Low molecular weight hyaluronic acid with peptide or protein

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai

Reexamination Certificate

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C514S002600, C514S021800, C514S054000, C514S412000, C514S420000, C514S576000, C514S777000, C536S055100, C424S078050

Reexamination Certificate

active

06180601

ABSTRACT:

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® (Kabi Pharmacia AB, Uppsala, Sweden) which has a average molecular weight of about 4 000 000 daltons. This product is produced as outlined in U.S. Pat No. 4,141,973 and is an ultrapure product. There are many literature references relating to the use of viscoelastic products of HA in ophthalmological application and the preparation of such products, including the preparation of chemically modified HA.
HA is known 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 U.S. Pat. No. 4,772,419 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/cm
2
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 (LMWHA) could be produced by acid or enzymatic hydrolysation and thereafter fractionation. These processes are known in the art. LMWHA is known as carrier 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 intra-articular injections. Fragments of HA as a 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. LMWHA 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 U.S. Pat. No. 5,079,236 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.
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 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 continuous 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 with 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 defined but there are difficulties in the administration of the drug in a defined amount over the whole 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 buccal composition can e.g. be stability problem due to a hydrophilic character of the base or calculation of the release time.
We have now found that when freeze-drying an aqueous solution of a peptide or protein and LMWHA 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. The claimed formulation is biocompatibile when applied on humans and is a perfect means 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. 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 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 of a layer characterised by a freeze-dried low-molecular weight hyaluronic acid containing at least one peptide or protein. The drug 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-like growth factor or functional analogues thereof, both IGF-I and IGF-II and by EGF is meant epidermal growth factor or functional analogues thereof.
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 activity as the peptide or protein and having at least 65% 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 carr

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