Title:  Microsphere encapsulation of gene transfer vectors

United States Patent:  6,048,551

Appl. No.:  824997

A controlled release delivery system includes a functional gene vector in a biodegradable polymeric microsphere encapsulating the vector. The present invention further provides a method of making a controlled release delivery system by encapsulating the functional gene vector in a biologically degradable polymeric microsphere.

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention provides a controlled release delivery system including a functional gene vector and a biodegradable polymer microsphere encapsulating the vector.

More specifically, the term "controlled release delivery system" means a drug delivery system providing controlled release either by time or by location or both. That is, the system is designed to allow release of the contents thereof by either controlled rate or a controlled time, or at a desired site. For example such systems have been developed in the past for immediate release of what may be considered a loading dose and then later release for maintenance over an extended period of time. Alternatively, or in combination, a coating may be applied, that is of the type which dissolves under specific ionic or acidic conditions so as to deliver the contained vector at a desired destination. For example, the coating may be of the type for dissolving in acidic conditions for delivery to the stomach. Such coatings, in general, are well known in the art. Examples of such coatings are which are insoluble in neutral pH (i.e., in the mouth) and are soluble in acidic pH to provide specific delivery to the stomach. Examples of such coatings include the Eudragit E series of copolymers--poly[butyl methacrylate, (2-dimethyl aminoethyl) methacrylate, methyl methacrylate].

Alternatively, the coating can be enteric in nature so as to either dissolve specifically in the small or the large intestine. Examples of such enteric coatings used in the prior art are cellulose acetate phthalate, hydroxymethylpropyl cellulcse, and polymethacrylates (Eudragit L and S series of copolymers).

By functional gene vector, it is meant that the present invention provides for delivery to a desired site. That is, a gene vector capable of incorporation and function within the target cell. Preferably, the present invention provides a gene vector selected from the group consisting of viruses, bacteriophage, plasmids, and purified DNA fragments.

More preferably, the present invention provides a means of delivery for recombinant adenoviruses, separately or in combination that are derived from the known adenoviral serotypes, such as serotypes 2, 5, 12, 40, and 41. Most preferably, the present invention provides a means of delivery for a recombinant adenovirus, serotype 2 or 5, which is replication-deficient. The replication-deficient adenovirus can contain the thymidine kinase (tk) gene from herpes simplex virus, type I, under control of the Rous Sarcoma Virus (RSV) promoter (Ad.RSVtk). Another virus suitable for the present invention is the replication-deficient adenovirus with the E.coli beta-galactosidase gene, lac Z, under control of the RSV promoter (Ad.RSVlacZ). A third example of a suitable recombinant adenovirus is the human interleukin 1 receptor antagonist gene under control of the RSV promoter, Ad.RSVIL-1ra. A fourth example of a suitable recombinant adenovirus contains the human IL-10 gene under control of the RSV promoter (Ad.RSVIL-10). Other recombinant adenoviruscs, derived from any of the known serotypes, and potentially with different promoter systems can be used by those skilled in the art. Other gene vectors can also be used by those skilled in the art.

The biodegradable microspheres encapsulating the vector of the present invention are preferably made utilizing polymers and copolymers selected from the group including poly (lactide-co-glycolide) (PLGA), hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, and the Ludragit R, L, and E series of polymers and copolymers.

Preferably, PLGA co-polymers are used as they have been well characterized and offer many advantages for sustained release of macromolecular preparations. As stated above, PLGA has well established biocompatability and has been shown to be safe in in vivo settings. (Redding, 1984). PLGA will degrade in vivo by acid or base catalyzed hydrolysis and results in production of lactic or glycolic acids with minimal inflammatory responses to the surrounding tissue (Mason et al., 1981, Visscher et al., 1987, Tice and Cowsar, 1984). Most importantly, hydrolysis rates can be adjusted by modifying the monomer ratios of the glycolic and lactic acid components (Miller et al., 1977). For example, lactide to glycolide ratios can range from 50:50 lactide to 100% to lactide:0% glycolide.

The microspheres of the present invention can range in size from one micron to 200 microns. Preferably, microspheres range in size between about one to one hundred fifty microns. Most preferably, the sizes range from ten to one hundred fifty microns. The smaller the microsphere diameter, the greater the surface area per unit mass. Hence, the smaller the microsphere, the faster the release rate of encapsulated drug. Thus, in vitro studies using microencapsulated norethisterone found that an increase in diameter of PLGA microspheres from 54 to 107 microns resulted in a 4 fold increase in the initial release rate of drug (Cowsar et al., 1985). Similar findings were reported for release of protein from PLGA microspheres (Yan et al., 1994). Factors most affecting the particle size of the microsphere include the initial concentration of the PLGA polymer and the method used to form the emulsion. The size of the microspheres can effect distribution, pharmacokinetics, and other factors as is well known by those skilled in the art.

The present invention can also be utilized to co-administer vectors and drugs which work in combination. For example, current protocols employ the systemic administration of ganciclovir (GCV) to tumors treated previously with AdRSVtk. Perez-Cruet et al. showed that with systemic doses of GCV above eighty milligrams per kilogram, it was possible to eradicate tumors in a rat model following even a single dose of Ad.RSVtk. However, the high toxicity of systemic GCV limits the dose which can be given in humans, (15 milligrams per kilogram), and thereby reduces efficacy. The present invention provides a means for providing a local concentration of GCV which can be increased to the level achieved by Perez-Cruet et al. thereby making treatment with virus more efficacious. The present invention further provides a means for delivering the dose intratumorally, thereby avoiding systemic toxicity. The present invention provides for encapsulation of the Ad.RSVtk virus, preferably utilizing PLG, designed for local delivery of both virus and high concentrations of GCV augmenting therapy.

By way of background, previous investigations have evaluated intrathecal administration of GCV for tk-mediated cell killing (Ram, 1994). While these studies showed little central nervous system (CNS) toxicity for this form of therapy, they also showed poor efficacy compared to treatment with systemically administered GCV. However, when utilizing the intrathecal route of administration for pharmaceutical agents, the dynamics of cerebral spinal fluid (CSF) flow must be taken into consideration. Anti-microbials administered into the CSF have been shown to attain different levels in various regions of the CNS depending on the location where the agents were introduced (Kaiser and McGee, 1975). A homogenous equilibrium of drug levels does not occur within the CSF. On the other hand, delivering agents directly to the desired location of action has been done effectively with chemotherapeutic agents (Brem et al., 1994, Judy et al., 1995, Walter et al., 1994), thus circumventing the effect of CSF regional concentration gradients. In this manner, encapsulated GCV can be delivered in accordance with the present invention directly to the tumor bed affording a high local sustained concentration of pro-drug thereby improving efficacy.

The present invention further provides, in general, a method of making a controlled release delivery system by encapsulating a functional gene vector in a biologically degradable polymeric microsphere. The step of encapsulating the functional gene vector is achieved by adding the functional gene vector to a polymeric solution and first forming a water-oil emulsion and then forming a water-oil-water emulsion of microspheres encapsulating the gene vector and then separating the formed microspheres from the remaining solution.

More specifically, the encapsulation process involves the formation of double emulsions consisting of oil-water layers by methods previously disclosed (Cowsar et al., 1985). FIG. 1A shows theoretical microspheres formation. PLGA microspheres theoretically form with the aqueous layer containing the adenoviral vector surrounded by polymer.

The molecule being encapsulated, such as viral vector, is dissolved in aqueous solution. As seen in the experimental section below, the addition of certain compounds, such as bovine serum albumin (BSA), enhances the overall release and release rate of adenovirus from microspheres. For encapsulation of adenovirus, Polymer, preferably (PLGAA 50:50), Birmingham Polymers, is dissolved in methylene chloride and virus is then added. However, other solvents can be utilized as exemplified in the experimental section below. Mixing can be accomplished by vortexing or sonification, resulting in the formation of an oil/water emulsion. The oil-water emulsion is then mixed with a second aqueous solution to form a water/oil/water double emulsion. The second aqueous solution can contain a surfactant, preferably polyvinyl alcohol (PVA) at concentrations ranging from 0.1% to 5% The PVA can be of low, medium or high viscosity grace. The PVA assists in maintaining the structural integrity of the microspheres. The water/oil/water double emulsion is further diluted into 0.1% PVA in water and gently stirred. To aid in removal of the organic phase, the water/oil/water double emulsion can be extracted with isopropanol or placed under a gentle vacuum with stirring. After a stirring period of 0.5 hours to 4 hours, the wet spheres are collected by either filtration or by gentle centrifugation and are then washed.

Although filtration sieves can be used to select microspheres based on size, several the experiments were done with an unsieved preparation. Using these methods, the sizes generally range from ten to one hundred fifty micrometers, as shown in FIG. 1B. FIG. 1B is a scanning electron micrograph of microspheres formed in accordance with the present invention. The scanning electron micrograph of microspheres followed encapsulation of adenoviral vectors. Microspheres were prepared in the standard fashion as set forth above and were fixed in osmium tetroxide vapor. Microscopy was performed with a Hitachi 2460N microscope at variable pressures between seventy and one hundred Pascals using a fifteen kilovolt beam. The scale bar on the micrograph equals two hundred microns.

For treatment of glioblastoma, the present invention can be used in conjunction with currently used protocols for tumor resection and stereotactic surgery (Bernstein et al., 1993) to prevent the regrowth of the tumor. The microspheres in the present invention are amenable to passage through a narrow gauge needle (>.about.400 microns) and can be delivered to the tumor bed with such a device.

The present invention can be used in the treatment of many other types of tumors in which resection of the tumor is a typical method of treatment. Further, it could be used in combination with or in place of tumor resection.

Claim 1 of 13 Claims

1. A controlled release delivery system comprising:

a functional gene vector; and

a biodegradable polymer microsphere encapsulating said vector, said microsphere consisting essentially of a biodegradable polymeric coating wherein said system includes a drug selected from the group consisting of ganciclovir, 5-flurocytosine, and 6-thioxanhine.

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