U.S. patent application number 10/491675 was filed with the patent office on 2004-12-09 for stable galenic freeze-dried pharmaceutical preparation of recombined carbohydrate-binding polypeptides.
Invention is credited to Gloger, Oliver, Muller, Bernd W., Witthohn, Klaus.
Application Number | 20040248778 10/491675 |
Document ID | / |
Family ID | 7701416 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248778 |
Kind Code |
A1 |
Gloger, Oliver ; et
al. |
December 9, 2004 |
Stable galenic freeze-dried pharmaceutical preparation of
recombined carbohydrate-binding polypeptides
Abstract
The invention relates to a method for the production of a
medicament containing a polypeptide comprising at least one
recombinant carbohydrate-binding polypeptide, or a functional
fragment or derivative of said carbohydrate-binding polypeptide in
a form stable for storage. The polypeptide mentioned comprises
polypeptides or functional derivatives thereof, which are fused
with cytotoxically effective peptides to give fusion proteins, or
which are linked to another polypeptide having a cytotoxic
activity. Moreover, the invention describes further formulating of
the disclosed medicaments to medicaments with different
pharmaceutical forms.
Inventors: |
Gloger, Oliver; (Aachen,
DE) ; Muller, Bernd W.; (Flintbeck, DE) ;
Witthohn, Klaus; (Overath, DE) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
7701416 |
Appl. No.: |
10/491675 |
Filed: |
April 2, 2004 |
PCT Filed: |
October 2, 2002 |
PCT NO: |
PCT/EP02/11093 |
Current U.S.
Class: |
435/69.1 ;
514/1.3; 514/19.3; 530/322; 530/395 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 9/0019 20130101; A61K 9/19 20130101; A61K 47/36 20130101; A61K
47/26 20130101; A61K 47/02 20130101; A61P 39/00 20180101; A61K
38/168 20130101; A61K 47/18 20130101; A61K 47/183 20130101 |
Class at
Publication: |
514/008 ;
530/322; 530/395 |
International
Class: |
C07K 009/00; A61K
038/14 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2001 |
DE |
101 49 030.5 |
Claims
1. A method for the production of a medicament containing a
polypeptide comprising at least one recombinant
carbohydrate-binding polypeptide or a functional fragment or
derivative of said carbohydrate-binding polypeptide wherein (a) the
polypeptide or a functional fragment or derivative of this
polypeptide which is fused to a cytotoxically effective peptide to
form a fusion protein; (b) the polypeptide or a functional fragment
or derivative of this polypeptide which is linked to another
polypeptide which has an enzymatic rRNA-N-glycosidase activity; (c)
the polypeptide or a functional fragment or derivative of this
polypeptide which is linked to another polypeptide in which an
enzymatic rRNA-N-glycosidase activity has been replaced by another
cytotoxic activity; or (d) the polypeptide or a functional fragment
or derivative of this polypeptide, which is linked to a fusion
protein, comprising a polypeptide with an enzymatic
rRNA-N-glycosidase activity and/or another cytotoxic activity; in a
form stable for long-time storage, moreover optionally containing a
pharmaceutically acceptable carrier, comprising the step of
cooling, freezing, spray drying or lyophilising while retaining the
pharmacological properties of the polypeptide in the solution,
wherein the solution is characterised in that the pH value of the
solution is higher than pH 6.0 and a buffer system contained in the
solvent guarantees that this pH-value is maintained.
2. The method of claim 1, wherein the recombinant
carbohydrate-binding polypeptide is the B-chain of a
ribosome-inactivating protein.
3. The method of claim 2, wherein the further polypeptide which is
linked to the recombinant carbohydrate-binding polypeptide is the
A-chain of a ribosome-inactivating protein.
4. The method of claim 2 and/or 3, wherein the
ribosome-inactivating protein is a ribosome-inactivating protein of
the type II.
5. The method of claim 4, wherein the ribosome inactivating protein
is a type II rViscumin.
6. The method of any one of claims 1 to 5, wherein the pH-value of
the solution is between 6.0 and 9.0.
7. The method of any one of claims 1 to 6, wherein the pH-value of
the solution is between 7.5 and 8.5.
8. The method of any one of claims 1 to 7, wherein the salt(s) of
the buffer system is/are used in a end concentration ranging from 5
mM to 200 mM.
9. The method of any one of claims 1 to 8, wherein the salt(s) of
the buffer system is/are used in an end concentration ranging from
100 mM to 200 mM.
10. The method of any one of claim 1 to 9, wherein the salt(s) of
the buffer system is/are selected from a group comprising:
TRIS/HCl, TRICIN/HCl, HEPES/HCl, ammonium carbonate buffer,
TRIS/glutamic acid and TRIS/aspartic acid.
11. The method of any one of claims 1 to 10, wherein the solution
contains one or more surfactants in order to stabilise the
pharmacological properties of the polypeptide.
12. The method of claim 11, wherein the surfactants are non-ionic
tensides and are used in a final concentration ranging from 0.01 to
5.0%.
13. The method of claim 12, wherein the non-ionic tensides are
selected from the group comprising: fatty alcohols, partial
glycerides, polysorbates, polyoxyethylene fatty acid ethers and
polyoxyethylene fatty acid esters, poloxameres
(polyoxypropylene-polyoxyethylene-block polymers), saccharide fatty
acid esters, polyoxyglycerol fatty acid esters and
phosphatides.
14. The method of claim 13, wherein the polysorbates are selected
from the group comprising Polysorbate 80, Polysorbate 20 and
polyoxyethylene sorbitol ether.
15. The method of claim 13, wherein the polyoxyethylene fatty acid
ethers and polyoxyethylene fatty acid esters are macrogol ethers or
macrogol esters.
16. The method of claim 13, wherein the poloxamere is Pluronic F68,
poloxamer 166 or poloxamer 188.
17. The method of claim 13, wherein the phosphatides are
lecithins.
18. The method of claim 11, wherein the surfactants are amphoteric
tensides and are used in a final concentration ranging from 0.01 to
5.0%.
19. The method of any one of claims 1 to 18, wherein one or more
lyoprotectors in a final concentration ranging from 0.1 to 20%
and/or cryoprotectors in a final concentration ranging from 0.01 to
1.0% are added to the solution for lyophilisation.
20. The method of claim 19, wherein the lyoprotectors are added in
a final concentration ranging from 4.0 to 10% and/or the
kryoprotectors are added in a final concentration ranging from 0.05
to 0.1%.
21. The method of claim 19 or 20, wherein the lyoprotectors are
selected from the group comprising: a) low molecular saccharides
such as glucose, trehalose and sucrose; b) hexites such as mannitol
(mannite) and sorbitol (sorbite); c) oligomeric and polymeric
saccharides such as cyclic beta-hydroxypropylcyclodextrin,
cyclodextrins, cellulose, starch, carboxyamylopektin, chitosan and
their derivatives; d) anorganic gelling agents such as bentonites,
and silicon dioxide; and e) synthetic polymers such as
polyvinylpyrrolidone and polyacrylates.
22. The method of claims 19 or 20, wherein the lyoprotector or the
lyoprotectors is dextrane or are dextranes.
23. The method of claims 19 or 20, wherein ionic substances are
used as cryoprotectors.
24. The method of claim 23, wherein the ionic substances are
selected from the group comprising sodium chloride, sodium
sulphate, potassium chloride and potassium sulphate.
25. The method of any one of claims 19 to 24, wherein the
lyoprotectors and cryoprotectors form amorphous structures during
lyophilisation.
26. The method of any one of claims 1 to 25, wherein the
stabilisers are amino acids which are used in a final concentration
of from 0.01 to 50 mg/ml.
27. The method of claim 26, wherein the amino acids are selected
from the group comprising acidic amino acids such as glutamic acid
and aspartic acid, the basic amino acid arginine and the neutral
amino acid valine.
28. The method of any one of claims 1 to 27, wherein the
polypeptide comprising at least one recombinant
carbohydrate-binding polypeptide or functional fragment or
derivative of this polypeptide is used in a final concentration of
from 10 ng/ml to 10 mg/ml.
29. The method of claim 28, wherein the polypeptide is used in a
final concentration of from 100 ng/ml to 1 mg/ml.
30. The method of any one of claims 1 to 29, moreover comprising
the further formulation or reconstitution of the medicament as
aqueous or non-aqueous solution.
31. The method of claim 30, wherein the medicament is further
formulated as injection solution, instillation solution or infusion
solution.
32. The method of any one of claims 1 to 29, moreover comprising
the further formulation or reconstitution of the medicament for
gastrointestinal, oral, nasal, pulmonary, dermal, transdermal or
local application.
33. The method of any one of claims 1 to 30, moreover comprising
the further formulation of the medicament into juice, capsules,
tablets, suppositories or gels.
34. The method of any one of claims 1 to 29, moreover comprising
the further formulation of the medicament into a powder for
inhalation which is administered by use of an inhalator.
35. A medicament, produced according to any one of the methods of
any one of claims 1 to 34.
Description
[0001] This application is a national stage filing under 35 U.S.C.
371 of PCT application PCT/EP02/11093, filed Oct. 2, 2002, which
claims the benefit of priority from German Application No. 101 49
030.5, filed Oct. 5, 2001, the specifications of each of which are
incorporated by reference herein in their entirety. PCT Application
PCT/EP02/11093 was published under PCT Article 21(2) in German.
[0002] The invention relates to a method for the production of a
medicament containing a polypeptide, comprising at least one
recombinant carbohydrate-binding polypeptide or a functional
fragment or derivative of said carbohydrate-binding polypeptide in
a form stable for storage. Said polypeptide comprises polypeptides
or functional derivatives thereof fused to cytotoxically effective
peptides to give fusion proteins or which are connected to a
further polypeptide having cytotoxic activity. The invention
further relates to the further formulation of the disclosed
medicaments to give medicaments of various dosage forms.
[0003] In recent years, medicinal research has uncovered a broad
spectrum of diseases that can be treated with recombinant proteins.
Examples of proteins of human origin are insulin, EPO and G-CSF the
dosage forms and kinds of application of which have been described
in various European patents. EP 0 430 200 B1 describes the
application of human proteins for subcutaneous and intramuscular
administration. Medicaments with stabilised human proteins which
contain, amongst others, urea or different amino acids, are known
from EP 0 306 824 B1. In this patent, EPO and G-CSF are given as
examples. EP 0 607 156 B1 describes the production of conserved
medicaments with human proteins for infusion or injection
purposes.
[0004] In general, the term "recombinant" refers to proteins which
are prepared using the recombinant DNA technique. These methods
comprise cloning of the gene encoding the protein in question,
inserting corresponding cDNA or genomic DNA in a suitable vector
system and transforming/transfecting said vectors in suitable host
organisms (bacteria or eukaryotic cells). If the cloned gene is
expressed in the host organism, the corresponding protein can be
recovered from the culture supernatant (if the protein expressed is
secreted) or from a homogenate of the host organism (if the
corresponding protein is expressed in an intracellular manner).
Methods for producing recombinant proteins have been described for
both animal and plant proteins. An example of the exact procedure
for producing a dimeric plant protein is described in EP 0 751 221
B1. This patent describes, amongst others, the unprecedented
successful cloning of the genes encoding the ML-subunits.
Furthermore, in this patent, the use of said dimeric plant proteins
produced recombinantly for the preparation of medicaments is
described, too.
[0005] The use of mistletoe extracts (extracts of Viscum album) as
a curative has been known for centuries. Active ingredients called
lectins have been identified as effective components of these
extracts. These lectins are proteins which recognise very specific
carbohydrate structures also in lipid- or protein-bound form and
which bind thereto. Mistletoe lectin which has been characterised
as ribosome-inactivating class II protein is pharmacologically
effective by the interplay of its two subunits. The B-chain of the
mistletoe lectin which has sequence motifs with specific
carbohydrate-binding properties is, in this case, responsible for
the transport of the protein to the target cell. In the target
cell, the A-subunit then blocks the ribosomal metabolism of the
cell due to its enzymatic rRNA-N-glycosidase activity and, in this
way, it triggers a programmed cell death (apoptosis) in said
cell.
[0006] The pharmaceutical preparations which have so far been known
from the state of the art generally contain human proteins,
humanised proteins, extracts containing plant proteins or proteins
isolated from plants. It is decisive for the effectiveness of
preparations containing proteins that the biological activity of
said proteins is maintained. For rViscumin, it is, for example, the
dimeric structure and the activities that are to be attributed to
the single chains and the specific pharmacological mode of action
of said molecules that are to be maintained. Maintaining these
biological activites strongly depends on the pH of the solution
containing the proteins (cf. FIG. 1). Furthermore, storage
conditions of the preparation in question influence the stability
of a drug/medicament.
[0007] The mode of action of the mistletoe plant and the extracts
obtained therefrom for treating diseases has been described in
European patent EP 0 602 686 B1. As explained in this
specification, mistletoe extracts have been used for therapeutic
purposes for centuries. Since the beginning of this century,
mistletoe preparations are used in cancer therapy with varying
success (Bocci, 1993; Gabius et al., Gabius & Gabius, 1994;
Ganguly & Das, 1994). Hajto et al. (1989, 1990) were able to
show that the therapeutic effects are mediated in particular by
so-called mistletoe lectins (viscumins, Viscum album agglutinins,
VAA). Apart from a cytotoxic effect, nowadays, in particular an
(unspecific) immunostimulation is discussed, the positive effects
of which are utilised for an accompanying therapy and for aftercare
of tumour patients. An increase in the life quality of such
patients is possibly mediated by the release of enogenous
endorphins (Heiny and Beuth, 1994).
[0008] Numerous tests in vitro (Hajto et al., 1990; Mnnel et al.,
1991; Beuth et al., 1993a) and in vivo (Hajto, 1986; Hajto et al.,
1989; Beuth et al., 1991; Beuth et al., 1992), as well as clinical
studies (Beuth et al., 1992) prove the increased release of
inflammatory cytokines (TNF-.alpha., IL-1, IL-6) mediated by
mistletoe lectin as well as an activation of cellular components of
the immune system (TH cells, NK cells).
[0009] Today a 60 kDa mistletoe lectin protein is considered an
active principle of the mistletoe extracts, wherein the mistletoe
lectin can be recovered from extracts in a biochemical manner
(Franz et al., 1977; Gabius et al., 1992). The ML protein consists
of two covalently S--S-coupled subunits, the A-chain of which is
responsible for an enzymatic inactivation of ribosomes (Endo et
al., 1988) and the B-chain of which is responsible for the
carbohydrate binding. The biological activity is correlated with
obtaining the lectin activity of the B-chain (Hajto et al.,
1990).
[0010] The use of a form of the medicament or a pharmaceutical
preparation with rViscumin as an active component is an interesting
an advantageous alternative for a plant preparation as it is now
possible to use a chemically classified substance as a medicament.
It is with regard to the high toxicity of the mistletoe lectin that
the use of recombinantly produced proteins makes a good tolerance
possible thanks to exact dosage. In this case, a form of the
medicament or a pharmaceutical preparation is of particular
advantage which is storage-stable over a long period of time, i.e.
several months and preferably at least one year. Storage of the
form of the medicament or the pharmaceutical preparation in said
storage-stable form should moreover be simple and should be
possible without sophisticated technology. Furthermore, it should
be possible to simply further formulate the form of the medicament
or the pharmaceutical preparation to a corresponding dosage form if
its storage-stable form is not the same as its dosage form. With
aqueous formulations according to the state of the art, storage
periods of less than 10 weeks (2.5 months) are realistic under
storage conditions of 2-8.degree. C. (fridge).
[0011] Therefore, the technical problem underlying the present
invention was to provide a method for producing a medicament or
pharmaceutical preparation in a stable form for long-term storage
which guarantees simple use both with regard to storage and
administration and, optionally, its preparation. In this case, the
medicament of the invention is to comprise at least one recombinant
carbohydrate-binding polypeptide or a functional fragment or
derivative of said polypeptide, furthermore, optionally, containing
a pharmacologically acceptable carrier.
[0012] This technical problem is solved by the embodiments
characterised in the claims.
[0013] As a consequence, the present invention relates to a method
for producing a medicament containing a polypeptide comprising at
least one recombinant carbohydrate-binding polypeptide or
functional fragment or derivative of said polypeptide in a form
stable for long-time storage, moreover, optionally, containing a
pharmaceutically acceptable carrier comprising the step of cooling,
freezing, spray drying or lyophilising while retaining the
pharmacological properties of the polypeptide in the solution,
wherein the solution is characterised in that the pH value of the
solution is higher than pH 6.0 and a buffer system contained in the
solvent guarantees that this pH-value is maintained.
[0014] Starting from the corresponding cloned genes, recombinant
polypeptides and proteins can be presented using conventional
methods of molecular biology. Amongst others, these have been
described in the textbook "Gentechnologie" (Old and Primrose, 1992)
or in the laboratory manuals "Methods for General and Molecular
Bacteriology" (Gerhardt et al., Chapter 18) or "Molecular Cloning:
A Laboratory Manual" (Sambrook et al. 1993).
[0015] In accordance with the invention, a "carbohydrate-binding
polypeptide" is a polypeptide which has the property that it
specifically binds to certain carbohydrates. Examples of such
carbohydrates are galactose, N-acetyl-galactosamine, modified
galactose, neuraminic acids, low-molecular saccharides and
oligosaccharides with terminal galactose and/or terminal
galactosamine moieties or modified galactose moieties or terminal
neuraminic acid moieties, and peptides and lipids having a
corresponding carbohydrate function. "Functional fragments or
derivatives of said polypeptide" of the invention are characterised
in that they also have a specificity for binding to the
above-mentioned carbohydrates.
[0016] The use of the polypeptides of the invention, such as e.g.
rVisumin and other plant, dimeric class II polypeptides of
ribosome-inactivating proteins (RIP II) for producing highly
effective medicaments has been described amongst others in EP 0 751
221 B1. However, said medicaments are preferred to be administered
one year after preparation at most.
[0017] Within the meaning of the invention, a medicament or
pharmaceutical preparation is considered storage-stable if it can
be stored over al long period of time, i.e. several months, that is
more than six months, without a significant change of the specific
properties of the pharmaceutical preparation and the polypeptide
and, therefore, the effectiveness of said medicament or preparation
being observed. In this context, a storage-stable form of the
medicaments or pharmaceutical preparations according to the
invention, which are stored over a period for 1, 2, 3, 4 or 5
years, is preferred. Preferably they can be stored under storage
conditions that are common in the market and to be adhered to by
distributors and applicants (2-8.degree. C. and/or ambient
temperature below 25.degree. C.) without a significant change in
the specific properties of the pharmaceutical preparation and the
polypeptide and, therefore, the effectiveness of said medicament or
preparation being observed. Thus, the invention relates to storage
and transport forms of the polypeptides described herein, which are
very well manageable.
[0018] The formulation of the medicament of the invention is
optionally effected in combination with a "pharmacologically
acceptable carrier" and/or diluent. Examples of particularly
suitable pharmacologically acceptable carriers are known to those
skilled in the art and comprise buffered saline solutions, water,
emulsions such as e.g. oil/water emulsions, various kinds of
detergents, sterile solutions, etc. Medicaments that comprise such
carriers can be formulated using known conventional techniques.
These medicaments can be administered to an individual in a
suitable dose. Administration can be effected orally or parentally,
e.g. intravenously, intraperitoneally, subcutaneously,
intramuscularly, locally, intranasally, intrabronchially or
intradermally or via a catheter at a site in an artery. The kind of
dosage is determined by the attending physician in accordance with
the clinical factors. The person skilled in the art knows that the
kind of dosage depends on various factors such as, e.g. the
patient's height or weight, body surface, age, sex or general
health, but also on the particular agent to be administered, the
duration and kind of administration and on other medicaments which
are possibly administered in parallel. A typical dose can, for
instance, range from 0.001 to 1000 .mu.g wherein doses below or
above this exemplary range are thinkable, in particular when the
aforementioned factors are taken into consideration. In general, if
the composition of the invention is administered regularly, the
dose should range from 10 ng to 10 mg units per day or per
application interval. If the composition is to be administered
intravenously, the dose should range from 1 ng and 0.1 mg units per
kilogram body weight per minute.
[0019] The composition of the invention can be administered locally
or systemically. Preparations for parenteral administration
comprise sterile aqueous or non-aqueous solutions, suspensions and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, plant oils such as, e.g. olive oil, and
organic ester compounds such as, e.g. ethyloleate, which are
suitable for injections. Aqueous carriers include water,
alcoholic-aqueous solutions, emulsions, suspensions, salt solutions
and buffered media. Parenteral carriers comprise sodium chloride
solutions, Ringer's dextrose, dextrose and sodium chloride,
Ringer's lactate and bound oils. Intravenous carriers comprise,
e.g. fluid supplements, nutrient supplements and electrolyte
supplements (such as, e.g. those based on Ringer's dextrose).
Furthermore, the composition of the invention can comprise
preservatives and other additives such as, e.g. anti-microbial
compounds, antioxidants, complex-forming agents and inert gasses.
Furthermore, depending on the intended use, compounds such as, e.g.
interleukins, growth factors, differentiation factors, interferons,
chemotactic proteins or an unspecific immunomodulary agent.
[0020] The buffer substances used are suitable to maintain the
adjusted pH within the ranges described during the phase of
cooling, freezing, spray drying or lyophilising. The buffer
substances are preferred to be selected in such a way that, with a
low buffer capacity, it is not possible for the pH to change to
lower values during freezing. By maintaining a high pH range during
lyophilisation, the stability of the polypeptide is guaranteed. A
low buffer capacity is moreover preferred for an injection solution
ready for application. In Example 1, a method for checking the pH
during cooling or freezing of pharmaceutical preparations is
described. By means of this or similar methods, buffer substances
can be determined which are suitable for the method of the
invention.
[0021] In the state of the art, a plurality of medicaments are
described which contain low-molecular, oligomeric compounds
(including peptides) and high-molecular compounds (including
polypeptides) in buffered solutions. In addition, for a plurality
of such medicaments which contain corresponding compounds that are
stable in a wide pH range methods for improving the storage
properties have been described and are known to the skilled person.
Examples thereof are methods comprising freezing, spray drying or
lyophilising of medicaments. Due to said pH-independent stability,
it has so far not been described that a specific control of the pH
during lyophilising or spray drying was necessary. Moreover,
conventional lyophilisation devices for producing medicaments und
pharmaceutical preparations have not been supplied with means for
controlling the pH.
[0022] When such known methods were used, it was surprisingly found
that the lectin properties of rViscumin and other plant dimeric
class II polypeptides of ribosome-inactivating proteins (RIP II)
can, under certain circumstances, be sensitive to the pH of the
particular solvent used in said method. Strong fluctuations of this
value and, in particular, a strongly acidic medium can lead to a
certain loss in specific lectin properties. Accordingly,
maintaining the pre-determined pH is a necessary feature of the
method of the invention. For maintaining these specific properties,
a pH control of the solution is necessary in all processing stages
in order to guarantee the stability of the polypeptide. in Example
1, a method for checking the pH during cooling or freezing of
pharmaceutical preparations has been described.
[0023] In a preferred embodiment, the method described comprises a
polypeptide containing
[0024] (a) the recombinant carbohydrate-binding polypeptide or a
functional fragment or derivative of this polypeptide which is
fused to a cytotoxically effective peptide to form a fusion
protein;
[0025] (b) the recombinant carbohydrate-binding polypeptide or a
functional fragment or derivative of this polypeptide which is
linked to another polypeptide which has an enzymatic
rRNA-N-glycosidase activity;
[0026] (c) the recombinant carbohydrate-binding polypeptide or a
functional fragment or derivative of this polypeptide which is
linked to another polypeptide in which an enzymatic
rRNA-N-glycosidase activity has been replaced by another cytotoxic
activity; or
[0027] (d) the recombinant carbohydrate-binding polypeptide or a
functional fragment or derivative of this polypeptide, which is
linked to a fusion protein, comprising a polypeptide with an
enzymatic rRNA-N-glycosidase activity and/or another cytotoxic
activity.
[0028] In accordance with this preferred embodiment of the
invention, the recombinant carbohydrate-binding polypeptide or a
functional fragment or derivative of said polypeptide is bound to
another peptide which has cytotoxic activity. Said binding of the
peptides can be both a covalent binding and a binding based on
other physico-chemical interactions. Examples of covalent binding
of the peptides of the invention comprise both peptide bonds which
are, amongst others, characteristic of fusion proteins and
disulfide bonds.
[0029] Within the meaning of the invention, the
carbohydrate-binding polypeptide or functional fragment or
derivative of said polypeptide permits an interaction of the
protein with the cell surface of the target cell. Subsequently, the
peptide having cytotoxic activity acts either directly on the cell
surface (e.g. by forming pores in the cell membrane) or after
absorption into the cell (e.g. by inhibiting or destroying the
protein biosynthesis, by inducing an apoptosis signal cascade or by
inhibiting or destroying the activity of the mitochondria). The
cytotoxic activity can be checked using various tests that are
known to the skilled person ("JAM test", cf. Matzinger (1991),
".sup.51Cr release test", "Propidium iodide staining of cells" or
"Annexin V test", cf. Dulat (2001)).
[0030] Examples of peptides having enzymatic rRNA-N-glycosidase
activity of ribosome-inactivating proteins (RIPs) are described,
amongst others, by Endo et al. (1988 and 1989) and in an overview
article by Peumans et al. (2001).
[0031] In another preferred embodiment of the method, the
recombinant carbohydrate-binding polypeptide is the B-chain of a
ribosome-inactivating protein.
[0032] In another embodiment which is preferred, too, the further
polypeptide which is linked to the recombinant carbohydrate-binding
polypeptide is the A-chain of a ribosome-inactivating protein.
[0033] In another embodiment which is furthermore preferred, the
B-chain and/or A-chain of the ribosome-inactivating protein
corresponds to the B-chain or A-chain of a ribosome-inactivating
protein of the type II. Said ribosome-inactivating type II-protein
is preferred to be rViscumin. Both the function and the recombinant
presentation of the holoenzyme rViscumin as an example of a
ribosome-inactivating protein have been described in EP 0 751 221
B1.
[0034] In another preferred embodiment of the method, it is
guaranteed that the pH of the solution is between 6.0 and 9.0, more
preferably, the pH of the solution is between 7.5 and 8.5. As
illustrated in the examples, a pH of 8.0 is particularly preferred.
A less preferred pH range of the solution is the range above pH 12
as in such high pH ranges, a deamidation is to be expected and, as
a consequence, the properties of the polypeptide as a medicinal
active agent would change. Without excluding higher pH ranges, in
the method of the invention, usually a pH of higher than 6.0 and
lower than pH 12 is to be selected. However, the person of skill in
the art can indeed also select pH ranges higher than pH 12. In this
case, it is however preferred that the pH of the medicament is
adjusted to a physiological pH range prior to administration to the
patient. A method for controlling the pH while carrying out the
method of the invention, is described in Example 1.
[0035] A method in which the salt or salts of the buffer system are
used in a final concentration ranging from 0.6% to 2.4% (5 mM to
200 mM) is preferred, too. Furthermore, a method in which the salt
or salts of the buffer system are used in a final concentration
ranging from 100 mM to 200 mM is furthermore preferred.
Accordingly, for instance, a final concentration for Tris base of
100 nM to 200 nM (1.2% to 2.4%) is preferred as in all studies
carried out in connection with this invention using optimised
formulations a loss in rViscumin of only 5% caused by the process
was observed. For the final concentration ranging from 20 mM to 100
mM a corresponding loss in the range of 10 to 15% was detected. As
shown in the Examples, for a final concentration below the optimum
concentration of 20 mM, a corresponding loss in the range of 10 to
20% was detected.
[0036] In connection with this invention, the expression "final
concentration" refers to a concentration of the solution in
mass/volume (m/v) which the person of skill in the art adjusts
before the cooling, freezing, spray drying or lyophilisation
process.
[0037] Moreover, a method is preferred in which the salt or salts
of the buffer system is/are selected from the group comprising:
TRIS/HCl, TRICIN/HCl, HEPES/HCl, ammonium carbonate buffer,
TRIS/glutamic acid and TRIS/aspartic acid. As is described amongst
others in the attached examples, said buffer systems guarantee that
a high pH is maintained during a freezing phase in the
corresponding solutions for the selected combinations of initial
substances. For this reason, the corresponding buffer systems play
a crucial role with regard to the stability of the polypeptide.
[0038] In another preferred embodiment of the method of the
invention, one or more surfactants are used for stabilising the
pharmacological properties of the polypeptide of the solution. Said
surfactants serve as wetting agents, thus reduce the surface
tension of a solution and favour wetting of lyophilisation products
with a reconstitution solution. In addition, these substances
occupy so-called "hot spots" on the walls of the preparation
vessels used and primary packing agents used to which, for
instance, rViscumin can preferably be bound as a hydrophobic
protein. In the absence of wetting agents, loss in protein or
protein activity during the production and packaging process and in
the pharmaceutical solutions is likely. Moreover, the addition of
wetting agents is advantageous to avoid loss after reconstitution
of the lyophilised powder. Such loss would lead to an inaccurate
dosage.
[0039] Non-ionic tensides are preferred to be used as surfactants,
these being used in a final concentration ranging from 0.01 to
5.0%.
[0040] Preferred non-ionic tensides are selected from the group
comprising: fatty alcohols, partial glycerides, polysorbates,
polyoxyethylene fatty acid ethers and polyoxyethylene fatty acid
esters, poloxameres (polyoxypropylene-polyoxyethylene-block
polymers), saccharide fatty acid esters, polyoxyethylene sorbitol
ethers and polyoxyethylene fatty acid ethers, polyoxy fatty acid
esters and phosphatides.
[0041] Preferred examples of polysorbates are selected from the
group comprising Polysorbate 80 and Polysorbate 20.
[0042] Moreover, preferred are polyoxyethylene fatty acid ethers
and polyoxyethylene fatty acid esters Macrogol ethers or Macrogol
esters, the poloxamer Pluronic F68, poloxamer 166 or 188 and the
phosphatides such as, e.g. lecithins. In this connection,
derivatives of lecithins from soy or chicken protein are also
comprised.
[0043] Amphoteric tensides which are used in a final concentration
ranging from 0.01 to 5.0% are also preferred as surfactants.
[0044] In another preferred embodiment of the method of the
invention, for lyophilisation, one or more lyoprotectors in a final
concentration ranging from 0.1 to 20% and/or cryoprotectors in a
final concentration ranging from 0.01 to 1.0% are added to the
solution. In this connection, lyoprotectors serve for the
protection of the substances during drying, whereas cryoprotectors
fulfil the corresponding task during freezing. The ranges of final
concentration mentioned herein for use of lyoprotectors and/or
cryoprotectors are deemed to be preferred, as a consequence, the
method of the invention also comprises ranges of final
concentration which are outside the preferred ranges of final
concentration. The lyoprotectors are preferred to be used in a
range of final concentration of 4.0 to 10%. In combination with the
lyoprotectors or also in their absence, the cryoprotectors are
preferred to be added to the solution at a final concentration
ranging from 0.05 to 0.1%.
[0045] Furthermore, lyoprotectors are preferred which are selected
from the group comprising:
[0046] a) low molecular saccharides such as glucose, trehalose and
sucrose;
[0047] b) hexites such as mannitol (mannite) and sorbitol
(sorbite):
[0048] c) oligomeric and polymeric saccharides such as cyclic
beta-hydroxypropylcyclodextrin, cyclodextrins, cellulose, starch,
carboxyamylopektin, chitosan and their derivatives;
[0049] d) anorganic gelling agents such as bentonites, and silicon
dioxide; and
[0050] e) synthetic polymers such as polyvinylpyrrolidones and
polyacrylates.
[0051] Dextranes having a molecular mass of 1,000 to 100,000 Da and
are preferred and more preferably 1,000 to 10,000 Da are used. As
documented in the examples described herein, dextranes are
preferred lyoprotectors which can preferably be used without
mannitol, which can, however, indeed be used together with other
lyoprotectors in the method of the invention. As shown in the
examples, dextranes can preferably also be used alone (without
other lyoprotectors) in the method of the invention. The fact that
dextranes can be used alone as lyoprotectors in the method of the
invention, in particular in the lyophilisation process, is
surprising as it has been described in the state of the art that
dextrane, as an accompanying adjuvant, can only contribute its
share in stabilising proteins (Carpenter et al., 1993, Carpenter et
al., 1999, Allison et al., 1999, Allison et al., 2000).
[0052] Ionic substances, too, are preferably used as
cryoprotectors. These ionic substances are, in turn, preferably
selected from the group consisting of sodium chloride, sodium
sulphate, potassium chloride and potassium sulphate. According to
the invention, sodium salts of edetic acid are also used. Said
salts contribute to a further stabilisation of the polypeptides due
to complex formation of metal cations taken up during the
production process.
[0053] According to the method of the invention, lyoprotectors and
cryoprotectors form amorphic structures during lyophilisation.
These lyoprotectors and cryoprotectors prevent the formation of
crystal grids (constant distances of atoms in a solid substance)
during the lyophilisation process. The absence of crystalline
structures in a solid substance can be detected by means of a
powder crystalline structure analysis (e.g. by diffraction of
x-rays).
[0054] In another preferred embodiment of the method, amino acids
are used as stabilisers. Preferably, they are used in a
concentration of 0.01 to 50 mg/ml. In addition to the stabilising
property, amino acids themselves can be used as buffer substances
according to the invention.
[0055] Furthermore, it is preferred that the amino acids are
selected from the group comprising acidic amino acids such as
glutamic acid and aspartic acid, the basic amino acid arginine and
the neutral amino acid valine.
[0056] In another embodiment of the method of the invention, the
polypeptide which comprises at least a recombinant
carbohydrate-binding polypeptide or a functional derivative or a
fragment of the recombinant carbohydrate-binding polypeptide is
used in a final concentration of 0.000001% (10 ng/ml) to 1.0% (10
mg/ml). In this case, a protein concentration of 0.00001% (100
ng/ml) 0.1% (1 mg/ml) is particularly preferred.
[0057] Another preferred embodiment of the method comprises the
further formulation or reconstitution of the medicament as an
aqueous or non-aqueous solution. Moreover, this includes the
further formulation of the medicament as an injection, instillation
or infusion solution. Depending on the ailments or diseases to be
treated, injection solutions according to the invention are
administered subcutaneously, intramuscularly, intravenously,
intracardially or intraperitoneally. Solutions for installation
into a body cavity are instilled, for example, into the urinary
bladder, depending on the ailment to be treated.
[0058] In another preferred embodiment of the method, the further
formulation or reconstitution of the medicament for
gastrointestinal, oral, nasal, pulmonary, dermal, transdermal or
local administration is also comprised.
[0059] Moreover, the further formulation of the medicament to give
a juice, capsules, tablets, suppositories or gels is preferred,
too.
[0060] The gels mentioned, which can be produced by further
formulation of the medicament of the invention, can be obtained
using inorganic and organic hydrogelling agents together with
aqueous or aqueous/alcoholic solutions. In this case, gelling
agents of natural, partially synthetic and synthetic origin are
comprised. These molecules have an, in part, extreme swelling
capability in common which leads to the formation of spreadable
gels.
[0061] Moreover, the further formulation of the medicament to give
a powder for inhalation which can be administered by use of an
inhalator is also preferred.
[0062] The invention furthermore relates to a medicament which is
prepared in accordance with one of the methods of the
invention.
[0063] The invention also relates to the use of a polypeptide for
producing such a medicament.
[0064] Depending on the further formulations, the administration of
the medicament of the invention can be carried out in various ways,
e.g. intravenously, intraperitoneally, subcutaneously,
intramuscularly, locally or intradermally. The attending physician
determines the kind of dosage in accordance with the clinical
factors. The skilled person knows that the kind of dosage depends
on various factors such as, e.g. the patient's height, body
surface, age, sex or general health, but also on the special agent
that is administered, the duration and kind of administration and
other medicaments which are possibly administered in parallel.
[0065] The figures show
[0066] FIG. 1 In FIG. 1 the change in the pH value of a buffer
solution dependent on the temperature is depicted. The buffer
solution corresponds to a 20 mM phosphate buffer and moreover
contains 0.1% sodium chloride. As described in example 1, this
buffer solution was cooled down in a commercially available
cryosate with temperature control. The pH value in the solution was
determined with specially suitable pH electrodes. The cooling down
rate in the depicted assay amounted to 1.2 K. The course of the
depicted curve shows that the cooling down of the buffer solution
from room temperature to the freezing point of the solution had no
significant effect on the pH value of this solution. If the
solution is cooled down to temperatures below their freezing point,
a marked decrease of the pH value of 8 to below 5 can be
observed.
[0067] FIG. 2 In FIG. 2, the stability of carbohydrate-specific
rViscumin dependent on the pH value and short-time storage at 2 to
8.degree. C., rViscumin in buffered saline solution is shown. The
buffer solution corresponds to a 20 mM phosphate buffer (ph 7.2)
which was adjusted to pH values of 3, 4, 5, 7, 8 and 9 with NaOH (1
M and 0.1 M) or HCl (10% or 1%). Moreover, the phosphate-buffered
solutions contain NaCl in a concentration of 0.7 to 0.9% for the
adjustment of the isotonicity of the solutions and low-molecular
polyvinylpyrrolidone in a concentration of 0.1 g/l for preventing
an adsorption of the polypeptide to the surface of the vessel.
[0068] In the assay shown in the figure it was observed that the
stability of the polypeptide rViscumin in the buffered solutions
decreases markedly when the pH value decreases. Below a pH value of
pH 6, no rVisumin with carbohydrate-specific properties is left
after a short storage period only.
[0069] FIG. 3 FIG. 3 shows the stability of carbohydrate-specific
rViscumin (rML) in a buffered stabilised solution and the
lyophilised powder (lyophilisation product) produced therefrom,
dependent on the temperature.
[0070] The buffer solution corresponds to a 200 mM Tris/HCl buffer
(pH 8.0), containing 8.0% (w/v) dextrane T10, 0.1% (w/v) NaCl and
0.1% (w/v) Polysorbate 80. rViscumin is contained in the solution
in a concentration of 2.0 .mu.g/ml. The solution is distributed,
treated and examined according to the assay described in example
3.
[0071] The result of the test shown in the figure shows that the
content of rViscumin in the buffered, stabilised solution decreases
markedly starting at a temperature of 40.degree. C. At 50.degree.
C., only 50% of the initial concentration of rViscumin with
carbohydrate-specific properties is detected. At 60.degree. C., no
carbohydrate-specific rViscumin is detected any more. Thus, the
disintegration temperature of rViscumin in the solution lies
between 40.degree. C. and 50.degree. C. The detected content of
rViscumin with carbohydrate-specific properties in the solid only
decreases very slowly as the temperature rises. At a temperature of
50.degree. C. a content of 94% and at a temperature of 60.degree.
C. a content of 91% of the initial content can still be
detected.
[0072] FIG. 4
[0073] FIG. 4 exemplifies the dependence of the stability of the
carbohydrate-binding activity of rViscumin in an aqueous solution
when the pH value changes.
[0074] FIG. 5
[0075] FIG. 5 shows the dependence of the carbohydrate-binding
activity of rViscumin in an aqueous solution and as lyophilised
powder with increasing temperature.
[0076] FIG. 6
[0077] FIG. 6 shows the influence which the adjuvants Pluronic F68
and Polysorbate 80 in their role as cryoprotectors have on the
process step freezing/thawing of an aqueous solution of rViscumin
in 100 mM TRIS buffer pH 8.0. The solution contains the
lyoprotector dextrane T1 in a concentration of 2% which is below
the preferred range.
[0078] FIG. 7
[0079] FIG. 7 shows the influence which the protein concentration
of an aqueous solution of rViscumin has on the lyophilisation
process.
[0080] FIG. 8
[0081] FIG. 8 shows the influence which the lyoprotector mannite
and a mixture of mannite together with a non-crystallising
kryoprotector has on rViscumin.
[0082] FIG. 9
[0083] FIG. 9 shows the suitability and the optimal range of the
cryoprotector dextrane T1 with regard to the stability of rViscumin
during lyophilisation.
[0084] FIG. 10
[0085] FIG. 10 shows the influence of different lyoprotectors on
the stability of lyophilised rViscumin preparations at an increased
temperature of 60.degree. C.
[0086] FIG. 11
[0087] FIG. 11 shows the storage stability of an aqueous
preparation of rViscumin (squares) over 10 weeks and of a
lyophilised culture (rhombuses) over 56 weeks at a storage
temperature of 2 to 8.degree. C.
EXAMPLE 1
Method for Verifying the pH Value during the Cooling Down or the
Freezing of Medicaments
[0088] rViscumin is a dimeric recombinantly produced plant protein
with sugar-specific binding activities. The pharmacological effect
of the protein, triggering apoptosis in cells, correlates with
obtaining the sugar-specific binding activity. Obtaining the sugar
specificity largely depends on the pH value of the surrounding
medium. If the pH value of the medium decreases, at a ph value of
below 6.0, the sugar binding activity of rViscumin decreases
markedly. This is also the case for pH changes during the freezing
process of the lyophilisation of aqueous preparations with
rViscumin. It is for this reason that the control of the pH of
aqueous buffer systems during freezing of rVisumin pharmaceutical
preparations in connenction with lyophilisation is necessary.
[0089] The problem can be solved by producing pharmaceutical
preparations of rViscumin or of its basic formulation without
active agent (combination of buffer salts) in a volume of 15 ml in
common freeze flasks (vials). The freeze flasks are placed into a
commercially available cryostate with controlled temperature
control. Special suitable pH electrodes (e.g. the
pressure-resistant Sure-Flow pHuture Probe with Converter Model
605--power supply for ISFET electrodes, Orion or frost-resistant
glass electrode, Schott Gerte GmbH, Hofheim) are used for pH
determination. The registration of the pH values is carried out
with commercially available pH-meters. A cooling down rate of 1.2 K
is suitable to picture the simulation of the cooling down rate of
lyophilisers. The pH values in the solution are determined
depending on the temperature.
[0090] FIG. 1 shows the temperature dependent course of the pH
value of a 20 mM sodium phosphate buffer (ph 8.0 at room
temperature).
[0091] With a decrease in temperature under 0.degree. C., phosphate
buffers show an erratic and strong decrease of the pH value as can
be proved by the example of the 20 mM phosphate buffer with 0.1%
(w/v) sodium chloride, measured with the described method. This
suggests a physical change of the buffer system. It is known that
with the temperature sinking, sodium monohydrogen phosphate
preferably crystallises from aqueous phosphate-buffered solutions
and thus causes this change in pH.
[0092] Aqueous preparations of rViscumin have already been
described in EP 0 751 221 B1. These aqueous preparations which are
suitable as medicaments are aqueous solutions buffered with
phosphate pH 7.2 and a rViscumin concentration of 100-200 ng/ml
and, e.g., have the following compositon:
1 rViscumin 100 ng Sodium monohydrogenphosphate dihydrate 3.56 mg
Sodium dihydrogenphosphate dihydrate 0.64 mg Sodium chloride 67.0
mg Poly(1-vinyl-2-pyrrolidone) K 17 0.5 mg Water for injection
purposes ad 1 ml.
[0093] When cooling down and freezing, phosphate buffers pH 7.2
show a decrease inhe pH value which is due to the sinking
temperature as has also been measured for phosphate buffer pH 8.0
and as has been described in FIG. 1. It is known to the person
skilled in the art that low initial values of the pH upon freezing
the aqueous solution lead to stronger shifts into the acid range as
the concentration of sodium dihydrogenphosphate in the solution is
increased. If preparations of the above-mentioned composition are
lyophilised this inevitably leads to low pH conditions below pH 6,
under which rViscumin is not stable and losses in activity occur
due to the denaturation of the protein, as has been depicted for
aqueous rViscumin preparations in FIG. 5.
[0094] The pH courses of the biological buffers TRIS/HCl,
TRICIN/HCl and Hepes/HCl pH 8.0 are shown and discussed in Gloger
O., Muller B. W., 2000.
[0095] With a decreasing temperature, buffer systems consisting of
TRIS/HCl, TRISIN/HCl and Hepes/HCl adjusted to the pH value 8.0
show a continuous low change in pH to larger pH values up to 9.0
(Gloger O., Muller B. W., 2000).
EXAMPLE 2
Stability of Carbohydrate-Specific rViscumin Depending on the pH
Value and Short-Time Storage
[0096] rViscumin in a concentration of 200 ng/ml is solved in
different buffers. Starting from a 20 mM phosphate buffer (pH 7.4)
buffers with the pH values 3, 4, 5, 7, 8 and 9 are produced with
NaOH (1M and 0.1 M) or HCl (10% or 1%). Moreover, the
phosphate-buffered solutions contain NaCl in a final concentration
of 0.7 to 0.9% for adjusting the isotonicity of the solution and
low-molecular polyvinylpyrrolidone in a concentration of 0.1 g/l
for preventing an adsorption of the polypeptide to the surface of
the vessel. The solutions are filtered for germs over a membrane
(pore size 0.2 .mu.m) and are stored in closed polyethylene
containers under controlled temperature conditions at 2 to
8.degree. C. Depending on the time, samples are taken. These
samples are diluted 1:10 with 20 mM phosphate buffer (pH 7.4) in
order to obtain uniform solutions for the determination of the
protein content with lectin activity by means of a specific
enzyme-coupled immunoassay using a glycoprotein and a specific
monoclonal antibody. Example 4 describes an example for an assay
for the determination of the protein content of a solution with
lectin activity. The assay shown in FIG. 2 indicates that the
stability of the polypeptide rViscumin decreases markedly in the
buffered solutions with decreasing pH value. Below a pH value of pH
6, no more rViscumin with lectin activity is left in the solutions
after a short storage period. The highest stability of rViscumin
while keeping the lectin activity is observed at high pH
values.
EXAMPLE 3
Stability of rViscumin (rML) Lyophilisation Product
[0097] rViscumin in a concentration of 2.0 .mu.g/ml is solved in a
bufferd, stabilised solultion containing 200 mM Tris/HCl buffer (pH
8.0), 8.0% (w/v) dextrane T10, 0.1% (w/v) NaCl and 0.1% (w/v)
Polysorbate 80. Part of this solution is transferred under aseptic
conditions by lyophilisation into a powder. To this avail, after
filtration for germs, 0.5 ml of the solution is filled into glass
vials through a 0.2 .mu.m filter, is partly closed with a
lyophilisation plug and is dried in a lyophiliser. The other part
is also filtered for germs, filled into glass vials and is closed
and stored until examination at 2 to 8.degree. C.
[0098] After lyophilisation the glass vials with the aqueous
solution as well as those with the solid (dried solution) are
placed into a controlled water bath with a temperature and time
control. The glass vials were subjected to the following
temperatures:
[0099] 5 minutes at 30.degree. C.
[0100] heating with a temperature increase of 1.5.degree.
C./minute
[0101] 5 minutes at 40.degree. C.
[0102] heating with a temperature increase of 1.5.degree.
C./minute
[0103] 5 minutes at 50.degree. C.
[0104] heating with a temperature increase of 1.5.degree.
C./minute
[0105] 5 minutes at 60.degree. C.
[0106] After adjusting the temperature, the protein content with
lectin activity in the selected samples of the solution and of the
solid was determined by means of a specific enzyme-coupled
immunoassay by using a glycoprotein and a specific monoclonal
antibody. An example of an assay for the determination of the
protein content of a solution with lectin activity is described in
example 4.
[0107] The assay shown in FIG. 3 shows that the content of
rViscumin in the buffered stabilised solution decreases markedly
starting at a temperature of 40.degree. C. At 50.degree. C., only
50% of the initial concentration of rViscumin with lectin activity
are detected. After heating the solution to 60.degree. C., it is no
longer possible to find rVisumin with lectin activity. Thus, the
disintegration temperature of rViscumin in solution lies between
40.degree. C. and 50.degree. C.
[0108] The content of rViscumin with lectin activity in the solid
only decreases very slowly as the temperature increases. At a
temperature of 50.degree. C., a content of 94% and at 60.degree.
C., a content of 91% of the initial content of rViscumin with
lectin activity is found. This shows that rVisumin is much more
stable in the lyophilised powder than in the solution.
EXAMPLE 4
Determination of the Protein Content of a Solution with Lectin
Activity
[0109] 100 .mu.l of a solution of 0.1 mg/ml Asialofetuin in
carbonate buffer pH 9.6 are placed in the wells of a 96 microtiter
plate with a high protein binding and are incubated for 16 hours at
ambient temperature. After washing three times with PBS containing
0.05 g/l Polysorbate 80, the wells of the microtiter plate are
incubated for 1 hour at ambient temperature with 200 .mu.l PBS
containing 10 g/l bovine serum albumine and 0.05 g/l polysorbate 80
(blocking of unspecific binding sites). After washing three times,
100 .mu.l of the rViscumin reference solution in the concentration
range of 10-200 ng/ml, 100 .mu.l of the test solution and for the
determination of the blank reading 100 .mu.l of the buffer (PBS
with 0.05 g/l Polysorbate 80), respectively, are placed into the
wells and are incubated at ambient temperature for two hours.
Afterwards, the wells of the microtiter plate are washed and 100
.mu.l of a solution of a specific monoclonal detection antibody
(anti-rViscumin A-chain IgG of mouse) in a concentration of 1
.mu.g/ml in PBS containing 0.05 g/l Polysorbate 80 and 0.1 g/l
bovine serum albumine are added and incubated at ambient
temperature for 1 hour. The wells of the microtiter plates are
washed three times and 100 .mu.l of a specific commercially
available peroxidase (POD)-coupled anti-IgG-mouse antibody in the
dilution according to the indications of the supplier are added and
are incubated at ambient temperature for 1 hour. The wells of the
microtiter plate are washed six times and subsequently 100 .mu.l of
a solution of a commercially available
ortho-phenylenediamine/H.sub.2O.sub.- 2 tablet in 25 ml citrate
buffer pH 5 are added and are incubated in the dark at room
temperature for 15 minutes. After 15 minutes 100 .mu.l of a 1 M
sulphuric acid are added to each well and the intensity of the
colouration of the solution is determined by absorption
measurement.
[0110] The content in the test solutions is determined in
comparison with the reference solutions.
EXAMPLE 5
Dextrane-Containing rViscumin Injection Solution 10 .mu.g/ml
(Lyophilisation Product)
[0111] In the following, different formulae for dextrane-containing
injection solutions are described.
[0112] To this avail, Polysorbate, Tris base and dextrane were
solved in 80% of the amount of water necessary for injection
purposes. Subsequently, the pH is adjusted to 8.0 with HCl (1 N).
rViscumin is added to this solution and is stirred well. The
residual water is used to fill up to the required set volume.
Subsequently, the solution is sterile filtered over a 0.2 .mu.m
filter. The solution is filled into glass vials under aseptic
conditions, is pre-closed with the lyophilisation plug and is dried
in the lyophiliser.
2 Formula with dextrane T1 rViscumin 0.01 mg Polysorbate 80 1 mg
Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 Dextrane T1 80 mg Water for
injection purposes ad 1.0 ml
[0113] Moreover, in the following the preparation of a rViscumin
injection solution is decribed which additionally comprises NaCl.
The latter is solved in water together with polysorbate, tris base
and dextrane.
3 Formula with dextrane T1 and NaCl rViscumin 0.01 mg Polysorbate
80 1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 NaCl 1 mg Dextrane
T1 80 mg Water for injection purposes ad 1.0 ml
[0114] The last example of this group of rViscumin preparations
moreover describes the preparation of an rViscumin injection
solution which also comprises Na-EDTA in addition to NaCl. These
are solved in water simultaneously with polysorbate, Tris base and
dextrane.
4 Formula with dextrane T1, NaCl and Na-EDTA rViscumin 0.01 mg
Polysorbate 80 1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0
Di-sodium-EDTA 0.01 mg NaCl 1 mg Dextrane T1 80 mg Water for
injection purposes ad 1.0 ml
[0115] In the examples shown, for the reconstruction of the
lyophilisation products, each of these is taken up in the amount of
water indicated.
EXAMPLE 6
.beta.-HP-Cyclodextrine-Containing rViscumin Injection Solution 10
.mu.g/ml (Lyophilisation Product)
[0116]
5 Formula with .beta.-HP-cyclodextrine rViscumin 0.01 mg
Polysorbate 80 1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0
Di-sodium-EDTA 0.01 mg .beta.-HP-cyclodextrine 80 mg Water for
injection purposes ad 1.0 ml
[0117] For the preparation of this injection solution, polysorbate,
Tris base, di-sodium edetine acid and
.beta.-hydroxypropyl-cyclodextrine are solved in 80% of the amount
of water necessary for injection purposes. Subsequently, the pH is
adjusted to 8.0 with HCl (1 N). rViscumin is added to this solution
and is stirred well. The residual water is used to fill up to the
required set volume. Subsequently, the solution is sterile filtered
over a 0.2 .mu.m filter. The solution is filled into glass vials
under aseptic conditions, is pre-closed with the lyophilisation
plug and is dried in the lyophiliser.
EXAMPLE 7
Aqueous rViscumin Solution 10 .mu.g/ml Containing Amino Acids
(Lyophilisation Product)
[0118] The production of the solutions is carried out according to
the process as described in example 4. Accordingly, polysorbate,
Tris base, sodium chloride and the amino acid(s) are solved in 80%
of the amount of water necessary for injection purposes. The
solutions are filled into glass ampouls or glass bottles under
aseptic conditions. The medicament is stable under storage
conditions of 2 to 8.degree. C.
6 Formula with glutamic acid rViscumin 0.01 mg Polysorbate 80 1 mg
Tris base 2.4 mg HCl (1 N) q.s. pH 8.0 NaCl 6.5 mg Glutamic acid
0.1 mg Water for injection purposes ad 1.0 ml Formula with glutamic
acid and valin rViscumin 0.01 mg Polysorbate 80 1 mg Tris base 2.4
mg HCl (1 N) q.s. pH 8.0 NaCl 6.5 mg Glutamic acid 0.1 mg Valine 10
mg Water for injection purposes ad 1.0 ml
[0119] If 80 mg dextrane T1 is added to the solutions prior to
filling them up, lyophilisation products, too, can be prepared.
EXAMPLE 8
Influence of Different Amino Acids on the Stability of
Carbohydrate-Specific rViscumin in Buffered Saline Solutions
[0120] In the description it is shown that representatives of the
amino acids with acidic, neutral and basic properties are able to
stabilise the polypeptide rViscumin in aqueous, buffered
solutions.
[0121] The assay summarised in the following table clarifies the
influence of amino acids on the stabilisation of rViscumin in
buffered, aqueous, saline solutions at a pH value of 8.0.
7 Concentration Content (%) Content (%) Amino acid mg/ml Initial
value 3 days storage no -- 100% 21.7% glutamic acid 0.1 100% 100%
10 100% 100% valine 0.1 100% 24.2% 10 100% 91.3% arginine 0.1 100%
74.2% 10 100% 30.5%
[0122] If an rViscumin solution is stored for three days at 2 to
8.degree. C., 22% of the carbohydrate-specific rViscumin can still
be detected after this period of time.
[0123] If, however, the acidic amino acid glutamic acid, which was
here used as an example of an acidic amino acid, is added to the
solution, 100% of the carbohydrate-specific polypeptide rViscumin
can be recovered after three days of corresponding storage. This
stabilising effect is observed in a concentration ranging from 0.1
to 10 mg/ml.
[0124] If neutral amino acids such as e.g. valine are added, a
stabilisation of the polypeptides in the aqueous solution can be
observed, too. With respect to this amino acid, the concentration
range which has a stabilising effect is at 10 mg/ml. After three
days of storage, 91% of the initial content of rViscumin is still
found.
[0125] Surprisingly, also with respect to the amino acids with
basic properties in the low concentration range of 0.1 mg/ml, a
stabilising effect on the protein could be observed. The recovery
of the protein in the corresponding solution with a content of 74%
is clearly above the content observed in the control preparation
amounting to 22%.
[0126] Thus, as additives, amino acids have a stabilising effect on
aqueous solutions and also as additives in dry preparations
(powder, lyophilisation product) of rViscumin.
EXAMPLE 9
Aqueous rViscumin Solution, Concentration to Infusion 200 .mu.g
[0127] An example of the preparation of a solution or of a
concentrate of rViscumin for infusion is described in the
following:
8 Formula with glutamic acid rViscumin 0.20 mg Polysorbate 80 10 mg
Tris base 24.1 mg HCl (1 N) q.s. pH 8.0 NaCl 65 mg Glutamic acid 1
mg Water for injection purposes ad 10 ml
[0128] The production of the solution is carried out according to
the procedure as described in example 4. Accordingly, polysorbate,
Tris base, sodium chloride and glutamic acid are solved in 80% of
the required amount of water for injection purposes. Subsequently,
the pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin is
added to this solution and is stirred well. The residual water is
used to fill up to the required set volume and the solution is
sterile filtered over a 0.2 .mu.m filter. The solution is filled
into glass bottles under aseptic conditions. The medicament is
stable under storage conditions of 2-8.degree. C.
[0129] If 800 mg dextrane T1 is added to the solution prior to
filling, it is also possible to produce a lyophilisation
product.
EXAMPLE 10
Aqueous rViscumin Instillation Solution 500 .mu.g
[0130] An example of the production of a solution of rViscumin for
the installation in a body cavity is described in the
following:
9 Formula with glutamic acid rViscumin 0.5 mg Polysorbate 80 500 mg
Trisbase 121.1 mg HCl (1 N) q.s. pH 8.0 NaCl 350 mg Glutamic acid 5
mg Water for injection purposes ad 50 ml
[0131] The production of the solution is carried out according to
the procedure as described in example 4. Accordingly, polysorbate,
Tris base, sodium chloride and glutamic acid are solved in 80% of
the required amount of water for injection purposes. Subsequently,
the pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin is
added to this solution and is stirred well. The residual water is
used to fill up to the required set volume and the solution is
sterile filtered over a 0.2 .mu.m filter. The solution is filled
into glass bottles under aseptic conditions. The medicament is
stable under storage conditions of 2-8.degree. C.
[0132] If 2.0 mg dextrane T1 is added to the solution prior to
filling, it is also possible to produce a lyophilisation
product.
EXAMPLE 11
Glucose-Containing rViscumin Solution 10 .mu.g/ml (Lyophilisation
Product)
[0133] As described above, in a preferred embodiment of the
invention sugar is added to the rViscumin solution. An example of
the production of such a solution, which is subsequently
lyophilised, is described in the following:
10 Formula with glucose and NaCl rViscumin 0.01 mg Polysorbate 80 1
mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 NaCl 1 mg Glucose 80 mg
Water for injection purposes ad 1.0 ml
[0134] Polysorbate, Tris base and glucose are solved in 80% of the
required amount of water for injection purposes. Subsequently, the
pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin is
added to this solution and is stirred well. The residual water is
used to fill up to the required set volume and the solution is
sterile filtered over a 0.2 .mu.m filter. The solution is filled
into glass vials under aseptic conditions, is preliminarily closed
with the lyophilisation plug and is dried in the lyophilisation
unit.
EXAMPLE 12
Sorbitol-Containing rViscumin Solution 10 .mu.g/ml (Lyophilisation
Product)
[0135] As is also described above, in other preferred embodiments
of the invention sorbitol is added to the rViscumin solution. An
example of the production of such a solution, which is subsequently
lyophilised, is described in the following:
11 Formula with sorbitol and NaCl rViscumin 0.01 mg Polysorbate 80
1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 NaCl 1 mg Sorbitol 80
mg Water for injection purposes ad 1.0 ml
[0136] Polysorbate, Tris base and sorbitol are solved in 80% of the
required amount of water for injection purposes. Subsequently, the
pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin is
added to this solution and is stirred well. The residual water is
used to fill up to the required set volume. Subsequently, the
solution is sterile filtered over a 0.2 .mu.m filter. The solution
is filled into glass vials under aseptic conditions, is
preliminarily closed with the lyophilisation plug and is dried in
the lyophilisation unit.
EXAMPLE 13
Chitosan-Containing rViscumin Solution 10 .mu.g/ml (Lyophilisation
Product)
[0137]
12 Formula with chitosan and NaCl rViscumin 0.01 mg Polysorbate 80
1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 NaCl 1 mg Chitosan
(low-molecular) 80 mg Water for injection purposes ad 1.0 ml
[0138] Polysorbate, Tris base and chitosan are solved in 80% of the
required amount of water for injection purposes. Subsequently, the
pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin is
added to this solution and is stirred well. The residual water is
used to fill up to the required set volume. Subsequently, the
solution is sterile filtered over a 0.2 .mu.m filter. The solution
is filled into glass vials under aseptic conditions, is
preliminarily closed with the lyophilisation plug and is dried in
the lyophilisation unit.
EXAMPLE 14
Aerosil-Containing rViscumin solution 100 .mu.g/ml (Lyophilised
Culture)
[0139]
13 Formula with silicum dioxide rViscumin 0.1 mg Polysorbate 80 10
mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 Silicium dioxide
(colloidal) 20 mg Dextrane T1 60 mg Water for injection purposes ad
1.0 ml
[0140] Polysorbate, Tris base and dextrane are solved in 80% of the
required amount of water for injection purposes. Subsequently, the
pH is adjusted to 8.0 with the help of HCl (1 N). rViscumin and the
colloidal silicium dioxide is added to this solution and is stirred
well. The residual water is used to fill up to the required set
volume. The solution is filled into glass vials, is preliminarily
closed with the lyophilisation plug and is dried in the
lyophilisation unit.
EXAMPLE 15
Povidone-Containing rViscumin Solution 10 .mu.g/ml (Lyophilisation
Product)
[0141]
14 Formula with polyvinylpyrrolidone and NaCl rViscumin 0.01 mg
Polysorbate 80 1 mg Tris base 24.2 mg HCl (1 N) q.s. pH 8.0 NaCl 1
mg Polyvinylpyrrolidone K17 80 mg Water for injection purposes ad
1.0 ml
[0142] Polysorbate, Tris base and polyvinylpyrrolidone are solved
in 80% of the required amount of water for injection purposes.
Subsequently, the pH is adjusted to 8.0 with the help of HCl (1 N).
rViscumin is added to this solution and is stirred well. The
residual water is used to fill up to the required set volume.
Subsequently, the solution is sterile filtered over a 0.2 .mu.m
filter. The solution is filled into glass vials under aseptic
conditions, is preliminarily closed with the lyophilisation plug
and is dried in the lyophilisation unit.
EXAMPLE 16
rViscumin Powder for the Preparation of a Solution, 10 mg rViscumin
Solution for Oral Uptake
[0143] Examples of the preparation of rViscumin powder, further
processed for the subsequent oral application as powder and
resolved in water prior to application are described in the
following:
15 Formula with dextrane 1. rViscumin 10 mg 2. Polysorbate 80 100
mg 3. Tris base 24 mg 4. HCl (1 N) q.s. pH 8.0 5. Dextrane T1 10 g
6. Sucrose 10 g
[0144] Positions 1 to 4 and parts of 5 (dextrane T1 serve as
lyoprotective substance in this formula) are solved with purified
water to 10 ml and are processed into a powder by lyophilisation.
This powder is storable. As in the above-identified examples, the
powder is mixed with the other substances and is filled into 100 ml
bottles. In order to prepare the solution, the solid is solved with
water to 100 ml.
[0145] Positions 1 to 5 and parts of 6 (sucrose serves as
lyoprotective substance in this formula) of the following formula
are solved with purified water to 10 ml and are processed into a
powder by lyophilisation. This powder is storable. As in the
above-identified examples, the powder is mixed with the other
substances and is filled into 100 ml bottles. In order to prepare
the solution, the solid is solved with water to 100 ml.
16 Formula with sucrose 1. rViscumin 10 mg 2. Polysorbate 80 100 mg
3. Tris base 120 mg 4. Glutamic acid 10 mg 5. HCl (1 N) q.s. pH 8.0
6. Sucrose 10 g 7. Flavours 0.1 mg 8. Sorbitol 10 mg 9. Water ad
100 ml
EXAMPLE 17
rViscumin Powder for the Preparation of a Solution, 10 mg rViscumin
Juice for Oral Uptake
[0146] An example of the preparation of rViscumin powder, further
processed for a subsequent oral application as powder for the
preparation of a juice and resolved in water prior to application
is described in the following:
17 Formula with sucrose 1. rViscumin 10 mg 2. Polysorbate 80 100 mg
3. Tris base 24 mg 4. HCl (1 N) q.s. pH 8.0 5. Sucrose 25 g 6.
Hydroxyethyl cellulose 400 700 mg 7. Xanthane gum 300 mg 8.
Flavours 0.1 mg 9. Glycerine 85% 1 g 10. Sorbitol 10 g
[0147] Positions 1 to 4 and parts of 5 are solved with purified
water to 10 ml and are processed into a powder by lyophilisation.
This powder is storable. In a known manner, the powder is mixed
with the other substances and is filled into 100 ml bottles. In
order to prepare the juice, the solid is filled up with water to
100 ml and is solved. After the swelling time has been observed,
the juice is suitable for uptake.
EXAMPLE 18
rViscumin tablets 0.1/0.5 mg 250 mg Tablet for Oral Uptake
[0148] Examples of the preparation of rViscumin tablets are shown
in the following:
18 Formula with dextrane/cellulose 1. rViscumin 0.1 mg 0.5 mg 2.
Soy lecithin 10 mg 10 mg 3. Tris base 24 mg 24 mg 4. HCl (1 N) q.s.
pH 8.0 q.s. pH 8.0 5. Dextrane T1 100 mg 100 mg 6. Cellulose,
microcrystalline 99 mg 99 mg 7. Highly disperse silicium dioxide
(Aerosil) 5 mg 5 mg 8. Cross-linked polyvinylpyrrolidone 5 mg 5 mg
(Kollidon CL) 9. Magnesium stearate 1 mg 1 mg
[0149] Positions 1 to 5 are solved with purified water to 2 ml and
are processed into a powder by lyophilisation. This powder is
storable. In a known manner, the powder is mixed with the other
substances to form the powder which is pressed into tablets. These
tablets can be coated with a common varnish which prevents the
release of the active agent in the stomach (retarded release).
19 Formula with sorbitol 1. rViscumin 0.1 mg 0.5 mg 2. Polysorbate
80 10 mg 10 mg 3. Tris base 24 mg 24 mg 4. HCl (1 N) q.s. pH 8.0
q.s. pH 8.0 5. Sorbitol 200 mg 200 mg 6. Highly disperse silicium
dioxide (Aerosil) 5 mg 5 mg 8. Sodium carboxymethyl cellulose 5 mg
5 mg (Tylopur) 9. Magnesium stearate 1 mg 1 mg
[0150] Positions 1 to 4 and a part of 5 are solved with purified
water to 2 ml and are processed into a powder by lyophilisation.
This powder is storable. In a known manner, the powder is mixed
with the other substances to form the powder which is pressed into
tablets. These tablets can be coated with a common varnish which
prevents the release of the active agent in the stomach (retarded
release).
20 Formula with dextrane 1. rViscumin 0.1 mg 0.5 mg 2. Polysorbate
80 5 mg 5 mg 3. Tris base 12 mg 12 mg 4. HCl (1 N) q.s. pH 8.0 q.s.
pH 8.0 5. Dextrane T1 40 mg 40 mg 6. Cellulose, microcrystalline 57
mg 57 mg 7. Highly disperse silicium dioxide (Aerosil) 5 mg 5
mg
[0151] Positions 1 to 5 are solved with purified water to 1 ml and
are processed into a powder by lyophilisation. This powder is
storable. In a known manner, the powder is mixed with the other
substances to form the powder which is filled into hard gelatine
capsules.
EXAMPLE 19
rViscumin Suppository 1 mg 250 Suppository for Introduction into
the Intestine
[0152] An example of the preparation of rViscumin suppositories is
shown in the following:
21 Formula with .beta.-HP-Cyclodextrine 1. rViscumin 1 mg 2. Soy
lecithin 100 mg 3. Tris base 24 mg 4. HCl (1 N) q.s. pH 8.0 5.
Disodium EDTA 10 mg 6. .beta.-HP-Cyclodextrine 160 mg 7. Sodium
stearate 50 mg 8. Macrogol 300 250 mg 9. Glycerol 85% 1.9 g 10.
Purified water ad 2.5 g
[0153] Positions 1 to 6 are solved with purified water to 2 ml and
are processed into a powder by lyophilisation. This powder is
storable. In a known manner, the powder is mixed with the other
substances to form a suppository. The admixing of rViscumin powder
solved in a mixture of purified water and glycerol 85% into the
suppository matrix is carried out at a controlled temperature. The
mass is pressed into forms and is left to solidify by cooling.
EXAMPLE 20
rViscumin Gel 1 mg Hydrophilic Gel for Dermal Application without
Conservation
[0154] An example of the preparation of a hydrophilic rViscumin gel
for dermal application is shown in the following:
22 Formula with .beta.-HP-Cyclodextrine 1. rViscumin 1 mg 2.
Poloxamer 166 100 mg 3. Tris base 24 mg 4. HCl (1 N) q.s. pH 8.0 5.
Disodium EDTA 10 mg 6. .beta.-HP-Cyclodextrine 160 mg 7. Sorbitan
monostearate (Arlacel 60) 200 mg 8. Macrogol-9-stearate 300 mg 9.
Glycerol 85% 500 mg 10. Medium-chain triglycerides 500 mg 11.
Purified water ad 10 g
[0155] Positions 1 to 6 are solved with purified water to 2 ml and
are processed into a powder by lyophilisation. This powder is
storable. In a known manner, the powder is mixed with the other
substances to form a gel. The admixing of rViscumin powder solved
in purified water into the gel matrix is carried out at a
temperature of below 30.degree. C.
[0156] If necessary, a conservation can be carried out with sodium
benzoate or PHB esters.
EXAMPLE 21
rViscumin Powder for Inhalation 0.1/0.5 mg 1 g Powder
[0157]
23 Formula with dextrane/cellulose 1. rViscumin 0.1 mg 0.5 mg 2.
Polysorbate 80 10 mg 10 mg 3. Tris base 24 mg 24 mg 4. HCl (1 N)
q.s. pH 8.0 q.s. pH 8.0 5. Dextrane T1 100 mg 100 mg 6. Cellulose,
microcrystalline 860 mg 860 mg 7. Sodium carboxymethyl cellulose 5
mg 5 mg
[0158] Positions 1 to 5 are solved with purified water to 2 ml and
are processed into a powder by lyophilisation. This powder is
storable. In a known manner, the powder is mixed with the other
substances to form a powder, micronised and is administered by
means of dry powder inhalators.
EXAMPLE 22
Influence of Selected Cryoprotectors on the Stability of
rViscumin
[0159] Preparations of rViscumin with the following
composition:
24 rViscumin 10 .mu.g Tris base 12.1 mg Hydrochloric acid 1 N for
adjustment of the pH to 8.0 Cryoprotector 1/10 mg Sodium EDTA 10
.mu.g Water for injection purposes ad 1 ml
[0160] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyopiliser at a cooling down rate of 3
K/hour, subsequently thawed and the carbohydrate-binding activity
of rViscumin in the solution is determined according to the method
as explained in example 4. Pluronic F68 and Polysorbate 80 are used
as cryoprotectors.
[0161] After thawing, a recovery of the rViscumin activity in the
range of 98 to 102% for both cryoprotectors in the two
concentrations is found (FIG. 6).
[0162] The two cryoprotectors Pluronic F68 and Polysorbate 80 are
suitable for stabilising rViscumin during freezing in the
lyophilisation process in the preferred range, as shown for the two
concentrations 0.1 to 1.0%,
EXAMPLE 23
Influence of the Protein Concentration on the Stability during
Lyophilisation
[0163] Preparations of rViscumin with the following
composition:
25 rViscumin 10/50/100 .mu.g Tris base 12.1 mg Hydrochloric acid 1
N for adjustment pH to 8.0 of the Polysorbate 80 1 mg Sodium EDTA
10 .mu.g Water for injection purposes ad 1 ml
[0164] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyopiliser at a cooling down rate of 3
K/hour and are subsequently dried.
[0165] Drying Programme:
[0166] Primary drying: 8 hours at -10.degree. C. and 80 kPa
pressure followed by an increase in temperature to 10.degree. C.
during 8 hours and 80 kPa pressure, secondary drying: 6 hours at
30.degree. C. and 10 kPa pressure.
[0167] When exclusively using the cryoprotector polysorbate 80,
which is suitable for the stabilisation of rViscumin during the
freezing process, the selected preparations with the different
concentrations of rVicumin show an insufficient stabilisation of
the protein after the termination of the lyophilisation process
(FIG. 7). The stability of rVisumin in the lyophilisation product
on the selection of the final concentration in the aqueous
solution. Thus, the recovery of the activity increases from 50% for
the concentration 10 .mu.g/ml to 80% for the concentration 100
.mu.g/ml. The example clearly shows that in all rViscumin
concentrations the addition of suitable lyoprotectors has an
advantageous effect on the stability of the lyophilised forms of
medicaments.
EXAMPLE 24
Influence of Mannitol (Mannite) and Mannitol/Dextrane on the
Stability of rViscumin
[0168] The preparations of rViscumin (10 .mu.g/ml) with the
following composition
26 Mannite/ Solution Mannite dextrane rViscumin 10 .mu.g 10 .mu.g
Mannite 20 mg 20 mg Dextrane T1 20 mg Tris base 12.1 mg 12.1 mg
Hydrochloric acid (1 N) for the adjustement of the pH to 8.0
Polysorbate 80 1 mg 1 mg Sodium EDTA 10 .mu.g 10 .mu.g Water for
injection purposes ad 1 ml ad 1 ml
[0169] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyophiliser at a cooling down rate of 3
K/hour and are subsequently dried.
[0170] Drying Programme:
[0171] Primary drying: 8 hours at -10.degree. C. and 80 kPa
pressure followed by an increase in temperature to 10.degree. C.
during 8 hours and 80 kPa pressure, secondary drying: 6 hours at
30.degree. C. and 10 kPa pressure.
[0172] By adding mannite in a suboptimal concentration of 2%, a
recovery activity for rViscumin of 61% is determined (FIG. 8).
Mannite is suitable for the stabilisation of rViscumin as it can
increase the stability of the lyophilised rViscumin solution 10
.mu.g/ml of 50% to 61%. A mixture of mannite 2% and dextrane T1 2%
results in a recovery of the activity of 74% after lyophilisation,
which leads to the conclusion that dextrane alone can also have a
positive effect on stability.
EXAMPLE 25
Influence of Dextrane T1 on the Stability of rViscumin
[0173]
27 rViscumin 10 .mu.g Dextrane T1 0/8/20/40/80 mg TRIS base 12.1 mg
Hydrochloric acid (1 N) for the pH to 8.0 adjustment of the
Polysorbate 80 1 mg Sodium EDTA 10 .mu.g Water for injection
purposes ad 1 ml
[0174] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyopiliser at a cooling down rate of 3
K/hour and are subsequently dried.
[0175] Drying Programme:
[0176] Primary drying: 8 hours at -10.degree. C. and 80 kPa
pressure followed by an increase in temperature to 10.degree. C.
during 8 hours and 80 kPa pressure, secondary drying: 6 hours at
30.degree. C. and 10 kPa pressure.
[0177] A recovery of 89% of the activity of rViscumin is detected
for the suboptimal concentration of 2% dextrane T1. The stability
of rViscumin with dextrane is significantly enhanced compared to
the results obtained when the mixture mannite/dextrane was used.
Beginning at a dextrane concentration larger than or equal to 4%,
stable, solid pharmaceutical preparations are obtained in the
lyophilisation process. Dextrane is suitable as lyoprotector for
rViscumin.
EXAMPLE 26
Influence of Further Lyoprotectors
[0178] The preparations of rViscumin (10 .mu.g/ml) with the
following composition:
28 rViscumin 10 .mu.g Lyoprotector 80 mg, except mannite 20 mg TRIS
base 12.1 mg Hydrochloric acid (1 N) for pH to 8.0 adjustment of
the Polysorbate 80 1 mg Sodium EDTA 10 .mu.g Water for injection ad
1 ml purposes
[0179] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyopiliser at a cooling down rate of 3
K/hour and are subsequently dried.
[0180] Drying Programme:
[0181] Primary drying: 8 hours at -10.degree. C. and 80 kPa
pressure followed by an increase in temperature to 10.degree. C.
during 8 hours and 80 kPa pressure, secondary drying: 6 hours at
30.degree. C. and 10 kPa pressure.
[0182] The suitability of the preparations with the lyoprotectors
in concentrations of 8% hydroxyethyl starch 450 (HES 450 8%), of 8%
.beta.-hydroxypropylcyclodextrine (.beta.-HP-CD 8%), of 8%
hydroxyethyl starch 130 (HES 130 8%) and of 8% dextrane T1 (TRIS
100 Dex T1 8%) and mannite in a concentration of 2% (w/v) (Man 2%)
is evident. The preparations which have been cited first show a
recovery of active rViscumin of above 60% after 8 hours at
60.degree. C., while the preparation with mannite only exhibits a
reduced stress stabililty under these conditions (FIG. 10).
[0183] The conditions for the distribution of medicaments can be
derived from these data with respect to stress stability. Dried
rViscumin medicaments do not have to be transported in a closed
cooling chain, as is necessary for the aqueous preparations.
EXAMPLE 27
Comparative Storage Stability of rViscumin Solution and rViscumin
Powder
[0184] The preparation of rViscumin (10 .mu.g/ml) with the
following composition:
29 rViscumin 10 .mu.g Dextrane T10 80 mg TRIS base 12.1 mg
Hydrochloric acid (1 N) pH to 8.0 for adjustment of the Polysorbate
80 1 mg Sodium EDTA 10 .mu.g Water for injection purposes ad 1
ml
[0185] are filled to 0.5 ml into freeze vials and are cooled down
to -35.degree. C. in the lyopiliser at a cooling down rate of 3
K/hour and are subsequently dried.
[0186] Drying Programme:
[0187] Primary drying: 8 hours at -10.degree. C. and 80 kPa
pressure followed by an increase in temperature to 10.degree. C.
during 8 hours and 80 kPa pressure, secondary drying: 6 hours at
30.degree. C. and 10 kPa pressure.
[0188] Subsequently, the vials are stored under controlled
conditions at 2 to 8.degree. C.
[0189] The preparation of rViscumin (1 .mu.g/ml) with the following
composition:
30 rViscumin 1 .mu.g Sodium monohydrogenphosphate dihydrate 17.8 mg
Sodium dihydrogenphosphate dihydrate 3.13 mg Sodium chloride 37.5
mg Polyvidone K 17 1 mg Sodium EDTA 1 mg Water for injection
purposes ad 1 ml
[0190] is filled into glass ampoules and is stored under controlled
conditions at 2 to 8.degree. C. This preparation is comparable to
the aqueous pharmaceutical preparations of rViscumin described in
EP 0 751 221 B1.
[0191] After a storage period of 52 weeks, rViscumin shows an
unchanged activity in the lyophilised powder. No loss in activity
can be detected. The aqueous preparation corresponding to the state
of the art only shows stability over a short storage period and
after 6 weeks of storage it has only an activity of 70% (FIG. 11).
The clear superiority of the lyophilised preparation is shown. From
these data, longer durations than 1 year for the forms of
medicaments of rViscumin in powder form can be concluded, while the
aqueous preparation formulated according to the state of the art
only has a shorter duration.
[0192] The examples given above explain the described
invention.
[0193] Various documents are cited in the text of this description.
The disclosure content of the cited documents (including all
manufacturers' descriptions and indications etc.) is herewith
incorporated in the description by reference.
[0194] Literature
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