NZ617197B2 - Lyophilized preparation of cytotoxic dipeptides - Google Patents
Lyophilized preparation of cytotoxic dipeptides Download PDFInfo
- Publication number
- NZ617197B2 NZ617197B2 NZ617197A NZ61719712A NZ617197B2 NZ 617197 B2 NZ617197 B2 NZ 617197B2 NZ 617197 A NZ617197 A NZ 617197A NZ 61719712 A NZ61719712 A NZ 61719712A NZ 617197 B2 NZ617197 B2 NZ 617197B2
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- New Zealand
- Prior art keywords
- melphalan flufenamide
- excipient
- solution
- melphalan
- lyophilized
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/05—Dipeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
- A61K38/105—Bombesin; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
Abstract
The disclosure relates to a lyophilized pharmaceutical preparation comprising melphalan flufenamide hydrochloride (J1) and at least one excipient selected from the group comprising sulfobutylether-B-cyclodextrin; Trehalose and Sucrose. The preparation may comprise two excipients. The disclosure also relates to a method of manufacture of this composition, a kit of parts comprising these features, the preparation of this pharmaceutical and the use of this pharmaceutical in the treatment of cancer. relates to a method of manufacture of this composition, a kit of parts comprising these features, the preparation of this pharmaceutical and the use of this pharmaceutical in the treatment of cancer.
Description
LYOPHILIZED PREPARATION OF CYTOTOXIC IDES
Technical field
The present ion is directed to lyophilized pharmaceutical preparations comprising
cytotoxic dipeptides or pharmaceutically acceptable salts thereof, methods for their
preparation, compositions comprising the lyophilized pharmaceutical preparations and
their use in the treatment of cancer.
Background art
Cancer is a disease which is difficult to cure and which may be fatal. Accordingly,
efforts to develop new ies for cancer are constantly ongoing in the ch
society. The vast majorities of cancers are present as solid tumors, e.g. lung cancer,
breast cancer, prostate cancer, while the rest are hematological and lymphoid
malignancies, e.g. leukemias and mas.
Chemotherapy is often used in attempts to cure or palliate the disease. As cancer cells
typically divide rapidly, chemotherapy usually acts by g rapidly dividing cells. In the
broad sense, most chemotherapeutic drugs work by impairing mitosis (i.e. cell on),
effectively targeting fast-dividing cells. As these drugs cause damage to cells they are
termed cytotoxic. Some drugs cause cells to undergo apoptosis (so-called
"programmed cell death"). Often ation herapy is used, when two or more
drugs having ent modes of action are used together in order to optimise the
antitumoral effect, to minimise side effects, and prevent resistance development. The
s obtained with chemotherapy vary according to tumor type. Some tumors are
very sensitive and the treatment has then a high probability of leading to cure.
Chemotherapeutic drugs can generally be divided into alkylating agents,
antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other
antitumor agents. The drugs affect cell division or DNA synthesis.
Alkylating agents, such as drugs derived from nitrogen mustard, that is bis(2-
chloroethyl)amine derivatives, are used as chemotherapeutic drugs in the treatment of
a wide variety of neoplastic diseases. Alkylating agents have the ability to covalently
attach alkyl groups to electronegative sites in cells. Thus, these agents act by impairing
cell on by g covalent bonds with heteroatoms in biologically important
molecules like RNA, DNA and proteins. Examples of alkylating agents are
rethamine, cyclophosphamide, chlorambucil, ifosfamide, temozolomide and
melphalan that chemically modify a cell's DNA.
WO01/96367 discloses alkylating di- and tripeptides and one or two additional amino
acids or amino acid derivatives. These derivatives were demonstrated to have an
improved efficacy on a variety of tumor types.
Melphalan, i.e. p-[bis -(2-chloroethyl)amino]phenylalanine, is a conjugate of nitrogen
mustard and the amino acid phenylalanine, which was synthesised in the mid 1950s
(US Patent No. 3,032,584). This classic alkylating substance soon became a valuable
drug in the chemotherapeutic field and is still of importance in the treatment of for
example myeloma. al use of melphalan in the treatment of late stage solid tumors
has, however, had limited cy. In the search for a more selective action on
malignant cells lan analogues have therefore been synthesised.
Larionov L. F., Cancer Res (1961), 21, 99-104 discloses various melphalan-related
derivatives.
STN registry files RN: 10606335, RN: 88 8-1, RN 7906504, RN:
7816061, RN: 7730463, RN: 7676219, RN: 7601650 and RN: 757941-
61-0 discloses s melphalan-related derivatives.
Koltun, M et al., Biopharmaceutics & Drug disposition (210), 31, 450-454 discloses
forms of melphalan.
Ma D Q et al., International l of Pharmaceutics , 189, 227-234 discloses
discloses forms of melphalan.
Murav’ev I et al., Farmatsiya (1978), 27, (2), 13-15 (with abstract in Chemical Abstracts
no. 1978:412066) discloses melphalan-related derivatives.
Lyophilization or freeze-drying is a method for dehydrating samples used to preserve or
increase stability or to stop degradation. Due to the low water content of lyophilized
ts, typically around 1-4%, the action of microorganisms and enzymes is inhibited
and the product life thereby increased. In lyophilization, the sample to be lyophilized is
dissolved in an aqueous solution and subsequently frozen after which the surrounding
re is reduced. The sample is then submitted to sublimation, optionally by the
application of heat, in order to sublime the frozen water directly from the solid phase to
the gas phase. The final water content in the product is very low, typically around 1% to
4%. Lyophilization is commonly used in the pharmaceutical field in order to increase the
shelf life of pharmaceutical products.
Summary of the invention
In general, lipophilic dipeptide ester derivatives suffer from a poor solubility in aqueous
solutions. Therefore, the use of c solvents, such as DMA (dimethylacetamide), is
necessary in order to dissolve such dipeptides. r, organic solvents are often
toxic and may also cause destruction of medical devices used for the administration of
the ides to subjects, such as cancer patients. Consequently, to me the
problems with dissolving and providing the cytotoxic dipeptides in an organic solvent,
there is a need for alternative ceutical preparations of cytotoxic dipeptides
having sufficient solubility in physiologically acceptable solutions.
The present invention refers to lyophilized preparations comprising melphalanyl-L-p-
fluorophenylalanine ethyl ester, also known as lan flufenamide, as well as
pharmaceutically able salt f, in particularly, melphalanyl-L-pfluorophenylalanine
ethyl ester hydrochloride, also known as melphalan flufenamide
hydrochloride, or J1.
In one , the invention provides a lized pharmaceutical preparation
comprising
(i) melphalan flufenamide hydrochloride (J1); and
(ii) at least one excipient selected from sulfobutylether-β-cyclodextrin; Trehalose;
and Sucrose.
Also described herein is a lyophilized pharmaceutical preparation comprising
(i) melphalan flufenamide, or a pharmaceutically acceptable salt thereof; and
(ii) at least one excipient selected from the group comprising
a polysorbate; a polyethylene glycol; β-cyclodextrin; α-cyclodextrin; ypropyl-
β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
succinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide; D-mannitol;
Trehalose; Sucrose and an amino acid.
Still an aspect of the present invention is a lyophilized pharmaceutical preparation
which is soluble in an aqueous solution.
Yet an aspect of the t ion is a method for the preparation of a lyophilized
pharmaceutical preparation of the invention, whereby:
a. melphalan flufenamide hydrochloride (J1) is dissolved in an organic t to
obtain a lan flufenamide hydrochloride (J1) solution;
b. water is added to the melphalan amide hydrochloride (J1) solution in order
to obtain an aqueous melphalan flufenamide hydrochloride (J1) solution, in a
concentration of about 0.2-3.0 mg/ml;
c. at least one excipient selected from sulfobutylether-β-cyclodextrin; and sucrose
is added to the lan flufenamide hydrochloride (J1) on; and
d. the aqueous melphalan flufenamide hydrochloride (J1) solution containing
excipient is subjected to lization.
Described herein is a kit of parts, comprising a first container comprising a lyophilized
pharmaceutical preparation as defined herein, and a second container comprising a
physiologically acceptable solution.
Still an aspect of the present invention is a lyophilized pharmaceutical preparation as
herein described, for use as a medicament.
Described herein is a kit of parts, for use as a medicament.
An aspect of the present invention is a lyophilized ceutical preparation of the
invention, for use in the ent and/or prevention of cancer, such as ovarian cancer,
lung cancer, bladder cancer, mesothelioma, le myeloma, breast , and/or
any solid or hematological cancer.
A kit of parts is herein described, for use in the treatment and/or prevention of cancer,
such as ovarian cancer, lung cancer, bladder , mesothelioma, multiple myeloma,
breast cancer, and/or any solid or hematological cancer.
Described herein is a method for the ent of and/or prevention of cancer, such as
ovarian cancer, lung cancer, bladder cancer, mesothelioma, le myeloma, breast
cancer, and/or any solid or hematological cancer, whereby a lyophilized
ceutical preparation as described herein,is stered in a eutically
effective dose to a subject in need thereof.
The invention also provides a use of an excipient selected from sulfobutylether-βcyclodextrin
; trehalose; and sucrose, in a lyophilized preparation of melphalan
flufenamide hydrochloride (J1), for decreasing the reconstitution time of the lized
preparation of melphalan flufenamide hloride (J1), when reconstituted in an
aqueous solvent.
The invention also provides a use of (i) melphalan flufenamide hydrochloride (J1); and
(ii) at least one excipient selected from sulfobutylether-β-cyclodextrin; Trehalose; and
Sucrose, in the preparation of a medicament.
Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this
invention pertains. Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the t invention, suitable
methods and materials are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by reference in their
entirety. In case of ct, the present specification, including definitions, will
supercede. In addition, the materials, methods, and es are illustrative only and
not intended to be limiting.
Other features and advantages of the invention will be apparent from the following
detailed description, drawings, examples, and from the claims.
The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification and claims which
e the term “comprising”, other features besides the features prefaced by this term
in each statement can also be present. Related terms such as ise” and
“comprised” are to be interpreted in similar manner.
In this specification where reference has been made to patent specifications, other
external documents, or other s of information, this is generally for the purpose of
providing a t for discussing the features of the invention. Unless specifically
stated otherwise, reference to such external documents is not to be construed as an
admission that such documents, or such sources of information, in any jurisdiction, are
prior art, or form part of the common general knowledge in the art.
In the description in this specification reference may be made to t matter that is
not within the scope of the claims of the current application. That subject matter should
be readily identifiable by a person skilled in the art and may assist in putting into
practice the invention as defined in the claims of this application.
Brief description of drawings
Figs. 1A-D contain graphs of four repeated dissolution speed measurements of
melphalan flufenamide lyophilized without ents by method A according to
Example 2. Samples were withdrawn at the indicated time points and the amount of
ved melphalan flufenamide was determined by HPLC. The y-axis shows the
amount of lan amide in mg/ml.
Figs. 2A-E contain graphs of dissolution speed measurements of melphalan
flufenamide lyophilized in the presence of excipients as indicated in the s by
method A according to Example 2. Samples were withdrawn at the indicated time
points and the amount of dissolved melphalan flufenamide was determined by HPLC.
The y-axis shows the amount of melphalan amide in mg/ml.
Fig. 3 is a graph of dissolution speed measurement of melphalan flufenamide without
excipients by method B according to Example 2. Samples were awn at the
indicated time points and the amount of dissolved melphalan flufenamide was
determined by HPLC. The y-axis shows the amount of melphalan flufenamide in mg/ml.
Figs. 4A-E contain graphs of ution speed measurements of melphalan
flufenamide lyophilized in the presence of excipients as indicated in the figures by
method B. Samples were withdrawn at the ted time points and the amount of
dissolved melphalan flufenamide was ined by HPLC. The y-axis shows the
amount of lan flufenamide in mg/ml.
Figs. 5 contain graphs of dissolution speed measurements as follows, A: melphalan
flufenamide lyophilized without Polysorbate 80; B lan flufenamide lyophilized in
the presence of 10% Polysorbate 80; C melphalan flufenamide lyophilized in the
presence of 50% Polysorbate 80; D melphalan flufenamide lized in the presence
of 100% Polysorbate 80. Amounts are relative to the amount of melphalan flufenamide.
The y-axis shows the amount ved melphalan flufenamide ve to the al
standard as determined using HPLC.
Fig. 6 is a photograph of glass tubes with melphalan flufenamide (J1) that following
lization is dissolved in a concentration of 1 mg/ml in a 5% glucose solution
ning 50% (mol) Polysorbate 80 (left) and no Polysorbate 80 (right).
Fig. 7 ns structural formulas for melphalan flufenamide (L-melphalanyl-L-p-
fluorophenylalanine ethyl ester), L-melphalanyl-L-p-fluorophenylalanine isopropyl ester
(JV28), L-prolinyl- L-melphalanyl- L-p-fluorophenylalanine ethyl ester (J3).
Detailed description of the invention
Non-lyophilized cytotoxic ides or pharmaceutically acceptable salts thereof may
have a low solubility in aqueous solutions, which may necessitate the use of organic
solvents, such as DMA (dimethylacetamide), for dissolving said dipeptides or
pharmaceutically able salts thereof. Therefore, when a cytotoxic dipeptide is to
be administered to a patient, the substance first has to be dissolved in an organic
solvent, such as DMA, and thereafter diluted in a solution for infusion before
administration to the patient. The patient is by this method exposed to organic solvents,
the exposure of which may be hazardous for the patient. Also, the organic solvent may
destroy the medical devices used for the administration of melphalan flufenamide to
subjects, such as cancer patients.
The present inventors have now surprisingly found that when certain cytotoxic
dipiptides or pharmaceutically acceptable salts thereof are lyophilized in the presence
of an excipient, the resulting lyophilized pharmaceutical preparation can have an even
higher solubility in a physiologically acceptable on. In fact, the solubility can be so
high that the step of dissolving the cytotoxic dipeptide or pharmaceutically acceptable
salt thereof in an organic solvent can be omitted and the cytotoxic dipeptide can be
ly dissolved in an aqueous, physiologically acceptable solution and administered
to a patient. Preferably, said cytotoxic dipeptide is melphalan flufenamide or a
ceutically acceptable salt thereof.
In previous preparations, melphalan flufenamide was obtained from synthesis as a
white powder in crystalline form. This lline form can only be dissolved in highly
acidic aqueous solutions, which for practical manufacturing purposes is ible. The
ce of excipients as such, did not sufficiently improve the solubility. Therefore,
usly melphalan flufenamide was instead dissolved in DMA (dimethylacetamide) in
a glucose solution. The ation is feasible but is unstable: 7% degradation/h.
Furthermore, dimerization occurs and the solution turns bright yellow. This preparation
was, however, unreliable and the rization rate varied in an unacceptable
manner.
Consequently, there is a need for fying alternative ways of providing a preparation
comprising melphalan flufenamide or a pharmaceutically acceptable salt thereof that is
soluble with increased stability. Further, the ation should be water-soluble to
avoid negative issues of having an organic solvent in the product that is ed to the
patient (such as DMA).
Described herein is a lyophilized pharmaceutical preparation comprising
(i) lan flufenamide, or a pharmaceutically acceptable salt thereof; and
(ii) at least one excipient selected from the group comprising a polysorbate; a
polyethylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- β-cyclodextrin;
sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium succinate;
propylene ; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose;
Sucrose; and an amino acid.
In one embodiment of this aspect, said excipient is selected from the group comprising
Polysorbate 80; PEG 400; lactose; benzyl alcohol; disodium succinate; propylene
glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose;
and histidine.
In another embodiment of this , said melphalan flufenamide is melphalan
flufenamide hydrochloride (J1).
Also described herein is a pharmaceutical preparation comprising
(i) melphalan flufenamide hydrochloride (J1); and
(ii) at least one excipient selected from the group comprising
a polysorbate; a hylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl-
β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
succinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide; D-mannitol;
Trehalose; e; and an amino acid.
In one embodiment of this aspect, said at least one excipient is a polysorbate or
polyethylene glycol.
In another embodiment of this aspect, said at least one excipient is Polysorbate 80.
In another embodiment of this , said at least one excipient has surfactant
properties. Such properties would increase the stability of the lyophilized
pharmaceutical preparation. Said at least one excipient having surfactant properties
may be polysorbate or polyethylene glycol, such as Polysorbate 80 or PEG400.
In another embodiment of this aspect the preparation comprises melphalan flufenamide
hloride (J1) and the excipient Polysorbate 80. The presence of the excipient
Polysorbate 80 would se the stability of the lyophilized ceutical
preparation. Further, the final preparation would be free, or ially free of organic
solvents, and therefore less toxic.
The invention provides a lyophilized preparation which is stable in dry form and soluble
in an aqueous solution without ce of an organic solvent. While it usly was
possible to prepare a lyophilized preparation of melphalan flufenamide alone, this
ation dissolved too slowly in aqueous solutions compared to the degradation
time. Incorporation of an excipient in the lyophilized melphalan flufenamide preparation
(via l solution in an organic solvent) improves the reconstitution time considerably,
but does not significantly alter the stability of reconstituted melphalan flufenamide. As a
result, the time window for the reconstituted melphalan flufenamide is widened, and this
improves the treatments of patients, e.g. by allowing for lower infusion rates, where
needed. A preparation “without presence of an organic solvent” could include trace
amounts of organic solvent, typically less than 0.5% (w/w).
The lyophilized ceutical preparation of melphalan flufenamide or a
pharmaceutically able salt thereof as described herein, is a white, fluffy powder
in contrast to a non-lyophilized melphalan flufenamide or a pharmaceutically acceptable
salt thereof, which can be in the form of a dense, slightly ish powder.
Typically, lyophilization comprises four steps, pretreatment, freezing, primary drying,
and secondary drying. In the pretreatment step, the substance to be lyophilized is made
ready for the lyophilization e.g. by ing a on having the desired concentration
or mixing the substance with further components in order to obtain an acceptable
result. The freezing step may be performed in a freeze-drying flask in a bath cooled e.g.
by mechanical eration, dry ice and ol, or liquid nitrogen. Freeze-drying
machines are available for lyophilization in a larger scale. Usually, the freezing
temperatures are between −50 °C and −80 °C.
In the primary drying step, the re is lowered to the range of a few millibars, and
heat may be supplied for the water to sublimate from the material. The amount of heat
necessary can be calculated using the ating molecules’ latent heat of
sublimation. The duration of this period depends, but may last for days in order to
preserve the materials structure.
The aim of the final secondary drying step is to remove any unfrozen water molecules.
In this phase, the temperature may be as high as above 0 °C, to break any physicochemical
ctions that have formed between the water molecules and the frozen
material.
In the context of the t invention, it is to be understood that melphalan
flufenamide or a pharmaceutically acceptable salt thereof, is lyophilized. The term “a
lyophilized pharmaceutical ation of a melphalan flufenamide or a
pharmaceutically acceptable salt thereof”, is therefore understood to mean that the
melphalan flufenamide or a pharmaceutically acceptable salt thereof is lyophilized.
Further aspects of the present invention provide lyophilized melphalan flufenamide or a
pharmaceutically acceptable salt thereof, a kit of parts comprising such melphalan
flufenamide, methods for the preparation of such melphalan flufenamide or a
pharmaceutically acceptable salt thereof, compositions comprising such lyophilized
melphalan amide or a pharmaceutically acceptable salt thereof and uses thereof.
“Lyophilization”, “lyophilized” y in the present context be used interchangeably
with “freeze-drying”, “freeze-dried” etc.
Examples of cytotoxic dipeptides that can be lyophilized as described herein are set
forth in WO01/96367. The N-terminus of a molecule should preferably not be protected
as amide or ate. This means that R4 in formula I therein should preferably not be
a protecting group, such as formyl, acetyl or propionyl, or benzoyl, as the protected
form of the compound in general has a lower cytotoxic activity than the corresponding
free form. Natural amino acids refer to amino acids that are normally ng and
exerting their functions in living sms. Modified amino acids refer to amino acids
that in some way have been modified into a different chemical structure and chemical
ition than a natural amino acid. An example of a l cyclic amino acid is
e. Examples of aromatic amino acids are phenylalanine, tyrosine, tryptophan, and
histidine.
The cytotoxic dipeptides, such as melphalan flufenamide, may also n unnatural
proportions of atomic isotopes at one or more of its atoms. For example, the
compounds may be radiolabeled with radioactive isotopes, such as for example tritium
(3H), deuterium (2H), iodine-125 (125 I) or -14 (14 C).
The cytotoxic dipeptide lan flufenamide clearly differs from melphalan:
? Difference in structure (melphalan flufenamide is an ethyl ester at the C-terminal
instead of the carboxylic acid in melphalan. Melphalan is y a zwitterion, but
lan flufenamide is not).
? Difference in size (melphalan flufenamide is a dipeptide, i.e. approximately twice
the size of melphalan).
? ence in lipophilicity, where melphalan flufenamide is clearly more lipophilic.
? Difference in stability in aqueous ons. Melphalan is 10 000 times more stable
in s solutions compared to J1. J1 is quickly hydrolyzed in water.
? Difference in degradation ys. The main ation pathway in melphalan
flufenamide involves hydrolysis of the ethyl ester, while the main ation in
melphalan relates to the reactivity of the o)alkyl groups.
Based on, but not limited to, the above differentiations, it is clear that teachings on
melphalan and, in particular preparations and formulations thereof, do not apply to
melphalan flufenamide and preparations and ations thereof.
The inclusion of at least one excipient (such as Polysorbate 80 with its tant
properties) provides lyophilized preparation that is stable as such and water-soluble
without the presence of an organic solvent at a sufficient rate compared to the
degradation rate, and is thereby useful in therapy and less toxic.
The lyophilized pharmaceutical preparation according to the invention may contain only
melphalan flufenamide or a ceutically acceptable salt thereof, or a mixture of
melphalan flufenamide with one or more different cytotoxic dipeptides or
pharmaceutically acceptable salts thereof. Further, the lyophilized pharmaceutical
preparation may n a mixture of two or more different pharmaceutically acceptable
salts.
Described herein is a lyophilized pharmaceutical preparation, comprising
(i) melphalan flufenamide; and
ii) a combination of two or more excipients selected from the group comprising a
polysorbate; a polyethylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- β-
cyclodextrin; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
succinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose;
Sucrose; and an amino acid.
Also described herein is a lyophilized pharmaceutical ation, comprising:
(i) melphalan flufenamide hydrochloride (J1); and
(ii) a combination of two or more ents ed from the group comprising a
polysorbate; a polyethylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- βcyclodextrin
; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
succinate; propylene glycol; Cremophor EL; Dimethyl ide; D-mannitol;
Trehalose; e; and an amino acid.
In one embodiment of this aspect, said combination of excipients is a mixture of
Polysorbate 80 and PEG400.
Pharmaceutically acceptable salts for all aspects of the present invention may be, for
instance, an acid-addition salt of a compound described herein which is sufficiently
basic, for example, an acid-addition salt with, for example, an inorganic or organic acid,
for example hydrochloric, hydrobromic, nitric, sulphonic, sulphuric, phosphoric,
trifluoroacetic, para-toluene sulphonic, 2-mesitylen sulphonic, citric, acetic, tartaric,
fumaric, lactic, succinic, malic, malonic, maleic, hanedisulphonic, adipic, aspartic,
benzenesulphonic, benzoic, ethanesulphonic or nicotinic acid.
In this document, when the term “melphalan flufenamide” is used, it is also intended to
include pharmaceutically acceptable salt(s) thereof, even if this is not explicitly stated.
As mentioned hereinbefore, when melphalan flufenamide or a pharmaceutically
acceptable salt thereof is lyophilized in the presence of a ceutically acceptable
ent, such as any one selected from a polysorbate; a polyethylene glycol; β-
cyclodextrin; α-cyclodextrin; hydroxypropyl- β-cyclodextrin; sulfobutylether-βcyclodextrin
; lactose; benzyl alcohol; disodium succinate; ene glycol; Cremophor
EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and an amino acid; an
unexpectedly high increase in solubility of the lyophilized pharmaceutical preparation
can be obtained, which enables the direct dissolution of the lized melphalan
flufenamide in an aqueous solution, such as a physiologically able on. This
is in contrast to a ophilized melphalan flufenamide which is not possible to
dissolve directly in an aqueous solution but that first has to be ved in an organic
solvent prior to dilution in an aqueous solution. It is therefore provided herein a
lyophilized pharmaceutical preparation comprising melphalan flufenamide or a
pharmaceutically acceptable salt thereof, wherein melphalan flufenamide is lyophilized
in the presence of an excipient. ably, said excipient is selected from polysorbate
or polyethylene glycol, such as Polysorbate 80 or PEG400.
Melphalan flufenamide or a pharmaceutically acceptable salt thereof may be lyophilized
in the ce of one or more of an excipient(s) (e.g. one, two, three, four, five, or
more excipients). Examples of excipients that can be used as described herein include,
without limitation, polysorbates such as Polysorbate 20, Polysorbate 40, Polysorbate
60, and Polysorbate 80; hylene glycols such as PEG 400 and PEG 300; βcyclodextrin
, α-cyclodextrin, sulfobutylether-β-cyclodextrin, hydroxypropyl- βcyclodextrin
, lactose, benzyl alcohol, disodium succinate, propylene glycol, Cremophor
EL, dimethyl ide, D-mannitol, trehalose, sucrose and amino acids such as
histidine.
In one aspect of the ion, the excipient is selected from any one of rbate 80;
PEG 400; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- β-cyclodextrin; sulfobutylether-
β-cyclodextrin; lactose; benzyl alcohol; disodium succinate; ene glycol; PEG 300;
Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and histidine.
In one aspect of the invention, the excipient is selected from Polysorbate 80; PEG 400;
lactose; benzyl alcohol; disodium ate; propylene glycol; PEG 300; Cremophor
EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and ine, or a combination
of two or more of said ents.
In one embodiment of this aspect, the ent is selected from Polysorbate 80 and
PEG 400, or a ation of said two excipients.
The amount of excipient such as Polysorbate 80, PEG 400 or β-cyclodextrin, is typically
about 10-100% by weight of the amount of melphalan flufenamide, such as 100, 90, 80,
70, 60, 50, 40, 30, 20 or 10 % by weight of the amount of melphalan flufenamide.
In yet an aspect of the invention, the amount of the excipient, such as Polysorbate 80,
PEG 400 or β-cyclodextrin, is typically about 10-50% by weight of the amount of
melphalan flufenamide, such as 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 % by weight of
the amount of melphalan flufenamide.
In one embodiment of this aspect, the ent represents Polysorbate 80 or PEG 400,
and the amount thereof is typically about 10-50% by weight of the amount of melphalan
flufenamide, such as 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 % by weight of the
amount of melphalan flufenamide.
Also described is a lyophilized pharmaceutical preparation comprising
(i) melphalan flufenamide, or a pharmaceutically acceptable salt thereof; and
(ii) at least one excipient ed from the group comprising
Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- β-
cyclodextrin; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
succinate; propylene glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide; D-
mannitol; Trehalose; Sucrose; and histidine;
wherein the amount of the excipient is about 10-100 % by weight of melphalan
flufenamide.
In one embodiment of this aspect, the at least one excipient selected from Polysorbate
80 and PEG 400.
In another embodiment of this aspect, melphalan flufenamide is represented by
melphalan flufenamide hydrochloride (J1).
Also bed is a lyophilized pharmaceutical ation comprising
(i) melphalan flufenamide, or a pharmaceutically acceptable salt thereof; and
(ii) at least one excipient selected from the group comprising
Polysorbate 80; PEG 400; odextrin; α-cyclodextrin; hydroxypropyl- βcyclodextrin
; utylether-β-cyclodextrin; e; benzyl l; disodium
succinate; propylene glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide; D-
mannitol; Trehalose; Sucrose; and histidine;
wherein the amount of the excipient is about 10-50 % by weight of melphalan
flufenamide hydrochloride (J1).
In one embodiment of this aspect, the at least one excipient selected from Polysorbate
80 and PEG 400.
In another embodiment of this aspect, melphalan flufenamide is represented by
melphalan flufenamide hydrochloride (J1).
Also described is a lyophilized pharmaceutical preparation sing
(i) melphalan flufenamide hydrochloride (J1); and
(ii) at least one excipient selected from the group comprising
Polysorbate 80; PEG 400; odextrin; odextrin; hydroxypropyl- βcyclodextrin
; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; um
succinate; propylene glycol; PEG 300; Cremophor EL; yl sulfoxide; D-
mannitol; Trehalose; Sucrose; and histidine;
wherein the amount of the excipient is about 10-100 % by weight of melphalan
flufenamide hydrochloride (J1).
In one embodiment of this aspect, the at least one excipient selected from Polysorbate
80 and PEG 400.
Also described is a lyophilized pharmaceutical preparation sing
(i) melphalan flufenamide hydrochloride (J1); and
(ii) at least one excipient selected from the group comprising Polysorbate 80; PEG 400;
β-cyclodextrin; α-cyclodextrin; hydroxypropyl-β-cyclodextrin; sulfobutylether-β-
cyclodextrin; e; benzyl alcohol; disodium succinate; propylene ; PEG
300; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and
histidine;
wherein the amount of the ent is about 10-50 % by weight of melphalan
flufenamide hydrochloride (J1).
In one embodiment of this aspect, the at least one excipient selected from rbate
80 and PEG 400.
In one embodiment of the invention, the amount of excipient, such as Polysorbate 80 or
PEG 400, may be up to the clinically acceptable amount.
In one embodiment of the invention, the amount of excipient, such as Polysorbate 80 or
PEG 400, may be up to the clinically acceptable .
When used as the only excipient, the amount of Polysorbate 80 or PEG 400, is e.g.
about 50% by weight of the amount of melphalan flufenamide hydrochloride (J1).
An aspect of the invention is a combination of the excipients Polysorbate 80 and
PEG 400.
An aspect of the invention is a combination of the excipients Polysorbate 80,
PEG 400 and β-cyclodextrin, such as 80% by weight of Polysorbate 80, 80% by weight
of PEG 400 and 50% by weight of β-cyclodextrin, of the amount of lan
flufenamide. A lyophilized pharmaceutical preparation of a melphalan derivative or a
pharmaceutically acceptable salt f, may in accordance with the invention
comprise one or more melphalan derivative(s) or a pharmaceutically acceptable salt(s)
thereof, and one or more ent(s) as defined herein.
As mentioned hereinbefore, one effect of the presence of an excipient during the
lyophilization is that the resulting lyophilized pharmaceutical preparation comprising
lan flufenamide, has an enhanced solubility in aqueous solutions, such as a
physiologically acceptable on, compared to when melphalan flufenamide is
lyophilized without an excipient as bed herein. In particular, the solubility in
aqueous ons of melphalan flufenamide when lyophilized in the presence of an
excipient(s) is higher compared to the solubility of the non-lyophilized product. This
increased solubility of melphalan flufenamide, in particular when lyophilized in the
ce of an excipient as herein described, compared to the non-lyophilized product,
has substantial advantages when it comes to administration of melphalan amide
to a patient.
Due to a low solubility of non-lyophilized melphalan flufenamide in aqueous
physiologically able solutions used for administration of the drug to a patient, it is
necessary to first dissolve the non-lyophilized lan flufenamide in an organic
solvent, such as DMA. Melphalan flufenamide is therefore often stored dissolved in
DMA. It has previously not been possible to directly dissolve the melphalan flufenamide
in an s solution, but organic solvents have had to be used. Once dissolved in
the organic solvent, this solution of melphalan flufenamide and organic solvent can be
dissolved in physiologically acceptable solutions for administration to a t.
As melphalan flufenamide is very toxic, in order to minimize the exposure of medical
personnel to such drugs, special devices for transferring the drugs after dissolution in
organic ts to the solution for administration, are used. These transfer devices are
often plastic tubings comprising polycarbonate. However, such tubings are sensitive to
and may be destroyed by organic solvents, such as DMA. Therefore, in the cases
where the drug to be administered is dissolved in such an organic solvent, it may not be
possible to use the transfer device, and the ved drug instead has to be ly
added to the physiologically acceptable solution used for administration just before the
time of administration to the patient. This can be hazardous for the medical staff, who
then are at risk being exposed to the toxic drug.
As mentioned above, lization of melphalan flufenamide increases its solubility in
physiologically acceptable solutions. This increase can be even more pronounced
when melphalan flufenamide is lyophilized in the presence of one or more excipients.
As described herein, when melphalan flufenamide is lyophilized in the presence of an
excipient as sed herein, the solubility of melphalan flufenamide can be increased,
in comparison to the non-lyophilized melphalan flufenamide. The use of an c
solvent, such as DMA, to first dissolve melphalan flufenamide can be d.
Melphalan flufenamide which has been lyophilized in the presence of at least one
excipient, such as a polysorbate which for e may be Polysorbate 80; a
polyethylene glycol which for example may be PEG 400 or PEG 300; β-cyclodextrin;
α-cyclodextrin; hydroxypropyl- β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose;
benzyl alcohol; disodium succinate; propylene glycol; Cremophor EL; Dimethyl
sulfoxide; D-mannitol; Trehalose; Sucrose; or an amino acid such as histidine; or a
combination of two or more of these excipients ; can be directly dissolved in a
physiologically acceptable solution, such as about 4.5-5.5 wt%, e.g. about 5%, glucose
solution or an s NaCl solution (e.g. about 0.9 wt% NaCl). Thereby, devices
comprising polycarbonate and which are used for the stration of melphalan
flufenamide are possible to use, zing the risk for exposing the medical personnel
to the drug. Also, in this way administering the toxic DMA to the patient is avoided. This
allows for directly preparing the solution comprising melphalan flufenamide at a
concentration suitable for administration to the patient. Alternatively, a concentrated
solution comprising a lyophilized pharmaceutical preparation of lan flufenamide
in a logically acceptable solution may first be prepared and then transferred to
the bag for infusion using the commonly used transfer devices.
Also, when melphalan flufenamide is dissolved in DMA, an adduct between the
melphalan amide and the DMA can be formed. By using a lyophilized
pharmaceutical preparation provided in accordance with the ion, it is possible to
dissolve the lyophilized melphalan flufenamide directly in a physiologically acceptable
solution, avoiding first dissolving the melphalan flufenamide in DMA. Thereby, the
formation of a DMA- melphalan flufenamide adducts can be avoided and neither the
adduct nor the DMA have to be administered to the patient.
Also described is a pharmaceutical composition comprising a lyophilized
pharmaceutical ation of melphalan flufenamide or pharmaceutically acceptable
salt thereof as defined herein, ally obtainable by the method for preparing such a
lyophilized preparation disclosed . Such a ceutical composition may
further comprise a physiologically acceptable solution, such as an aqueous NaCl (e.g.
about 0.9 wt%) or glucose solution (e.g. about 5 wt%, such as about 5 wt%,
glucose). This pharmaceutical composition may be a concentrated on intended for
dilution before administration to a subject or as a solution enabling direct administration
to a patient.
Due to the sed solubility of melphalan flufenamide after lyophilization in the
presence of one or more excipients as described herein, it is possible to prepare a
dissolved melphalan flufenamide solution, such as a pharmaceutical composition
sing a melphalan flufenamide or pharmaceutically acceptable salt thereof, which
is substantially free from organic solvents such as DMA, dichloromethane,
tetrahydrofuran, 2-methyl tetrahydrofuran, ethyl acetate, acetone, dimethylformamide,
acetonitrile, dimethyl sulfoxide, dioxane, diethyl ether, acetic acid, n-butanol,
isopropanol, anol, tert-butanol, sec-butanol, methanol, ethanol, and acetic acid.
By “substantially free” is in this document meant that the pharmaceutical composition
comprises only trace amounts of an organic solvent, such as less than about a total of
about 0.1 wt% of an organic t. In one aspect, the lyophilized preparation or the
pharmaceutical composition does not contain any measurable amounts of an c
solvent. Such preparations would be less toxic and therefore more tolerated by a
patient, ie giving less side effect such as ng, nausea or other general symptoms
when infused.
In one aspect of the invention, there is ed a lyophilized pharmaceutical
preparation of the invention, which is free, or substantially free from organic solvents.
The ceutical compositions described herein may consist of a lized
pharmaceutical preparation as disclosed herein, comprising melphalan amide or
pharmaceutical salt thereof, and the physiologically acceptable solution, such as a
glucose solution. As disclosed hereinbefore, the melphalan derivative may be
melphalan flufenamide or a mixture of melphalan flufenamide and one or more different
cytotoxic dipeptides, either lyophilized together or separately.
The pharmaceutical composition may be obtainable by dissolving melphalan
flufenamide or a pharmaceutical salt thereof in a physiologically able solution. A
method for preparing a pharmaceutical composition comprising the step of ving
the lyophilized pharmaceutical preparation comprising melphalan flufenamide or a
pharmaceutically acceptable salt thereof in a logically able on is
therefore also provided herein.
The wording a “physiologically acceptable solution” is herein defined, is an aqueous
solution, such as a NaCl solution (such as about 0.9 wt-% NaCl) or glucose on,
such as about 4.5-5.5 wt-% glucose, e.g. about 5 wt-%, or another physiologically
acceptable solution. Any such solution may optionally be buffered.
A pharmaceutical composition comprising lyophilized melphalan flufenamide and a
physiologically acceptable solution for direct administration to a subject, generally
comprises melphalan flufenamide at a concentration of about 1 mg/ml or less, such as
about 0.2 mg/ml. However, the pharmaceutical composition may comprise melphalan
flufenamide in a tration of up to about 4 mg/ml for dilution in a physiologically
acceptable solution before administration to a patient.
Described herein is a method for preparing a lyophilized pharmaceutical preparation,
whereby:
a) melphalan flufenamide, or a pharmaceutically acceptable salt thereof, is
dissolved in an organic solvent to obtain a melphalan amide solution;
b) water is added to the melphalan flufenamide solution in order to obtain an
aqueous melphalan flufenamide solution, in a tration of about 0.2-3.0
mg/ml;
c) at least one ent selected from the group comprising a polysorbate;
a hylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- βcyclodextrin
; sulfobutylether-β-cyclodextrin; lactose; benzyl l; disodium
succinate; propylene glycol; Cremophor EL; Dimethyl ide; D-mannitol;
Trehalose; Sucrose and an amino acid is added to the melphalan flufenamide
solution; and
d) the aqueous lan flufenamide solution containing excipient(s) is
subjected to lyophilization.
Also described is a method, whereby:
a) melphalan amide, or a pharmaceutically acceptable salt f, is
dissolved in an organic solvent;
b) water is added to the solution obtained in step a) in order to obtain a solution
of said melphalan flufenamide or a pharmaceutically acceptable salt thereof, in
a concentration of about 0.2-3.0 mg/ml;
c) at least one excipient selected from the group comprising a polysorbate; a
polyethylene glycol; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- βcyclodextrin
; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium
ate; propylene glycol; Cremophor EL; Dimethyl sulfoxide; D-mannitol;
Trehalose; Sucrose and an amino acid is added to the on obtained in
step b); and
d) the solution obtained in step c) is subjected to lyophilization.
The organic solvent may be ed from any one of ethanol, ethanol containing acid,
glycerin, propylene , benzyl alcohol, dimethylacetamide (DMA), N-methyl
pyrrolidone, isopropanol, n-butanol, tert-butanol, methyl tert-butyl ether, propylene
glycol, dimethylsulfoxide, tetrahydrofuran, yl tetrahydrofuran, acetone,
dimethylformamide, acetonitrile, dioxane, acetic acid, lactic acid, propionic acid, nbutanol
, isopropanol, n-propanol, tert-butanol, sec-butanol, methanol, and a mixture of
ethanol and water. Preferably, said organic solvent is ethanol.
The excipient may be ed from the group comprising Polysorbate 80; PEG 400; β-
cyclodextrin; odextrin; hydroxypropyl- β-cyclodextrin; sulfobutylether-βcyclodextrin
; lactose; benzyl l; disodium succinate; propylene glycol; PEG 300;
Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and histidine.
Preferably, said excipient is selected from Polysorbate 80 and PEG 400.
The melphalan flufenamide in said methods is ably melphalan flufenamide
hydrochloride (J1).
An aspect of the present invention is a method for the ation of a lyophilized
pharmaceutical preparation as herein described, whereby
a) melphalan flufenamide, or a pharmaceutically acceptable salt thereof, is dissolved
in an organic solvent;
b) water is added to the solution obtained in step a) in order to obtain a on of
said melphalan flufenamide or a pharmaceutically acceptable salt thereof, in a
concentration of about 0.2-3.0 mg/ml;
c) at least one excipient as herein defined, is added to the on obtained in step b);
d) the solution obtained in step c) is subjected to lyophilization.
Preferably, said c solvent is ethanol.
An aspect of the present invention is a method for the preparation of a lyophilized
pharmaceutical preparation as herein described, y
a) melphalan flufenamide hloride (J1), is dissolved in an organic solvent;
b) water is added to the solution obtained in step a) in order to obtain a solution of
said melphalan flufenamide hydrochloride (J1), or a pharmaceutically acceptable salt
thereof, in a concentration of about 0.2-3.0 mg/ml;
c) at least one excipient as herein d, is added to the solution obtained in step b);
d) the solution ed in step c) is subjected to lyophilization.
Examples of organic solvents useful for dissolving melphalan flufenamide,
or a pharmaceutically acceptable salt thereof in step a), may be any one selected from
ethanol, ethanol containing acid, glycerin, propylene glycol, benzyl alcohol,
dimethylacetamide (DMA), N-methylpyrrolidone, isopropanol, n-butanol, tert-butanol,
methyl utyl ether, propylene glycol, dimethylsulfoxide, tetrahydrofuran, yl
tetrahydrofuran, acetone, ylformamide, itrile, dioxane, acetic acid, lactic
acid, propionic acid, n-butanol, isopropanol, n-propanol, tert-butanol, sec-butanol,
methanol, and a mixture of ethanol and water.
Also described is a method for the preparation of a lyophilized pharmaceutical
preparation as herein described, whereby
a) melphalan flufenamide, or a pharmaceutically acceptable salt thereof, is dissolved in
an organic solvent selected from any one of ethanol, ethanol ning acid,
glycerin, propylene glycol, benzyl alcohol, dimethylacetamide (DMA), N-methyl
pyrrolidone, isopropanol, n-butanol, tert-butanol, methyl tert-butyl ether, propylene
glycol, dimethylsulfoxide, tetrahydrofuran, yl ydrofuran, acetone,
dimethylformamide, acetonitrile, dioxane, acetic acid, lactic acid, propionic acid, n-
butanol, isopropanol, n-propanol, tert-butanol, sec-butanol, methanol, and a mixture
of ethanol and water;
b) water is added to the solution obtained in step a) in order to obtain a solution of
said melphalan flufenamide or a pharmaceutically acceptable salt f, in a
concentration of about 0.2-3.0 mg/ml;
c) at least one excipient as herein defined, is added to the solution obtained in step b);
d) the solution obtained in step c) is subjected to lization.
Also described is a method for the preparation of a lyophilized pharmaceutical
preparation as herein described, whereby
a) lan flufenamide hydrochloride (J1), is dissolved in an organic solvent
selected from any one of l, ethanol containing acid, glycerin, propylene glycol,
benzyl alcohol, dimethylacetamide (DMA), N-methylpyrrolidone, isopropanol, nbutanol
, tert-butanol, methyl tert-butyl ether, propylene glycol, dimethylsulfoxide,
tetrahydrofuran, 2-methyl ydrofuran, acetone, dimethylformamide, acetonitrile,
dioxane, acetic acid, lactic acid, propionic acid, n-butanol, isopropanol, n-propanol,
tert-butanol, sec-butanol, methanol, and a mixture of ethanol and water;
b) water is added to the solution obtained in step a) in order to obtain a solution of
said lan amide hloride (J1), or a pharmaceutically acceptable salt
thereof, in a concentration of about 0 mg/ml;
c) at least one excipient as herein defined, is added to the solution obtained in step b);
d) the solution obtained in step c) is subjected to lyophilization.
Also described is a method for the preparation of a lyophilized pharmaceutical
preparation as herein described, whereby
a) melphalan flufenamide hydrochloride (J1), is dissolved in an organic solvent;
b) water is added to the solution obtained in step a) in order to obtain a solution of said
melphalan flufenamide hydrochloride (J1), in a concentration of about 0.2-3.0 mg/ml;
c) at least one excipient as herein defined, is added to the solution obtained in step b);
d) the solution obtained in step c) is subjected to lyophilization;
n said at least one excipient is selected from Polysorbate 80 and .
When ethanol containing acid is used for dissolving melphalan flufenamide or a
ceutically acceptable salt thereof in step a) in the method above, the acid can
be HCl, in a concentration of for example 5-20 mM, or the HCl concentration may for
example be 10 mM, in the ethanol.
When melphalan flufenamide or a pharmaceutically acceptable salt thereof is dissolved
in ethanol and water, the concentration of ethanol may be about
-100 vol-%, such as 10-90 vol-%, 50-90 vol-%, or about 70 vol-%.
The water used for dissolving and/or diluting samples of a lyophilized pharmaceutical
preparation in ance with the present invention, is e or purified water, or
water for injection (WFI).
When l is used for dissolving lan flufenamide or pharmaceutically
acceptable salt thereof, the solution obtained in step a) is diluted in step b) so that the
concentration of ethanol, is about % by volume, such as about 2, 5, 10, 20, 30,
40, 50, 60, 70, 80, 90 or 100%, or such as 5-15 %, or such as 5, 6, 7, 8, 9, 10, 11, 12,
13, 14 or 15 %. Typically, the concentration of ethanol after the dilution step b) is about
The solution obtained in step b) may be sterile filtered before the lyophilization step c).
The lyophilization step c) ses the typical freezing and primary and secondary
drying steps as described herein. Information about how lyophilization is performed
may be found e.g. in Rey, L. and May, J. Freeze Drying/Lyophilization of
Pharmaceutical and Biological Products (2010 ), ISBN 978-1439B2575-4. In the
freezing step, the sample is for example frozen in a bath of dry etone at a
temperature of about -70 oC to -90 oC, such as about -70 oC, -75 oC, -78 oC, -80 oC,
-82 oC, -85 oC, -88 oC or -90 oC for example for 10 minutes to 120 minutes.
Alternatively, the sample may be frozen in a freezer at a temperature about
-14 oC to -25 oC, such as -14 oC, -16 oC, -18 oC, -20 oC, -22 oC, or -25 oC, for example for
about 10 min to 24 hours. It is also possible to freeze the sample in liquid nitrogen.
Step c) may be performed by applying conventional techniques for lyophilization, see
e.g. Rey, L. and May, J. Freeze Drying/Lyophilization of Pharmaceutical and Biological
Products (2010 ), ISBN 978-1439B2575-4.
For example, in the primary drying step, the re can be d to about to about
0.1 mbar to 50 mbar, such as 1 mbar to 10 mbar. The temperature is typically below
0oC, such as -50 to 0 oC, or -20 to -1 oC, e.g. -50, -40, -30, -20, -10, or -5 oC. This phase
may for example last for 4 hours to 48 hours, e.g. 12 hours to 24 hours.
In the final secondary drying step, when most of the water has ated, the
temperature may be as in the y drying step or above 0 °C.
When one or more ents as defined herein are to be present during the
lization, these can be added in step b) prior to or after diluting the solution
obtained in step a) and prior to performing the lyophilization. The excipients may be
added in powder form but are generally added as an aqueous solution. The excipients
can therefore be present during the lyophilization.
The present invention is also directed to a lyophilized pharmaceutical preparation as
defined herein obtainable by the above disclosed method.
Also described is a kit of parts comprising:
(i) a first container comprising a lized pharmaceutical preparation comprising
melphalan flufenamide as described herein; and
(ii) a second container comprising a physiologically acceptable solution, such as a NaCl
solution (such as about 0.9 wt% NaCl) or a glucose solution, such as about 4.5-5.5
wt% glucose solution, e.g. about 5 wt% glucose solution, or other physiologically
acceptable solution.
Such a kit may also se a device for mixing the ts of the two containers
with each other and/or for transferring the resulting mixture to a device, such as a bag
comprising a glucose solution, for the administration to a patient.
Such a kit may consist of the first container comprising a lyophilized pharmaceutical
preparation comprising melphalan flufenamide as described herein and the second
container comprising the physiologically acceptable solution. Melphalan amide in
the kit may also be in admixture with a pharmaceutically acceptable carrier and/or
excipient. One example is 5% glucose with e.g. 1% albumin or another protein or
compound. The amount of physiologically acceptable solution may either be a small
amount in order to prepare a concentrated solution of the lyophilized pharmaceutical
preparation sing melphalan flufenamide, or a larger amount in order to enable
the preparation of a solution having the desired concentration for stration to a
patient. Alternatively, the kit may comprise both a container comprising a
physiologically acceptable solution for preparing a trated solution of the
lyophilized pharmaceutical preparation and a second container, such as a bag for
on, comprising a larger amount of a physiologically acceptable on for
preparation of the more d solution for administration to a subject.
A lyophilized pharmaceutical preparation, pharmaceutical composition or kit provided
herein may comprise only melphalan flufenamide or a pharmaceutically acceptable salt
thereof as an antitumoral agent. However, melphalan flufenamide may also be
combined with one or more moral agents, such as other antitumoral substances
such as gemcitabine, etoposide, doxorubicine or taxanes or other therapeutically
effective substances. When combined with other moral agents these may either
be mixed with melphalan flufenamide or pharmaceutically acceptable salt thereof
before lyophilisation and consequently lyophilized er with melphalan flufenamide
or ceutically acceptable salt f or ed with the lyophilized melphalan
flufenamide or pharmaceutically acceptable salt thereof after lyophilisation, such as in a
kit or a pharmaceutical composition. Lyophilized melphalan flufenamide may also be
mixed with one or more antitumoral substances in dry form, even though not
lyophilized, after lyophilisation of melphalan flufenamide or pharmaceutically acceptable
salt thereof.
Melphalan flufenamide provided herein have a cytotoxic activity and may therefore be
used in the tion and/or treatment of cancer as described elsewhere (see e.g.
WO 01/96367). A reduction of tumor cell survival of these compounds was in WO
67 demonstrated for different hematological and/or solid tumors, e.g. lung
cancer, myeloma, lymphoma, leukemia, breast cancer, and ovarian carcinoma. Further,
these compounds were in WO 01/96367demonstrated to circumvent melphalan
resistance. These nds may therefore be used in the prevention and/or
treatment of , reducing tumor growth and/or killing tumor cells. Thus, the
compounds may be used for curing and/or prolonging the survival of patients afflicted
with cancer diseases.
Also ed herein is the lyophilized pharmaceutical preparation, kit or
pharmaceutical composition as disclosed and claimed herein, for use as a medicament.
The invention is also directed to such a lyophilized pharmaceutical preparation, kit or
pharmaceutical composition, for use in the treatment and/or prevention of cancer, such
as ovarian cancer, lung cancer, bladder , mesothelioma, multiple myeloma,
breast cancer and/or any other solid or hematological cancer.
An aspect of the present invention is the use of a lyophilized pharmaceutical
preparation, or ceutical composition as disclosed and claimed herein, for the
preparation of a medicament for the treatment and/or prevention of cancer, such as
ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, breast
cancer and/or any other solid or hematological cancer.
Also described is a lyophilized pharmaceutical preparation, kit or pharmaceutical
composition comprising melphalan flufenamide hloride (J1) in combination with
another drug useful in the treatment of cancer, for use in ent and/or prevention of
cancer, such as ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple
a, breast cancer and/or any other solid or hematological .
Also described is a method for the treatment of and/or prevention of cancer, such as
n cancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma, breast
cancer and/or any other solid or hematological cancer. The method can comprise the
administration of a lyophilized ceutical preparation, a kit or a pharmaceutical
composition as provided herein in a therapeutically effective dose to a subject in need
thereof. The subject is typically a human or a domestic .
Also described is a method for the treatment of and/or prevention of , such as
ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiple a, breast
cancer and/or any other solid or hematological cancer, wherein the lyophilized
pharmaceutical preparation, a kit or a pharmaceutical composition comprising
melphalan flufenamide hydrochloride (J1) is provided in a eutically effective dose
to a subject in need thereof, in combination with another drug, useful in the treatment of
cancer. The subject is lly a human or a domestic animal.
The administration of a lyophilized pharmaceutical preparation, a kit or a
pharmaceutical composition to a subject in need thereof may take place by intravenous
injections. It is also le to administer lyophilized melphalan flufenamide or a
pharmaceutical composition comprising such lyophilized melphalan flufenamide in body
cavities, such as instillation in the bladder, or in peritoneal or pleural cavities.
Melphalan flufenamide or a ceutically acceptable salt thereof may be
administered in an amount of about 20-130 mg, such as 30-75 mg, for example 50 mg
total amount of melphalan flufenamide per stration. The pharmaceutical
composition or kit provided herein comprising melphalan flufenamide may therefore
have an amount of lized melphalan flufenamide such that this amount can be
administered.
Lyophilized melphalan flufenamide or a pharmaceutically acceptable salt thereof may
be administered daily, every second or third day, weekly, every second, third or 4th
week or even as a high single dose (e.g. before transplantation) depending on the
subject and cancer form to be treated.
The g “prevention” as used herein, is intended to include therapy in a patient that
has been subjected to chemotherapy against any cancer form as herein described, and
who is subjected to continued therapy with the aim of preventing any methastasis
ing from said cancer.
Also bed is the use of an excipient selected from the group comprising
Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin; hydroxypropyl- β-cyclodextrin;
sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium succinate; propylene
glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose;
and histidine, in a lyophilized ation of melphalan flufenamide, or a
pharmaceutically acceptable salt thereof, for decreasing the reconstitution time of the
lyophilized preparation of melphalan flufenamide, or a pharmaceutically acceptable salt
thereof, when reconstituted in an aqueous solvent.
Said melphalan flufenamide, or a pharmaceutically acceptable salt thereof, is preferably
melphalan flufenamide hydrochloride (J1).
Said excipient is preferably selected from Polysorbate 80 and PEG 400.
Said melphalan flufenamide, or a pharmaceutically acceptable salt f, is ably
dissolved in ethanol prior to subjecting said melphalan flufenamide to said excipient.
In this document “lyophilization”, “freeze-drying”, “lyophilized”, “freeze-dried”, and the
like may be used interchangeably.
Polysorbate 80 (having the chemical name Polyoxyethylene 20 sorbitan monooleate
and the CAS registry number 90056) is cially available from e.g. Fluka or
Sigma-Aldrich.
PEG 400 has the empirical formula HOCH2(CH 2OCH 2)mCH 2OH, where m is 8.7, and
the avarage lar weight is 380-420, and is commercially available from e.g.
Fluka or Sigma-Aldrich.
PEG 300 has the empirical formula HOCH2(CH 2OCH 2)mCH 2OH, where m is 6.4, and
the avarage molecular weight is 285-315, and is cially available from e.g.
Fluka or Aldrich.
Cremophor EL® is a trade mark sold by Sigma-Aldrich, and is Polyoxyethylene castor
oil having the CAS ry Number 617916.
Exemplary cytotoxic ides that can be used as described herein are also disclosed
in WO01/96367 and can have the formula V
N COR1
R4HN
wherein
R1 is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH2, alkylamino, cycloalkylamino or
arylamino;
R3 is NH2, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH2CH2Cl)2, NO2, F, CF3 or H; and
R4 is a l or modified cyclic or aromatic amino acid, or H; as well as
pharmaceutically acceptable salts thereof.
Also, cytotoxic peptides that can be used as described herein include peptides of the
formula I or V, wherein R3 is F. ides are examples of peptides of the formula I or
V, wherein R1 is alkyloxy; R3 is F, CF3, H, OH, O-alkyl, NO2, N(CH2CH2Cl)2, NH-acyl or
NH2; and R4 is H.
Tripeptides are example of peptides of the formula I or V, wherein R1 is alkyloxy; R3 is
F, CF3, H, OH, O-alkyl, l, NO2, N(CH2CH2Cl)2 or NH2; and R4 is a natural or
modified cyclic or aromatic amino acid.
Melphalan flufenamide, or a pharmaceutically acceptable salt thereof, may be prepared
as disclosed in WO 01/96367, which disclosure is incorporated by nce. Example
1 of WO 01/96367 ses a synthetic procedure for making melphalan flufenamide
(L-melphalanyl-L-p-fluorophenylalanine ethyl , as well as its hydrochloride salt -
melphalan flufenamide hydrochloride J1 (L-melphalanyl-L-p-fluorophenylalanine ethyl
ester, compound J1), which sure is incorporated herein.
The dipeptide tives disclosed in WO01/96367 can be synthesised from toxycarbonyl
(Boc)-protected melphalan as disclosed therein and can be lyophilized
and used as described herein. Also, WO01/96367 discloses the preparation of
tripeptide derivatives, in which Boc-protected amino acids were coupled to the
melphalan containing dipeptide derivative using EDC/NMM/HOBt as coupling reagents
(EDC is triethylamine or 1-[3-dimethylamino)propyl]ethylcarbodiimide hydrochloride,
NMM is N-methylmorpholine and HOBt is 1-hydroxybenzotriazole). Such tripeptide
derivatives may be lyophilized and used as described herein.
Examples of derivatives of melphalan that can be lized and used as described
herein in all aspects include, without limitation, melphalan flufenamide, L-melphalanyl-
L-p-fluorophenylalanine isopropyl ester (JV28), inyl- L-melphalanyl- L-pfluorophenylalanine
ethyl ester (J3) (Fig. 7) and pharmaceutically acceptable salts
thereof. These compounds are disclosed previously in WO01/96367, which also
provides methods for their preparation. Melphalan flufenamide, JV28 and J3 may be
transformed into melphalan in the body. In WO 01/96367, these derivatives were
demonstrated to have an increased cell killing activity against tumors, even when used
at lower concentrations than melphalan. In addition, melphalan ance could be
circumvented.
The invention will be further described by way of the following examples, which do not
limit the scope of the invention.
mental section
Example 1: Lyophilization of melphalan flufenamide hydrochloride (J1) under
different conditions
It this experiment lyophilization of melphalan flufenamide hydrochloride (J1), under
various conditions was tested.
Example 1A
Weighed amounts of J1 were dissolved in various volumes of zed water in
ultrasound bath with slight g to get clear solutions. The samples were frozen in a
bath of dry ice - acetone (-78 oC, samples A1-A3) or in a r at -16 oC (samples B1-
B3). -drying was then conducted for 16 h at a re of 1 mbar at room
temperature with a dry ice-acetone (-78 oC) trap between the drying flask and the
pump.
The visual appearance after drying was as summarized in Table 1.
Table 1. Six different solutions of J1 at various freeze trations or temperatures.
Exp no mg J1 mL water conc (mg/mL) appearance after drying
J1A1 2.4 6 0.4 white fluffy
J1A2 2.7 27 0.1 white fluffy – some incompletely dry
J1A3 2.5 10 0.25 white fluffy
J1B1 2.7 6.75 0.4 white solid
J1B2 2.5 25 0.1 light yellow powder
J1B3 2.8 11.2 0.25 white fluffy
Example 1B
Samples of the dried compounds were dissolved in 50% s acetonitrile and
analyzed by HPLC (ACE-column, C8, 50x3 mm, 10-97% CH 3CN in 3 min, 1 mL/min). In
one case (J1A1) the aqueous solution was analyzed by HPLC before freeze-drying
(J1A1-start). The purities after drying were as summarized in Table 2.
Table 2. Purity after freeze drying. Rt= ion time
Exp no J1: Rt 2.27 (%) Rt 1.87 (%) Rt )
J1A1-start 88 12
J1A1 79 21
J1A2 80 20
J1A3 41 45 14
J1B1 34 42 25
J1B2 36 43 21
J1B3 79 21
Example 1C
Next it was tested to use either slightly acidic water (for example 0.01% HCl) to
enhance the speed of dissolution or to first dissolve J1 in ethanol, before adding water
(neutral or slight acidic).
Three samples of J1 were prepared by ving melphalan flufenamide (ca 3 mg) in
70% aq. ethanol (0.5 mL). The solutions were diluted with 5 mM HCl to give a
concentration of 0.4 mg/mL. Since melphalan flufenamide dissolved rapidly in aq.
ethanol it was not necessary to use ultrasound bath or heating to get a clear solution.
The solutions were then frozen in a bath of dry ice – acetone (-78 oC) trap between the
drying flask and the pump. The visual appearance after drying was as summarized in
Table 3.
Table 3. Three replicates of J1 dissolved in ethanol and acid.
Exp no mg J1 mL HCl conc (mg/mL) appearance after drying
J1C1 3.0 7.0 0.4 white solid some of which adhered to the glass
J1C2 3.2 7. 0.4 white solid some of which adhered to the glass
J1C3 2.9 6.75 0.4 white solid some of which adhered to the glass
Example 1D
Two HPLC runs were done on each sample: one from the solid nd that could
be removed from the flask and one by dissolving the remainder in compound in the
flask (table 4).
Table 4. Purity after freeze drying.
Run 1
Exp no J1: Rt (%) Rt(%) Rt(%)
J1C1 2.25 (97%) 2.32 (3%)
J1C2 2.24 (97%) 1.87 (1%) 1.98 (1%)
J1C3 2.22 (99%) 1.87 (1%)
Run 2
Exp no J1: Rt (%) Rt(%) Rt(%)
J1C1 2.25 (100%)
J1C2 2.25 (95%) 1.88 (3%) 1.98 (2%)
J1C3 2.25 (97%) 1.87 (3%)
In conclusion, by dissolving J1 in 70% l, diluting with 5mM HCl and freezedrying
three samples were obtained with purity > 95%.
Example 1E
It was then tested to omit the acid and instead dilute the ethanol with deionized water.
Three samples of J1 were prepared by ving J1 (ca 3 mg) in 70% aq. ethanol (0.5
mL) at room temp. The solutions were diluted with zed water to give a
concentration of 0.4 mg/mL. The solutions were then frozen in a bath of dry ice-acetone
(-78 oC). Freeze-drying was then conducted over 16 h at a pressure of 1 mbar at room
temperature with a dry ice-acetone (-78 oC) trap between the drying flask and the
pump. The visual appearance after drying was as summarized in Table 5 and the
purities in Table 6.
Table 5. Three replicates of J1 dissolved in ethanol and water.
Exp no mg J1 mL water conc (mg/mL) appearance after drying
J1D1 3.15 7.37 0.4 white fluffy solid
J1D2 3.11 7.27 0.4 white fluffy solid
J1D3 3.17 7.42 0.4 white fluffy solid
Table 6. Purity after freeze drying.
Exp no J1:R t (%)
J1D1 2.26 (ca 100%)
J1D2 2.26 (ca 100%)
J1D3 2.25 (ca 100%)
By dissolving J1 in 70% ethanol, diluting with water and freeze-drying; three ate
samples were obtained with the same purity as the starting al.
e 2: Effect of excipients on the dissolution rate of lyophilized melphalan
flufenamide
In this experiment the effect on speed of dissolution by adding excipients to the freezedrying
process of melphalan flufenamide hydrochloride (J1) was tested. The following
excipients were used, all of which are common formulation agents Generally
Considered As Safe (GRAS) according to the FDA (US Food and Drug stration):
− D-mannitol, trehalose and sucrose;
− Trizma hydrochloride and L-histidine;
− Polysorbate 80, β-cyclodextrin;
J1 was used in all experiments.
D-Mannitol, was bought from Sigma no. 33440;
D-(+)-Trehalose dihydrate, was bought from Sigma no. T9449-25 g;
Trizma hydrochloride, was bought from Sigma no. T3253-100 g;
odextrin hydrate, was bought from Sigma no. 856088-5 g;
Polysorbate 80, was bought from Fluka 59924-100 g.
Freeze-drying was performed on a Leybold Lyovac GT2 equipment. LCMS (Liquid
chromatography-mass spectrometry) was run on a -system using acetonitrile-
0.1% trifluoroacetic acid in water as eluent. An ACE-column C8, 50 x 3 mm and a
gradient 10-97% itrile in 3 min was used. The filter vials were from Whatman,
Mini-UniPrep, 0.45 µm.
(i) Method A, freeze-drying
Melphalan flufenamide (30.1 mg) was ved in 5 mL of 70% ethanol with 1 mM HCl,
total dissolution within 12 min at 18-19 °C. The solution was diluted with water (70 mL)
and distributed (10 mL) into 250 mL round ed flasks with and without excipient
(eg β-cyclodextrin, 9 mg). When all material had dissolved, the solutions were frozen by
immersion in a dry-ice/acetone bath at -78 °C. The frozen ons were then freezedried
at <0.1 mbar overnight and room temperature, evaporation keeping the samples
frozen until dryness.
(ii) Method A, speed of dissolution measurement
A 5% glucose solution (10 mL) was added in one portion at 18.5-19 oC to the freezedried
material and stirred with a magnet. Aliquots (ca 0.3 mL) was taken with 1-mL
syringe at various times and filtered through a filter vial (0.45 m). The filtrate (8 L)
was ed by HPLC.
(iii) Method B, freeze-drying
Melphalan flufenamide (10.2 mg) was dissolved in 1.67 mL of 70% ethanol with 5 mM
HCl, total dissolution within 5 min at 25 °C. The solution was diluted with water (23.3
mL) and distributed (10 mL) into flasks with and without excipients (e.g. β-cyclodextrin,
9 mg). The on of J1 and ent was dispensed into plastic vials with a fitting
insert 0.45 µm filter (0.25 mL to each vial). The vials were frozen by immersion in a dryice
/acetone bath at -78 °C and then kept at -20 °C overnight in a rack fitting the vials.
The frozen vials were covered by alumina foil to prevent cross-contamination and kept
in the rack led to -20 °C, while exposing the rack in a desiccator to <0.1 mbar
overnight, evaporation keeping the samples frozen until dryness.
(iv) Method B, speed of dissolution measurement
A 5% glucose solution (0.5 mL) was added, which contained an internal standard (3-
ybenzoic acid, 0.08 mg/mL). After various times (15 s - 12 min) the contents of
the vials were filtered, the te directly transferred to glass vials to prevent leaking of
undissolved material into the filtrate and 8 µL of the filtrate injected into the LCMS.
Determination of dissolution speed
In a first approach, Method A, aqueous solutions of J1 with different additives were
freeze-dried in round bottomed flasks. To each freeze dried compound, a glucose
solution was added with controlled stirring. Small aliquots were withdrawn with a
syringe at specific times and filtered through a 0.45 µm GHP syringe filter. The degree
of dissolution of J1 in the filtrate was then determined by HPLC. This method was used
with freeze-dried melphalan flufenamide alone and together with D-mannitol, trehalose,
sucrose, rbate 80 and β-cyclodextrin. The result of these tests showed that J1
was completely dissolved within 2-4 min regardless of excipient (see Fig. 1, no
excipients, and Fig. 2, with excipients. See also Table 7). In fact, the dissolution rate for
J1 lyophilized with excipients was ly faster than could be measured using this
method.
Table 7. Excipient additions to J1 (4 mg) on freeze-drying, Method A.
Freeze dried material Ratio Number of
J1:additive (mg ments
J1:mg ve)
D-Mannitol 4:2 1
D-Mannitol 4:10 2
Trehalose 4:2 1
Trehalose 4:10 2
e 4:10 1
odextrin 4:9 1
β-Cyclodextrin 4:18 2
Polysorbate 80 4:0.05 1
Polysorbate 80 5 2
To improve the precision and enable measurement of dissolution at shorter intervals,
Method B was developed. In this method, aqueous solutions of melphalan flufenamide
and excipients (see Table 2) were added to 2 mL c vials and freeze-dried. Then a
e solution with internal standard 3-methoxybenzoic acid was added without
stirring. After varying times (15 s-6 min) the contents of the vial was filtered with a 0.45
µm GHP vial insert, the filtrate transferred to a glass vial and the degree of dissolution
of melphalan flufenamide hydrochloride (J1) determined by HPLC with internal
standard. The lack of stirring made possible a slower dissolution process, both more
clinically relevant and easier to measure the kinetics of.
With this method the dissolution kinetics of freeze-dried J1 could be followed to
complete dissolution after 3-4 min (see Fig. 3, no ents and Fig. 4, with ents,
see also Table 8).
Table 8. ents additions to J1 (4 mg) on freeze-drying, Method B.
Freeze dried Ratio J1:additive Number of
material (mg J1:mg ments
additive)
J1:Mannitol 4:20 1
J1:Trehalose 4:20 1
J1: β-Cyclodextrin 4:9 1
J1-Polysorbate 4:5 1
J1:Trizma HCL 4:8 1
The speed of dissolution of J1, with and without ves, determined with Method A
and Method B, are summarized in Table 9.
Table 9. Summary of dissolution times of J1 with and without additives, Methods A and
B.
Ratio
J1:addative
Freeze dried (mg J1:mg Time (min) Time (min)
material additive Method A Method B
J1 without
additives <2 3-4
J1:Trehalose 4:2 <2
J1:Trehalose 4:10 <2
J1:Trehalose 4:20 0.5-1
J1:Sucrose 4:10 <2
J1:Mannitol 4:2 <2
J1:Mannitol 4:10 <2
J1:Mannitol 4:20 0.5-0.75
J1: β-
extrin 4:9 <2 0.75-1
J1: β-Cyclodextrin 4:18 <2
J1:Polysorbate 4:0.05 <2
J1:Polysorbate 4:0.265 <2
J1:Polysorbate 4:5 0.25-0.5
J1:Trizma HCL 4:8 >12
Purity and recovery of J1
A sample of melphalan flufenamide hloride (J1) was dissolved in 50% aq.
acetonitrile and analyzed immediately with LCMS (Liquid tography-mass
spectrometry), showing only one peak (>99%). The purity of J1 directly after dissolution
in 70% ethanol containing 1 mM HCl or 5 mM HCl was found to be ca 97%, with a
minor byproduct of ca 3%. The amount of this byproduct increased if the solution was
left at room temperature.
The results demonstrate that the speed of dissolution of freeze-dried J1 in glucose
solution with stirring was faster than could be ed (Method A), not enabling the
effect of excipient additions to be seen. Using a more clinically relevant Method B
without stirring, the dissolution of freeze-dried melphalan flufenamide in e
solution could be followed to teness after 3-4 minutes. Addition of excipients dextrin
, Polysorbate 80, Mannitol and Trehalose to the melphalan flufenamide
solution before freeze-drying all gave complete dissolution below 1 minute. The fastest
dissolution was given by Polysorbate 80 addition, giving complete dissolution at the first
time-point 15 seconds.
e 3: Test of effect of concentration of the ent Polysorbate 80 on the
dissolution rate of melphalan flufenamide
The following was performed to test the amount of the ent Polysorbate 80 to be
added in the freeze-drying process of melphalan flufenamide and to maximize the
dissolution rate in a 5% glucose solution. 0, 10, 50 and 100 % weight, in relation to
melphalan amide of Polysorbate 80 was used. The experiments were run in
duplicate.
Melphalan flufenamide hydrochloride (J1) was used in all experiments.The Polysorbate
80 used was bought from Fluka, 59924-100 g.
Freeze-drying was done on a Leybold Lyovac GT2 equipment. LCMS was run on a
HP1100-system using acetonitrile-0.1% trifluoroacetic acid in water as eluent. An ACE-
column C8, 50 x 3 mm and a gradient 10-97% acetonitrile in 3 min was used. The filter
vials were from Whatman, Mini-UniPrep, 0.45 µm.
General preparation of 2 mg/mL stock solution of melphalan flufenamide before freezedrying
was performed as s:
11.0 mg melphalan flufenamide was suspended in 10 mM solution of HCl in absolute
EtOH (0.5 mL). The mixture was stirred for 30 minutes before 0.2 mL water was added.
The mixture was stirred for 10 minutes at room temperature (clear solution) before it
was added to a 0 0C solution of water (4.8 mL). 0.25 mL of the solution was transferred
to a plastic vial containing 10%, 50% or 100% weight rbate 80. The vial was
shaken, cooled and freeze-dried.
A 5% glucose solution with an internal standard 3-methoxybenzoic acid was prepared
by ving 3-methoxybenzoic acid (1.2 mg) in water (15 mL). The mixture was d
for 1 hour before 750 mg of glucose was added while stirring. 0.5 mL of the 5% glucose
on was added to each freeze-dried plastic vial and the mixtures were filtered, at
different time-points, transferred to a glass vial and the dissolution of J1 was
ined by HPLC.
Determination of dissolution rate
J1 (11 mg) was suspended in EtOH (0.5 mL) and stirred for 30 minutes at room
temperature before water (5 mL) was added. The solution was divided into 4 different
flasks containing 0%, 10%, 50% or 100 %weight (in relation to J1) of Polysorbate 80.
The solutions were transferred to 2 mL plastic vials and freeze-dried overnight.
A 5% glucose solution with an internal standard 3-methoxybenzoic acid was added to
each vial without stirring and the mixtures were filtered through a 0.45 µm GHP vial
insert at different time-points (2 – 300 seconds). The filtrate was ately
transferred to a glass vial to prevent leaking from undissolved material. The amount of
dissolved J1 ve to the internal standard was determined using HPLC.
s
The speed of dissolution of J1 (1 mg/mL in 5% glucose solution) with, and without
Polysorbate 80, is summarized in Table 10 and depicture in Fig. 5.
Table 10.
Time to achieve
Freeze-dried material steady state
(1 mg/mL) dissolution
(seconds)
J1 without additive 300-600
J1 with 10% Polysorbate 80 30-60
J1 with 50% Polysorbate 80 30-60
J1 with 100% Polysorbate 80 30-60
Table 10 shows that all samples containing freeze-dried J1 and the ent
Polysorbate 80 dissolves much faster than J1 -dried in the absence of excipient.
Special attention was devoted to the sample containing 10% Polysorbate 80 and the
oints in this experiment were:
immediate filtration, 2 seconds, 15 seconds, 30 seconds and 5 minutes. In the first
oint where the sample was ed immediately, approximately 40% was
dissolved and after 2 seconds approximately 70% was dissolved. Full dissolution was
achieved after 30-60 seconds.
The dissolution rate of freeze-dried J1 at 1 mg/mL containing varying amounts of
Polysorbate 80 in a 5% glucose on was under 1 minute for all samples. The
lowest amount of Polysorbate 80 for rapid ution was between 10 and 50 % by
weight.
Example 4: Test of effect of trations of the excipients Polysorbate 80, PEG
400 and β-cyclodextrin on the dissolution rate of melphalan flufenamide
This example was performed to study the effect of different concentrations of the
excipients Polysorbate 80, PEG 400 and β-cyclodextrin added in the freeze-drying
process of melphalan flufenamide to maximize the solubility and speed of dissolution in
a 5% glucose solution towards the long-term goal of developing a lyophilized material,
stable to storage and with facile preparation for dosing.
Melphalan flufenamide hydrochloride (J1) was used in all experiments.
Polysorbate 80 used was bought from Fluka (59924-100 g), β-cyclodextrin from h
(856088) and PEG 400 from nt (100316).
Freeze-drying was done on a Leybold Lyovac GT2 equipment. LCMS was run on a
HP1100-system using acetonitrile-0.1% trifluoroacetic acid in water as eluent. An ACE-
column C8, 50 x 3 mm and a gradient 10-97% acetonitrile in 3 min was used. The filter
vials were from Whatman, niPrep, 0.45 µm.
General preparation of 2 mg/mL stock solution of melphalan flufenamide for
freeze drying
11.1 mg lan flufenamide was suspended in 10 mM on of HCl in absolute
EtOH (0.5 mL). The mixture was stirred for 30 minutes before 0.2 mL water was added.
The mixture was stirred for 10 minutes at room temperature (clear solution) before it
was added dropwise to a 0 0C solution of water (4.8 mL). 0.25 mL or 0.5 mL of the
solution was erred to a c vial containing the excipients. The vial was shaken,
cooled and freeze-dried.
Solubility ment
A 5% glucose solution with an internal standard was prepared by dissolving 3-
methoxybenzoic acid (1.2 mg) in water (15 mL). The mixture was stirred for 1 hour
before 750 mg of glucose was added while stirring. 0.2 mL of the 5% glucose solution
was added to each freeze-dried plastic vial and the mixtures were shaken for 10-15
seconds and filtered after 5 minutes. The filtrate was transferred to a glass vial and the
solubility of melphalan flufenamide was determined by HPLC and a calibration curve.
Determination of solubility
A 2 mg/mL stock solution of melphalan flufenamide hydrochloride (J1) was used as in
us ments.
For a solubility of 2.5 mg/mL of J1 in 5% glucose solution, 0.25 mL of the stock solution
was dispensed into 2 mL plastic vials containing a e of the excipients determined
by experimental design and the mixtures were immediately cooled and freeze-dried.
The high/low levels of each excipient (in weight-% relative to melphalan flufenamide)
were as follows: rbate 80 (8%-80%), PEG 400 (80%-400%) and β-cyclodextrin
(10%-50%). The highest amount of each ent was ined from FDA Inactive
Ingredient database of registered IV-administered drugs. β-cyclodextrin is on FDA's
GRAS (Generally Recognized As Safe) list but no recommendations are given for
intravenous injections to our knowledge, which caused a fairly conservative high level
to be set. The weight percent of each excipient in on to melphalan flufenamide
hydrochloride (J1) (weight) is shown in Table 11.
Table 11. Weight percentage of each excipient in relation to J1.
Experiment Polysorbate 80 PEG 400 β-cyclodextrin
No [%] [%] [%]
1 8 400 10
2 8 80 10
3 8 400 50
4 8 80 50
80 400 10
6 80 80 10
7 80 400 50
8 80 80 50
9 44 240 30
44 240 30
11 44 240 30
As is trated in other experiments herein, the dissolution rate of J1 increased
markedly with the addition of Polysorbate 80 in the freeze drying process. Three
experiments were performed to attempting to reach a solubility of 5 mg/mL. A stock
solution of J1 was added to 3 different plastic vials (exp 12, 13 and 14) containing
Polysorbate 80 (10%, 50% and 100% weight in relation to melphalan flufenamide). The
mixtures were immediately cooled and freeze-dried.
A 5% glucose solution with an internal standard (3-methoxybenzoic acid) was added to
each vial and the vials were shaken and allowed to stand for 5 s. The mixtures
were filtered through a 0.45 µm GHP filter vial and the filtrate was ately
transferred to a glass vial to prevent leaking from undissolved al. The amount of
dissolved J1 was determined using HPLC and a calibration curve.
Results
The solubilities of J1 in mg/mL with high/low levels of the excipients Polysorbate 80,
PEG 400 and β-cyclodextrin are summarized in Table 12.
Table 12. Solubility of J1 in mg/mL.
Experiment Polysorbate 80 PEG 400 β-cyclodextrin Solubility of J1
[%] [%] [%] [mg/ml]
1 8 400 10 1.9
2 8 80 10 0.9
3 8 400 50 2
4 8 80 50 1.4
80 400 10 2
6 80 80 10 1.5
7 80 400 50 2
8 80 80 50 1.9
9 44 240 30 1.9
44 240 30 1.8
11 44 240 30 1.7
12 10 x x 1
13 50 x x 1.2
14 100 x x 1.4
* x x x 0.67
16* x x x 0.25
17 50 80 100 1.2
*Experiments 15-16 did not use freeze-dried J1, fine powder was used in Experiment
and larger lumps were used in Experiment 16.
The results provided in Table 12 demonstrates that the solubility of J1 increased in all
experiments containing excipients compared to not freeze-dried J1 (entry 15 and 16).
The large discrepancy in the solubility of not freeze-dried J1 is probably due to different
particle size in the batch, since entry 15 was a suspension of fine white powder, while
entry 16 was larger lumps giving lower dissolution rate and hence lower solubility of J1
in 5 minutes. The precision of the analysis is shown in center experiments 9-11 (1.9,
1.8 and 1.7) with identical excipient concentrations. The 3 samples with Polysorbate 80
as the excipient (10, 50 and 100%) exhibited a solubility of 1.0, 1.2 and 1.4 mg/mL,
respectively.
The entries with a e of the excipients rbate 80, Peg 400 and β-cyclodextrin
ted l ations with solubilities at or close to 2.0 mg/mL. The t
determined solubilities 2.0 (entries 3, 5 and 7) were only attainable with high levels of
PEG 400, giving liquids or semisolids after freeze drying.
The samples with lower amount of PEG 400 (entries 2, 4, 6 and 8) formed a white fluffy
powder after freeze drying, with the highest determined solubility of 1.9 mg/mL in entry
8. This prompted testing if a higher solubility could be obtained by lowering the amount
of PEG 400 and increasing the amount of β-cyclodextrin. An additional sample (row 17
in Table 12) was -dried containing 50% Polysorbate 80, 80% of PEG 400 and
100% of β-cyclodextrin. The solubility of J1 with this mixture of excipients was 1.2
mg/mL.
The results demonstrate that the maximum solubility of J1 with ations of
excipients is close to 2 mg/mL.
With experiment 13 it was shown that a on of J1 with 50% Polysorbate gave a
solubility of approximately 1.2 mg/mL alone, sufficient for a 1.0 mg/mL formulation and
allowing the exclusion of PEG 400 and β-cyclodextrin.
Visual mation experiments
To confirm the dissolution in a more clinically relevant setting, a larger scale experiment
in transparent glass vials instead of plastic vials was performed. Vial 1 contained a
solution 4.8 mg melphalan flufenamide hydrochloride (J1) and 2.4 mg Polysorbate 80.
As a control vial 2 contained 4.8 mg melphalan flufenamide hydrochloride (J1) and no
rbate 80. The vials were freeze-dried overnight.
To each vial containing the -dried melphalan J1 as white fluffy material, 4.70 mL
of a 5% glucose solution was added to give a concentration of J1 of 1.02 mg/mL. The
mixtures were shaken for 10-15 seconds and the test tube containing J1 and 50%
Polysorbate 80 showed a clear solution after 15 seconds, see Fig. 6, left vial. The
nce tube with freeze-dried J1 without the Polysorbate showed small particles and
was not totally dissolved after 30 minutes, see Fig. 6, right vial. LC-MS analysis
revealed that the purity of melphalan amide after 30 minutes was >95%, in both
vials.
The results provided herein trate that the solubility of J1 in 5% glucose solution
could be enhanced using a mixture of the ents rbate 80, Peg 400 and βcyclodextrin
to 1.9 mg/mL. Such a mixture of excipients with J1 ed in a fluffy white
solid on lyophilization.
Lyophilization of J1 with 50 %-weight Polysorbate 80, resulted in a white fluffy solid that
is rapidly dissolved in 5% glucose solution. The saturation concentration 1.2 mg/mL is
sufficient to use in a clinical setting for dosing preparation at 1.0 mg/mL.
Example 5: Stability test
The purpose of the first part of this study was to investigate the dissolution rate of
melphalan flufenamide hydrochloride (J1) (freeze-dried together with Polysorbate 80) in
% glucose solution.
The dissolution speed of J1 (freeze-dried) in 5% glucose solution containing
Polysorbate 80 will be measured in another experiment.
Finally the dissolution speed of non-freeze dried J1 in 5% glucose solution containing
Polysorbate 80 will be measured.
The second part is an investigation of the degradation of J1 in two different
preparations at ed temperature. The first preparation was a freeze dried solid
containing polysorbate 80 and the second was a 25 mg/ml solution of J1 in N,N-
dimethylacetamide (DMA). The degradation was followed for 1 month at +40 ºC, using
two preparations
(i) Determination of dissolution rate.
A 5% glucose solution was added to each plastic vial containing J1. The vials were
were shaken and filtered at different time-points. The filtrate was transferred to glass
vials and the amount of ved J1 was determined by HPLC.
(ii) Accelerated stability study design.
10 Vials with freeze-dried J1 and Polysorbate 80, and 10 vials of J1 solution in DMA,
were stored at 40°C for 1 month. Two vials of the freeze-dried material (named freeze
dried 1 and 2 in table below) and one vial of the DMA solution (named DMA in table 1
below) was taken out from the 40 °C chamber and stored at -20°C and ed at the
same time for assay and purity of J1. Sample times were 0, 1, 3, 10 and 30 days. Each
freeze dried vial contained 0.25 mg of J1. The 25 mg/ml solution in DMA was from
Oncopeptides.
(iii) Analysis and s
The freeze dried s were dissolved in 500 µl DMA in Whatman 0.45 µm filter
vials. The samples were vortexed briefly before ng the two parts of the vial
together and thus filtrating the sample. The 25 mg/ml solution samples were diluted
with DMA by aliquoting 20 µl solution to HPLC vials and diluting with 980 µl DMA. 4µl
were injected in the chromatographic system.
The ity was evaluated as the ve purity, since there was a slight variation in
the amount of J1 in the freeze dried vials. By using relative purity, each sample is
standardised against itself and the effect of g J1 amount is minimised on the
stability result.
The speed of dissolution of J1 in 5% glucose in the presence of PS is summarized in
table 13 :
Table 13: y of dissolution experiments.
Time (min) to Content in plastic Solution
reach steady state vial
dissolution
Dissolution 1 freeze-dried 5% glucose
exp. 1 J1+Polysorbate 80
Dissolution 1 freeze-dried J1 5% glucose +
exp. 2 Polysorbate 80
Dissolution 1 - 2 non-freeze-dried J1 5% e+
exp. 3 Polysorbate 80
ity test results
Table 14. Results of stability test at 40 ºC, comparison between DMA solution and
freeze-dried J1 at +40 ºC / Ambient Relative Humidity
Freeze dried 2 Freeze dried 2 Freeze dried Avg DMA
Day ve [%] Relative [%] Relative [%] Relative [%]
0 98.80 98.74 98.77 96.81
1 98.77 98.69 98.73 95.76
3 98.71 98.77 98.74 95.43
98.55 98.66 98.61 92.22
98.32 98.42 98.37 86.90
The results in table 14 show that the freeze dried material is essentially unchanged
during the test period. Only a small change in purity can be observed. Also the
dissolution rate of freeze-dried J1 at 1 mg/mL in a 5% glucose solution was under 1 min
in the presence of Polysorbate 80. The ution rate of non-freeze-dried J1 at 1
mg/mL in a 5% glucose solution containing Polysorbate 80 was estimated to 1-2 min.
J1 in DMA on degraded significantly during storage at + 40 ºC for one month. The
ve amount decreased from about 96.8 % to 86.9%. J1 stored as a freeze dried
solid only showed a small degradation from 98.7 % to 98.3 % during the same period of
time.
Other embodiments
It is to be understood that while the invention has been described in conjunction with
the detailed description thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention, which is defined by the scope of the appended
claims. Other aspects, advantages, and modifications are within the scope of the
following claims.
Claims (31)
1. A lyophilized pharmaceutical preparation comprising 5 (i) melphalan flufenamide hydrochloride (J1); and (ii) at least one excipient ed from sulfobutylether-β-cyclodextrin; Trehalose; and Sucrose. 10
2. A lyophilized pharmaceutical preparation according to claim 1, sing melphalan flufenamide hydrochloride (J1) and the excipient sulfobutylether-βcyclodextrin.
3. A lized pharmaceutical ation according to claim 1, comprising 15 melphalan flufenamide hydrochloride (J1) and the excipient sucrose.
4. A lized pharmaceutical preparation according to claim 1, comprising melphalan flufenamide hydrochloride (J1) and the excipient trehalose. 20
5. A lyophilized pharmaceutical preparation according to any one of claims 1-4, wherein the amount of excipient is about 10-100 by weight of said melphalan flufenamide hloride (J1).
6. A lyophilized pharmaceutical preparation according to any one of claims 1-5, 25 further comprising a physiologically acceptable solution, said physiologically acceptable solution being a glucose solution.
7. A lyophilized pharmaceutical preparation according to claim 1 or 2, comprising 25 mg melphalan flufenamide hydrochloride (J1) and 12.5 mg sulfobutylether-β- 30 cyclodextrin.
8. A lyophilized pharmaceutical ation according to claim 1 or 4, comprising 25 mg melphalan flufenamide hydrochloride (J1) and 12.5 mg sucrose.
9. A lyophilized pharmaceutical preparation according to claim 1 or 4, comprising 25 mg melphalan flufenamide hydrochloride (J1) and 12.5 mg trehalose.
10. A lyophilized pharmaceutical preparation according to any one of claims 1-9, 5 which is free, or ntially free, from ts.
11. A lyophilized pharmaceutical preparation ing to any one of claims 1-10, for use as a medicament. 10
12. A lyophilized pharmaceutical preparation according to any one of claims 1-10, for use in the treatment and/or prevention of cancer.
13. A lyophilized pharmaceutical preparation for use according to claim 12, wherein said cancer is any one of ovarian cancer, lung cancer, bladder , mesothelioma, 15 multiple myeloma, breast cancer or hematological cancer.
14. A method for ing a lyophilized pharmaceutical preparation according to any one of claims 1-10, whereby: a. melphalan flufenamide hydrochloride (J1) is dissolved in an organic t to 20 obtain a melphalan flufenamide hydrochloride (J1) solution; b. water is added to the melphalan flufenamide hydrochloride (J1) solution in order to obtain an aqueous melphalan flufenamide hydrochloride (J1) solution, in a concentration of about 0.2-3.0 mg/ml; c. at least one excipient selected from sulfobutylether-β-cyclodextrin; and sucrose is 25 added to the melphalan flufenamide hydrochloride (J1) solution; and d. the aqueous melphalan flufenamide hydrochloride (J1) solution containing excipient is subjected to lization.
15. The method according to claim 14, whereby: 30 a. melphalan flufenamide hloride (J1) is dissolved in an c solvent; b. water is added to the on obtained in step a. in order to obtain a solution of said melphalan flufenamide hydrochloride (J1) in a concentration of about 0.2-3.0 mg/ml; c. at least one excipient selected from sulfobutylether-β-cyclodextrin; trehalose; and 35 sucrose is added to the solution obtained in step b.; and d. the on obtained in step c. is subjected to lyophilization.
16. A method according to claim 14 or 15, wherein the organic solvent is selected from any one of l, ethanol containing acid, glycerin, propylene glycol, benzyl alcohol, dimethylacetamide (DMA), N-methylpyrrolidone, isopropanol, n-butanol, tert- 5 l, methyl tert-butyl ether, propylene glycol, dimethylsulfoxide, ydrofuran, 2- methyl tetrahydrofuran, acetone, dimethylforamide, acetonitrile, dioxane, acetic acid, lactic acid, propionic acid, n-butanol, isopropanol, n-propanol, tert-butanol, sec-butanol, methanol, and a mixture of l and water. 10
17. A method according to claim 14 or 15, wherein the c solvent is ethanol.
18. A method according to any one of claims 14-17, wherein the excipient is sulfobutylether-β-cyclodextrin. 15
19. A method according to any one of claims 14-17, wherein the excipient is sucrose.
20. A method according to any one of claims 14-17, n the excipient is trehalose.
21. Use of an excipient selected from sulfobutylether-β-cyclodextrin; trehalose; and sucrose, in a lyophilized preparation of melphalan amide hydrochloride (J1), for decreasing the reconstitution time of the lyophilized preparation of melphalan flufenamide hydrochloride (J1), when reconstituted in an aqueous t.
22. The use according to claim 21, wherein said excipient is sulfobutylether-βcyclodextrin.
23. The use according to claim 21, wherein said excipient is Sucrose.
24. A use ing to claim 22 or 23, wherein said melphalan flufenamide hydrochloride (J1) is dissolved in ethanolprior to subjecting said melphalan flufenamide hydrochloride (J1) to said excipient.
25. Use of (i) melphalan amide hydrochloride (J1); and (ii) at least one excipient selected from sulfobutylether-β-cyclodextrin; Trehalose; and Sucrose, in the preparation of a medicament. 5
26. The use according to claim 25, n the medicament is for the treatment and/or tion of cancer.
27. The use according to claim 26, wherein said cancer is any one of n cancer, lung cancer, bladder cancer, elioma, multiple myeloma, breast cancer 10 or hematological cancer
28. The use according to any one of claims 25-27, wherein the medicament is a pharmaceutical preparation as defined in any one of claims 1 to 10. 15
29. A lyophilized pharmaceutical preparation according to any one of claims 1-10, substantially as herein described with reference to any example thereof.
30. A method as according to any one of claims 14-20, substantially as herein described with reference to any example thereof
31. A use according to any one of claims 21-28, substantially as herein described with reference to any example thereof
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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SE1150371-1 | 2011-04-28 | ||
SE1150371 | 2011-04-28 | ||
US201161535126P | 2011-09-15 | 2011-09-15 | |
US61/535,126 | 2011-09-15 | ||
PCT/EP2012/057577 WO2012146625A1 (en) | 2011-04-28 | 2012-04-25 | Lyophilized preparation of cytotoxic dipeptides |
Publications (2)
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NZ617197A NZ617197A (en) | 2015-06-26 |
NZ617197B2 true NZ617197B2 (en) | 2015-09-29 |
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