High concentration chitosan-nucleic acid polyplex compositions

Cross-reference of related applications This application claims the benefit of US Patent Application No. 60 / 976,376 filed on September 28, 2007, and is fully incorporated herein by reference.

Technical field The present invention relates to a homogeneous chitosan-nucleic acid complex. The present invention further relates to a method for concentrating a chitosan-nucleic acid complex in solution, and a highly concentrated preparation of a homogeneous chitosan-nucleic acid complex.

background Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine and D-glucosamine. Chitosan can form a complex with nucleic acids and is used as a DNA delivery vehicle for transfecting cells.

There are difficulties in producing a concentrated solution of the chitosan-nucleic acid complex. Increasing the concentration of chitosan and nucleic acid in the mixed solution results in precipitation and undesired variation in the size of the chitosan-nucleic acid complex produced. In addition, increasing the concentration of chitosan-nucleic acid complex in the prepared solution results in complex aggregation and precipitation from the solution.

The use of concurrent flow mixing to produce homogeneous particles containing DNA and condensing agents (eg, polycationic carbohydrates) is described in US Pat. No. 6,537,813. To produce such particles, the DNA solution and condensing agent solution are introduced simultaneously and separately into the fluid stream through a mixer, including a static or dynamic mixer, which provides mixing and particle formation. Can be done. It has been reported that it is important to maintain proper proportions of DNA and condensing agents throughout the introduction and mixing steps, and significant deviations from charge neutrality are incomplete condensation or particles during the process. It can result in any of the agglomerations.

<p> Outline of the invention The present inventor has overcome obstacles in the field of producing highly concentrated chitosan-nucleic acid complex compositions, which have been found to have many applications in the fields of research and medicine. In a preferred embodiment, the composition comprises a constant size polyplex that does not aggregate or precipitate despite high concentrations and exhibits stability under various conditions. Moreover, the preferred compositions of the present invention are also isotonic when formed. Achieving isotonicity while maintaining the stability of the polyplex is highly desirable for pharmaceutical and therapeutic applications. In addition, the inventor has discovered a method of producing a chitosan-nucleic acid complex by an in-line mixing step that does not require a static or dynamic mixer and is improved by not including the mixer. In addition, these methods can be used to produce stable polyplexes with charges that are significantly out of neutral (ie, zeta potential). Surprisingly, we also found that the size and homogeneity of the particles did not change the mixing ratio of DNA and chitosan in the mixed solution, or substantially without changing the concentration of DNA and chitosan. It has been found that it can be adjusted by varying the volume of feedstock for in-line mixing.</p><p> Thus, in one embodiment, the invention provides a composition comprising a hydrated chitosan-nucleic acid complex. The composition is highly enriched with chitosan-nucleic acid polypeptide, and it has a nucleic acid concentration of greater than about 0.5 mg / ml, where the composition is substantially free of polyplex precipitates.</p><p> In a preferred embodiment, the composition is a dispersion system comprising chitosan-nucleic acid polypeptide particles.</p><p> In a preferred embodiment, the composition is isotonic. In other embodiments, the composition is hypertonic or hypotonic.</p><p> In a preferred embodiment, the composition is at least about 0.6 mg / ml, more preferably at least about 0.75 mg / ml, more preferably at least about 1.0 mg / ml, more preferably at least about 1.2 mg / ml. , And most preferably have a nucleic acid concentration of at least about 1.5 mg / ml.</p><p> In a preferred embodiment, the composition further comprises an aggregation inhibitor. In a preferred embodiment, the aggregation inhibitor is sugar, preferably sucrose.</p><p> In a preferred embodiment, the composition comprises a counterionic concentration of less than about 80 mM, more preferably less than about 60 mM, more preferably less than about 40 mM, more preferably less than about 20 mM. Preferably, the counter anion is an acetate ion.</p><p> In a preferred embodiment, the chitosan-nucleic acid complex of the composition has an average polydispersity of less than about 0.5, more preferably less than about 0.4, more preferably less than about 0.3, more preferably less than about 0.2. It has a polydispersity index) (PDI).</p><p> In a preferred embodiment, the polyplex is at least about 2: 1, more preferably at least about 5: 1, more preferably at least about 10: 1, more preferably at least about 15: 1, and more preferably at least. It has an N: P ratio of about 20: 1.</p><p> In a preferred embodiment, the polyplexes, on average, are less than about 3000, more preferably less than about 2000 , more preferably less than about 1500, more preferably less than about 1000, more preferably about 500. It contains less than, more preferably less than about 100, more preferably less than about 50 chitosan molecules having monomeric units of glucosamine.</p><p> In a preferred embodiment, the polyplex is less than about 500 kDa, more preferably less than about 250 kDa, more preferably less than about 150 kDa, more preferably less than about 100 kDa, more preferably less than about 50 kDa, more preferably about. Contains chitosan with an average molecular weight of less than 25 kDa.</p><p> In a preferred embodiment, the polyplex of the composition is less than about 750 nm, more preferably less than about 500 nm, more preferably less than about 250 nm, more preferably less than about 200 nm, and most preferably less than about 150 nm. Has an average diameter.</p><p> In a preferred embodiment, the composition essentially comprises a chitosan-nucleic acid complex and an aggregation inhibitor.</p><p> In another embodiment, the composition consists essentially of a chitosan-nucleic acid complex.</p><p> In one embodiment, the invention provides a method for enriching the dispersion of a chitosan-nucleic acid complex. The method provides a non-concentrated dispersion of the chitosan-nucleic acid complex and concentrates the non-concentrated dispersion of the chitosan-nucleic acid complex using a concentrating means, preferably tangential flow filtration, as a result. Includes producing a concentrated dispersion of chitosan-nucleic acid complex. Preferably, the non-concentrated dispersion system comprises an aggregation inhibitor. This concentration step substantially increases the concentration of the chitosan-nucleic acid complex while substantially maintaining the concentration of small molecules (eg, aggregation inhibitors), resulting in a composition that is concentrated into the chitosan-nucleic acid complex. Bring things.</p><p> In a preferred embodiment, the concentrating means is at least 2-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, and Most preferably, the dispersion is concentrated at least 10-fold.</p><p> In a preferred embodiment, the aggregation inhibitor is a saccharide, preferably sucrose.</p><p> In a preferred embodiment, the non-concentrated dispersion of the chitosan-nucleic acid complex comprises a counter anion, preferably acetate ion, and the concentrating means does not selectively concentrate the counter anion. The enrichment of the counter anion is not as great as the enrichment of nucleic acid in the enriched dispersion system. Preferably, the concentrated dispersion system has a counter anion concentration that is not substantially higher than the concentration of the non-concentrated dispersion system.</p><p> In a preferred embodiment, the nucleic acid concentration in the concentrated dispersion is at least about 0.5 mg / ml, more preferably at least about 0.6 mg / ml, more preferably at least about 0.75 mg / ml, more preferably at least about 1.0 mg / ml. ml, more preferably at least about 1.2 mg / ml, and most preferably at least about 1.5 mg / ml.</p><p> In a preferred embodiment, in-line mixing is used to prepare a non-concentrated dispersion of the chitosan-nucleic acid complex.</p><p> In a preferred embodiment, the non-concentrated dispersion is at least 10 mL, more preferably at least about 50 mL, more preferably at least about 500 mL, more preferably at least about 1 L, more preferably at least about 2 L, more preferably at least about 3 L, It has a volume of at least about 4 L, more preferably at least about 5 L, and more preferably at least about 10 L.</p><p> In a preferred embodiment, the non-concentrated dispersion chitosan-nucleic acid complex has an average PDI of less than about 0.5, more preferably less than about 0.4, more preferably less than about 0.3, more preferably less than about 0.2. ..</p><p> In a more preferred embodiment, the concentrated dispersion chitosan-nucleic acid complex has an average PDI of less than about 0.5, more preferably less than about 0.4, more preferably less than about 0.3, more preferably less than about 0.2. ..</p><p> In a preferred embodiment, the polyplex is at least about 2: 1, more preferably at least about 5: 1, more preferably at least about 10: 1, more preferably at least about 15: 1, and more preferably. It has an N: P ratio of at least about 20: 1.</p><p> In a preferred embodiment, the polypeptides average less than about 3000, more preferably less than about 2000, more preferably less than about 1500, more preferably less than about 1000, more preferably about 500. It contains less than, more preferably less than about 100, more preferably less than about 50 monomer units of glucosamine.</p><p> In a preferred embodiment, the polyplex is less than about 500 kDa, more preferably less than about 250 kDa, more preferably less than about 150 kDa, more preferably less than about 100 kDa, more preferably less than about 50 kDa, more preferably. It has an average molecular weight of less than about 25 kDa.</p><p> In a preferred embodiment, the polyplex of the composition is less than about 750 nm, more preferably less than about 500 nm, more preferably less than about 250 nm, more preferably less than about 200 nm, and most preferably less than about 150 nm. Has an average diameter.</p><p> In one embodiment, the invention provides a concentrated dispersion of chitosan-nucleic acid polyplexes produced by the methods disclosed herein.</p><p> In one embodiment, the invention provides a method for adjusting the properties of a chitosan-nucleic acid complex formed by mixing a chitosan solution and a nucleic acid solution. In one embodiment, the invention provides a method for adjusting the diameter of a chitosan-nucleic acid complex. In another embodiment, the invention provides a method for regulating the zeta potential of a chitosan-nucleic acid complex. The method relates to varying the volume of nucleic acid or nucleic acid solution used to produce the complex without altering the ratio of nucleic acid to chitosan in the mixed solution, or the concentration of nucleic acid and chitosan. In a preferred embodiment, the present invention relates to in-line mixing of chitosan and nucleic acid feedstock solutions.</p><p> In one embodiment, the invention provides a pharmaceutical composition comprising a hydrated chitosan-nucleic acid complex and having a nucleic acid concentration of greater than about 0.5 mg / ml, wherein the chitosan-nucleic acid complex is therapeutic. Contains nucleic acid constructs.</p><p> In a preferred embodiment, the pharmaceutical composition is at least about 0.6 mg / ml, more preferably at least about 0.75 mg / ml, more preferably at least about 1.0 mg / ml, more preferably at least about 1.2 mg / ml. , And most preferably have a nucleic acid concentration of at least about 1.5 mg / ml.</p><p> In a preferred embodiment, the pharmaceutical composition comprises an aggregation inhibitor. In a preferred embodiment, the aggregation inhibitor is a saccharide, preferably sucrose.</p><p> In a preferred embodiment, the pharmaceutical composition has a counterionic concentration of less than about 80 mM, more preferably less than about 60 mM, more preferably less than about 40 mM, more preferably less than about 20 mM. Preferably, the counter anion is an acetate ion.</p><p> In a preferred embodiment, the pharmaceutical composition is isotonic. In other embodiments, the pharmaceutical composition is hypertonic or hypotonic.</p><p> In one embodiment, the invention is a method for preparing a concentrated chitosan-nucleic acid polyplex dispersion, in which a chitosan solution and a nucleic acid solution are mixed in-line to form a non-concentrated chitosan-nucleic acid dispersion, followed by The method is provided which comprises concentrating the non-concentrated chitosan-nucleic acid dispersion system using TFF and resulting in the production of a concentrated chitosan-nucleic acid polypeptide dispersion system.</p><p> In a preferred embodiment, the TFF comprises at least 2-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold the dispersion system. , And most preferably at least 10-fold concentrate.</p><p> In a preferred embodiment, the concentration of nucleic acid in the concentrated dispersion is at least about 0.5 mg / ml, more preferably at least about 0.6 mg / ml, more preferably at least about 0.75 mg / ml, more preferably at least about 1.0 mg. / ml, more preferably at least about 1.2 mg / ml, and most preferably at least about 1.5 mg / ml.</p><p> In a preferred embodiment, the concentrated dispersion has a counterion concentration of less than about 80 mM, more preferably less than about 60 mM, more preferably less than about 40 mM, more preferably less than about 20 mM. Preferably, the counter anion is an acetate ion.</p><p> Preferably, the concentrated dispersion system comprises an aggregation inhibitor. Preferably, the aggregation inhibitor is a saccharide, preferably sucrose.</p><p> Preferably, the concentrated dispersion system is isotonic. In other embodiments, the concentrated dispersion system can be hypertonic or hypotonic.</p>

<figref num="1">FIG. 1 shows a plan of a specific process block for producing a 1 L batch of chitosan-nucleic acid polyplex dispersion and then performing TFF enrichment.</figref><figref num="2">FIG. 2 shows a plan diagram of the in-line mixing process on a small scale. The syringes are polypropylene latex free and can be scaled up to 60 mL each. Two precision syringe pumps drive the syringe. The tubes are platinum-cured silicone, each with a 1/16 inch ID. The mixed junction shown can be T-shaped as well as Y-shaped. The mixed junction material of the structure is polypropylene.</figref><figref num="3">FIG. 3 shows a planned diagram of an in-line mixing process on a moderate scale for the preparation of a 10 L chitosan-nucleic acid polyplex dispersion system. All containers were expanded according to smaller batch sizes. The diameter of the tube is 0.48 cm (3/16 inch). The flow rate of the pump is adjusted so that the mixed volume ratio of DNA: chitosan is 2: 1.</figref><figref num="4">FIG. 4 shows a plan diagram of the TFF enrichment process.</figref><figref num="5">FIG. 5 shows a graph of the results of in vitro transfection for polyplexes manufactured on a moderate scale process and mixed in different ratios.</figref><figref num="6">FIG. 6 shows a graph of stability at room temperature for small scale mixing with 10% sucrose and polyplexes prepared by fast TFF.</figref><figref num="7">FIG. 7 shows a graph showing the effect of volume modification of DNA and chitosan feedstock on polyplex diameter and PDI. Adjusting the volume ratio adjusts the particle size. The compositions are listed in Table 10. All final products were N40-c75.</figref>

Detailed explanation "Chitosan-nucleic acid complex", "chitosan-nucleic acid complex particle", or "polyplex" means a complex containing a plurality of chitosan molecules (each of which is a polymer of a glucosamine monomer) and a plurality of nucleic acid molecules. Chitosan monomers include derivatives containing chitosan bound to a ligand. "Derivatives" were associated with a wide range of categories of chitosan-based polymers, including covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, and other units. , Or will be understood as containing a chitosan-type polymer bound to other sites. Derivatives are often based on modifications of the hydroxyl or amino groups of glucosamine. Examples of chitosan derivatives are, but are not limited to, trimethylated chitosan, PEGylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-bound chitosan, arginine-modified chitosan, Includes uronic acid-modified chitosan and the like. Further teachings on chitosan derivatives include, for example, "Non-viral Gene. Therapy , K.Taira, K.Kataoka, T.Niidome (editors), Springer-Verlag Tokyo, 2005, ISBN4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol.52 (23) ), Pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55 (2004) 77-93.

As used herein, the "average weight" of a chitosan polymer is the weight of the average molecular weight.

"Counter-anion" means an anion capable of electrostatically interacting with a charged chitosan amine. Preferred counter anions include acetate and chloride ions.

Chitosan may be prepared as disclosed in US Patent Application No. 11 / 694,852, filed March 30, 2007, expressly incorporated herein by reference in its entirety. Chitosan derivatives can be used as well, including chitosan derivatives containing ligand sites.

Chitosan-Nucleic Acid Polyplex Composition

In one embodiment, the invention provides a chitosan-nucleic acid complex composition comprising a hydrated chitosan-nucleic acid complex. The composition is highly concentrated in a chitosan-nucleic acid complex having a nucleic acid concentration of greater than about 0.5 mg / ml. The composition is substantially free of polyplex precipitation. As used herein, "substantially free" of polyplex precipitation means that there are substantially no particles in the composition that can be observed in visual inspection.

In a preferred embodiment, the chitosan-nucleic acid complex composition is at least about 0.6 mg / ml, more preferably at least about 0.75 mg / ml, more preferably at least about 1 mg / ml, more preferably at least about 1.2. It has a nucleic acid concentration of mg / ml, and most preferably at least about 1.5 mg / ml. In a preferred embodiment, the composition is substantially free of uncombined nucleic acids.

In a preferred embodiment, the chitosan-nucleic acid complex composition is a dispersion system. In a preferred embodiment, the dispersion is isotonic. Achieving isotonicity while maintaining the stability of the polyplex is highly preferred in the formulation of pharmaceutical compositions, and these preferred compositions are highly preferred for pharmaceutical formulations and therapeutic applications. is there.

In other embodiments, the composition can be hypertonic or hypotonic.

In a preferred embodiment, the chitosan-nucleic acid complex composition further comprises an aggregation inhibitor. The aggregation inhibitor partially or completely reduces the aggregation and / or precipitation of the polyplex, and the chitosan-nucleic acid polyplex is subjected to concentration means, preferably through tangential flow filtration (TTF). Provides enrichment. Aggregation inhibitors other than sucrose, such as other sugars that can reduce polyplex precipitation and provide a concentrated chitosan-nucleic acid polypeptide, can be used, but a highly preferred aggregation inhibitor is sucrose. Examples of other aggregation inhibitors include, but are not limited to, trehalose, glycerol, fructose, glucose, and other reducing and non-reducing sugars.

In a preferred embodiment, the aggregation inhibitor used is sucrose. The concentration of sucrose in the chitosan-nucleic acid complex dispersion system is preferably about 3% by weight to 20% by weight. Most preferably, the sucrose concentration provides an isotonic composition.

The chitosan-nucleic acid complex composition is preferably homogeneous in terms of polyplex size. Therefore, in a preferred embodiment, the chitosan-nucleic acid complex of the composition has a low average polydispersity (PDI). In a particularly preferred embodiment, the chitosan-nucleic acid polypeptide dispersion system has a PDI of less than about 0.5, more preferably less than about 0.4, more preferably less than about 0.3, more preferably less than about 0.2.

The chitosan-nucleic acid complex is preferably of a size that is substantially stable in the composition. In a preferred embodiment, the compositions of the invention are at room temperature for 6 hours, more preferably 12 hours, more preferably 24 hours, more preferably 48 hours, with an average diameter of less than 100%, more preferably less than 50%. , More preferably contains a polyplex that increases by less than 25%.

The chitosan-nucleic acid complex is preferably of a size that is substantially stable under cooling conditions. In a preferred embodiment, the compositions of the invention have an average diameter of less than 100%, more preferably 2-8 ° C for 6 hours, more preferably 12 hours, more preferably 24 hours, more preferably 48 hours. It comprises a polyplex that increases by less than 50%, more preferably less than 25%.

The chitosan-nucleic acid complex of the composition is preferably of a substantially stable size under freeze-thaw conditions. In a preferred embodiment, the compositions of the invention are thawed from freezing at -20 ° C to -80 ° C and then at room temperature for 6 hours, more preferably 12 hours, more preferably 24 hours, more. It comprises a polyplex in which the average diameter increases by less than 100%, more preferably less than 50%, more preferably less than 25% in 48 hours.

The chitosan-nucleic acid complex contains a nucleic acid component and a chitosan component. In some cases, it may include an alternative backbone or a nucleic acid analog that may have other modifications or sites bound for any various purpose, eg, for stability and protection purposes. The nucleic acids of the invention will generally contain phosphodiester bonds. Other possible nucleic acid analogs include those with a non-ribose backbone. In addition, naturally occurring nucleic acids, analogs and mixtures thereof can be produced. Nucleic acids can be single-strand or double-stranded, or contain both moieties of double-stranded or single-stranded sequences. Nucleic acids include, but are not limited to, DNA, RNA, and any combination of nucleic acids deoxyribonucleotides and ribonucleotides, and uracil, adenine, thymine, cytosine, guanine, inosin, xathanine, hypoxathanine. ), Isocytosine, Isoguanine, etc., including hybrids containing any combination of bases. Nucleic acids include any form of DNA and any form of DNA, including triple-stranded, double-stranded, or single-stranded, antisense, siRNA, ribozyme, deoxyribozyme, polynucleotide, oligonucleotide, chimera, and derivatives thereof. Contains RNA.

In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. Therapeutic nucleic acids include therapeutic RNA, which is an RNA molecule capable of exerting a therapeutic effect on mammalian cells. Therapeutic RNA includes antisense RNA, siRNA, short hairpin RNA, and enzymatic RNA. Therapeutic nucleic acids include triple-stranded molecules, protein-binding nucleic acids, ribozymes, deoxyribozymes, and nucleic acids intended to form small nucleic acid molecules.

Therapeutic nucleic acids also include nucleic acids encoding therapeutic proteins, including cytotoxic proteins and prodrugs; ribozymes; antisense or its complements; or other such molecules.

In a preferred embodiment, the nucleic acid component comprises a therapeutic nucleic acid construct. The therapeutic nucleic acid construct is a nucleic acid construct capable of exerting a therapeutic effect. The Therapeutic Nucleic Acid Construct may include a nucleic acid encoding a Therapeutic protein and a nucleic acid for producing a transcript that is a Therapeutic RNA. Therapeutic RNA is an RNA molecule capable of exerting a therapeutic effect in mammalian cells. Therapeutic RNA includes antisense RNA, siRNAs, short hairpin RNA, and enzymatic RNA. Therapeutic nucleic acids include triple-stranded molecules, protein-binding nucleic acids, ribozymes, deoxyribozymes, and nucleic acids intended to form small nucleic acid molecules. Therapeutic nucleic acids can be used to influence gene therapy by helping to replace or enhance defective genes, or to compensate for the lack of a particular gene product by encoding a therapeutic product. .. Therapeutic nucleic acids can also inhibit the expression of endogenous genes. Therapeutic nucleic acids can encode all or part of the translation product and can function by recombining with DNA already present in the cell, thereby replacing the defective portion of the gene. It can also encode a portion of a protein and exert its effect by co-suppression of the gene product. In a preferred embodiment, the therapeutic nucleic acid is selected from those disclosed in US Patent Application No. 11 / 694,852.

In a preferred embodiment, the average polyplex is less than about 3000, more preferably less than about 2000, more preferably less than about 1500, more preferably less than about 1000, more preferably about 500. Includes chitosan molecules with less than, more preferably less than about 100, more preferably less than about 50 glucosamine monomer units.

In a preferred embodiment, the polyplex is less than about 500 kDa, more preferably less than about 250 kDa, more preferably less than about 250 kDa, more preferably less than about 150 kDa, more preferably less than about 100 kDa, more preferably about. Includes chitosan with an average weight of less than 50 kDa, more preferably less than about 25 kDa.

In a preferred embodiment, the polyplex of the composition has an average diameter of less than 750 nm, more preferably less than 500 nm, more preferably less than about 250 nm, more preferably less than about 200 nm, and most preferably less than about 150 nm. Have.

In one embodiment, the chitosan component has an average molecular weight of 3 kDa to 250 kDa.

In one embodiment, the chitosan component has an average molecular weight of 250 kDa or greater.

In one embodiment, the chitosan component has an average molecular weight of 3 kDa or less.

In one embodiment, the chitosan-nucleic acid complex is about 2: 1 to about 100: 1, more preferably about 5: 1 to about 90: 1, more preferably about 10: 1 to about 90 :. It has an N: P ratio of 1.

In one embodiment, the chitosan-nucleic acid complex has an N: P ratio of 90: 1 or greater.

In one embodiment, the chitosan-nucleic acid complex has an N: P ratio of 10: 1 or less.

In one embodiment, the chitosan-nucleic acid complex has an average zeta potential of +30 mV to +50 mV at pH 5.

In one embodiment, the chitosan-nucleic acid complex has an average zeta potential of +30 mV or less at pH 5.

In one embodiment, the chitosan-nucleic acid complex has an average zeta potential of +50 mV or greater at pH 5.

In one embodiment, the chitosan-nucleic acid complex has an average diameter of less than 225 nm.

In one embodiment, the chitosan-nucleic acid complex has an average diameter of 225 nm or greater.

In one embodiment, the composition has a pH of less than 6.5, more preferably less than 6.0, and most preferably 4.5-5.5.

In one embodiment, the composition has a pH of 6.5 or higher.

In one embodiment, the composition has a pH of 4.5 or less.

In one embodiment, the polyplex chitosan molecule is greater than about 70%, more preferably greater than about 75%, more preferably greater than about 80%, more preferably greater than about 85%, more preferably about. It has a degree of deacetylation of greater than 90%, more preferably greater than about 95%, and most preferably at least 98%.

In one embodiment, the polyplex chitosan molecule has a degree of deacetylation of 70% or less.

In one embodiment, the composition consists essentially of a chitosan-nucleic acid complex and an aggregation inhibitor. Such compositions may include counter anions and other excipients such as parabens.

In another embodiment, the composition consists essentially of a chitosan-nucleic acid complex. Such compositions may include counter anions and other excipients such as parabens.

In a particularly preferred embodiment, the chitosan-nucleic acid complex is selected from those disclosed in US Patent Application No. 11 / 694,852.

Production method

In a preferred embodiment, the high concentration chitosan-nucleic acid complex of the present invention is produced by enriching the non-concentrated dispersion of the chitosan-nucleic acid complex.

The non-concentrated chitosan-nucleic acid complex composition preferably has a nucleic acid concentration of less than 0.5 mg / ml.

The non-concentrated dispersion of the chitosan-nucleic acid complex can be prepared by other methods, eg, the method of forming a mixed solution by dropping a nucleic acid or chitosan solution onto the other, but preferably by in-line mixing. However, in-line mixing is a large volume homogeneous chitosan-nucleic acid complex having an average PDI of preferably less than 0.5, more preferably less than about 0.4, more preferably less than about 0.3, more preferably less than about 0.2. Providing manufacturing.

In-line mixing is a well-known process in which two (or more) fluid streams are integrated into a single stream. In-line mixing is illustrated below. For further disclosure regarding in-line mixing, see, for example, US Pat. Nos. 6,251,599 and 6,537,813 (each of which is incorporated herein by reference in its entirety).

Mixers such as static mixers and dynamic mixers can be used, but such devices result in an increase in the PDI of the complexes formed by the methods of the invention. Therefore, in a preferred embodiment of the invention, in-line mixing is done without the use of such a mixer.

In the present invention, tangential flow filtration (TFF) is the preferred means for concentrating the non-concentrated dispersion of the chitosan-nucleic acid complex. In the operation of the TFF, pressure is applied to the membrane to force a portion of the liquid into the membrane, but the chitosan-nucleic acid polypeptide dispersion system uses a pump across the surface of the semipermeable membrane. And be pushed in. Numerator smaller than the pores are transported through the pores and recovered as a permeate. Osmotic solutions include, but are not limited to, salts, ions, sugars and microbial preservatives. Molecules that are too large to pass through the membrane pores, including the chitosan-nucleic acid complex, are retained in the stream and recirculated as retentates. Using TFF, the polyplex concentration can be increased many times, resulting in a highly concentrated polyplex dispersion system. In a preferred embodiment, the highly concentrated polyplex dispersion system is isotonic.

In a preferred embodiment, the non-concentrated chitosan-nucleic acid complex dispersion system comprises a saccharide, preferably sucrose. As described below, sucrose has been found to be an aggregation inhibitor that prevents particle aggregation during the concentration step. In addition, sucrose is an effective antifreeze, and an exemplary frozen polyplex dispersion with a DNA concentration of about 1 mg / mL and containing up to 15% sucrose is at least one month. Is stable.

The use of TFF to concentrate non-concentrated chitosan-nucleic acid complex is illustrated below.

Modifying the properties of the chitosan-nucleic acid complex by manipulating the volume of feedstock

The properties of the chitosan-nucleic acid complex formed by the mixture of nucleic acids and nucleic acids, while maintaining a substantially constant chitosan: nucleic acid ratio and a substantially constant mixed concentration of chitosan and nucleic acids. It was surprisingly found that it could be adjusted. In particular, it has been found that by changing the volume ratio of the chitosan and nucleic acid feedstock solutions, the properties of the resulting polyplex formed by mixing the two solutions can be changed.

Thus, in one embodiment, the invention provides a method for adjusting the properties of a chitosan-nucleic acid complex formed by mixing a chitosan and nucleic acid solution. In one embodiment, the invention provides a method for adjusting the diameter of a chitosan-nucleic acid complex. In another embodiment, the invention provides a method for regulating the zeta potential of a chitosan-nucleic acid complex. The method modifies the volume of the nucleic acid or chitosan solution used to produce the complex without substantially altering the ratio of nucleic acid to chitosan in the mixed solution, or the concentration of nucleic acid to chitosan. Regarding to do. In a preferred embodiment, the method relates to in-line mixing of chitosan and nucleic acid feedstock solutions.

Powder formulation

The chitosan-nucleic acid complex composition of the present invention comprises a powder. In a preferred embodiment, the present invention provides a dry powder chitosan-nucleic acid polypeptide composition. In a preferred embodiment, the dry powder chitosan-nucleic acid complex composition is produced through dehydration of the chitosan-nucleic acid complex dispersion system of the present invention.

how to use

In addition to therapeutic applications, the present invention is generally useful whenever nucleic acid stabilization is desired (eg, for increased transfection efficiency) and increased nucleic acid concentration is desired. is there. Nucleic acid stability is important whenever a procedure is taken to compromise the structural integrity and functionality of a nucleic acid in a laboratory setting.

Pharmaceutical product

The present invention also provides a "pharmaceutically acceptable" or "physiologically acceptable" formulation comprising the chitosan-nucleic acid complex composition of the present invention. Such formulations may be administered to a subject in vivo to carry out the procedure.

The terms "pharmaceutically acceptable" and "physiologically acceptable" as used herein are preferably to an object that does not cause excessive side effects (eg, nausea, abdominal pain, headache, etc.). Carriers, diluents, excipients, etc. that can be administered. Such preparations for administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical compositions are made from carriers, diluents, excipients, solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, etc. that are compatible with subject administration. obtain. Such formulations may be included in (coated or uncoated) tablets, (hard or soft) capsules, microbeads, emulsions, powders, granules, crystals, suspensions, syrups, or elixirs. Among other additives, auxiliary active compounds and preservatives may also be present in, for example, antibacterial agents, antioxidants, chelating agents, and inert gases.

The pharmaceutical composition can be formulated to fit its intended route of administration. For example, for oral administration, the composition can be combined with excipients and used in the form of tablets, lozenges, capsules, eg geratin capsules. Pharmaceutically compatible binders and / or auxiliary materials may be included in oral formulations. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacant gum, or gelatin; excipients such as starch or lactose; disintegrants. , For example alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or stereote; fluidity promoters such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, Methyl salicylate, or fragrance.

The formulation may also include a carrier for protecting the composition from rapid degradation or removal from the body, eg, a release controlled formulation comprising an implant and a microencapsulated delivery system. For example, time-delaying materials such as glycerin monostearate or glyceryl stearate alone can be used, or in combination with waxes.

Suppositories and other rectally administrable formulations (eg, those that can be administered by enema) are also considered. For further intrarectal delivery, for example, Song et al., Mucosal drug delivery: membranes, methodsologies, and applications, Crit.Rev.Ther.Drug.Carrier Syst., 21: 195-256, 2004; Wearley, Recent progress in See protein and peptide delivery by noninvasive routes, Crit.Rev.Ther.Drug.Carrier Syst., 8: 331-394, 1991.

Additional pharmaceutical compositions suitable for administration are known in the art and are applicable to the methods and compositions of the invention (eg, Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., See Easton, Pa .; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)) ..

Administration

Any number of routes of administration are possible, and the choice of specific route will be partially dependent on the target tissue. Syringes, endoscopes, cannulas, intubation tubes, catheters, and other items can be used for administration.

A satisfactory result is to prevent or inhibit the progression or exacerbation of the disease or condition, but the dose or "effective amount" for treating the subject is preferably one, some or all of the symptoms. Is sufficient to relieve measurable or detectable. Thus, in the case of a condition or disease that can be treated by expressing a Therapeutic nucleic acid in a target tissue, the amount of Therapeutic RNA or Therapeutic protein produced to ameliorate the condition that can be treated by the methods of the invention. It depends on the condition and the desired result, and can be easily confirmed by those skilled in the art. The preferred amount will depend on the condition to be treated, the desired therapeutic effect, and the individual subject (eg, bioavailability, gender, age, etc. in the subject). Effective amounts can be confirmed by measuring the associated physiological effects.

Veterinary applications are also intended in the present invention. Therefore, in one embodiment, the present invention provides a method for treating a non-human mammal, which comprises administering the chitosan nanoparticles of the present invention to the non-human mammal in need of treatment.

Oral administration

The compounds of the present invention can be administered orally. Oral administration may be involved in swallowing, resulting in the compound entering the gastrointestinal tract. The compositions of the present invention can also be administered directly into the gastrointestinal tract.

Suitable formulations for oral administration are solid formulations such as tablets, capsules containing microparticles, liquids or powders, lozenges (including those containing liquids), chew, multiparticles and nanoparticles, gels, films, ovles. (ovule), and includes spray.

Liquid formulations include suspensions, solutions, syrups, and elixirs. Liquid formulations can be prepared by solid reconstitution.

Tablet dosage forms generally include a disintegrant. Examples of disintegrants are sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, crospovidone, polyvinylpyrrolidin, methyl cellulose, microcrystalline cellulose, lower alkyl substituted hydroxypropyl cellulose, starch, pregelatinized starch and alginate. Contains sodium. Generally, the disintegrant will contain 1% to 25% by weight, preferably 5% to 20% by weight, in the dosage form.

Binders are commonly used to impart cohesiveness to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, saccharides, polyethylene glycol, natural and synthetic rubbers, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose. Tablets also include diluents such as lactose (monohydrate, spray-dried monohydrate, anhydride, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and calcium hydrogen phosphate dihydrate. May include Japanese products.

The tablets also optionally contain surfactants such as sodium lauryl sulfate and polysorbate 80, as well as flow promoters such as silicon dioxide and talc. In the presence, the surfactant may contain 0.2% to 5% by weight of the tablet, and the flow accelerator may contain 0.2% to 1% by weight of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate and sodium lauryl sulfate. Lubricants generally contain 0.25% to 10% by weight, preferably 0.5% to 3% by weight of tablets.

Other possible ingredients include antioxidants, colorants, flavors, preservatives, and flavoring agents.

The tablet mixture can be compressed either directly or by a roller to form a tablet. The tablet mixture or a portion of the mixture may instead be wet, dry, or melt granulated, melt condensed, or extruded. The final formulation may contain one or more layers and may or may not be coated; it may be encapsulated.

The formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol.1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

Consumable oral films for human or veterinary use are typically in the form of flexible, water-soluble or water-swellable thin films. It can dissolve rapidly or can be mucosally adherent, and can typically contain membrane-forming polymers, binders, solvents, moisturizers, plasticizers, stabilizers or emulsifiers, viscosity modifiers and solvents. Some compounds in the formulation may perform one or more functions.

In the present invention, multiparticulate beads containing the composition of the present invention are similarly included.

Other possible ingredients are antioxidants, colorants, flavors, and flavor enhancers, preservatives, saliva secretagogues, coolants, co-solvents (including oils), palliatives, fillers, defoamers. , Surfactants, and flavoring agents.

The films of the present invention are typically prepared by evaporative drying of a peelable backing support or an aqueous thin film coated on paper. This can be done by a drying oven or tunnel, typically a combined coating dryer, or lyophilization or vacuuming.

Solid formulations for oral administration can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Including.

Other suitable emission techniques are known, such as high energy dispersion, as well as permeability and coated particles.

Parenteral administration

The compounds of the present invention can also be administered directly into the bloodstream, intramuscularly, or into internal organs. Suitable means for parenteral administration are intravenous, intraarterial, intraperitoneal, subarachnoid, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Including administration. Suitable devices for parenteral administration include needle (including microneedle) insertion devices, needle-free insertion devices, and infusion techniques.

Parenteral formulations are usually aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers, but in some applications they may be sterile non-aqueous solutions or suitable vehicles such as sterile. It can be suitably formulated as a dry form used in combination with pyrogen-free water.

The production of parenteral preparations under sterile conditions, for example by lyophilization, can be easily accomplished by using standard pharmaceutical techniques well known to those of skill in the art.

The solubility of compounds used in the production of parenteral solutions can be increased by using suitable formulation techniques, such as in combination with solubility enhancers.

Formulations for parenteral administration can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and planned release. Thus, the compounds of the invention can be formulated as solids, semi-solids, or thixotropic liquids for administration as implant depots that provide controlled release of the active compound.

Topical administration

The compounds of the present invention can also be administered topically to the skin or mucous membranes, i.e. to the skin or transdermally. Topical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, bandages, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and Contains microemulsion.

Other means of topical administration include electroporation, ion electrophoresis, phonophoresis, sonophoresis and microneedle or needle-free (eg, Powderject , Bioject , etc.) injections.

Formulations for topical administration can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and planned release.

Inhalation / intranasal administration

The compounds of the present invention are also typically in the form of dry powders (either as a mixture, such as a dry mixture mixed with lactose, or as mixed component particles), with the use of suitable high pressure gases. Or without, it can be administered intranasally or by inhalation from a dry powder inhaler or as an aerosol spray from a pressurized vessel, pump, spray, sprayer or nebulizer.

Capsules, blisters and cartridges for the use of inhalers or insufflators are compounds of the invention, suitable powder bases such as lactose or starch, and performance modifiers such as L. -Can be formulated to contain a powder mixture of leucine, mannitol, or magnesium stearate.

Formulations for inhalation / intranasal administration can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and planned release.

Rectal / intravaginal administration

The compounds of the present invention can be administered rectally or intravaginally. It can be administered in the form of suppositories, pessaries, or enemas. Cocoa butter is a conventional suppository base, but various alternatives can be used as suitable.

Formulations for rectal / intravaginal administration can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and planned release.

Eye / aural administration

The compounds of the invention are also administered directly to the eye or ear, typically in the form of an infusion. Other formulations suitable for ophthalmic and intraoural administration are ointments, biodegradable agents (eg, absorbent gel sponge, collagen) and non-biodegradable agents (eg, silicone), implants, wafers, lenses and particles. Includes a particulate system. The formulation can also be delivered by ion electrophoresis.

Ophthalmic / intraoural formulations can be formulated for immediate and / or controlled release. Controlled release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and planned release.

<p> Example 1: Concentration to&gt; 1 mg / mL after production of a polyplex of 250 μg / mL or less</p><p> See Figure 1 and the following for exemplary descriptions of reagents, concentrations, and ratios.</p><p> Simple small scale in-line mixer, syringe pump and high speed peristaltic pump Tested using pump), silicone tubing and polypropylene T junction. This step is performed using N / P using a 23-mer / 98% DDA chitosan (ie, a chitosan polymer having an average of 23 monomers (glucosamine) and 98% deacetylated). It was used to produce a polymer with a final DNA concentration of 250 μg / mL or less, with a ratio of 20. The results showed that the particle size and PDI of the polyplex could be tightly regulated by adjusting the feedstock concentration, volume, mixing ratio, and flow rate of the DNA and chitosan solutions. In addition, the incorporation of sucrose into DNA and chitosan feedstocks has been found to support the manufacturing process by reducing particle size and PDI. The inclusion of sucrose also prevented particle agglutination during the concentration step. Frozen polyplexes with a DNA concentration of about 1 mg / mL and containing 15% or less sucrose were stable within at least 1 month. In addition, the in-line mixing step was easily scaled up from 50 mL to 2 L with low relative standard deviation (RSD) values: particle size = ± 9%, PDI = ± 6%.</p><p><tables num="1"><img file="JP2010540468A_D0001.tif" /></tables></p><p><tables num="2"><img file="JP2010540468A_D0002.tif" /></tables></p><p> Polyplex naming convention</p><p> The term relates to chitosan type, N / P ratio, acetic acid content, pH, and DNA concentration. Table 1 shows an example of detailed explanation.</p><p><tables num="3"><img file="JP2010540468A_D0003.tif" /></tables></p><p> In-line mixing process flow</p><p> Figure 1 shows a typical process block for manufacturing in 1 L batch and then performing TFF enrichment.</p><p> Small scale in-line mixing</p><p> A simple small scale in-line mixer was tested using a syringe pump, 1/16 inch ID silicone tubing; and a T-shaped 3 / 32-inch ID polypropylene junction. Figure 2 shows the setting scheme using a 3 mL volume syringe. Note that the maximum volume of the syringe in this setting is 60 mL. This step was used to produce a polyplex with a final DNA concentration of 150 μg / mL with a N / P ratio of 20 using a 24-mer / 98% DDA chitosan.</p><p> Moderate scale in-line mixing</p><p> A simple moderate scale in-line mixer was tested using a peristaltic pump, a 3/16 inch ID silicone tube; and a 3 / 16-inch ID polypropylene junction. Figure 3 shows the setting scheme using the Y junction. Note that the maximum output volume in this setting is limited only by the volume of the feedstock container. This step was used to produce a polyplex with a final DNA concentration of 150 μg / mL with a N / P ratio of 20 using a 24-mer / 98% DDA chitosan. DNA and chitosan feedstock were mixed in a 2: 1 volume ratio to produce a homogeneous polyplex formulation.</p><p> TFF process</p><p> Hollow fiber filters were rinsed and washed prior to performing the TFF test according to the manufacturer's operating instructions. To perform the concentration, the TTF system was set up as shown in the schematic (Figure 4) and residual water was removed. After closing the penetrant valve and fully opening the back pressure valve, the DNA-chitosan polypeptide was added to the product reservoir. The pump was turned on to start concentration, the permeate valve was fully opened, and then the back pressure valve was adjusted to the target filter inlet pressure. During the concentration step, the mass of the recovered penetrant was weighed and used to determine when the target DNA concentration was achieved. See equation below:</p><p><maths num="1"><img file="JP2010540468A_D0004.tif" /></maths></p><p> After the target volume reduction was achieved, the concentration step was stopped by closing the penetrant valve and opening the back pressure valve completely. After removing the retention line and recovering the final product, this post-TFF product was submitted for analytical testing and for DNA concentration determination by picogreen assay. Residuals were stored at 4 ° C until the analytical test was completed and then used immediately or frozen for storage.</p><p> Analytical test</p><p> Particle size</p><p> Particle size measurements were performed using a Zethasizer Nano light scatterer. Generally, samples were diluted 20-fold with 10 mM NaCl (minimum 0.4 mL) and loaded into disposable cuvettes. The Zetasizer was programmed to incubate the sample at 25 ° C for 3 minutes, followed by 3 measurements for 3 minutes. Z-average diameter and polydispersity (PDI) were reported using standard deviation (n = 3). Zetasizer was also programmed to describe the composition of the sample in terms of viscosity and index of refraction.</p><p> Zeta potential</p><p> Zeta potential measurements were performed using a Zetasizer Nano light scatterer. In general, undiluted samples were loaded into the Zetasizer folded capillary cell (minimum 0.8 mL). The Zetasizer was programmed to incubate the sample at 25 ° C for 3 minutes and then repeat the measurements (the number of iterations was automatically determined by the Zetasizer software). Zeta potential values were reported using the standard deviation (n = 3). Zetasizer was also programmed to describe the final composition of the sample in terms of viscosity and permittivity.</p><p> Short-term stability due to freezing</p><p> For short-term stability testing, the final polyplex product was frozen and stored at a suitable temperature (-20 ° C, -30 ° C, or -80 ° C). In some cases, the samples were rapidly frozen in a dry ice / ethanol bath and then stored at a suitable temperature. Samples were thawed to room temperature at a suitable time and analyzed as described.</p><p> Quantification of DNA using PicoGreen</p><p> Prior to DNA measurements using the PicoGreen assay, total DNA must be released from the polyplex by chitosanase. After release, the DNA was subjected to DNA degradation with a suitable restriction enzyme to linearize the supercoiled DNA plasmid.</p><p> Degradation by chitosanase</p><p> To ensure complete release of DNA, the polyplex was first diluted to 50 μl in 150 mM NaOAc at pH 5.5, 37 ° C. to obtain a concentration of chitosan of 0.909 to 1.818 mM (C). For (24,98) -N20-c1000 particles, the sample is usually diluted to 1/70 and 1/35 to achieve the target chitosan concentration). A 50 μl diluted polyplex was degraded with 50 μl 4.44 U / mL chitosanase at 37 ° C for 2 hours (stock chitosanase concentration was 62 U / mL and cooled 50 mM NaOAc, pH 5.5, Diluted at 37 ° C).</p><p> Decomposition by EcoR1</p><p> After incubation^*X μL of chitosanase-degraded sample was added to 5 μL of EcoR1 buffer and made into a final volume of 50 μL with milliQ water (sample volume X μL was adjusted to a final DNA concentration of 4 ng / μL). Normally, for a C (24,98) -N20-c1000 particle sample, the sample volume X is 25 μL). EcoR1 samples were then incubated for 30 minutes at 37 ° C.</p><p> PicoGreen Assay</p><p> The PicoGreen Quant-iT ds DNA HS assay kit was supplied with two buffers (A and B) and two standards (1 and 2). Buffer A was diluted 1:20 with buffer B to prepare solution "A / B". Standards 1 and 2 were diluted 20-fold (10 μL to 200 μL) with solution A / B. The final concentrations for Standards 1 and 2 were 0 and 10 ng / μL, respectively.</p><p> Samples digested with 10-20 μL EcoR1 to a final volume of 200 μL with solution A / B, vortexed quickly, incubated for 2 minutes at room temperature, and then on a Qubit Fluorometer according to the manufacturer's operating instructions. Fluorescence was measured.</p><p> Gel electrophoresis</p><p> Samples were subjected to gel electrophoresis to verify DNA capture into the polyplex. 1-5 μL of sample aliquots (target volume of 800 ng of DNA) were mixed with 2 μL of Tracklt loading buffer and finally made into a volume of 10 μL with water. The standard lane was loaded with a supercoiled DNA ladder. The sample was analyzed on a 0.8% agarose gel containing ethidium bromide (50 μg / mL) at 120 V for 45 minutes. Gels were imaged using the FluorChem Imaging System.</p><p> In vitro transfection</p><p> In vitro transfection of 293T-K cells with a polyplex formulation was generally performed by two steps: preparing the cells and then transfecting them.</p><p> Maintenance of 293T-K cells</p><p> The 293T-K cell line was obtained with the courtesy of Dr. Kieffer's laboratory at UBC and was prepared as follows. Human renal cells are transformed with SV40 T antigen; grown in high glucose, Dalbeco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin / streptomycin; and 80 It was maintained at a culture density of less than%.</p><p> Preparation of cells for transfection</p><p> For transfection, cells were prepared as follows. 6-well tissue culture plates (3 x 10) on the day before transfection⁵293T-K cells were added to 3 mL of complete medium (high glucose DMEM + 10% FBS + pen / strep) in cells / wells. On the day of transfection, cells 1X were washed with phosphate buffered saline (PBS), cells were trypsinized with 1 ml of 0.05% trypsin, 1 ml of complete medium was added, and hemocytometer was performed. Cell counts were determined by counting 10 μL using a vessel. Cells are about 50% confluent (about 7 x 10)⁵If it was (cells / wells), transfection proceeded (if the cells were too low or too dense, transfection did not proceed).</p><p> Cell transfection</p><p> Transfection was performed as follows. First, the medium was removed from each well and 1 mL Opti-mem (adjusted to pH 5.0 with HCl and filtered through a 0.2 μm filter) was then added to each well and gently agitated. It was then removed (six wells were washed at once to prevent dislodging of cells). An additional 1 mL of Opti-mem (pH 5.0) was then carefully added to each well without damaging the cells. Polyplex samples were then added to each well (target amount 2 μg DNA), stirred and incubated at 37 ° C for 2 hours. After incubation, medium was removed and replaced with 2 mL complete medium and reincubated at 37 ° C. At the required time, the supernatant was removed and stored at -20 ° C for subsequent SEAP assays.</p><p> SEAP assay</p><p> The SEAP assay was performed using the SEAP chemiluminescent assay kit. All reagents for the assay were equilibrated at 25 ° C for 30 minutes prior to use. The standard for the assay was prepared by dissolving placental alkaline phosphatase in 1X dilution buffer from a kit supplemented with 0.1% bovine serum albumin and 50% glycerol to 1 mg / mL, followed by 10-fold serial dilution with DMEM. Diluted with to make 0.01 pg / μL. The standard and thawed samples are then diluted 1/4 with dilution buffer, inactivated by heating at 65 ° C for 30 minutes, incubated on ice for 2 minutes and centrifuged (16100 x rcf, 2). (Min, room temperature), and the supernatant was transferred to a new tube. After equilibration at 25 ° C for 5 minutes, 50 μL of sample and standard were added in pairs to each well of the Microlite-1 plate. Inactive buffer (50 μL) is then added to each well and pipetted up and gently without bubble formation. down), mixed, and incubated for 5 minutes. Substrate / enhancer reagents were prepared in a 1:19 ratio of substrate to enhancer during the 5-minute incubation. Substrate / enhancer was then added to each well, incubated for 20 minutes, and the plate was measured using a luminometer for an integration time of 1 second.</p><p> result</p><p> High-speed in-line mixing of C (23,98) -N40-c75 + TFF</p><p> A moderate scale in-line mixing system was used to prepare polyplexes at varying flow rates of 90, 210, 270, and 420 mL / min. The mixing system also used an in-line static mixing device to evaluate the usefulness of the device. The system was also completely free of excipients. In this study, the starting DNA concentration of the polyplex was 0.075 mg / mL, which was then concentrated to 0.25 mg / mL by TFF.</p><p> For in-line stationary mixers, the flow rate was selected based on the calculated Reynolds number, which determines the minimum number of factors required to achieve optimum solution mixing at a given flow rate (Table 2). ). The following equation is based on the manufacturer's operating instructions.</p><p><maths num="2"><img file="JP2010540468A_D0005.tif" /></maths></p><p> Table 2: Calculated Reynolds numbers for in-line stationary mixers</p><p><tables num="4"><img file="JP2010540468A_D0006.tif" /></tables></p><p> Table 3 shows that an increase in the final mixing flow rate results in an increase in Z-average particle size and PDI. Concentration with TFF showed no significant effect on particle size, PDI, zeta potential, viscosity (Table 3) or in vitro transfection (Figure 5).</p><p> Table 3: Size, PDI, and zeta potential of mixed polyplexes in different proportions on a moderate scale</p><p><tables num="5"><img file="JP2010540468A_D0007.tif" /></tables></p><p> In-line mixing and TFF precipitation</p><p> To achieve a DNA concentration of at least 1 mg / mL, we sought to mix the polyplex with 0.15 mg / mL DNA and then 1 mg / mL with TFF. However, during this study, significant precipitation was observed after the concentration step (data not shown). This occurred in the small scale mixing step (3 mL / min and 23 mL / min) and in the moderate scale mixing step (210 mL / min and 420 mL / min).</p><p> Sucrose content during polyplex formation to prevent precipitation</p><p> To ameliorate the problem of precipitation in TFF, we mixed all excipients (about 10 wt% sucrose, 0.09 wt% methylparaben, 0.01 wt% propylparaben) before mixing to form a polyplex. , Chitosan and DNA feedstock. No static mixer was used. The inclusion of excipients prevented particle agglutination and precipitation during the TFF concentration step. Furthermore, it was found that the stability of PDI and particles was improved when the stationary mixer was not included.</p><p> Small scale batch with updated process</p><p> Mixing and recirculation rate of 90 mL / min (shear rate 7200s)^-1) Was used to produce small scale batches using 10% sucrose from DNA and chitosan feedstock. After TFF, the particle size was less than 200 nm and no precipitation occurred. The particle size is summarized in Table 4.</p><p> Table 4</p><p><tables num="6"><img file="JP2010540468A_D0008.tif" /></tables></p><p> After 18 hours at room temperature, the size leveled off at about 140 nm, and no precipitation was observed after 3 days at room temperature (Fig. 6). In addition, the post-TFF sample did not precipitate after freezing / thawing at either -20 ° C or -80 ° C. After 24 hours at room temperature, the thawed particles leveled off at about 160 and 150 nm at -20 ° C and -80 ° C, respectively (Table 5). In addition, an in vitro transfection assay showed that the batch had biological efficacy (data not shown).</p><p> Table 5: 10 Small scale mixing with 10% sucrose and high speed TFF: Freeze-thaw</p><p><tables num="7"><img file="JP2010540468A_D0009.tif" /></tables></p><p> Moderate scale batch in updated process</p><p> A moderate-scale confirmation batch was produced using all of the above process changes: filtration of DNA stock prior to mixing with sucrose solution; 10% sucrose into DNA and chitosan feedstock prior to mixing. Containing, and recirculation rate 90 mL / min (sucrose rate 7200 s)^-1) TFF enrichment. In addition, control batches excluding sucrose and paraben excipients were produced.</p><p> Precipitation was rapidly observed in non-excipient (control) batches during and after TFF (data not shown). However, no precipitation was observed in the excipient batch (Table 6) until the DNA concentration exceeded approximately 1.5 mg / mL.</p><p> Table 6: Parameters of Polyplex batch containing excipients</p><p><tables num="8"><img file="JP2010540468A_D0010.tif" /></tables></p><p> Medium scale 0.8L demonstration batch</p><p> We tested the in-line mixing process and the expandability of TFF to a batch size of 0.8 L. The polyplex was mixed to a volume of 0.8 L and then concentrated to 1.1 mg / mL by TFF. This test included testing the homogeneity of the mixing process by collecting multiple 5 mL aliquots of the mixed polyplex during the early, middle and final stages of the mixing process. TFF process is 850 cm²Using a cartridge (on the other hand the previous small scale TFF test is 73 cm²The cartridge was used) and included a hold time test of the product concentrated at 4 ° C before transferring to a vial and freezing at -30 ° C.</p><p> Homogeneity test of in-line mixing process</p><p> Multiple 0.5 mL samples were collected from 25 mL at the beginning, middle and end of the mixing step and homogeneity during the mixing step was determined. The results showed that the initial mixed sample started at about 120 nm and stabilized at 80 nm after 15 ml mixing.</p><p> Table 7 Homogeneity of in-line mixing on a moderate scale</p><p><tables num="9"><img file="JP2010540468A_D0011.tif" /></tables></p><p> All samples were previously frozen at -30 ° C, thawed and then analyzed. "B" indicates a 5 mL sample from the beginning of the mixing step in the order of 1-5 (initially 25 ml). "M" indicates a 5 mL sample from the middle of the mixing step (starting at about 400 ml) in the order 1-5. "E" indicates a 5 mL sample from the end of the mixing step (starting at about 750 ml) in the order 1-5.</p><p> Retention time test for final TFF product</p><p> After the TFF process was completed, the final TFF product was dispensed (in a 20 ml amber glass vial) into a 9 x 10 mL aliquot and sealed. The vials were then subjected to various retention times to mimic typical manufacturing retention conditions.</p><p><tables num="10"><img file="JP2010540468A_D0012.tif" /></tables></p><p> The thawed product results showed good stability of the product at 4 ° C., 24 hours retention time (Table 8). For samples at "4 hours, 3 ° C, then -30 ° C", the stability at 7 hours after thawing also showed particle stability with an increase of less than 10% compared to the starting material.</p><p> Table 8</p><p><tables num="11"><img file="JP2010540468A_D0013.tif" /></tables></p><p> All samples were pre-frozen at -30 ° C, thawed and then analyzed.</p><p> 2L engineering run on a moderate scale</p><p> We tested the in-line mixing process and the extensibility of TFF to a batch size of 2 L. The polyplex was mixed to a volume of 2 L and then concentrated to 1.1 mg / mL by TFF. This test included a repeat test of the homogeneity of the mixing step. The study included repeatedly testing the homogeneity of the mixing process by collecting multiple 5 mL aliquots of the mixed complex at the beginning (25 mL of waste), middle and end of the mixing process. .. TFF process is 850 cm²Cartridges were used and included overnight retention of the final concentrated product at 4 ° C before transferring to vials and freezing at -30 ° C.</p><p> In general, the physics of the polyplex were well within the expected range and limits (Table 9).</p><p> Table 9</p><p><tables num="12"><img file="JP2010540468A_D0014.tif" /></tables></p><p> All samples were either fresh or pre-frozen (as instructed) at -30 ° C, thawed and then analyzed.</p><p> Expandability of in-line mixing with excipients</p><p> The following is a summary of the physical properties of a polyplex batch produced by in-line mixing in two different scales and four batch volumes (Table 10). All batches were C (24,98) -N20-c150-pH4.8-Suc15% -Pbn0.1%. The mixing ratio was 2: 1. The overall flow rates for the small and moderate scale mixes were 23.3 ml / min and 210 mL / min, respectively (at these rates, the linear flow rates through the tubes were equal for the two mix scales).</p><p> Table 10 Physical characteristics of scale-expanded mixed batches</p><p><tables num="13"><img file="JP2010540468A_D0015.tif" /></tables></p><p> Test of DNA capture by gel electrophoresis</p><p> Several polyplex preparations were tested for the presence of free DNA to determine if all DNA was captured by chitosan. Overall, free DNA was not observed in the formulations tested (not listed).</p><p> In vitro transfection of polyplex</p><p> In vitro transfection was performed on the two formulations and the in-line mixture was compared to the polyplex produced by the infusion method (eg, US Patent Application No. 11 / 694,852). The average transfection efficiency of the in-line mixing method was about 11% higher than that of the infusion method.</p><p><tables num="14"><img file="JP2010540468A_D0016.tif" /></tables></p><p> The final polyplex formulation was C (23,98) -N40-Ac10.5-pH4.8-c75. For in-line mixing batches, the DNA: chitosan volume mixing ratio was 2: 1 and the output flow rate was 3 mL / min.</p><p> Changing the feedstock volume ratio changes the polyplex size and PDI.</p><p> Compositions were prepared according to Table 11 using the methods described herein.</p><p> Table 11</p><p><tables num="15"><img file="JP2010540468A_D0017.tif" /></tables></p><p> The particle size and PDI of the preparation were analyzed as described herein. The results are shown in FIG. 7, and the results are obtained by changing the particle size by changing the feedstock volume while substantially maintaining the mixing ratio of chitosan and DNA and maintaining the mixing concentration of chitosan and DNA. Prove that it can be adjusted.</p><p> All citations are hereby incorporated by reference in their entirety.</p>