 32 IU of MGL per ml of
finished product while maintaining good stability.
[0238] In another study, three murine finished products
RC-MGL-PLP1, RC-MGL-PLP2 and RC-MGL-PLP3 were prepared according to
the following methods: [0239] RC-MGL-PLP1: co-encapsulation of MGL
and of PLP from a suspension containing 3 mg/ml of MGL and
.about.30 .mu.M of PLP. The final product was taken up in
SAG-Mannitol, 6% BSA supplemented with final 10 .mu.M PLP. [0240]
RC-MGL-PLP2: co-encapsulation of MGL and of PLP from a suspension
containing 3 mg/ml of MGL and .about.30 .mu.M of PLP. The finished
product was taken up in SAG-Mannitol 6% BSA. [0241] RC-MGL-PLP3:
this product stems from a co-encapsulation of MGL and PLP from a
suspension containing 3 mg/ml of MGL and .about.124 .mu.M of PLP.
The final product was taken up in SAG-Mannitol 6% BSA.
[0242] In a third study, a murine finished product RC-MGL-PLP4 was
prepared from a new batch of MGL according to the following
methods: [0243] RC-MGL-PLP4: co-encapsulation of MGL and the PLP
from a suspension containing 5 mg/ml of MGL and .about.35 .mu.M of
PLP. The finished product was taken up in SAG-Mannitol 6% BSA.
[0244] Finally in a fourth study, a murine product RC-MGL-PLP5 was
prepared from a third batch of MGL according to the following
methods: [0245] RC-MGL-PLP5: co-encapsulation of MGL and PLP from a
suspension containing 6 mg/ml of MGL and .about.100 .mu.M of PLP.
The finished product was taken up in SAG-Mannitol 6% BSA.
[0246] The hematologic and biochemical characteristics of the three
finished products at D0 (after their preparation) are detailed in
the table 5 below. The encapsulation yields are satisfactory and
vary from 18.6% to 30.5%.
TABLE-US-00005 RC- RC- RC- RC- RC- MGL- MGL- MGL- MGL- MGL- PLP1
PLP2 PLP3 PLP4 PLP5 Hematolo- Hematocrit (%) 50.0 49.6 50.0 50.0
50.0 gical data Corpuscle volume (fl) 46.3 46.5 46.8 42.4 45.6
Corpuscle hemoglobin (g/dl) 24.7 24.0 24.2 27.4 25.1 RC
concentration (10.sup.6/.mu.l) 6.5 6.9 6.6 7.2 6.0 Total hemoglobin
(g/dl) 14.8 15.4 15.0 16.6 13.8 Extracellular Hb (g/dl) 0.1 0.1 0.1
0.2 0.05 mgl Intra-erythrocyte concentration of 0.97 0.94 0.79 1.01
1.36 MGL (mg/ml of RC) Intra-erythrocyte activity of MGL 12.8 12.4
8.8 5.0 8.6 (IU/ml of RC)* Extracellular activity (%) 0.92% 0.97%
1.32% 1.18% 2.23% Intracellular activity (%) 99.08% 99.03% 98.68%
98.82% 97.77% Encapsulation yield of MGL (%) 18.6% 30.5% 22.6%
19.4% 22.7% PLP Intra-erythrocyte concentration of ND 13.4 71.4
10.2 ND PLP (.mu.mol/l of RC) Intracellular PLP fraction (%) ND
99.5 98.7 98.1 ND Extracellular PLP fraction (%) ND 0.5 1.3 1.92 ND
PLP encapsulation yield (%) ND 44.8 57.4 30.7 ND *Calculated from
the specific activity of each batch.
EXAMPLE 6. PRODUCTION OF HUMAN RCS ENCAPSULATING METHIONINE GAMMA
LYASE AND PLP ACCORDING TO THE INDUSTRIAL METHOD
[0247] A pouch of leukocyte-depleted human Red Cell RCs (provided
by the "Etablissement Francais du Sang") is subject to a cycle of
three washes with 0.9% NaCl (washer Cobe 2991). The freeze-dried
MGL is re-suspended with 0.7% NaCl and added to the erythrocyte
suspension in order to obtain a final suspension with a hematocrit
of 70%, containing 3 mg/ml of MGL and .about.30 .mu.M of PLP
(stemming from the formulation of MGL). The suspension is
homogenized and it is proceeded with encapsulation according to the
method described in EP 1 773 452. The suspension from the resealing
is then incubated for 3 h at room temperature in order to remove
the most fragile RCs. The suspension is washed three times with a
0.9% NaCl, 0.2% glucose solution (washer Cobe 2991) and then
re-suspended with 80 ml of preservation solution (AS-3). The
encapsulated MGL level is assayed like in Example 6, see table 6
below.
TABLE-US-00006 J0 J1 J7 Hematocrit (%) 52.0 51.6 52.7 Corpuscle
volume (fl) 91.0 92.0 88.0 Corpuscle hemoglobin (g/dl) 30.3 29.8
31.6 RC concentration (10.sup.6/.mu.l) 6.00 5.92 5.98 Total
hemoglobin (g/dl) 16.4 16.2 16.6 Extracellular Hb (g/dl) 0.119
0.197 0.280 Osmotic fragility (g/l) 1.17 Hemolysis (%) 0.7% 1.2%
1.7% Total MGL concentration (mg/ml) 0.36 0.35 MGL supernatant
concentration 0.01 0.01 (mg/ml) MGL intra-erythrocyte concentra-
0.68 0.67 tion (mg/ml, 100% Ht) Extracellular activity (%) 1.3%
1.4% Intracellular activity (%) 98.7% 98.6% Encapsulation yield (%)
19.7%
EXAMPLE 7
Additional Abbreviations
[0248] RPMI: Le Roswell park memorial institute medium
I. Operating Conditions
I.1 Test Item
I.1.1. L-Asparaginase
[0249] Description: Medac.RTM. (Germany), E. Coli L-asparaginase 10
000 IU.
[0250] One concentration of L-asparaginase (2.2 IU/mL) was prepared
by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS)
1.times.. Concentration of L-asparaginase was diluted 11-fold to
obtain final concentration of 0.20 IU/mL (IC50).
I.1.2. Methionine-.gamma.-Lyase (MGL)
[0251] Description: P. Putida methionine-.gamma.-lyase (MGL)
produced in E. Coli.
[0252] One concentration of MGL (3.85 IU/mL) was prepared by serial
dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1.times..
Concentration of MGL was diluted 11-fold to obtain final
concentration of 0.35 IU/mL (IC50).
I.2 Cell Lines
I.2.1. Description
[0253] Name: NCI-N87 cell line Description: Human gastric carcinoma
cell line (adherent) Supplier and reference number: ATCC,
CRL-5822
I.2.2. Culture Conditions
[0254] Cells were cultivated in a RPMI media supplemented with 10%
(v/v) of foetal bovine serum, 100 IU/mL of penicillin and 100
.mu.g/mL of streptomycin. Subculturing was performed according to
PO-CELL-002 and PO-CELL-005.
I.2.3. Colorimetric Kit
Name: Cell Counting Kit-8 (CCK-8)
[0255] Supplier and reference number: Fluka 96992
[0256] Principle: the CCK-8 reagent contains a highly water-soluble
tetrazolium salt WST-8. WST-8 is reduced by dehydrogenases in cells
to give a yellow colored product (formazan) which is soluble in the
tissue culture medium. The amount of the formazan dye generated by
the activity of dehydrogenases in cells is directly proportional to
the number of living cells. The colorimetric test was performed
according to PO-CELL-004.
II. Cytotoxicity Assay
II.1 Method
[0257] Two thousand five hundred cells in 100 .mu.L/well were
dispensed in 96-well flat bottom plates (cf. number of plates in
raw data). In addition, two wells were filled with culture medium
for blank control on each plate. All empty wells were filled with
culture medium in order to minimize evaporation and condensation.
On day 0 (D0), 10 .mu.L of IC50 concentrations of L-asparaginase or
MGL were added to the corresponding wells. Controls (blank wells
and control plate) received 10 .mu.L of DPBS 1.times.. On day 4
(D4), medium was removed from wells and replaced by fresh medium
and 10 .mu.L of DPBS 1.times. or 10 .mu.L of IC50 concentrations of
L-asparaginase (for cells previously incubated with MGL) or MGL
(for cells previously incubated with L-asparaginase) added to the
corresponding wells. Controls (blank and positive control) received
10 .mu.L of DPBS 1.times.. Then, plates were incubated for 4 more
days in the incubator. At the end of the incubation period (D8), 10
.mu.L of CCK-8 solution were added to each well according to
PO-CELL-004 and plates incubated for 4 hours. Optical density (OD)
was then determined at 450 nm using a microplate reader.
II.2 Internal Controls
[0258] Controls were performed in duplicate.
II.2.1. Blank Wells
[0259] As above in Example 1.
II.2.2. Viability Control (Positive Control)
[0260] As positive control for the NCI-N87 cell line (100% cell
viability), cells were cultivated in the culture medium (100 .mu.L)
without L-asparaginase nor MGL, but with 10 .mu.L of the diluent
(DPBS 1.times.).
II.3 Determination of Cell Viability
[0261] As above in Example 1.
III. Results
III.1 Internal Control
[0262] Internal controls were acceptable when it was not specified
in raw data.
III.2 IC50 Calculations with L-Asparaginase or MGL Alone
[0263] Percentages of cell viability with drug alone (MGL or
L-asparaginase) were controlled in each experiment of drugs
combination. Fifty percent of cell viability are expected at half
of the test (D4) because IC50 value used here for enzymes were
previously validated in single treatment at D4.
III.2.1. Sequential Addition of L-Asparaginase and MGL
[0264] The experiment with sequential treatment of L-asparaginase
and MGL was done twice with duplicate data. All quality controls
(blank and positive control) were accepted in experiments.
[0265] Details of % of cell viability calculations and graphical
representation are presented below in table 7 and FIG. 2.
TABLE-US-00007 TABLE 7 % of cell viability for controls and enzyme
association % cell viability at D8 Mean SD Cells alone 100 0 Cells
+ IC50 L-aspa D0 56 8 Cells + IC50 MGL D0 45 4 Cells + IC50 L-aspa
D0 + IC50 MGL D3 44 0 Cells + IC50 MGL D0 + IC50 L-aspa D3 25 6
[0266] Results indicated that enzyme association with MGL added at
IC50 dose before the addition of L-asparaginase at IC50 dose (cf.
FIG. 2) permitted to reduce cell viability of: [0267] 55% compared
to IC50 L-asparaginase (IC50 control for L-asparaginase), [0268]
44% compared to MGL (IC50 control for MGL), [0269] 43% compared to
enzyme association with L-asparaginase added in first at IC50
dose.
IV. Conclusion
[0270] Sequential enzyme association demonstrated that cell
mortality could be increased with an addition of MGL at IC50 dose
followed 4 days later by the addition of L-asparaginase at IC50
dose.
[0271] We can hypothesize that Met deprivation induced by MGL
enzyme activity makes NCI-N87 gastric cells more sensitive to
L-asparaginase activity. Moreover, the roles of L-asparaginase and
MGL have to be discussed considering their known respective
effect.
[0272] Indeed, L-asparaginase is known to trigger apoptosis in
leukaemia cells (Ueno et al., 1997), therefore, it could probably
plays a role of cytotoxic agent. MGL being known for blocking cell
division in S or G2 phase of the cell cycle probably acts more as a
cytostatic agent.
EXAMPLE 8
I. Additional Abbreviations
[0273] F12K: Kaighn's modification of ham's F-12
II. Operating Conditions
II.1 Test Item
II.1.1. L-Asparaginase
[0274] Description: Medac.RTM. (Germany), E. Coli L-asparaginase 10
000 IU.
[0275] One concentration of L-asparaginase (2.97 IU/mL) was
prepared by serial dilutions in Dulbecco Phosphate Buffered Saline
(DPBS) 1.times.. Concentration of L-asparaginase was diluted
11-fold to obtain final concentration of 0.27 IU/mL (IC50).
II.1.2. Methionine-.gamma.-Lyase (MGL)
[0276] Description: P. Putida methionine-.gamma.-lyase (MGL)
produced in E. Coli.
[0277] One concentration of MGL (1.43 IU/mL) was prepared by serial
dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1.times..
Concentration of MGL was diluted 11-fold to obtain final
concentration of 0.13 IU/mL (IC50).
II.2 Cell Lines
II.2.1. Description
[0278] Name: AGS cell line Description: Human gastric
adenocarcinoma cell line (adherent) Supplier and reference number:
ATCC, CRL-1739
II.2.2. Culture Conditions
[0279] Cells were cultivated in a F12K media with L-glutamine
supplemented with 10% (v/v) of foetal bovine serum, 100 IU/mL of
penicillin and 100 .mu.g/mL of streptomycin. Subculturing was
performed according to PO-CELL-002 and PO-CELL-005.
II.2.3. Colorimetric Kit
Name: Cell Counting Kit-8 (CCK-8)
[0280] Supplier and reference number: Fluka 96992
[0281] Principle: the CCK-8 reagent contains a highly water-soluble
tetrazolium salt WST-8. WST-8 is reduced by dehydrogenases in cells
to give a yellow colored product (formazan) which is soluble in the
tissue culture medium. The amount of the formazan dye generated by
the activity of dehydrogenases in cells is directly proportional to
the number of living cells. The colorimetric test was performed
according to PO-CELL-004.
III. Cytotoxicity Assay
III.1 Method
[0282] One thousand cells in 100 .mu.L/well were dispensed in
96-well flat bottom plates (cf. number of plates in raw data). In
addition, two wells were filled with culture medium for blank
control on each plate. All empty wells were filled with culture
medium in order to minimize evaporation and condensation. On day 0
(D0), 10 .mu.L of IC50 concentrations of L-asparaginase or MGL were
added to the corresponding wells. Controls (blank wells and control
plate) received 10 .mu.L of DPBS 1.times.. On day 4 (D4), medium
was removed from wells and replaced by fresh medium and 10 .mu.L of
DPBS 1.times. or 10 .mu.L of IC50 concentrations of L-asparaginase
(for cells previously incubated with MGL) or MGL (for cells
previously incubated with L-asparaginase) added to the
corresponding wells. Controls (blank and positive control) received
10 .mu.L of DPBS 1.times.. Then, plates were incubated for 4 more
days in the incubator. At the end of the incubation period (D8), 10
.mu.L of CCK-8 solution were added to each well according to
PO-CELL-004 and plates incubated for 4 hours. Optical density (OD)
was then determined at 450 nm using a microplate reader.
III.2 Internal Controls
[0283] Controls were performed in duplicate.
III.2.1. Blank Wells
[0284] As above in Example 1.
III.2.2. Viability Control (Positive Control)
[0285] As positive control for the AGS cell line (100% cell
viability), cells were cultivated in the culture medium (100 .mu.L)
without L-asparaginase nor MGL, but with 10 .mu.L of the diluent
(DPBS 1.times.).
III.3 Determination of Cell Viability
[0286] As above in Example 1.
IV. Results
IV.1 Internal Control
[0287] Internal controls were acceptable when it was not specified
in raw data.
IV.2 IC50 Calculations with L-Asparaginase or MGL Alone
[0288] Percentages of cell viability with drug alone (MGL or
L-asparaginase) were controlled in each experiment of drugs
combination. Fifty percent of cell viability are expected at half
of the test (D4) because IC50 value used here for enzymes were
previously validated in single treatment at D4.
IV.2.1. Sequential Addition of L-Asparaginase and MGL
[0289] The experiment with sequential treatment of L-asparaginase
and MGL was done twice with duplicate data. All quality controls
(blank and positive control) were accepted in experiments.
[0290] Details of % of cell viability calculations and graphical
representation are presented below in table 8 and FIG. 3.
TABLE-US-00008 TABLE 8 % of cell viability for controls and enzyme
association % cell viability at D8 Mean SD Cells alone 100 4 Cells
+ IC50 L-aspa D0 101 1 Cells + IC50 MGL D0 106 1 Cells + IC50
L-aspa D0 + IC50 MGL D3 88 2 Cells + IC50 MGL D0 + IC50 L-aspa D3
79 6
[0291] Results indicated that enzyme association with MGL added at
IC50 dose before the addition of L-asparaginase at IC50 dose (cf.
FIG. 3) permitted to reduce cell viability of: [0292] 22% compared
to IC50 L-asparaginase (IC50 control for L-asparaginase), [0293]
26% compared to MGL (IC50 control for MGL), [0294] 10% compared to
enzyme association with L-asparaginase added in first at IC50
dose.
[0295] Moreover, for precision here, IC50 control for
L-asparaginase or MGL (used and validated initially at D4) returned
to 100% of cell viability after 8 days of culture with renewal of
media at D4/half of the test. Indeed, remaining viable cells at D4
could re-growth with addition of "fresh" nutrients. Results were
conform for IC50 controls (enzyme alone) reaching 50% of cell
viability at D4.
V. Conclusion
[0296] Sequential enzyme association demonstrated that cell
mortality could be increased with an addition of MGL at IC50 dose
followed 4 days later by the addition of L-asparaginase at IC50
dose.
[0297] We can hypothesize that Met deprivation induced by MGL
enzyme activity makes AGS gastric cells more sensitive to
L-asparaginase activity. Moreover, the roles of L-asparaginase and
MGL have to be discussed considering their known respective effect.
Indeed, L-asparaginase is known to trigger apoptosis in leukaemia
cells (Ueno et al., 1997), therefore, it could probably plays a
role of cytotoxic agent. MGL being known for blocking cell division
in S or G2 phase of the cell cycle probably acts more as a
cytostatic agent.
EXAMPLE 9
I. Additional Abbreviations
[0298] A.M.: Ante meridiem ERY-ASP: L-asparaginase encapsulated
into red blood cells ERY-MET: Methionine gamma-lyase encapsulated
into red blood cells IG: Intragastric injection (gavage) IU:
International Unit corresponding to pmol/min
IV: Intravenous
[0299] ND: Not determined
PN: Pyridoxine
[0300] TGI: Tumor growth inhibition
II. Objective of In Vivo Study
[0301] The objective of this study is to determine if combination
of methioninase-loaded erythrocytes (ERY-MET) with
L-asparaginase-loaded erythrocytes (ERY-ASP) can improve the
antitumor activity observed with ERY-MET alone in a NCI-N87 gastric
tumor subcutaneous xenograft mouse model.
III. Operating Conditions
[0302] NCI-N87 cells were cultivated at ERYTECH Pharma and prepared
at 5.10.sup.7 cells/mL in DPBS 1.times. for injection. Four groups
of 10 or 12 female NMRI nude mice (groups 1, 2, 3 and 4) were
subcutaneously injected with the cell line at the fixed
concentration of 5.10.sup.6/100 .mu.L. ERY-MET and ERY-ASP
injections were administrated (I.V. route) respectively at 108
IU/kg (8 mL/kg) and 200 IU/kg (4-5.4 mL/kg). Group 2 received 3
injections of ERY-MET on days 7, 14 and 21. Group 3
(ERY-ASP/ERY-MET) received 1 injection of ERY-ASP on day 7 and
then, 2 injections of ERY-MET on days 21 and 28. Group 4
(ERY-MET/ERY-ASP) received 2 injections of ERY-MET on days 7 and 14
and then 1 injection of ERY-ASP on day 21. Group 1 was administered
with the preservative solution of ERY-MET (SAG mannitol/plasma) at
8 mL/kg on days 7, 14 and 21.
[0303] Oral administrations (gavage) of PN co-factor was performed
6 hours after each ERY-MET injection (Day 7+6 h, Day 15+6 h, Day
21+6 h for group 2; Day 21+6 h, Day 28+6 h for group 3; Day 7+6 h,
Day 15+6 h for group 4) and once a day (A.M.) for the other days
(without ERY-MET administration) until Day 20 (for group 4), Day 27
(for group 2) or Day 34 (for group 3).
IV. Results
[0304] Tumor volume regression associated to ERY-MET/ERY-ASP
combination appeared different to this observed for ERY-MET arm;
indeed at D37, mice ERY-MET displayed a mean tumor volume of
298.3.+-.36.2 mm.sup.3 and mice ERY-MET/ERY-ASP displayed a mean
tumor volume of 189.7.+-.29.8 mm.sup.3 corresponding to
respectively 37% and 57% of mean tumor volume reduction while mice
given vehicle (control) had a mean tumor volume of 441.5.+-.56.6
mm.sup.3. Percentage of tumor growth inhibition (TGI*) were
calculated for the enzyme association ERY-MET/ERY-ASP vs control
(vehicle group) or vs ERY-MET group according to the following
formula:
100 - ( Tumor .times. .times. Volume enzyme .times. .times.
association .times. .times. at .times. .times. Day .times. .times.
X Tumor .times. .times. Volume vehicle .times. .times. or .times.
.times. ERY - MET .times. .times. alone .times. .times. at .times.
.times. Day .times. .times. X .times. 100 ) ##EQU00002##
[0305] Results are presented below in the table 9 below:
TABLE-US-00009 TABLE 9 TGI calculations for the association
ERY-MET/ERY-ASP % TGI for ERY-MET/ ERY-ASP treatment vs control vs
ERY-MET alone Day 7 ND** ND** Day 20 41% 33% Day 37 57% 36% **Not
determined (not relevant) due to low volume measure disparity at
the beginning of the study (D7 is the first time point of tumor
volume measure).
[0306] In order to assess significance between groups and
efficiency of ERY-MET/ERY-ASP treatment compared to ERY-MET alone
on NCI-N87 gastric tumors, a two-way ANOVA test was performed with
GraphPad Prism software (version 5.04) on tumor growth measures.
Analysis comparing vehicle (control), ERY-MET and ERY-MET/ERY-ASP
treatment indicating significance between groups at D37 with a P
value inferior to 0.0001 (cf. FIG. 4) revealing efficacy of the
combination ERY-MET/ERY-ASP 16 days after last injections for
treatment against gastric tumors. With the reverse scheme of
administration ERY-ASP/ERY-MET treatment compared to ERY-MET alone
on NCI-N87 gastric tumors, two-way ANOVA test (cf. FIG. 4) revealed
no significance between groups for three time points of follow-up
(D7/D20/D37) with a P value>0.05.
V. Conclusion
[0307] ERY-MET was combined to ERY-ASP with 2 scheme of
administrations: 1-ERY-ASP (D7)-ERY-MET (D21/D28) and 2-ERY-MET
(D7/D15)-ERY-ASP (D21). Positive response compared to ERY-MET alone
seems to appear when ERY-MET was administrated (twice) before
ERY-ASP. This significance of result is supported by the obtaining
of a P value inferior to 0.0001 at D37 on individual tumor volume
measure.
* * * * *