Note: Descriptions are shown in the official language in which they were submitted.
2179294
W 0 95118635 PCT/SE95/00011
1
LOW MOLECULAR WEIGHT HY T TRC~NT~ A~ WTTH PEPTI1~E OR
PROTEIN
' S
The invention relates to a freeze-dried soft, flexible and continuous
matrix of low-molecular weight hyaluronic acid or salt thereof
containing at least one peptide or protein, useful as pharmaceutical
composition.
Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan
consisting of a linear polymer of repeating units of glucuronic acid and
N-acetyl-glucosamine. The molecular weight can vary over a wide
range depending on the source. HA is present in several tissues of
animals, and in some organs, such as rooster combs, in concentrations
high enough for commercial scale extraction. Such tissue contains HA of
a wide range of molecular weights and during a complex series of
extraction, purification and sterilisation steps, high molecular weight
chains are more or less degraded resulting in a final product having a
considerably narrower molecular weight range.
The critical parameters determining the characteristics of the final
product in this respect are the molecular weight distribution of HA in
the raw material, the degree of degradation of HA chains during the
purification and sterilisation process and the effectiveness of removing
low molecular weight HA.
A commercial available hyaluronic acid product is HEALON~ (ICabi
Pharmacia AB, Uppsala, Sweden) which has a average molecular weight
of about 4 000 000 daltons. This product is produced as outlined in
USA 4 141 973 and is an ultrapure product. There are many literature
references relating to the use of viscoelastic products of HA in
ophthalinological application and the preparation of such products,
including the preparation of chemically modified HA.
l
WO 95118635 ~ 17 9 2 ~ 4 PCT/SE95/00011
2
HA is know in slow release formulations and in WO 9005522 HA is
mentioned as a slow release carrier together with a binding protein for
e.g. GH or IGF.
In US 4772419 a shaped article based upon cross-linked, possible
derivatized HA or salt thereof, which is a substantially unswollen
water-swellable state has a dry matter content of at least 65 percent
by weight and a tensile strength greater than 100 N/cm2 is disclosed.
HA is of high molecular weight, i.e. about 3 000 000 Da. The article
could be produced by freeze-drying. Thin sheets of paper-like
structure or cellophane-like structures were obtained. The article could
be used for preventing the adhesion and accretion of tissues.
Low molecular weight hyaluronic acid (I,MW~iA) could be produced by
acid or enzymatic hydrolysation and thereafter fractionation. These
processes are known in the art.
LMWHA is known as Garner for pharmaceutical active agents and also
for pharmaceutical activity itself. .
In EP 138-572 a product comprising HA with Mw of 50 000 -
100 000 is stated as useful for wound healing and HA with a Mw of
500 000 - 730 000 is useful for intraocular and infra-articular
injections. Fragments of HA as carrier for drugs, e.g. EGF, in eyedrops is
also disclosed.
In EP 197 718 HA with different Mw between 30 000-730 000 is
useful in the ophthalmic and dermatologic field. LM~NHA with EGF is
mentioned as example.
HA with Mw of 500-800 000 together with water for cosmetic and skin
disorder is known from GB 2 228 736.
In US S 079 236 HA with Mw 50 000-200 000 for treatment of
osteoarthritis and joint function is disclosed and in JP 1 290 631 HA
with Mw 50 000-3 000 000 for treatment of arthris, diabetic
retinopathy is claimed.
WO 9316732 and WO 9316733 disclose HA or fragments thereof
(e.g. < 750 000) and a drug e.g. anti-inflammatory NSAID, diclofenac,
naproxen, anti-cancer, especially useful topically for skin.
2179284
W O 95118635 PCTISE95/00011
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In GB 2 235 204 is disclosed that a readily water-soluble film or
sheet for cosmetic use is formed when hyaluronic acid is freeze-
dried in vacuo. The hyaluronic acid used has a molecular weight of
1 200 000, giving a viscous solution in water. The layer containing
' 5 magnesium-L-ascorbil phosphate is used as a cosmetic sheet for a
face mask. Skin moisture, skin tension and whitening effect was
shown for this composition.
EP 522 491 discloses a freeze-dried composition comprising
hyaluronic acid and a polypeptide, which is administered by
injection after reconstitution of the composition.
Our claimed composition comprises low molecular weight
hyaluronic acid and peptide or protein, which gives unexpected
advantageous effect when used for administration of a drug.
For the production of a matrix, which is soft, flexible and continous and
preferably in the form of a layer, special binding forces and
interactions within the molecule are needed. Hyaluronic acid with a
high molecular weight has a special structure of the molecule, which
cannot be compared the molecular structure of the low molecular
weight hyaluronic acid. A person skilled in the art could not foreseen
how the low molecular weight hyaluronic acid could react when freeze-
dried.
When administrating a drug topically, a problem is to know how much
drug is released during a certain time, so that the patient always
receives the right dosage per time unit.
When giving the drug dropwise on an ulcer, the total amount is well
defmedbut there are difficulties in the administration of the drug in a
defined amount over thewhole surface and this method requires
normally clinical care.
When giving the drug in a paste-base, the exact amount of the drug is
- difficult to calculate and apply. Difficulties for sublingual or buccai
composition can e.g. be stability problem due to a hydrophilic character
of the base or calculation of the release time.
CA 02179294 2005-03-24
51067-58
4
We have now found that when freeze-drying an aqueous solution of a
peptide or protein and LNiWHA which is not cross-linked, a layer in
the form of a cake is formed with a structure like a wowen or a filter
paper. The "paper" is porous, massive and homogenous.
This "paper" can be cut in a desired form, can be torn and is easily
handled. For this "paper" the exact amount of the drug per area is
known. This means that the dosage can be accurate when the area of
the "paper" is known.
We have also found that when applying this "paper" topically,
subligunally or buccally, the whole amount of the drug is quickly
released.
The drug is stable and keeps the activity within this formulation
during storage.
IS The claimed formulation is biocompatibile when applied on humans
and is a perfect mean for treatment of ulcers of different kind.
The "paper" or "cake" can be applied directly to the ulcer or in the
mouth. The drug will be thereby be quickly released by the pus or the
saliva.
~0 We have also found that the drug can be present in a high
concentration when freeze-dried together with low molecular weight
HA.
The present invention relates thus to a freeze-dried soft, flexible and
~S continuous matrix of low-molecular weight hyaluronic acid or salt
thereof containing at least one peptide or protein..
The molecular weight of the low molecular weight hyaluronic acid is
preferably between 50 000 and 200 000 Da.
The invention also relates to pharmaceutical compositions in the form
30 of a layer characterised by a freeze-dried low molecular weight
hyaluronic acid containing at least one peptide or protein. The peptide
or protein could be e.g. GH, IGF-I, IGF-II or EGF or mixtures thereof.
By GH is meant growth hormone or functional analogues thereof, by
IGF is meant insulin-Iike growth faetor or functional analogues thereof,
3 S both IGF-I and IGF-II and by EGF is meant epidermal growth factor or
functional analogues thereof.
Pharmaceutical compositions of the invention may be contained in a
commercial package together with instructions for the use thereof.
W095/18635 217 9 2 9 4 pCTISE95/00011
An antibiotic agent can be mixed with a growth hormone or growth
factor when applied to a wound.
By functional analog is meant a substance having the same biological
5 activity as the peptide or protein and having at least 65 96 homology
with the peptide or protein.
The invention relates also to a process for the manufacture of the
matrix or the pharmaceutical composition, which is characterised by
freeze-drying a water solution of low-molecular weight hyaluronic acid
and the peptide or protein in a layer. This freeze-drying and further
production of the pharmaceutical article must be sterile.
The invention also relates to the use of freeze-dried low molecular
weight hyaluronic acid in the form of a layer as Garner for peptide or
protein
This use is preferably for accurate dosing of the drug.
The invention also relates to the use of the claimed pharmaceutical
composition for the manufacture of a medicament for wound healing
and to a method for accurately obtaining a pre-determined dosage of a
topically administerable peptide or protein which is characterised by
freeze-drying a water solution of low-molecular weight hyaluronic acid
and the peptide or protein to form a layer.
By low molecular weight is meant less than 1 000 000 D and
preferably between 50 000 and 200 000 D.
The layer can be between 1-40 mm and is preferably 2-12 mm.
GH can be in a concentration of 1-200 IU/ml and is preferably 5-120
lU/ml.
pH cari be between 6.0 and 8.2 in the water solution prior to freeze-
~g~
Growth hormone is here used as an example for the usefulness of the
invention, but is not limiting the scope of protection by the claims.
R'O 95!18635 PCT/SE95J00011
2179294
6
STABILITY OF PROTEINS
The stability of proteins depends on the chemical and physical
properties of the protein. _
Different degradation pathways are known such as deamidation,
oxidation, cleavage and aggregation.
Deamidation and oxidation are common chemical reactions comprising
changes of the primary structure of the protein. Deamidation occurs
especially in aqueous solutions but low temperature and low pH of the
solutions suppress the deamidation reaction.
Different forms of aggregation result from the physical instability of
the protein. Aggregates can be soluble or insoluble and binding of both
the forms can be covalent or non covalent.
The aggregates can give opalescent solutions but there can also be
non-visible aggregation-which only can be shown chemically.
The prevention of covalent aggregation in protein formulations is of
importance since such processes are irreversible and could result in
the production of inactive species which in addition also may be
immunogenic.
Changes in the primary structure may also give rise to conformational
changes which can be the cause of self association of the protein,
aggregation.
The non covalent aggregation-occurring under certain conditions can
lead to precipitation and loss of activity.
However, by monitoring these degradation reactions, it is possible to
prove indirectly that the drug (in the examples GH) retains full
biological activity. (Bristow A F et al. Pharmeuropa, Human Growth
Hormone, Vol.3, 1-49, March 1991)
WO 95!18635 217 9 2 9 4 " PCTISE95100011
7
METHODS
Isoelectric focllsing~F) with densitometric evaluation
IEF is a method according to which the extent of deamidation can be
evaluated.
The separation of hGH components is carried out in a pH gradient,
which is established between two electrodes and stabilised by carrier
ampholytes. The proteins migrate until they align themselves at their
isoelectric point in the gradient, at which a protein possesses no net
overall charge and will therefore concentrate as migration ceases.
Thus the separation is obtained according to charge. The relative
distribution of charged hGH forms are quantified by densitometric
scanning of Coomassie Blue stained polypeptides.
The higher percentage of the monomer, the less deamidation.
PolYpeDtides size distribution (SDS-PAGE)
Proteins in preparations of somatropin, hGH, were denatured by
sodium dodecyl sulphate (SDS) to yield negatively charged molecular
complexes of SDS-protein. Separation was then obtained according to
molecular size by electrophoresis in polyacrylamide gels (PAGE) in the
presence of SDS. The relative polypeptide size distribution of hGH was
quantified by desitometric scanning of the silver stained polypeptide
bands.
Visual inspection
The appearance of the solutions were eye-inspected according to Ph.
Eur. 2nd Ed. The scale is I to IV, and I is the most clear.
EXAMPLES
Hyaluronic acid with a molecular weight of about 150 000 dalton has
been produced from Na- hyaloronate. 2.51 g of Na- hyaloronate
(Pharmacia AB, Sweden) was solved in 500 ml of water in argon
atmosphere.
R'O 95!18635 ~ ~ 9 z 9 ~ PCT/SE95100011
8
16 ml HCl was added and the mixture was thereafter stirred during 2
hours at 22-23 °C. pH was <l. The solution was neutralised to pH 7.0
with 0.5 M NaOH. Thereafter 0.37 M HCl was added and the solution
was stirred during 5 hours at 45 °C. in argon atmosphere. pH 7.0 was
S then achieved with 0.5 M NaOH.
The solution was dialysed by using a dialyse tube with destilled water.
The used tube was 130885/10 30M with a cut off 12-14 x103 D.
The molecular weight of hyaluronic acid was analysed in the solution
and the hyaluronic acid was freeze-dried.
The freeze-drying was performed during 30 hours in a rotation freeze-
drier at -5°C to -50°C.
Ex~.~
Hyaluronic acid with a molecular weight (LMWHA) of 150 000 in water
is mixed with growth hormone (GH, Genotropin~ from Pharmacia AB,
Sweden) so that each ml comprises 6.5 mg LMWHA and 110 IU GH.
10 ml of the solution is placed in a Petri dish with diameter of 70
mm with cover. The solutions are freeze-dried according to the
following scheme:
Freezing: 0--S°C during 3 hours
-45 °C during 26 hours
1st drying: -30°C during 28 hours at 0.1 mBar
2S
2nd drying +25°C during 5-6 hours at 0.1 mBar
After storage at S-8°C during one month the cake is dissolved in 2
ml
destillated water and analyzed. The following result were obtained:
(Table 1)
217929
WO 95!18635 PCT/SE95100011
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Table 1
Tests:
1. dissolving time (min) 5
' S 2. clarity II
3. SDS-PAGE
aggregates ('~) 0.6
GH (~Y6) 98.8
Fragment (96) 0.7
4. IEF
Main component (9b) gg
deamidation (96) 0
Example 3
Hyaluronic acid with a molecular weight (LMWHA) of 150 000 is mixed
with growth hormone (GH) ( Genotropin~ from Kabi Pharmacia AB,
Sweden) in the following way: 65 mg hyaluronic acid was mixed with
2.65 ml of Genotropin~, 76 IU/ml. and diluted to 10 ml with destiIled
water, so that each ml comprises 6.5 mg LMWHA and 20.1 !U GH.
10 ml of the solution is dispensed in Petri dish with the diameter of
70 mm diameter with cover. The solutions are freeze-dried as
described in Example 2.
The freeze-dried cake is as a filter paper which can be bent and be cut.
The diameter is 6.0 cm and the thickness is 0.5 cm.
1 cm2 of the cake is formulated to contain 7.1 IU Genotropin~ and 2.3
mg hyaluronic acid, 150 000 dalton.
1 cm2 of the cake is cut out and analysed. See Table 2
WO 95118635 217 9 2 ~ 4 PCT~E95/00011
Table 2
Months
0 1 1
5 Tests: 5C 30C
1. Dissolving time (min ) 1 2 3
2. SDS-PAGE
aggregates (4b) 0.5 2.9 4.5
GH (4f~) 99 96.2 94.5
10 Fragment (95) 0.5 0.8 1.0
3. IEF
Main component (45) 99 97 94
deamidation (~Yo) 1 1 0
The results for the claimed formulation confirms that a drug in a
freeze-dried matrix of low-molecular weight hyaluronic acid can be
stored in room temperature for at least one month in 30°C.
This result was surprising, as proteins and especially GH normally are
unstable and not possible to store in room temperature for such a long
time.
By a biological assay, nephelometry, the amount of GH per area-unit
was determined. It was found that the growth hormone was uniformly
(homologeously) dispersed in the cake.
These results demonstrates indirectly that growth hormone retains full
biological activity, since little or no degradation was observed after
storage of growth hormone formulated with freeze-dried low
molecular hyaluronic acid.
