Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1996-09-30
2002-11-05
Kishore, Gollamudi S (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S001210, C424S009321, C424S009510
Reexamination Certificate
active
06475515
ABSTRACT:
This application is a 371 of PCT/EP94/03964 filed Nov. 30, 1994.
The invention relates to a process for increasing the stability of liposome suspensions that contain hydrophilic active ingredients, which is characterized in that the active ingredient that is to be encapsulated in the liposomes is metered such that 5 to 95% by weight (preferably 30 to 70% by weight) of the active ingredient is present in unencapsulated form.
The process according to the invention especially relates to those methods in which this unencapsulated active ingredient is not separated.
Hydrophilic active ingredients are to be defined as those substances according to the invention—especially pharmaceutical agents and pharmaceutical agent mixtures—that dissolve at room temperature by at least 1% by weight and preferably by at least 10% by weight in water.
Stability is to be defined according to the invention in that the product that is produced according to the process of the invention can be stored for at least 3 months, but preferably more than 6 months, at 4-8° C., without pharmaceutically unacceptable quality changes occurring.
Of course, the invention also relates to the liposome suspensions that are produced according to the process of the invention.
In recent years, liposomes have become increasingly important as potential pharmaceutical vehicles. In this connection, the possibility of using them primarily for selective transport of a pharmaceutical substance to the site of action (so-called drug targeting) is emphasized (Rubas, W., Schreier, H., Liposomen: Fortschritte in Herstellungs-Technologie und Therapie, Pharmazie Unserer Zeit [Liposomes: Improvements in Production-Technology and Therapy, Pharmaceutics of Our Time] 20, 255-270 (1991)). In targeting, a distinction is made between so-called “passive targeting” because of the liposomes being taken up in cells of the reticulo-endothelial system (RES; e.g., liver and spleen) and “active targeting,” in which surface-modified liposomes are directed to the target tissue via the introduction of “homing devices” (e.g., antibodies).
In addition to this purpose, the liposomes can be used as reservoirs for the sustained release of pharmaceutical substances, or the liposomes can protect the pharmaceutical substances from quick hydrolytic or enzymatic degradation in the organism.
Liposomes are suitable for encapsulating both hydrophilic and lipophilic pharmaceutical substances. While quantitative inclusions can be obtained with lipophilic pharmaceutical substances, complete inclusion of hydrophilic pharmaceutical substances is not achieved with conventional processes. In the case of some mechanical dispersion processes, when using high lipid concentrations (up to 400 mg/ml) for certain hydrophilic substances, inclusion levels of up to 70% can be achieved. This value lies near the theoretical maximum, which can be calculated at about 74% assuming extremely close sphere packing of spheres of like size. In most cases, however, only inclusion capacities in the range of between 10 and 55% for the various liposome production processes are cited for hydrophilic pharmaceutical substances. In this case, only the so-called remote or active loading technique represents an exception (Mayer, L. D.; Bally, M. B.; Hope, M. J.; Cullis, P. R., Biochim. Biophys. Acta 816, 294-302 (1985)), in which ionizable, hydrophilic pharmaceutical agents are quantitatively conveyed to the interior of the liposome via pH gradients.
With respect to the use of such liposome preparations as pharmaceutical agents, it appears desirable for various reasons to avoid leaving a portion of pharmaceutical substance unencapsulated. Primarily in the case of encapsulation of toxic pharmaceutical substances (e.g., cytostatic agents), which often have fewer side effects in liposomal form, the overall load on the organism should be minimized by removing the unencapsulated pharmaceutical substance.
Up until now, therefore, in the standard processes for liposome production, measures have been taken to remove the unencapsulated, hydrophilic pharmaceutical substances. For this purpose, standard physical separating processes, such as, for example, centrifuging, exclusion chromatography (GPC), or membrane processes, such as dialysis or ultrafiltration, are generally used. A common feature of all these processes is that they are technically expensive and, moreover, can drastically alter the properties of the liposomes (New, R. R. C., Preparations of Liposomes, in: New, R. R. C. (Editors), Liposomes: A Practical Approach, Oxford University Press, New York, 1990, p. 91 ff).
In this way, liposome suspensions can be produced in which an approximately 100% inclusion is present immediately after separation. Due to instabilities, e.g., because of osmotic effects, however, a considerable reduction in the enclosed portion is noted even within a short time. In the case of methotrexate (MTX)-containing liposomes, in which the separation of the unencapsulated portion was carried out by centrifuging twice, the MTX-release (6° C., away from light) was, for example, about 0.2%/day (Stricker, H.; Mentrup, E.; Krotz, R.; Zeller, W. J.; Sturm, V.; Wowra, B, Eur. J. Pharm. Biopharm. 37, 175-177 (1991)). Even more drastic inclusion losses were described for liposomal dideoxyinosine triphosphate, where the inclusion losses depending on the lipid composition of the liposomes was up to 60% within one month (4° C.) (Betageri, G. V., Drug. Devel. Ind. Pharm. 19, 531-539 (1993)).
Owing to this fact, various groups were tested to determine whether, by freeze-drying, corresponding liposome suspensions can be converted to a form that is stable in storage. For this purpose, an attempt was made by suitable measures to achieve complete retention of the inclusion during freeze-drying and subsequent resuspension. In these studies, it was noted that the liposome structure can be obtained only in the presence of cryoprotectors (e.g., saccharose, trehalose) (Crow, J. H.; Crowe., L. M., et al., Biochim. Biophys. Acta 947, 367-384 (1988)).
In the presence of those additives, however, to date it has not been possible to stabilize liposome suspensions with hydrophilic pharmaceutical substances, so that the inclusion that existed before freeze-drying (>90%) was maintained. The inclusions that are obtained after resuspension are considerably below 60% (Talsma, H., Crommelin, D. J. A., Liposomes as Drug Delivery Systems, Part III: Stabilization, Pharmaceutical Technology International 5, 36-42 (1993)).
In connection with the freeze-drying of liposomes, an alternative process for the production of liposomes with hydrophilic pharmaceutical substances was recently described. In the case of these methods, referred to as “dehydration-rehydration-methods,” (Kirby, C.; Gregoriadis, G., Biotechnology 2, 979-984 (1984)), an aqueous, pharmaceutical substance-containing phase is mixed with a liposome dispersion, and the mixture is freeze-dried. After rehydration of corresponding lyophilizates with small amounts of resuspension agents, MLV dispersions with inclusion capacities of up to 72% are obtained. In this connection, however, the inclusion capacities that can be achieved depend to an extreme extent on the type of pharmaceutical substance to be encapsulated. Thus, for liposomes that are produced by these methods, with nonionic x-ray contrast media, an inclusion capacity of less than 7% was described (Seltzer, St. E., Gregoriadis, G.; Dick, R., Invest. Radiol. 23, 131-138 (1988)).
With respect to providing liposomal preparations with hydrophilic pharmaceutical substances, there are still limitations with regard to determining the limited storage stability of such preparations. Thus, the corresponding liposome preparations, from which the unencapsulated pharmaceutical substance was separated before storage, had problems with respect to determining the retention of pharmaceutical substances (“leakage”). Because of leakage, the encapsulated portion of pharmaceutical substance generally decreased very quickly. It previously also was not possible to p
Leike Jens
Rossling Georg
Sachse Andreas
Kishore Gollamudi S
Millen White Zelano & Branigan P.C.
Schering Aktiengesellschaft
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