WO2010142660A2

WO2010142660A2 - Drug delivery system

Info

Publication number: WO2010142660A2
Application number: PCT/EP2010/057965
Authority: WO
Inventors: Michael Keller; Geoffrey Gogniat
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2009-06-09
Filing date: 2010-06-08
Publication date: 2010-12-16
Anticipated expiration: 2011-12-09
Also published as: WO2010142660A3

Abstract

The present invention provides pharmaceutically active formulations in the form of microparticles of defined size comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid that functions as both an amphipathic emulsifier and siRNA binding entity. Methods for making the microparticles are also provided. The microparticle formulations are especially useful for pulmonary delivery of siRNA and are therefore useful for treating diseases of the lung.

Description

DRUG DELIVERY SYSTEM

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecular biology, medicine, oncology and gene silencing. In particular, the present invention relates to pharmaceutically active formulations comprising microparticles of short interfering RNA (siRNA), a highly hydrophobic biodegradable polymer such as poly-lactide-co-glycolic acid (PLGA), and a cationic lipid, as well as a process for making and delivering the formulations to the lung.

BACKGROUND OF THE INVENTION

Short interfering RNAs (siRNAs) are double stranded ribonucleotide sequences of 19-27 base pairs in length, responsible for RNA interference (RNAi), the process of sequence specific post-transcriptional gene silencing. Delivery of siRNA in vivo is known to be difficult due to poor intracellular uptake and susceptibility to degradation following uptake. In an effort to increase uptake and effective delivery of siRNA to target cells, nano and micro-sized carriers such as cationic liposomes, polymers and lipids have been used.

The presently available nano and micro-sized carriers comprising siRNAs suffer from a number of drawbacks. In the first instance, many of the techniques available for preparing siRNA compositions result in siRNA bound only to the outside of the microparticles. Further, known processes for preparing microparticles with siRNA involve first preparing the microparticles followed by attaching the siRNA to the microparticles in a number of steps. Additionally, procedures available for producing microparticles comprising siRNA do not quantitatively encapsulate siRNA into microparticles of defined size.

There is a need therefore for a method of producing a microparticle comprising siRNA in a reduced number of steps and where the siRNA is quantitatively encapsulated into a microparticle of a defined size. SUMMARY OF THE INVENTION

The present invention provides pharmaceutically active formulations in the form of microparticles of defined size comprising siRNA , a highly hydrophobic biodegradable polymer, and a cationic lipid which acts as an emulsifier and siRNA binding entity. The siRNA and cationic lipid are encapsulated into the microparticle.

Preferably, the cationic lipid is DC-Choi or DOTAP and the highly hydrophobic biodegradable polymer is poly-lactide-co-glycolic acid (PLGA).

The siRNA present in the microparticle is a double stranded oligonucleotide of about 19-23 bp in length and is preferably targeted to a pathogenic or endogenous sequence present in lung tissue.

The formulations of the present invention are useful for pulmonary delivery and are therefore useful in treating pulmonary diseases. Preferably, the formulation is in the form of an inhalable powder such as a powder spray.

The present invention also provides a method for producing the microparticle formulations of the present invention. The method comprises the steps of: a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a water/oil/water double emulsion containing the microparticles; and c) removing the organic solution.

In order to improve the resulting particle size of the microparticle, a secondary emulsifier may be added to the water/oil/water emulsion of step b). Preferably, the secondary emulsifier is polyvinyl alcohol (PVA).

In a preferred embodiment, spray drying is performed after the water/oil/water double emulsion is obtained.

In accordance with the present invention, there is also provided a method of delivering a pharmaceutically active formulation to the lung. The method comprises the steps of a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a water/oil/water double emulsion; c) spray drying the water/oil/water double emulsion of step b) to obtain the microspheres comprising encapsulated siRNA; and d) delivering the microspheres to the lung by use of a powder spray device.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically depicts the general procedure of the double emulsion process for generating the siRNA microparticles of the present invention.

Figure 2 illustrates the general process of forming the water/oil/water (w1/o/w2) double emulsion used in making the microparticles of the present invention.

Figure 3a is a photograph of a gel electrophoreses analysis of Novartis star^® PLGA based microspheres.

Figure 3b is a photograph of a gel electrophoreses analysis of Alkermes^® PLGA based microspheres.

Figure 4 graphically depicts results of an in vitro cellular toxicity assay (measured in relative units of WST- 1 levels) performed on human lung epithelial cells incubated with different quantities of siRNA microparticles prepared using lipoplexes from commercial suppliers (siRNA-Luc, Luc 8548) as well as those made according to the present invention.

Figures 5a and 5b are SEM photographs of Alkermes^® and Novartis star^®, respectively, PLGA/DC-chol microspheres prepared by the double emulsion procedure followed by filtration of the microspheres and lyophilisation.

Figure 6 is an SEM photograph of Novartis star^® PLGA/DC-chol microspheres prepared by the emulsion procedure followed by spray drying. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutically active formulations in the form of microparticles of defined size comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid that functions as both an amphipathic emulsifier and siRNA binding entity. Methods for making the microparticles are also provided.

As used herein, the term "pharmaceutical composition" means a mixture or solution containing at least one therapeutic compound or nucleic acid molecule to be administered to a mammal, e.g., a human. As used herein, the term "therapeutic compound" means any nucleic acid or amino acid sequence having a therapeutic or pharmacological effect, and which is suitable for administration to a mammal. This term can refer to short interfering ribonucleic acids ("siRNAs").

As used herein, the term "short interfering RNA" or "siRNA" means double stranded ribonucleotide sequences of typically 15-50 base pairs and preferably 19-27 base pairs in length that are highly negatively charged and soluble predominantly in water. siRNA may be composed of either two annealed ribonucleotide sequences or a single ribonucleotide sequence that forms a hairpin structure. One of ordinary skill would understand that siRNA is responsible for RNA interference, the process of sequence-specific post-transcriptional gene silencing in animals and plants. siRNAs are generated by ribonuclease III cleavage from longer double-stranded RNA (dsRNA) which are homologous to the silenced gene or by delivering synthetic RNAs to cells. Techniques for the design of such molecules for use in targeted inhibition of gene expression are well known to one of skill in the art.

The compositions and methods of the present invention are an improvement over the prior art since the siRNA is incorporated within the microparticles rather than only bound to the outside. A further advantage is that the siRNA microparticles may be formulated in as little as one step (Figure 1 ). In addition, the siRNA may be quantitatively encapsulated into a neutral biodegradable polymer resulting in microparticles of defined size.

The microparticles of the present invention are especially useful for pulmonary delivery of siRNA and therefore useful for treating pulmonary diseases. Due to the methodology used to generate the pharmaceutical composition, the described processes are industrially scalable. In a first aspect of the invention, there are microparticle compositions comprising a highly hydrophilic siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid (also referred to herein as a cationic amphiphile). The cationic lipid functions as both an amphipathic emulsifier and siRNA binding entity. The siRNA is a highly negatively charged double stranded RNA oligonucleotide of approximately 19-23 bp in length and about 46 negative charges.

The above-described microparticles of tailored sizes may be used as microparticles in pharmaceutical compositions to mediate the down regulation (silencing) of a specific target gene defined by the choice of the siRNA sequence.

Examples of biodegradable hydrophobic polymers which may be used in the compositions and methods of the present invention, include, e.g., polylactides, polycarprolactone, polymers of lactide and glycolide, polymers of lactide and carprolactone, polymers of lactide and 1 ,4-dioxan-2-one, polyorthoesters, polyanhydrides, polyphosphazines, poly(amino acid)s and polycarbonates. Preferably, the hydrophobic polymer is poly-lactide-co-glycolic acid (PLGA) having an LG molar ratio of 50:50, MW 40,000-75000.

Examples of cationic lipids which function as emulsifiers and siRNA binding entities (also referred to herein as cationic amphiphiles) and which may be used in the present invention, include for example, cholesteryl-3(beta)N-dimethyl aminoethyl (DC- Chol), Λ/-[1-(2,3-dimyristyloxy)propyl-Λ/, N,N,N,-trimethyl-2-bis[(1-oxo-9- octadecenyl)oxy]-(Z,Z)-1 propanaminium methyl sulfate, 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), and N[1-(2,3-dioleyloxy) propyl]-N,N,N- trimethylammonium chloride (DOTMA), and dimethyldioctadecylammonium bromide (DDAB). Preferably, DC-chol or DOTAP is used.

Pharmaceutically acceptable excipients may additionally be used in the compositions of the present invention. Examples of pharmaceutically acceptable excipients that may be used in the present invention include lipid oxidation inhibitors (U.S. Pat. No. 5,605,703 and WO 9202208), stabilizing agents such as sterols, polyethyleneglycol (PEG) or tocopherol, antioxidants, such as α-tocopherol or its acetate salt; vitamin E; β-carotene; carotenoids, such as α-carotene, lycopene, lutein, zeaxanthine, and the like; and buffering agents such as citrate buffer, tris-buffer, phosphate buffer and the like; or acidifying agents, such as citric acid, maleic acid, oxalic acid, succinic acid, tartaric acid, hydrochloric acid, hydrobromic acid, phosphoric acid and the like. The use of siRNAs is well known in the art. In the pharmaceutical compositions and methods of the present invention, at least one siRNA is targeted to a specific gene sequence of interest leading to a down-regulation of the protein encoded by this mRNA. Any drugable or non-drugable gene of interest may be targeted by designing an siRNA sequence homologous to the mRNA of interest. With the entire human genome now sequenced, any portion thereof may serve as a target sequence in designing an siRNA for use in a pharmaceutical compositions of the present invention. For instance, siRNA could be designed to target an oncogene such as BCI-2, resulting in downregulation and prevention of cancer cell proliferation. Sequences of many oncogenes are known and readily available. Examples include EphA2, focal adhesion kinase (FAK), β₂ - adrenergic receptor (β₂AR), ESR1 , tumor suppressor protein pRB, MDR-1 , NKkappaB and Nek2.

Thus, the sequence for an siRNA for use in the present invention may be obtained from plasma DNA or the human genome. siRNA may be generated by ribonuclease III cleavage from longer double-stranded RNA (dsRNA) which are homologous to the silenced gene or by delivering synthetic RNAs to cells. The target gene sequence may be drugable or non-drugable. siRNAs for use in the pharmaceutical compositions and methods of the present invention may also be derived from known anti-sense sequences.

The siRNA for use in the present invention are double stranded ribonucleotide sequences of typically 15 to 50 base pairs in length that are highly negatively charged and soluble predominantly in water. Preferably, the siRNA sequence of the present invention ranges from about 19 to about 27 base pairs in length and is highly homologous or 100% homologous to the target sequence. The siRNA may be blunt ended or else have base pair overhangs. The siRNA further may include a repeating amino acid sequence consisting of serine-aspartic acid-threonine and/or phosphorothioate backbone modifications.

Various types of RNA may serve as siRNA for use in a pharmaceutical compositions and methods of the present invention. For example, double stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) or combinations thereof, may be used.

Examples of siRNAs that may be used in the pharmaceutical compositions and methods of the present invention include. e.g, those targeted to sequences involved in respiratory diseases, and can include both pathogen and endogenous sequences. siRNAs targeted to viral pathogen sequences include but are not limited to respiratory syncytial virus (RSV) sequences, as well as PIV, SARS-SCV and influenza. With respect to influenza, siRNAs targeted to sequences common to all flu genomes, including those of avian origin such as the H5N1 strain can be used in the compositions of the present invention. siRNAs targeted to IKK2 (IKK-beta), a gene involved in inflammatory responses, as well as siRNAs targeted to pro-inflammatory cytokines such as TNF-alpha and IL-1beta may also be used in the microparticles of the present invention in order to provide in vivo silencing of target genes in the lung for the treatment of respiratory disease. Additionally, siRNAs directed to sequences involved in asthma, chronic obstructive pulmonary disorder (COPD), cystic fibrosis, and idiopathic pulmonary fibrosis (IPF) may be used. siRNAs targeted to sequences involved in lung cancer, such as sequences of the pituitary transforming gene (PTTG) may also be used as siRNAs in microparticle formulations of the present invention.

In another aspect of the invention, there are provided methods for preparing the microparticle compositions comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid. In accordance with this aspect of the invention, highly hydrophilic siRNA, for example, about 46 negative charges, is encapsulated into a highly hydrophobic polymer scaffold through assistance of a cationic lipid. The cationic lipid functions as both an amphipathic emulsifier and siRNA binding entity and is itself incorporated into the microparticles. This process is illustrated in Figure 1.

In one embodiment of the process, microparticles comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid are prepared by a double emulsion process. The process comprises the steps of: a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a water/oil/water double emulsion containing the microparticles; and c) removing the organic solvent to obtain the microparticles.

The siRNA is dissolved in water or any appropriate aqueous buffer solution. One of ordinary skill would understand how to select and prepare an aqueous buffer solution in the present invention. Examples of suitable aqueous buffers include any salt of acetic acid, including sodium acetate and potassium acetate, succinate buffer, phosphate buffer, citrate buffer, HEPES, PBS and any others known to the art. The organic solvent used in the present invention is selected depending on the solubility of the siRNA and polymer in the solvent, the degree to which the solvent is miscible in water and the toxicity of the solvent. Examples of organic solvents which may be used to dissolve the hydrophobic biodegradable polymer include any solvent with a low boiling point and which is not miscible with water, such as chloroform and dichloromethane (DCM), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), dimethylformamide, various alcohols such as ethanol, glycols, glycerin, propylene glycol, and various polyethylene glycols

The organic solvent may be removed by methods known in the art e.g. evaporation. Preferably, after removal of the organic solvent, the microparticles are filtered and lyophilized to a white powder.

In an alterative embodiment of the invention, and in order to better control the process and the resulting size of the microparticle, a secondary emulsifier is preferably added to the water/oil/water double emulsion of step b). The secondary emulsifier does not incorporate into the microparticle. Examples of secondary emulsifiers which may be used include, PEG such as PEG 20O00, pluronic F68 and polyvinyl alcohol (PVA). Preferably, PVA is used as the secondary emulsifier.

In a preferred embodiment, after step b), the water/oil/water emulsion is spray dried to obtain microspheres. By using spray drying technology, the microsphere size can be tightly controlled by the flow rate, the concentration of the three component mix, and the inlet/outlet temperature. This process results in increased homogeneity of the microspheres. Thus, spray drying allows the morphology, size, density and flowability of the powder to be tailored and adapted for direct passive targeting within the lung tissue.

The present invention also provides a method of preparing microspheres having siRNA encapsulated within. The method comprises the steps of a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a double water/oil/water emulsion; c) spray drying the water/oil/water double emulsion of step b) to obtain the microspheres.

Microparticles prepared by the methods of the present invention, may range in size up to 500 nanometers, preferably 2-20 micrometers, and most preferably smaller diameters of 1-2 micrometers. The molar ratios between the hydrophobic polymer, siRNA and cationic amphiphile, may be adapted in order to obtain microparticles with differential surface potential. In the working examples, due to the stoichiometric choice of PLGA, siRNA and DC-chol, overall negatively charged particles were engineered that are optimal for pulmonary applications. A preferred ratio of hydrophobic polymer : siRNA : cationic amphiphile is 5:25:50 (mass), to quantitatively encapsulate the siRNA.

In one embodiment of the invention, the pharmaceutical compositions of the present invention can be formulated into a variety of suitable formulations and administered orally, in aerosol form, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, interperitoneally, rectally, topically and vaginally. One preferred aspect of the invention is to use a device, such as a powder spray (e.g. Penn Century^® DP4) to mediate the microparticle powders to the lung of rodents.

In accordance with the present invention, there is also provided a method of delivering an siRNA targeted to the lung. The method comprises the steps of a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a double water/oil/water emulsion; c) spray drying the water/oil/water double emulsion of step b) to obtain the microspheres comprising encapsulated siRNA; and d) delivering the microspheres to the lung by use of a powder spray device.

The invention is further described in the following examples, which are not meant in any way to limit the scope of the invention.

Example 1 Process of formulation of PLGA/siRNA microspheres using WJoIw₂ double emulsion process that quantitatively incorporates the emulsifier.

An aqueous solution containing 5mg of siRNA (Luciferase) solution (1mg/ml_) is added within 10min to a solution of 1 mg of PLGA dissolved in 2OmL dichloromethane (DCM). This first wi/o phase is then added to a solution of 20OmL DC-Choi at 9500 rpm within 30min. The DCM is evaporated overnight under a flow hood and the W₁ZoZw₂ emulsion is lyophilized (Alnmemo 2390-5, MultiTemp Scientific AG, Kloten) to obtain a white powder. Different PLGA were tested and analyzed by gel electrophoresis (GE) and HPLC as indicated in Table 1. Units are mg/ml.

^*with siRNA (FVII)

HPLC analysis indicated that no free siRNA was detected in the permeate. All siRNA was found in the filtered microspores (filtrate). These results are confirmed by gel data (Figures 3a and 3b).

Example 2 Process of formulation of PLGA/siRNA microspheres using w_Λlolw₂ double emulsion process

A secondary emulsifier (e.g. PVA) is added to the DC-chol solution of Example 1 to homogenize the particles. Table 2 indicates the secondary emulsifiers were tested:

Table 2

The microparticles were evaluated for in vitro cellular toxicity and compared to lipoplexes from commercial suppliers (siRNA-Luc, Luc 8548). H1291 human lung epithelial cells were incubated with differential quantities (0-15OnM) of siRNA formulated as microspheres and the impact on the WST-1 production quantified. As can be seen by the result of the WST-1 assay (commercial kit utilizing cell proliferation reagent WST-1 , Roche^®' Basel), the microparticles exhibited no toxicity (constant level of WST-1 production) whereas the liposomal siRNA controls reduced the WST-1 levels substantially.

Example 3

Scale up of formulation process of PLGA/siRNA microspheres using spray drying technology

To analyze the microparticles by SEM a scale-up by a factor 5 was performed. 25ml siRNA were dissolved in 1OmL of water and added to a solution of PLGA (5mg) dissolved in DCM (2OmL). After homogenization this Wi/o phase was added to a solution of DC-Choi (50mg) in water in mixed during 30min at 9500rpm. The encapsulation stayed very high with different PLGA tested (LA/GA ratio, M_w, Supplier). However, the microspheres generated by this process are not very homogenous as can be seen from the SEM pictures, Figures 5a and 5b. As a result, spray drying as described in Master and Keith, Spray Drying Handbook, G Godwin, London, publisher (incorporated by reference herein as if fully set forth), was used to obtain better homogenization and smaller particles. For this purpose, 500mg of DC-chol dissolved in water were emulsified with 50mg PLGA in 1OmL DCM. The parameters used with the Spray dryer (Bϋchi 290) are indicated in Table 3:

Table 3

Figure 6 is an SEM picture with NVS star PLGA/DC-Chol microspheres prepared by the emulsion procedure followed by spray drying. The SEM picture clearly shows improved homogeneity in particle size and shape.

Claims

Claims:

1. A pharmaceutical composition in the form of microparticles of defined size, said microparticles comprising: siRNA, a highly hydrophobic biodegradable polymer, and a cationic amphiphile, wherein said siRNA and cationic lipid are encapsulated into the microparticle.

2. The pharmaceutical composition of claim 1 wherein the cationic lipid is DC- chol or DOTAP.

3. The pharmaceutical composition of claims 1 or 2 wherein the highly hydrophobic biodegradable polymer is poly-lactide-co-glycolic acid (PLGA).

4. The pharmaceutical composition of claim 1 wherein the siRNA is a double stranded oligonucleotide of about 19-23 bp in length.

5. The pharmaceutical composition of claim 1 in the form of an inhalable powder.

6. The pharmaceutical composition of claim 3 wherein the ratio of hydrophobic polymer : siRNA : cationic amphiphile is 5:25:50 (mass),

7. The pharmaceutical composition of claim 5 wherein the inhalable powder is dispensable as a powder spray.

8. A method for producing microparticles of defined size comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid, wherein said siRNA and cationic lipid are encapsulated into the microparticles, said method comprising: a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a water/oil/water double emulsion containing the microparticles; and c) removing the organic solvent solution.

9. The method of claim 8 further comprising d) filtering the microparticles.

10. The method of claim 9 further comprising lyophilizing the microparticles to a white powder.

11. The method of claim 8 wherein the hydrophobic biodegradable polymer is poly-lactide-co-glycolic acid (PLGA).

12. The method of claim 8 wherein the cationic lipid is DC-chol or DOTAP.

13. The method of claim 8 which further comprises adding a secondary emulsifier to the water/oil/water double emulsion of step b).

14. The method of claim 13 wherein the secondary emulsifier is polyvinyl alcohol (PVA).

15. The method of claim 8 wherein the organic solvent is dichloromethane (DCM).

16. A method for producing microspheres of defined size comprising siRNA, a highly hydrophobic biodegradable polymer, and a cationic lipid, wherein said siRNA and cationic lipid are encapsulated into the microspheres, said method comprising: a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a double water/oil/water emulsion; and c) spray drying the water/oil/water double emulsion of step b) to obtain the microspheres.

17. A method of delivering a pharmaceutically active formulation to the lung, said method comprising: a) combining an siRNA/aqueous solution with a hydrophobic biodegradable polymer dissolved in an organic solvent solution and homogenizing to form a water/oil emulsion; b) combining the water/oil emulsion of step a) with a cationic lipid to obtain a water/oil/water double emulsion; c) spray drying the water/oil/water double emulsion of step b) to obtain microspheres comprising encapsulated siRNA and d) delivering the microspheres to the lung by use of a powder spray device.