Lipophilic modified nucleic acid medicine composition and application thereof

Abstract

The invention discloses a lipophilic modified nucleic acid medicine composition and application thereof, the lipophilic modification is saturated or unsaturated C4-C30 alkyl, the composition comprises an absorption enhancer, and the method is that the lipophilic modified nucleic acid medicine is delivered to the brain through the nasal mucosa. Nucleic acid drugs are transmitted into the olfactory bulb region through a neural pathway, namely, cross-olfactory mucosa, bypass BBB and directly enter the brain, the drug concentration content of the olfactory bulb is far higher than that of other tissues of the brain, and the tissues except the olfactory bulb are uniformly distributed, so that the olfactory bulb can be used as a drug reservoir and gradually and slowly spread to the other tissues of the brain; the long-time drug effect can be maintained by one-time drug administration, so that the drug administration frequency can be reduced. Compared with intrathecal injection, by adopting the composition disclosed by the invention for nasal administration and using 1 / 2 of intrathecal administration dosage, the same intrathecal steady-state distribution as intrathecal distribution can be achieved, so that the method disclosed by the invention is safer, noninvasive and efficient.

Description

Technical Field

[0001] This invention relates to a nucleic acid drug composition, more specifically to a lipophilic modified nucleic acid drug composition, and also to the use of the composition. Background Technology

[0002] Small nucleic acid drugs (including siRNA, antisense oligonucleotides, etc.) are a rapidly developing new treatment approach in recent years. They can provide highly specific potential treatment options for refractory neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and various central nervous system tumors by intervening in the expression of specific genes. However, their clinical application faces a fundamental core challenge: how to efficiently and safely deliver these large, hydrophilic, and negatively charged biological agents to the target sites in the brain.

[0003] 1. The main obstacle to drug administration through the central nervous system: the blood-brain barrier. The central nervous system is protected by a tight blood-brain barrier. This barrier, composed of tight junctions between cerebral vascular endothelial cells, the basement membrane, and astrocyte terminales, effectively prevents over 98% of macromolecular drugs and the vast majority of hydrophilic compounds from entering the brain parenchyma from the bloodstream. Due to their inherent physicochemical properties, small nucleic acid drugs, when administered systemically via intravenous or oral routes, are almost unable to effectively penetrate the blood-brain barrier, resulting in extremely low drug concentrations in the brain, failing to reach the levels required for treatment.

[0004] 2. Current mainstream solution: Invasive drug delivery methods To overcome the blood-brain barrier, the main route for delivering small nucleic acid drugs to the central nervous system in preclinical studies and early clinical explorations currently relies on invasive physical methods. Invasive drug delivery methods share prominent problems such as complex operation, high cost, poor patient tolerance, high risk of infection, and difficulty in home-based or long-term repeated administration, which seriously limit the clinical application prospects and patient accessibility of small nucleic acid drugs in the field of central nervous system diseases.

[0005] 3. The urgent need for non-invasive alternative routes and the advantages of nasal drug delivery Therefore, developing a novel, non-invasive delivery strategy that is highly patient-compliant and can bypass the blood-brain barrier has become a key technological bottleneck that urgently needs to be overcome in the field of small nucleic acid neurotherapy.

[0006] In recent years, nasal drug delivery has received widespread attention as a highly promising solution. This route utilizes the unique anatomical and physiological connection between the nasal cavity and the cranial cavity: Olfactory pathway: Drugs can enter the olfactory bulb and even deeper brain regions directly through the olfactory epithelium of the nasal cavity, via endocytosis-axonal transport-transsynaptic transmission of olfactory neurons, or along the surrounding perineal space.

[0007] Trigeminal nerve pathway: Drugs can be taken up by the abundant trigeminal nerve endings in the nasal mucosa and transported retrogradely to areas such as the brainstem and trigeminal nucleus.

[0008] Indirect effects after systemic absorption: Some drugs are absorbed into the systemic circulation through the nasal mucosa. Although they still face the blood-brain barrier, nasal administration can avoid the first-pass effect of the liver and improve bioavailability.

[0009] In summary, compared with invasive methods, nasal drug delivery has potential advantages such as being non-invasive, convenient, allowing for self-administration, facilitating long-term treatment, and enabling direct delivery into the brain by bypassing the blood-brain barrier. However, direct nasal delivery of naked small nucleic acid drugs still faces challenges such as degradation by nasal mucosal enzymes, rapid clearance by mucociliary mucosa, limited penetration efficiency, insufficient intracerebral delivery volume, and weak targeting. Existing technologies generally employ the form of small nucleic acid drugs in the form of nanomicelles or nanoparticles, or link transmembrane peptides to small nucleic acid drugs to facilitate penetration through the nasal mucosa into the brain. However, both methods still have certain drawbacks. Although nanocarriers can provide some protection, the complex physiological environment of the nasal cavity (such as the mucus layer, enzymatic activity, and rapid ciliary clearance) can still lead to carrier capture, degradation, or premature clearance. Nanomicelles / nanoparticles have limited permeability in nasal mucus, and their stability can be affected by mucus pH, ionic strength, and enzyme activity, resulting in significant loss of small nucleic acids before reaching the absorptive epithelium. While the conjugation of transmembrane peptides can enhance cellular uptake, their drug-linked conjugation may be unstable and prone to breakage in vivo. Furthermore, transmembrane peptides themselves may be rapidly degraded by proteases, thereby weakening their effect in promoting transmembrane transport.

[0010] Brain targeting is poor. Although Alnylam has modified specific siRNAs, the siRNAs modified by VP and C16 have the best activity in the central nervous system region, knocking down mRNA by up to 90% and 75% in the spinal cord and brain, respectively. This drug requires intrathecal administration. In addition, an article published in the journal Nature Biotechnology, "Expanding RNAitherapeutics to extrahepatic tissues with lipophilic conjugates," showed that the conjugation of 2'-O-hexadecyl (C16) to siRNA can achieve safe, effective and durable silencing in the central nervous system (CNS), eyes and lungs of rodents and non-human primates, with broad cell type specificity.

[0011] Therefore, this invention aims to address the shortcomings of existing technologies by providing a novel nasal delivery system or formulation strategy to significantly improve the efficiency, stability, and targeting of small nucleic acid drugs delivered to the brain via the nose, thereby providing a safe, effective, and patient-friendly non-invasive treatment option for central nervous system diseases. Summary of the Invention

[0012] In view of this, the present invention provides a composition and method for non-invasive and efficient delivery of small interfering nucleic acid drugs to the brain.

[0013] To address the technical problem that small nucleic acid drugs are difficult to deliver to the brain non-invasively in existing technologies, this invention provides a method for non-invasively delivering small nucleic acid drugs to the brain.

[0014] According to a first aspect of the present invention, a composition is provided, the composition comprising: a) Nucleic acid drugs, including antisense oligonucleotide drugs, messenger RNA, small interfering nucleic acid, small activating RNA drugs, non-coding RNA drugs, plasmid DNA drugs, and nucleic acid aptamer drugs, wherein the nucleic acid drugs have lipophilic modifications, wherein the lipophilic modifications are saturated or unsaturated C4~C30 hydrocarbon groups; Preferably, the lipophilic modification is a C4-C30 alkyl or alkenyl group. More preferably, the lipophilic modification is a chain-like C6-C18 alkyl or alkenyl group. More preferably, the lipophilic modification is a C16 alkyl group; Specifically, when the small nucleic acid drug is a small interfering nucleic acid, one of the sense or antisense strands of the small interfering nucleic acid contains at least one lipophilic modification, while the other strand of the small interfering nucleic acid does not contain a lipophilic modification. The lipophilic modification is a saturated or unsaturated C4-C30 hydrocarbon group. Preferably, the lipophilic modification is a C4-C30 alkyl or alkenyl group. More preferably, the lipophilic modification is a chain-like C6-C18 alkyl or alkenyl group. More preferably, the lipophilic modification is a C16 alkyl group; b) An absorption enhancer, wherein the absorption enhancer is dodecyl-β-D-maltoside; c) A diluent or buffer, wherein the diluent is water and the buffer is PBS buffer; The content of the absorption enhancer, based on the total weight of the composition, is 0.1~0.5% (w / w). The pH value of the composition is 5.0~7.0. In one specific embodiment, the sense or antisense strand of at least one lipophilic modified small interfering nucleic acid forms a double strand with the strand that does not contain lipophilic modification; or the molar excess of the strand containing at least one lipophilic modification relative to the other strand is less than 1%, or the molar equivalents of the two strands are equal; or the molar excess of the strand that does not contain lipophilic modification relative to the other strand.

[0015] In one specific embodiment, the sense strand of the small interfering nucleic acid contains at least one lipophilic modification, while the antisense strand does not contain a lipophilic modification; optionally, the antisense strand has a molar excess relative to the sense strand. Optionally, the antisense chain has at least about 0.1% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 0.2% molar excess relative to the right chain; Optionally, the antisense chain has at least about 0.3% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 0.4% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 0.5% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 1% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 2% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 3% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 4% molar excess relative to the justice chain; Optionally, the antisense chain has at least about 5% or more molar excess relative to the justice chain.

[0016] In one specific embodiment, the antisense strand of the small interfering nucleic acid contains at least one lipophilic modification, while the sense strand of the small interfering nucleic acid does not contain a lipophilic modification; optionally, the sense strand has a molar excess relative to the antisense strand. Optionally, the justice chain has at least about 0.1% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 0.2% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 0.3% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 0.4% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 0.5% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 1% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 2% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 3% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 4% molar excess relative to the antisense chain; Optionally, the justice chain has at least about 5% or more molar excess relative to the antisense chain.

[0017] In one specific embodiment, the composition is used for nasal administration; Particularly preferred is administration via the nasal olfactory region; More preferably, the drug is administered via targeted spraying or precise constant-flow dripping into the nasal olfactory region.

[0018] In one specific embodiment, the lipophilic modification is a saturated or unsaturated C4~C30 hydrocarbon group; Preferably, the lipophilic modification is a C4-C30 alkyl or alkenyl group; More preferably, the lipophilic modification is a chain-like C6-C18 alkyl or alkenyl group; More preferably, the lipophilic modification is a C16 alkyl group; Particularly preferably, the lipophilic modification is attached to the 2'-ribose position of a small interfering nucleic acid residue; more specifically, the lipophilic modification is attached to the 2'-ribose position of the sixth nucleotide residue of the positive strand.

[0019] In one specific embodiment, the small interfering nucleic acid includes at least one modified nucleotide that is not a 2'-deoxynucleotide.

[0020] In one specific embodiment, at least one modified nucleotide that is not a 2'-deoxynucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl alcohol modified nucleotides, cyclohexenyl modified nucleotides, and nucleotides including 2-hydroxymethyl-tetrahydrofuran-5-phosphate.

[0021] In one embodiment, the small interfering nucleic acid may further include a modified nucleotide comprising a 5'-phosphate ester or a 5'-phosphate ester analog.

[0022] In one specific embodiment, the 5'-phosphate analog modification is a phosphate ester modification; preferably, the 5'-phosphate analog modification is... , , or The aforementioned structure replaces the 4'-CH2OH group of the ribose in the 5'-terminal nucleotide. Most preferably, the 5'-phosphate analog modification is a 5'-(E)-vinylphosphonate modification.

[0023] In one specific implementation, the sense strand and the antisense strand each independently have 15-40 nucleotides.

[0024] In one specific implementation, almost all nucleotides of the positive strand are modified.

[0025] In one specific implementation, the length of the justice chain is 21 nucleotides.

[0026] In one specific implementation, almost all nucleotides of the antisense strand are modified.

[0027] In one specific implementation, seven or fewer nucleotides in the antisense strand are 2'-deoxynucleotides; Optionally, six or fewer nucleotides in the antisense strand are 2'-deoxynucleotides; Optionally, five or fewer nucleotides in the antisense strand are 2'-deoxynucleotides; Optionally, four or fewer nucleotides in the antisense strand are 2'-deoxynucleotides; Optionally, three or fewer nucleotides in the antisense strand are 2'-deoxynucleotides; Optionally, two or fewer nucleotides in the antisense strand are 2'-deoxynucleotides.

[0028] In one embodiment, the antisense strand is 23 nucleotides in length. In another embodiment, the antisense strand is 19 nucleotides in length. In one embodiment, the antisense strand is 20 nucleotides in length. In another embodiment, the antisense strand is 21 nucleotides in length. In one embodiment, the antisense strand is 22 nucleotides in length.

[0029] In one specific implementation, both the sense strand and the antisense strand are 19 nucleotides in length.

[0030] In one specific implementation, both the sense strand and the antisense strand are 20 nucleotides in length.

[0031] In one specific implementation, both the sense strand and the antisense strand are 21 nucleotides in length.

[0032] In one specific implementation, both the sense strand and the antisense strand are 22 nucleotides in length.

[0033] In one specific implementation, both the sense strand and the antisense strand are 23 nucleotides in length.

[0034] In one embodiment, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long. In another embodiment, the sense strand is 20 nucleotides long and the antisense strand is 22 nucleotides long. In yet another embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0035] In one specific embodiment, the absorption promoter is dodecyl maltoside, preferably dodecyl β-D-maltoside; more preferably, dodecyl β-D-maltoside is substantially pure; even more preferably, dodecyl β-D-maltoside may contain a certain proportion of dodecyl α-D-maltoside, for example, 5% to 10% by weight of dodecyl α-D-maltoside.

[0036] In one specific embodiment, the small interfering nucleic acid has the positive strand of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or other free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and / or, The small interfering nucleic acid has the antisense strands described in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, or other free acid or sodium salt forms having at least 85% identical nucleotides to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0037] In one specific embodiment, the sense strand of the small interfering nucleic acid is SEQ ID NO:1 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:1, and the antisense strand is SEQ ID NO:2 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:2, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand.

[0038] In one specific embodiment, the sense strand of the small interfering nucleic acid is SEQ ID NO:3 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:3, and the antisense strand is SEQ ID NO:4 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:4, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand.

[0039] In one specific embodiment, the sense strand of the small interfering nucleic acid is SEQ ID NO:5 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:5, and the antisense strand is SEQ ID NO:6 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:6, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand.

[0040] In one specific embodiment, the sense strand of the small interfering nucleic acid is SEQ ID NO:7 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:7, and the antisense strand is SEQ ID NO:8 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:8, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand.

[0041] In one specific embodiment, the small interfering nucleic acid has a sense strand and an antisense strand as shown below;

[0042] Another aspect of the present invention provides a kit comprising: a) Compositions comprising the above description; b) Optionally, it also includes a nasal mucosa activator; said nasal mucosa activator is hyaluronidase; c) Optionally, it also includes means for administering the composition to a subject and / or means for administering a nasal mucosal activator to a subject.

[0043] In another aspect, the present invention provides the use of the above-described composition or kit in preparing a formulation that reduces the expression of a target gene; said target gene is amyloid precursor protein, amyotrophic lateral sclerosis (ALS), superoxide dismutase (SOD), or Huntington's gene; preferably, the disease associated with said amyloid precursor protein is cerebral amyloid angiopathy or Alzheimer's disease, more preferably, said Alzheimer's disease is early-onset Alzheimer's disease, familial Alzheimer's disease, early-onset familial Alzheimer's disease, or late-onset Alzheimer's disease. Preferably, said composition is administered by nasal spray, nasal drops, intravenous, subcutaneous, intrathecal, or oral administration; said kit is administered by nasal spray or nasal drops.

[0044] The present invention also provides the use of the above-described composition or kit in reducing target genes via nasal administration to the brain.

[0045] In another aspect, the invention provides a method for treating a subject suffering from a disease that can be mitigated by reducing the expression of a target gene. The method comprises administering an effective amount of the composition of the invention to the subject, thereby treating the subject.

[0046] In one specific implementation, the subject is a human being.

[0047] In one specific implementation, the target gene is the amyloid precursor protein (APP), amyotrophic lateral sclerosis (ALS), superoxide dismutase (SOD1), or Huntington's (HTT) gene.

[0048] In one specific implementation, the subject suffers from an app-related disease. In some embodiments, the app-related disease is cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD); specifically, the Alzheimer's disease (AD) is early-onset Alzheimer's disease (EOAD), familial Alzheimer's disease, early-onset familial Alzheimer's disease (EOF AD), or late-onset Alzheimer's disease.

[0049] Preferably, when the subject is a primate, the step of activating the nasal mucosa is also included; Preferably, the nasal administration method includes the following steps: Step 1) First, apply the nasal mucosa activator to the nasal olfactory mucosa; allow it to act for a certain period of time; Step 2) Reabsorb or wash away the nasal mucosa activator and its residual liquid; Step 3) Apply the solution of the small interfering nucleic acid drug.

[0050] In one specific embodiment, in step 1), the nasal mucosa activator is hyaluronidase; In one specific implementation, after the application of hyaluronidase in step 1), the action time is 1 to 30 minutes, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or any value between any two values.

[0051] The beneficial effects of this invention are as follows: 1. The technical solution of the present invention allows nucleic acid drugs to be rapidly absorbed, reaching peak absorption at the olfactory bulb in 3-10 minutes.

[0052] 2. The technical solution of the present invention can enter the olfactory bulb area through olfactory nerve conduction, that is, bypass the BBB and directly enter the brain across the olfactory mucosa; the distribution of the solution in the tissues outside the olfactory bulb is uniform, and the effect can be sustained.

[0053] 3. With this formulation, after administration via the olfactory region, the drug concentration in the olfactory bulb is much higher than in other brain tissues. Therefore, the olfactory bulb can act as a drug reservoir, gradually and slowly diffusing to other brain tissues. A single administration via the olfactory region can maintain the drug effect for a long time, thus reducing the frequency of administration and improving patient compliance.

[0054] 3. Compared with intrathecal administration in the prior art, the composition of the present invention, delivered via nasal and olfactory mucosa to the brain via a brain-targeted delivery method, achieves the effect of 0.9 mg intrathecal administration with a dosage of 0.5 mg. Therefore, the technical solution of the present invention uses a lower dosage and has a non-invasive administration route, resulting in higher safety. Attached Figure Description

[0055] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0056] Figure 1 This is the average drug concentration-time curve of each tissue in rats after first administering hyaluronidase and then FD10, as shown in Example 4.

[0057] Figure 2 This is the mass spectrum of the justice chain in Example 5; Figure 3 This is the mass spectrum of the antisense chain in Example 5; Figure 4 Example 6 shows the plasma drug-time curve of a single nasal-brain targeted precise delivery of 2 mg / dose in rats; Figure 5 Example 6 shows the drug delivery curve of 2 mg / dose to brain tissue in rats via a single nasal-brain targeted delivery. Figure 6 Example 8 shows the drug delivery curve of 0.5 mg / dose to brain tissue in rats via a single nasal-brain targeted delivery. Figure 7Example 9 shows the drug delivery curve of 1 mg / dose to brain tissue in rats via a single nasal-brain targeted delivery. Figure 8 The drug-time curves of the olfactory bulb on the administration side are obtained at different drug doses (0.5 mg / dose, 1.0 mg / dose, 2 mg / dose) and at different detection time points (1 min, 3 min, 10 min, 30 min and 60 min). Figure 9 This is a schematic diagram of the structure of small nucleic acid drug-1; Figure 10 This is a schematic diagram of the structure of the small nucleic acid drug-2; Figure 11 This is a schematic diagram of the structure of the small nucleic acid drug-3; Figure 12 This is a schematic diagram of the structure of the small nucleic acid drug-4. Detailed Implementation

[0058] The measurement method of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0059] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0060] Terminology Explanation: In this invention, the absorption rate constant refers to the absorption rate constant of rat nasal perfusion. The determination method employs methods known to those skilled in the art. The method for determining rat nasal perfusion in this invention is as follows: A rat respiratory pathway is established through tracheal intubation; then, an esophageal cannula is inserted, and a peristaltic pump provides the driving force for drug circulation, establishing an in vivo nasal circulation pathway. The drug flows through the esophageal cannula across the nasal mucosa, returns to a constant-temperature collection bottle, and then enters the next cycle via the peristaltic pump. In the rat nasal circulation pathway, the drug is partially absorbed through the nasal mucosa. The remaining drug content in the collection bottle of the circulation system is determined by HPLC or other detection methods, and the amount of drug absorbed by the rat nasal mucosa is calculated. In this patent application, the absorption rate constant is determined using three rats, and the average value is calculated. (Reference: 1) Huang CH, KimuraR, Nassar RB, Hussain A. Mechanism of nasal absorption of drugs I:Physicochemical parameters influencing the rate of in situ nasal absorption of drugs in rats. J Pharm Sci. 1985 Jun;74(6):608-11.

[0061] In this invention, FD10 is used as a model drug. FD10 is a fluorescently labeled dextran with a linear molecular weight of approximately 10 kD. The method of administering nucleic acid drugs to the brain via the nasal olfactory region is optimized.

[0062] In this invention, DDM refers to dodecyl-β-D-maltodextrin.

[0063] Example 1 Solution preparation Preparation of 0.5% EDTA-2Na-pH6 citrate isotonic buffer: Weigh 500mg EDTA-2Na, 708mg NaCl and 100g 20mM citrate buffer (pH6) into a sample vial, sonicate to dissolve, add 400μL 2M sodium hydroxide solution, adjust the pH to 6, and set aside. Batch number AF25112601.

[0064] 0.1 mg / mL FD10 sample preparation: Add 7 mg of FD10 (FITC-Dextran 10) labeled dextran, 70 mg of DDM, and 70 mL of pH 6.8 buffer to the sample vial. Dissolve slowly, mix well, and set aside. Batch number: YY25112602. Preparation of 2750U / mL hyaluronidase-0.5% EDTA-2Na-pH6 citrate isotonic solution: Weigh 18.5g of 0.5% EDTA-2Na-pH6 citrate isotonic buffer into a sample vial, measure 100μL of hyaluronidase, mix well, and the solution is ready. Batch number: AY25112601.

[0065] Example 2 The absorption rate constant was determined using a rat nasal perfusion model. Sample YY25112602 (100 μg / mL FD10) was used for nasal perfusion in rats at sampling times of 30, 45, 60, 90, and 120 min. The calculated absorption rate constant was 7.37 × 10⁻⁶. -4 .

[0066] Example 3 Before nasal perfusion in rats, the nasal mucosa was pre-circulated for 5 min with AY25112601 hyaluronidase mucosal activator to activate it. Then, nasal perfusion was performed using 100 μg / mL FD10 sample at sampling time points of 30, 45, 60, 90, and 120 min. The calculated absorption rate constant was 2.07 × 10⁻⁶. -3 .

[0067] Example 4 First, 50 μL of nasal mucosa activator AY25112601 solution was administered to rats. After approximately 5 minutes, residual fluid in the rat's nasal cavity was removed, and the rats were transferred to a drug delivery box. Solution YY25112602 was placed in a nasal drug delivery device (the applicant's intelligent integrated nasal drug delivery system for small animals, as disclosed in CN202021777601.0 and CN202010148279.3). The prescribed dose of drug was administered nasally to the rats. In this embodiment, the rat nasoencephalogram was administered a single dose of 10 mg / kg, with the nasal delivery method being a constant-rate administration over 2 minutes under air anesthesia. Pharmacokinetics and distribution were measured at fixed time points after administration. The sampling methods for plasma, olfactory bulb (drug delivery side), hypoolfactory bulb (drug delivery side), and hemisphere (contralateral side) were employed using methods commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 1 below. Figure 1 As shown; Table 1

[0068] Example 5: Delivery of Mivelsiran to the brain via the olfactory region of the nose 1. Prepare a nasoencephalogenic solution for Mivelsiran (i.e., small nucleic acid drug-1) to be delivered to the brain via the nasal olfactory region; Preparation of PBS buffer: Add 80g of sodium chloride, 200g of potassium chloride, 36.31g of disodium hydrogen phosphate dodecahydrate, and 2.42g of potassium dihydrogen phosphate to a reagent bottle. Add 9L of ultrapure water and sonicate for 5 minutes until completely dissolved. Add an appropriate amount of phosphate solution to adjust the pH to 6.8-7.0. Make up the volume to 10L with ultrapure water. Filter aseptically using a 0.22μm filter membrane to obtain the PBS buffer.

[0069] Preparation of 1 mg / mL DDM-pH7.0 PBS buffer sample: Add 35 mg of DDM and 35 mL of the freshly prepared PBS buffer to the sample vial, sonicate for 1 min to completely dissolve and mix, and you will get 1 mg / mL DDM-pH7.0 PBS buffer.

[0070] Preparation of Mivelsiran-DDM solution (50 mg / mL): For example, dissolve 50 mg of FAM-labeled Mivelsiran in 1 mL of 1 mg / mL DDM-pH7.0 PBS buffer to obtain a Mivelsiran solution containing the absorption enhancer DDM.

[0071] In this embodiment, Mivelsiran is labeled with FAM, making detection more intuitive and accurate; the justice chain of Mivelsiran is shown in SEQ ID NO:1. In this embodiment, the 5' end of the justice chain is labeled with FAM, solely for the purpose of making detection more intuitive. The mass spectrum is shown below. Figure 2 As shown; The Mivelsiran antisense strand is shown in SEQ ID NO:2, and its mass spectrum is shown in [image missing]. Figure 3 As shown.

[0072] 2. Administration The newly prepared Mivelsiran-DDM solution was placed in a nasal delivery device (the small animal intelligent integrated nasal delivery system developed by the applicant company, as disclosed in CN202021777601.0 and CN202010148279.3) and administered a prescribed dose of drug to rats via the nose. In this embodiment, the rats received a single nasoencephalometric injection of 2 mg / dose, administered via a 2-minute constant-rate or pulsatile nasal delivery method. Pharmacokinetics and distribution were measured at fixed time points after administration. The plasma and tissue samples were collected using methods commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 2 below. Table 2

[0073] Experimental conclusion: After delivery via the nasal olfactory region, the formulation of this patent application, C max（脑） / Cmax（血浆） AUC 0-t（脑） / AUC 0-t（血浆） The proportion of drugs is relatively high, with only a small amount entering the bloodstream and most entering various brain tissues through neural pathways; the ratio of the olfactory bulb to other brain tissues is about 3, suggesting that this prescription has a relatively stable and clear distribution characteristic in the brain. Example 6: Nasal infusion of C16-SOD1 solution containing an absorption enhancer into the brain.

[0074] A solution containing an absorption enhancer, C16-SOD1 ((small nucleic acid drug-4)), was prepared using methods conventional to those skilled in the art. For example, 50 mg of FAM-labeled C16-SOD1 is dissolved in 1 mL of 1 mg / mL DDM-pH7.0 PBS buffer to obtain a solution of C16-SOD1 containing the absorption enhancer DDM, i.e., small nucleic acid drug-4, at a concentration of 50 mg / mL.

[0075] In this embodiment, C16-SOD1 is marked with FAM, making the detection more intuitive and accurate; A newly prepared solution of C16-SOD1 containing an absorption enhancer was placed in a nasal delivery device (the applicant's intelligent integrated nasal delivery system for small animals, as disclosed in CN202021777601.0 and CN202010148279.3) and administered a prescribed dose of drug to rats via the nose. In this embodiment, the rats received a single nasoencephalometric injection of 2 mg / dose, administered via a 2-minute constant-rate or pulsed nasal delivery method; three rats were paralleled at each time point; pharmacokinetics and distribution were measured at fixed times after administration. The plasma and tissue sampling methods employed were those commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 3 below. Figure 4 and Figure 5 As shown; Table 3

[0076] The pharmacokinetic parameters calculated from Table 3 are shown in Table 4 below: Table 4

[0077] Note: Data at 1 minute was not included in the calculation of T1 / 2 and AUC.

[0078] Experimental conclusion: After delivery via the nasal olfactory region, the formulation of this patent application, C max（脑） / C max（血浆） AUC 0-t（脑） / AUC 0-t（血浆）The proportion of drugs is relatively high, with very little entering the bloodstream and most entering various brain tissues through neural pathways; the ratio of the olfactory bulb to other brain tissues is about 4, suggesting that this prescription has a relatively stable and clear distribution characteristic in the brain.

[0079] Seven days later, the rats were euthanized, and samples were taken to detect pharmacokinetics and distribution. Plasma and tissue samples were collected using methods commonly used by those skilled in the art; as shown in Table 5 below: Table 5

[0080] Experimental conclusion: Seven days after administration, the drug concentration in the cortex on the administration side and the contralateral hemisphere remained at 8 µg / g. The content in the olfactory bulb decreased after administration. Therefore, the olfactory bulb acts as a drug reservoir, slowly and steadily releasing the drug into the brain. The brain can maintain an effective drug concentration for a long time, reduce the frequency of administration, and improve compliance.

[0081] Example 7 The solution prepared in Example 6 was administered to the olfactory region of mice using a method similar to that in Example 6. In this example, the dosage was 0.5 mg / dose, and three mice were administered in parallel. Samples were taken 3 minutes after administration to detect pharmacokinetics and distribution. Plasma and tissue sampling methods were employed using methods commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 6 below. Table 6

[0082] Experimental conclusion: After delivery via the olfactory region, the formulation of this patent application results in almost no drug entering the bloodstream. Most of the drug enters various brain tissues through neural pathways. At an extremely low dosage of 0.5 mg / dose, the intracranial drug concentration can reach 8 µg / mL, and the content in the olfactory bulb on the administration side is 47.45 µg / g, which can act as a drug reservoir, releasing the drug slowly and steadily into the brain. Therefore, it can maintain an effective drug concentration for a long time and reduce the frequency of administration.

[0083] Example 8 The solution prepared in Example 6 was administered to the olfactory region of rats using a similar method. In this example, the dosage was 0.5 mg / dose, and three rats were administered in parallel. Samples were taken at 3 mins, 10 mins, and 30 mins after administration to detect pharmacokinetics and distribution. Plasma and tissue sampling methods were employed using methods commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 7 below. Figure 6 As shown; Table 7

[0084] Example 9 The solution prepared in Example 6 was administered to the olfactory region of rats using a similar method. In this example, the dosage was 1 mg / dose, and three rats were administered in parallel. Samples were taken at 3 mins, 10 mins, and 30 mins after administration to detect pharmacokinetics and distribution. Plasma and tissue sampling methods were employed using methods commonly used by those skilled in the art. The average drug concentrations in plasma and brain tissue are shown in Table 8 below. Figure 7 As shown; Table 8

[0085] The drug-time curves of the olfactory bulb at different administration doses (0.5 mg / dose, 1.0 mg / dose, 2 mg / dose) and different detection time points (1 min, 3 min, 10 min, 30 min, and 60 min) are shown below. Figure 8 As shown.

[0086] Experimental conclusion: The drug concentration in the olfactory bulb varies with different dosages, but the content in the brain tissue remains the same, indicating that after the composition of this patent application is administered, the olfactory bulb can act as a drug reservoir to maintain a stable concentration in the brain tissue.

Claims

1. A composition, characterized in that, include: a) A small interfering nucleic acid having a sense strand and an antisense strand, wherein one of the sense strand or the antisense strand contains at least one lipophilic modification, while the other strand of the small interfering nucleic acid does not contain a lipophilic modification; the lipophilic modification is a saturated linear C16 modification; b) Absorption enhancer; the absorption enhancer is dodecyl β-D-maltodextrin; c) A diluent or buffer, wherein the diluent is water and the buffer is PBS buffer; The content of the absorption enhancer, based on the total weight of the composition, is 0.1~0.5% (w / w). The pH value of the composition is 5.0 to 7.

0.

2. The composition according to claim 1, characterized in that, The sense or antisense strand of the at least one lipophilic modified small interfering nucleic acid forms a double strand with the strand that does not contain lipophilic modification. Or, the molar excess of the chain containing at least one lipophilic modification relative to the other chain is less than 1%, or the molar equivalents of the two chains are equal; or the molar excess of the chain not containing lipophilic modification relative to the other chain; and / or, The sense strand of the small interfering nucleic acid contains at least one lipophilic modification, while the antisense strand does not contain any lipophilic modification. The antisense chain is molar excess relative to the justice chain; Alternatively, the antisense strand of the small interfering nucleic acid contains at least one lipophilic modification, and the sense strand does not contain a lipophilic modification, wherein the sense strand is molarly in excess relative to the antisense strand.

3. The composition according to claim 1, characterized in that, The lipophilic modification is located at the 2'-ribose position of the sixth nucleotide residue of the positive strand.

4. The composition according to claim 1, characterized in that, The positive strand of the small interfering nucleic acid is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or other free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and / or, The antisense strand of the small interfering nucleic acid is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or other free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:

8.

5. The composition according to claim 4, characterized in that, The sense strand of the small interfering nucleic acid is SEQ ID NO:1 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:1, and the antisense strand is SEQ ID NO:2 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:2, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand; or, The sense strand of the small interfering nucleic acid is SEQ ID NO:3 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:3, and the antisense strand is SEQ ID NO:4 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:4, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand; or, The sense strand of the small interfering nucleic acid is SEQ ID NO:5 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:5, and the antisense strand is SEQ ID NO:6 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:6, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand; or, The sense strand of the small interfering nucleic acid is SEQ ID NO:7 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:7, and the antisense strand is SEQ ID NO:8 or another free acid or sodium salt form having at least 85% identical nucleotides to SEQ ID NO:8, wherein the lipophilic modification is performed at the 2'-ribose position of the sixth nucleotide residue of the sense strand.

6. A reagent kit, characterized in that, The kit includes, a) comprising the composition according to any one of claims 1 to 5; b) Optionally, it also includes a nasal mucosa activator; said nasal mucosa activator is hyaluronidase; c) Optionally, it further includes means for administering the composition of any one of claims 1 to 5 to the subject and / or means for administering the nasal mucosa activator to the subject.

7. The use of the composition of any one of claims 1 to 5 or the kit of claim 6 in the preparation of a medicament for reducing the expression of a target gene; wherein the target gene is amyloid precursor protein, amyotrophic lateral sclerosis (ALS), superoxide dismutase (SOD), or Huntington's gene.

8. The application according to claim 7, characterized in that, The composition according to any one of claims 1 to 5 is administered by nasal spray, nasal drops, intravenous, subcutaneous, intrathecal, or oral administration; and / or, The kit described in claim 6 is administered via nasal spray or drops.

9. The application according to claim 7, characterized in that, The diseases associated with the amyloid precursor protein are cerebral amyloid angiopathy or Alzheimer's disease.

10. The application according to claim 9, characterized in that, The Alzheimer's disease mentioned refers to early-onset Alzheimer's disease, familial Alzheimer's disease, early-onset familial Alzheimer's disease, or late-onset Alzheimer's disease.

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