CN1980641A - Microspheres containing oligonucleotides and their use in the preparation of medicaments for treating type 1 diabetes - Google Patents

Abstract

The present invention relates to microspheres comprising oligonucleotides for treating type 1 diabetes, the oligonucleotides comprising about 30 weight percent to about 100 weight percent of the microspheres, based on the total weight of the microspheres, the microspheres having an average particle size of no greater than about 50 microns. The oligonucleotides are targeted to bind to primary transcripts CD40, CD80, CD86 and combinations thereof.

Description

Microspheres containing oligonucleotides and their use in the preparation of medicaments for treating type 1 diabetes

Cross References to Related Applications

Provisional Patent Application Serial No. 60/570,273, filed May 12, 2004, and Provisional Patent Application Serial No. 60/625,483, filed November 5, 2004.

Background of the invention

bright technical field

The present invention broadly relates to microsphere delivery of AS-oligonucleotides to induce tolerance in dendritic cells, especially in a non-obese diabetic (NOD) mouse model. More particularly, the present invention relates to drug delivery technology by utilizing microspheres prepared under conditions of complete water solubility, wherein antisense (AS) oligonucleotides are introduced into the microspheres. These microspheres were used as an antisense approach to prevent autoimmune diabetic symptoms in vivo and in situ in NOD mice.

Background of the invention

Microparticles, microspheres and microcapsules are solid or semi-solid particles having a diameter of less than 1 mm, more preferably less than 100 microns, which can be formed from a variety of materials, including synthetic polymers, proteins and polysaccharides. Microspheres have been used in many different applications, mainly separations, diagnostics and drug delivery.

These microspheres can be prepared from synthetic polymers, natural polymers, proteins and polysaccharides using a number of different techniques including phase separation, solvent evaporation, emulsification and spray drying. Typically polymers form the supporting structure of these microspheres, and the drug of interest is incorporated into the polymer structure. Exemplary polymers used to form microspheres include, for example, Ruiz et al. in U.S. Patent No. 5,213,812, Reid et al. in U.S. Patent No. 5,417,986, Tice et al. in U.S. Patent No. 4,530,840, Tice et al. 5,075,109, Singh et al. in U.S. Patent No. 5,102,872, Boyes et al. in U.S. Patent No. 5,384,133, Tice et al. in U.S. Patent No. 5,360,610 and Southern Research Institute's European Patent Application Publication No. 248,531. PLGA); block copolymers such as tetronic 908 and poloxamer 407 described in U.S. Patent No. 4,904,479 by Illum; and polyphosphazenes described in U.S. Patent No. 5,149,543 by Cohen et al. Microspheres prepared using these polymers exhibit low loading efficiencies, often enabling only a small percentage of the drug of interest to be incorporated within the polymer structure. It is therefore often necessary to administer large quantities of microspheres in practice to achieve a therapeutic effect.

For years, biochemists have commercially available spherical beads or particles as a tool. For example, antibodies bound to beads form relatively large particles that are specific for a particular ligand. Large antibody-coated particles are often used to cross-link receptors on the cell surface for cell activation, they are bound to a solid phase for immunoaffinity purification, and use tissue or tumor-specific antibodies bound to the particles Targeting the agent to the desired location can be used to deliver a therapeutic agent that is slowly released over time.

A disadvantage of currently available microparticles or beads is that they are difficult and expensive to produce. Microparticles produced by these known methods have a broad particle size distribution, often lack uniformity, and fail to show long-term release kinetics when the active ingredient is present at high concentrations. Furthermore, the polymers used in these known methods need to be dissolved in organic solvents to form microparticles. They must therefore be produced in special equipment designed to handle organic solvents. These organic solvents can denature proteins or peptides contained in microparticles. Residual organic solvents can cause toxicity when administered to humans or animals.

In addition, few microparticles are available that are sufficiently small to fit through a pinhole, the size typically used to administer therapy or suitable for administration by inhalation. For example, particles prepared using polylactic-glycolic acid (PLGA) are large in size and tend to agglomerate. When used for injection, a size selection step leading to product loss is necessary to remove particles that are too large. PLGA particles of an appropriate size for injection must be administered through a large gauge needle to accommodate the large particle size, often causing patient discomfort.

Typically, many of the currently available microparticles are activated in aqueous media to release their contents, so they must be lyophilized to prevent prior release. Additionally, particles such as those prepared using the PLGA system exhibit erosion and diffusion based release kinetics. In such systems, an initial rupture or rapid release of the drug is observed. This disruptive effect can lead to deleterious side effects to patients who have been administered the particles.

Target drug-loaded microparticles prepared using lipids are known. For example, lipids arranged in a bilayer membrane surrounding multiple aqueous compartments to form particles can be used to encase water-soluble drugs for continuous delivery, as described by Sinil Kim in US Patent No. 5,422,120. These particles are typically greater than 10 microns in size and are designed for intra-articular, intrathecal, subcutaneous and epidural administration. Alternatively, liposomes have been used to deliver small molecules intravenously. Liposomes are spherical particles composed of single or multiple phospholipid and cholesterol bilayers. Liposomes are 30 microns or larger in size and can carry various water-soluble or fat-soluble drugs. Issues including the purity of lipid components, potential toxicity, vesicle heterogeneity and stability, excess uptake, and manufacturing or shelf-life difficulties have hindered liposome technology.

One goal of the pharmaceutical community is to deliver nucleic acids to cells of animals for diabetes treatment. For example, nucleic acids can be delivered relatively efficiently to cells in culture (in vitro), but when nucleic acids are delivered to animals (in vivo), nucleases lead to high rates of nucleic acid degradation.

In addition to protecting nucleic acids from nuclease digestion, nucleic acid delivery vehicles must exhibit low toxicity, must be efficiently taken up by cells and have a defined, easily prepared formulation. As clinical trials have shown, the viral vectors used for delivery can lead to severely harmful and even fatal immune responses in the body. Additionally, these methods have the potential to induce mutagenic effects in vivo. Delivery via packaging of nucleic acids in lipoplexes of different formulations, such as liposomes or cationic lipoplexes, is generally ineffective in vivo and may produce toxic effects. Complexes of nucleic acids with various polymers or with peptides have shown inconsistent results, and the toxicity of these formulations has not been resolved. Nucleic acids have also been encapsulated into polymer matrices for delivery, but in these instances the particles have a wide range of sizes and have not been proven useful for therapeutic applications.

Therefore, there is a need to solve the problem of nucleic acid delivery, as well as for the development of microspheres and the development of new methods for making microspheres. Details regarding microspheres can be found in Scott et al. US Patent No. 6,458,387, Woiszwillo et al. These and all references identified herein are incorporated herein by reference.

Summary of the invention

According to the present invention, the DNA delivered to dendritic cells is delivered as microspheres. It is believed that this mode of delivery prevents nucleases from accessing the nucleic acids inside the microspheres. Microsphere delivery of AS-oligonucleotides, in particular, was performed in the NOD murine model to induce tolerance in dendritic cells. Microspheres were prepared using aqueous conditions and incorporated antisense (AS) oligonucleotides. These microspheres were used in vivo and in situ to inhibit gene expression and prevent autoimmune diabetic symptoms in NOD mice.

In a preferred aspect of the invention, three AS-oligonucleotides targeting CD40, CD80 and CD86 primary transcripts were synthesized and an aqueous solution of the oligonucleotide mixture was prepared and mixed with the polymer solution. After processing, microspheres comprising oligonucleotides are provided and administered to NOD mice.

These and other aspects, objects, features and advantages of the present invention, including various combinations, will be apparent and clearly understood by consideration of the following detailed description.

Brief description of the drawings

In the course of the description, reference will be made to the accompanying drawings, in which:

Figure 1 is a schematic diagram of the role of dendritic cells in the autoimmune destruction of pancreatic insulin-producing beta-cells in type 1 diabetes;

Figure 2 is a diagram of a plasmid vector comprising a β-galactosidase gene;

Figure 3 shows photomicrographs providing evidence of transfection of NIH 3T3 fibroblasts with plasmid DNA microspheres;

Fig. 4 is the photomicrograph of the agarose electrophoresis gel after contacting DNAase of naked plasmid DNA and two plasmid DNA microsphere preparations according to the present invention;

Figure 5 is a bar graph of β-galactosidase activity in four different plasmid DNA applications;

Figure 6 is a scanning electron micrograph of microspheres of AS-oligonucleotides and poly-L-lysine polycations;

Figure 7 is a scanning electron micrograph micrograph of microspheres of AS-oligonucleotides and poly-L-ornithine polycation; and

Figure 8 is a graph summarizing the incidence of diabetes in three groups of NOD mice treated with microspheres and following other methods for delivery of the three primary transcripts.

Description of the preferred embodiment

As required, detailed technical solutions of the present invention are disclosed here; however, it can be understood that the technical solutions disclosed here are merely examples of the present invention, and they can be embodied in various forms. Therefore, specifics disclosed herein are not to be interpreted as limiting, but merely as a basis for claiming claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriate manner. sexual basis.

Optimal protocol for preventing autoimmune insulin-dependent diabetes mellitus by preparing and injecting antisense (AS)-oligonucleotide microspheres targeting the primary transcripts of CD40, CD80 and CD86 described here. These oligonucleotides were designed to induce immune tolerance to prevent destruction of insulin-producing β-cells in the NOD mouse model. The events leading to the destruction of these β-cells are illustrated in Figure 1 . This exemplifies how type 1 diabetes is manifested by autoimmune destruction of pancreatic insulin-producing β-cells in NOD mice and humans. At clinical onset, humans have 10-20% remaining beta-cell mass. Saving of this residual amount can result in a residual insulin level suitable for regulating glucose levels. Microparticles of the invention are provided to interfere with the autoimmune destruction of β-cells as illustrated in FIG. 1 .

It is understood that dendritic cells (DC) can be activated to become potent antigen presenting cells found in all tissues and present in high concentrations subcutaneously. These antigen-presenting dendritic cells act as triggers of immune responses by T cells, especially in the lymph nodes.

Figure 2 is a map of the plasmid vector containing the β-galactosidase gene, which can be used to transfect NIH 3T3 fibroblasts. Cells producing blue color by reaction with added β-galactosidase x-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside), NIH 3T3 fibroblasts with plasmid DNA In vitro evidence of microsphere transfection is shown in Figure 3.

Figure 4 illustrates the ability of microspheres to protect DNA in solution. This is an agarose gel showing nuclease protection conferred by microspheres of plasmid DNA typically produced as noted here. In plasmid samples 1, 2 and 3, naked plasmid DNA was exposed to DNAse, with smearing indicating that degradation of the plasmid DNA occurred upon application of the three levels of DNAase. In the pellet 1 and pellet 2 samples, the plasmid DNA microsphere preparation was exposed to DNAase, and there was no tailing indicating that the microsphere preparation protected the plasmid DNA from degradation.

Figure 5 reports β-galactosidase activity in four different applications of plasmid DNA. Naked plasmid DNA applications showed extremely low levels. Using lipofectamine, a commercial cationic lipid as a delivery vehicle, plasmid DNA cationic lipoplex applications showed slightly higher levels. A substantially greater activity was manifested in both pDNA microspheres, microsphere 1 corresponding to particle 1 of FIG. 4 and microsphere 2 corresponding to particle 2 of FIG. 4 .

In the preparation of microspheres for autoimmune therapy of diabetes in mice, three AS-oligonucleotides were dissolved in aqueous solution and conjugated to a water-soluble polymer and polycation. The solution is typically incubated at about 60-70°C, then cooled to 23°C to remove excess polymer. Microspheres are believed to be generated containing the following three AS-oligonucleotide sequences, where the asterisks indicate thiolation:

Seq ID 1: CD 40-AS: 5′C*AC*AG*C C*GA*GG*C*AA*A

GA*C*AC*CA*T*GC*AG*GG*C*A-3′

Seq ID 2: CD80-AS: 5′-G*GG*AA*A G*CC*AG*G A*AT*CT*A

G*AG*CC*AA*TG G*A-3′

Seq ID 3: CD86-AS: 5′-T*GG*GT*G C*TT*CC*G T*AA*

GT*T C*TG*GA*A C*AC*G*T*C-3′

More particularly, nucleic acids generally comprise between about 30 and about 100 weight percent microspheres having an average particle size of no greater than about 50 microns. They are generally prepared as follows. An aqueous solution of the oligonucleotide mixture is prepared by mixing aliquots of three oligonucleotide solutions, each solution containing one of the three types of oligonucleotides. Solutions containing these three types of oligonucleotides were prepared. The solution preferably contains about 10 mg/ml oligonucleotide. These were mixed with an aliquot of a stock solution of 10 mg/ml polycation solution in a polycation:oligonucleotide volume ratio of about 1:1 to about 4:1. Polymer solutions of polyvinylpyrrolidone and/or polyethylene glycol are prepared and mixed with other solutions. Multiple heatings, coolings, centrifugations and washings provide an aqueous suspension which can usually be solidified and lyophilized to form a dry powder comprising microspheres of oligonucleotides and polycations.

Microspheres according to the invention are useful virus-free delivery vehicles for plasmid DNA and antisense oligonucleotides and other nucleic acids. They allow in vitro delivery of β-galactosidase plasmid DNA into 3T3 fibroblasts. Microspheres protect plasmid DNA from nuclease activity. High levels of β-galactosidase activity were expressed after transfection with microsphere preparations.

Microspheres containing antisense oligonucleotides of interest downregulate surface cell antigens CD40, CD80, and CD86, which are known to be critical in the activation of autoimmune responses that lead to the destruction of pancreatic insulin-producing β-cells important. This can be done by subcutaneous injection into the dendritic cells located under the skin. Studies in NOD mice have shown that it can effectively prevent the autoimmune destruction of β cells. DNA and oligonucleotide microspheres are effective in vitro and in vivo transfection vehicles. Dendritic cells appear to take up oligonucleotide microspheres and suppress expression of the surface cell antigens CD40, CD80 and CD86. Antisense oligonucleotide microspheres effectively prevent the development of diabetes in NOD mice.

The following examples further illustrate certain features and advantages of the present invention. The examples are not to be considered limiting of the invention or otherwise.

Example 1

Three AS-oligonucleotides targeting CD40, CD80 and CD86 primary transcripts were synthesized by a DNA synthesizer at the University of Pittsburgh (Pittsburgh, PA). The AS-oligonucleotide sequence is:

Seq ID 1: CD 40-AS: 5′C*AC*AG*C C*GA*GG*C*AA*A

GA*C*AC*C A*T*G C*AG*GG*C*A-3′

Seq ID 2: CD80-AS: 5′-G*GG*AA*A G*CC*AG*G A*AT*CT*A

G*AG*CC*A A*TG G*A-3′

Seq ID 3: CD86-AS: 5′-T*GG*GT*G C*TT*CC*G T*AA*

GT*T C*TG*GA*A C*AC*G*T*C-3′

An aqueous solution of the oligonucleotide mixture was prepared by mixing aliquots of three oligonucleotide solutions, each containing one oligonucleotide, to form a 10 mg/ml solution of the three oligonucleotides. solution. Poly-L-Lysine·HBr at 10 mg/ml in diH2O was prepared (Poly-L-Lysine·HBr up to 50,000 by Bachem, King of Prussia, PA). Poly-L-lysine·HBr was added to the oligonucleotide solution at a volume ratio of 1:1. Vortex the mixture gently. 12.5% PVP (polyvinylpyrrolidone, 40000 Daltons, Spectrum Chemicals, Gardena, CA) and 12.5% PEG (polyethylene glycol, 3350 Daltons, Spectrum A 25% polymer solution from Chemicals, Gardena, CA) was prepared, pH = 5.5. The polymer solution was added in a volume ratio of 2:1 as follows: 750 μl of AS-oligonucleotide, 0.75 ml of poly-L-lysine·HBr, 3.0 ml of PEG/PVP, for a total volume of 4.50 ml.

The batch was incubated at 70°C for 30 minutes and then cooled to 23°C, upon cooling the solution became cloudy and precipitated. The suspension was then centrifuged to remove excess PEG/PVP. The resulting pellet was washed by resuspension in deionized water, followed by centrifugation and removal of the supernatant. The washing process was repeated three times. The suspension in water was lyophilized to form a dry powder comprising microspheres of oligonucleotides and poly-L-lysine.

Figure 6 shows a scanning electron micrograph (SEM) of a 1:1 ratio of poly-L-lysine:oligonucleotide species. The size of the assembled microspheres was 0.5-4 μm, with an average particle size of about 2.5 μm. Precipitation of an unknown substance was also observed. Additional studies by HPLC confirmed that the precipitate consisted of residual PEG/PVP, mainly PVP.

Example 2

The AS-oligonucleotides targeting CD40, CD80 and CD86 primary transcripts are the AS-oligonucleotide sequences in Example 1. An aqueous solution of the oligonucleotide mixture was prepared by mixing aliquots of three oligonucleotide solutions, each containing one oligonucleotide, to form a 10 mg/ml solution of the three oligonucleotides. solution. A solution of the oligonucleotide mixture is prepared. Poly-L-Ornithine·HBr (Poly-L-Ornithine·HBr 11,900(vis) by Sigma) was prepared at 10 mg/ml in diH2O. Poly-L-ornithine·HBr was added to the oligonucleotide solution, and the mixture was vortexed gently. 12.5% PVP (polyvinylpyrrolidone, 40000 Daltons, Spectrum Chemicals, Gardena, CA) and 12.5% PEG (polyethylene glycol, 3350 Daltons, Spectrum A 25% polymer solution from Chemicals, Gardena, CA) was prepared, pH = 5.5. Add the polymer solution. Incubation and washing were then performed as described in Example 1. Prepare a total volume of 6.0ml, including 1.5ml of AS-oligonucleotide, 1.5ml of poly-L-ornithine·HBr, and 3ml of PEG/PVP.

Figure 7 shows the SEM of a 1:1 ratio of poly-L-ornithine:oligonucleotide species. The size of the assembled microspheres was 0.2-8 μm, with an average particle size of about 2 μm. Precipitation of unknown substances was also observed. Complementary HPLC studies were able to confirm that the precipitate consisted of residual PEG/PVP, mainly PVP.

Example 3

In vivo experiments were performed using the NOD mouse model of type 1 diabetes. Type 1 diabetes is manifested by autoimmune destruction of the insulin-producing β-cells of the pancreas as explained in Figure 1 . AS-oligonucleotides were used in three applications to interfere with autoimmune destruction of β-cells. The goal is to interfere with dendritic cell function by targeting primary transcripts of CD40, CD80, and CD86, which encode dendritic cell surface proteins required for T cell activation. Dendritic cells with low levels of CD40, CD80 and CD86 are known to promote a network of suppressive immune cells in vivo. These cascades can lead to insufficient T cell reactivity to β-cells in vivo.

In the first group of experimental animals, dendritic cells were proliferated in vitro from bone marrow progenitor cells of NOD mice. Combinations of three AS-oligonucleotides targeting primary transcripts of CD40, CD80 and CD86 were added to cells in tissue culture medium. After culture, AS-oligonucleotide-transfected dendritic cells were injected into 5-8 week old related recipients (not yet diabetic). This is a known ex vivo delivery method.

In parallel, AS-oligonucleotide microspheres were injected directly into additional NOD mice of the same age. Single injections were performed on each of these treated mice. Another group of these NOD mice was not treated and served as a kind of control.

Figure 8 shows that control, untreated NOD mice were all diabetic by 23 weeks of age. Groups of ex vivo AS-oligonucleotide transfected and reinfused dendritic cells (AS-ODNDC) showed a delayed onset of diabetes, with 20% remaining diabetes-free, suggesting that glucose levels were maintained in the non-diabetic range. Of the microsphere-injected NOD mice in vivo, 71% remained diabetes-free at 43 weeks.

It is clear that the technical solutions of the present invention that have been described are some explanatory illustrations for the application of the principles of the present invention, and those skilled in the art can make various modifications without departing from the connotation and extension of the present invention. The various features described herein can be used in any combination and are not limited to the specific combinations specifically listed herein.

sequence listing

<110> Baxter International Inc. (BAXTER INTERNATIONAL INC.)

BAXTER HEALTHCATE S.A.

Children's Hospital of Pittsburgh (CHILDREN'S HOSPITAL OF PITTSBURGH)

<120> microspheres containing oligonucleotides and their use in the preparation of drugs for the treatment of type 1 diabetes

(OLIGONUCLEOTIDE-CONTAINING MICROSPHERES, THEIR USE FOR THE

MANUFACTURE OF A MEDICAMENT FOR TREATING DLABE)

<130>SCT064887-47

<141>2005-05-12

<160>3

<210>1

<211>31

<212>DNA

<213>Mus musculus

<400>1

cacagccgag gcaaagacac catgcagggc a 31

<210>2

<211>29

<212>DNA

<213>Mus musculus

<400>2

gggaaagcca ggaatctaga gccaatgga 29

<210>3

<211>30

<212>DNA

<213>Mus Musculus

<400>3

tgggtgcttc cgtaagttct ggaacacgtc 30

Claims (12)

1. the microsphere that is used for the treatment of type 1 diabetes that comprises oligonucleotide, described oligonucleotide accounts between about 30 percetages by weight of described microsphere and about 100 percetages by weight based on the microsphere gross weight, and the particle mean size that described microsphere has is not more than about 50 microns.

2. the microsphere of claim 1, wherein said oligonucleotide targeting is in conjunction with being selected from the primary transcript of CD40, CD80 and CD86 and their combination.

3. the microsphere of claim 2, wherein said oligonucleotide is selected from Seq ID 1, SeqID 2 or Seq ID 3 and their combination.

4. one kind is used for the method for nucleic acid delivery to the individuality of suffering from type 1 diabetes with the microsphere form, that the route of administration that wherein is used to send described microsphere is selected from is intravenous, muscle, subcutaneous, partial, Intradermal, endoperitoneal, mouthful, lung, eye, approach nose or rectum.

5. one kind is used to protect the pancreas beta cell of non-obese diabetes mice to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 1.

6. one kind is used to protect the pancreas beta cell of non-obese diabetes mice to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 2.

7. one kind is used to protect individual pancreas beta cell to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 1.

8. one kind is used to protect individual pancreas beta cell to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 2.

9. one kind is used to protect people's pancreas beta cell to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 1.

10. one kind is used to protect people's pancreas beta cell to avoid the destructive method of autoimmune, comprises the microsphere of subcutaneous injection according to claim 2.

11. one kind is used to protect individual pancreas beta cell to avoid the method that autoimmune is destroyed and type 1 diabetes is fallen ill, comprises the microsphere of subcutaneous injection according to claim 1.

12. one kind is used to protect individual pancreas beta cell to avoid the method that autoimmune is destroyed and type 1 diabetes is fallen ill, comprises the microsphere of subcutaneous injection according to claim 2.

Images