Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – Coated – impregnated – or colloidal particulate
Reexamination Certificate
1999-12-27
2003-07-15
Lovering, Richard D. (Department: 1712)
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
Coated, impregnated, or colloidal particulate
C264S004100, C264S004600, C424S278100, C424S491000, C424S492000, C424S493000, C427S213350, C427S430100, C514S962000
Reexamination Certificate
active
06592844
ABSTRACT:
The invention relates to microspheres, films and coatings made from proteins or modified proteins and in particular to improvements in the methods of preparation thereof which result in more stable products. The microspheres, films and coatings produced in accordance with the invention are suitable for a variety of biomedical applications.
The term “microspheres” is generally employed to describe colloidal particles which are substantially spherical and have a diameter in the range 10 nm to 2 mm. Particles having a diameter of less than 1 &mgr; are sometimes called “nanoparticles”. Microspheres made from a very wide range of natural and synthetic polymers have found use in a variety of biomedical applications. They can be labelled with markers (labels or sensing devices) and transported through various media both in-vitro and in-vivo. The labels may be chemical fluorescent, magnetic or radioactive and thus they may, by appropriate sensing equipment, be observed when in use. The sort of applications for which microspheres have been used are diagnostic screening, cell separation, immunoassays, studies of phagocytosis and blood flow, studies of cell motility, haemoperfusion and extracorporeal therapy, drug delivery devices, targeted drug delivery, cell encapsulation and endovascular embolisation.
An important property which microspheres must possess for biomedical applications is biocompatibility. They should be as resistant as possible to attack from the immune system in-vivo. Further, for many applications it is important that the microspheres be biodegradable and/or resorbable in the body once their function has been discharged. Also, in other cases, they should be small enough for easy introduction into the body.
For these reasons, naturally occurring polymer materials such as proteins have been the subject of much study for the preparation of microspheres. Nanoparticles as small as 100 nm can be prepared from, for example, albumin using certain preparation techniques and this is very useful for, among other things, injectable preparations.
Because of their biocompatability some proteins have been used in making coatings for artificial prostheses which will be introduced into the human body and therefore in contact with body fluids. As with microspheres any such coating should be as resistant as possible to attack from the immune system and furthermore should not be thrombolytic i.e. should cause only minimal platelet activation.
It is known that the blood biocompatibility of arterial prostheses is improved by coating the surfaces with albumin as demonstrated by Kottke-Marchant et al in Biomaterials 1989 10 147-155. Indeed albumin has been a particular material of choice for both coatings and microspheres because it is non-antigenic, biodegradable and readily available.
A number of methods are known for preparing protein microspheres and films and protein coatings for prostheses but certain drawbacks are associated with them all.
For example, a well-known method of preparing protein microspheres is suspension cross-linking. In this process an aqueous solution of protein is added to an immiscible liquid or oil phase. Droplets of protein are dispersed by high speed stirring and then hardening or stabilization of the droplets to form microspheres is brought about by heating of the suspension to, for example, a temperature above 80° C. or alternatively by chemical cross-linking employing a cross-linking agent such as glutaraldehyde. Various methods of preparation of albumin microspheres by the suspension cross-linking technique are described by Arshady in Journal of Controlled Release, 14 (1990) 111-131.
A disadvantage of preparing microspheres by the suspension cross-linking technique is that it is difficult to produce microspheres less than 500 nm in size, although nanospheres of about 100 nm diameter have been prepared using high power ultra-sonication. A further disadvantage is that the cross-linking agents used are often toxic which is not conducive to biocompatability.
Apart from suspension cross-linking protein microspheres, especially gelatin microspheres, have been prepared by coacervation or controlled desolvation. This procedure has also been used to prepare albumin microspheres in the size range 0.1 to 5 &mgr;m by Knop, et al (1975) using ethanol as the coacervation agent added to an aqueous solution of albumin. Ishizak et al (1985) have prepared albumin microspheres in the size range 0.5 to 1.5 &mgr;m using isopropyl alcohol as the coacervation agent. A similar technique has been developed to prepare 200-500 nm nanoparticles by adding acetone to an aqueous solution of human serum albumin and heating the colloidal system (Chen et al, 1994, J. Microencapsulation, 11, 395-407). In an alternative approach, chemical crosslinking agents such as glutaraldehyde have been used to harden the microspheres (Lin et al (1993) J. Drug Targeting 1 pp 237-243).
Coacervation methods for preparing protein microspheres are simpler to perform than suspension cross-linking methods and the particles are less toxic. A disadvantage however is that the particles are not particularly stable and aggregate easily to form larger microspheres. It has been difficult, to date, to use coacervation methods to prepare protein nanospheres around 200 nm or less which may have potential use as injectable preparations.
Albumin microparticles (2-10 &mgr;m) have also been prepared by spray drying followed by heat stabilisation at 100° C. or 150° C. for 6-24 hours (Pavanetto et al., J. Microencapsulation, 11, 445-454). The main advantage of spray drying is that albumin microparticles are free of oil residues or organic solvent and the process is useful for continuous operation.
The problems of toxicity of cross-linking agents and of stability also arise in the case of protein coatings to be applied to artificial prostheses. In particular, in the physiological environment the coating is subject to wear leaving areas of the prosthesis material exposed which may lead to an unwanted immune response or clot formation.
There is thus a continuing need for improved methods of preparation of protein microspheres, films and coatings which do not have the various disadvantages out-lined above. The present inventors have developed new methods of preparing protein microspheres, films and coatings which meet this goal and all the methods are based on the simple observation that when the pH of an aqueous albumin solution is lowered to about 4.4 to 4.7 with an &agr;-hydroxy acid, e.g. lactic acid, a rapid and extensive precipitate of albumen forms and this precipitate is unusually stable. The same effect is observed with other proteins although the pH range at which precipitation occurs varies.
Thus in its first aspect the invention provides a process for stabilizing a microsphere, film or coating made from at least one protein or modified protein which comprises preparing said protein microsphere, film or coating in the presence of an aqueous solution of at least one &agr;-hydroxy acid or a derivative or analogue thereof.
It is to be understood that herein the term protein is intended to include peptides, polypeptides, metalloproteins, glycoproteins and lipoproteins and the term “modified protein” refers to proteins modified so as to have an additional molecule attached thereto, that would not naturally be associated with the protein. For example, the modified protein might consist of the protein conjugated to another organic polymer such as polyethylene glycol, polylactide or other polymer which can influence the surface characteristics of the microsphere, film or coating advantageously from a biocompatability point of view.
Preferred proteins which may be used in the process of the invention are albumen, gelatin, zein, casein, collagen or fibrinogen. Particularly preferred is albumen, either human serum albumen or ovalbumen.
Preferred &agr;-hydroxy acids for use in the invention are glycolic acid, lactic acid, hydroxybutyric acid or mixtures of two or more thereof. Particularly preferred is lactic acid. By &agr;-hydroxy
Coombes Allan G. A.
Davis Stanley S.
Lin Wu
O'Hagen Derek T.
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Lovering Richard D.
Robins Roberta L.
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