# US 10,246,708 B2 · Dosages and methods for delivering lipid formulated nucleic acid molecules

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- Number: US 10,246,708 B2
- Document ID: US10246708B2
- Status: Granted
- Kind: B2
- Application number: 14/889,352
- Filed: 2014-05-06
- Published: 2016-05-05
- Granted: 2019-04-02
- Earliest claimed: 2013-05-06
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Pia Kasperkovitz; Jared Gollob
- Web page: https://patentgrep.com/patent/US10246708B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- A61K 9/5123: {Organic compounds, e.g. fats, sugars}
- A61K 9/5146: {obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides}
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61M 5/31533: {Dosing mechanisms, i.e. setting a dose (administrating mechanisms A61M5/31565)}
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 37/00: Drugs for immunological or allergic disorders
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/335: Modified T or U
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2320/35: based on a specific dosage / administration regimen
- C12N 2320/53: reducing unwanted side-effects
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)}
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Methods, kits and devices for dosing a subject to reduce a hypersensitivity response to a lipid-formulated nucleic acid (e.g., RNA) molecule are disclosed.

## Claims

### Claim 1

A method of reducing an infusion-related response (IRR), or a hypersensitivity reaction, or both, in a subject, to a composition comprising a lipid formulation and a nucleic acid molecule, wherein the nucleic acid molecule comprises a double stranded RNA (dsRNA) molecule, said method comprising administering to a subject:
a first dose of said composition;
a second dose of said composition;
wherein the amount of said composition administered in said first dose is no more than 1/10 of the amount of said composition administered in said second dose; and
wherein one or both of a)-b) of the following conditions is met:
a) the first dose is administered over a time period that is no more than ½ of the time period over which the second dose is administered; or
b) the rate of administration of said first dose is no more than ½ of the rate of administration of said second dose.

### Claim 2

A method of reducing the expression of a target gene, or treating a disorder related to the target gene, in a subject, the method comprising:
administering to the subject a first dose and a second dose of a composition, said composition comprising a lipid formulation and a nucleic acid molecule, wherein the nucleic acid molecule comprises a double stranded RNA (dsRNA) molecule, and wherein said first and second doses are administered in an amount sufficient to reduce expression of a target gene, or treat the disorder, in the subject;
wherein the amount of said composition administered in said first dose is no more than 1/10 of the amount of said composition administered in said second dose; and
wherein one or both of a)-b) of the following conditions is met:
a) the first dose is administered over a time period that is no more than ½ of the time period over which the second dose is administered; or
b) the rate of administration of said first dose is no more than ½ of the rate of administration of said second dose.

### Claim 3 (depends on 1)

The method of claim 1, wherein:
(i) the rate of administration of said first dose about 5% to about 50%, about 10% to about 40%, or about 20% to about 40% of the rate of administration of the second dose;
(ii) the total amount of said composition administered in said first dose is about 0.5% to 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 10%, or about 5% to about 10% of the total amount of said composition, or the amount of the composition administered in said second dose; or
(iii) both (i) and (ii).

### Claim 4 (depends on 1)

The method of claim 1, wherein:
(i) the rate of administration of the lipid formulation in said first dose is about 0.05 μg/min/kg to about 50 μg/min/kg or about 0.1 μg/min/kg to about 25 μg/min/kg;
(ii) the rate of administration of the lipid formulation in said second dose is about 0.5 μg/min/kg to about 500 μg/min/kg, about 1 μg/min/kg to about 250 μg/min/kg, about 10 μg/min/kg to about 150 μg/min/kg, or about 50 μg/min/kg to about 100 μg/min/kg; or
(iii) both (i) and (ii).

### Claim 5 (depends on 1)

The method of claim 1, wherein:
(i) the rate of administration of the nucleic acid molecule in said first dose is about 0.01 μg/min/kg to about 5 μg/min/kg[H] or about 0.02 μg/min/kg to about 2.5 μg/min/kg;
(ii) the rate of administration of the nucleic acid molecule in said second dose is about 0.1 μg/min/kg to about 50 μg/min/kg, about 0.2 μg/min/kg to about 25 μg/min/kg, about 0.5 μg/min/kg to about 10 μg/min/kg, or about 1 μg/min/kg to about 5 μg/min/kg; or
(iii) both (i) and (ii).

### Claim 6 (depends on 1)

The method of claim 1, wherein:
(i) the amount of the lipid formulation administered in said first dose is about 0.5 μg/kg to about 1000 μg/kg or about 1 μg/kg to about 500 μg/kg;
(ii) the amount of the lipid formulation administered in said second dose is about 20 μg/kg to about 50000 μg/kg, about 100 μg/kg to about 25000 μg/kg, about 500 μg/kg to about 10000 μg/kg, or about 1000 μg/kg to about 5000 μg/kg; or
(iii) both (i) and (ii).

### Claim 7 (depends on 1)

The method of claim 1, wherein:
(i) the amount of the nucleic acid molecule in said first dose is about 2 μg/kg to about 30 μg/kg;
(ii) the amount of the nucleic acid molecule in said second dose is about 2 μg/kg to about 1000 μg/kg, about 5 μg/kg to about 750 μg/kg, about 10 μg/kg to about 500 μg/kg, or about 50 μg/kg to about 300 μg/kg; or
(iii) both (i) and (ii).

### Claim 8 (depends on 1)

The method of claim 1, wherein:
(i) the second dose is administered over a time period that is at least 3 or 4 times greater than the time period over which the first dose is administered;
(ii) the first dose is administered over a time period that is no greater than ¼ or ⅕ of the time period over which the total dose is administered;
(iii) the first dose is administered over a time period that is between 5% and 50%, between 10% and 45%, between 15% and 40%, between 20% and 35%, or between 25% and 30% of the time period of administration of the second dose;
(iv) the first dose is administered over a time period that is between 5 minutes and 60 minutes, between 10 minutes and 50 minutes, between 20 minutes and 40 minutes, between 5 minutes and 30 minutes, or between 10 minutes and 20 minutes;
(v) the second dose is administered over a time period that is between 30 minutes and 180 minutes, between 40 minutes and 120 minutes, between 45 minutes and 90 minutes, or between 50 minutes and 65 minutes; or
(vi) no more than 1, 10, 20, 30, 60, or 180 minutes separates the completion of the administration of the first dose and the initiation of the administration of the second dose.

### Claim 9 (depends on 1)

The method of claim 1, further comprising administering to the subject one or more additional doses of the composition.

### Claim 10 (depends on 1)

The method of claim 1, wherein said first and second doses are administered by intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intravenously by infusion, at a substantially constant rate via a pump or a sustained or controlled release formulation, or as a gradient for multiple rates.

### Claim 11 (depends on 10)

The method of claim 10, wherein:
(i) the flow rate of administration of the first dose is no more than ½ or ⅓ of the flow rate of administration of said second dose;
(ii) the flow rate of administration of the first dose is about 0.5 to 1.5 mL/min, about 0.8 to 1.3 mL/min, about 1 to 1.2 mL/min, about 1 mL/min or 1.1 mL/min;
(iii) the flow rate of administration of the second dose is about 2 to 4 mL/min, about 2.5 to 3.7 mL/min, about 3 to 3.5 mL/min, about 3 mL/min or 3.3 mL/min;
(iv) both (ii) and (iii); or
(v) the total volume of infusion is about 100 to 300 mL, about 150 to 250 mL, about 180 mL or 200 mL.

### Claim 12 (depends on 1)

The method of claim 1, wherein the IRR and/or hypersensitivity reaction is an acute hypersensitivity reaction during dose administration or occurs after administration of the second dose is completed.

### Claim 13 (depends on 1)

The method of claim 1, further comprising evaluating the subject after administration of the first dose, the second dose, or both, for one or more of the following:
(i) the presence of one or more of the following: a skin reaction, a hemodynamic change, a change in blood pressure, a respiratory problem, pain, or one or more of fever, chills, nausea, vomiting or neurological changes;
(ii) a change in a complement biomarker chosen from one or more of complement activation, or a change in one or more complement factors chosen from Bb or C3ab, wherein an increase the level of a complement biomarker is indicative of an IRR and/or hypersensitivity reaction;
(iii) a change in thromboxane levels or thromboxane B2 in plasma, wherein an increase in the level of thromboxane or thromboxane B2 is indicative of an increased hypersensitivity reaction; or
(iv) a change in one or more cytokines chosen from interferon-alpha, interferon-gamma, or tumor necrosis factor-alpha, interleukin 1beta, interleukin 1 receptor antagonist (IL-1RA), interleukin-6, interleukin-8, interleukin-12, interleukin-18, interferon inducing protein-10, granulocyte colony stimulating factor, or C-reactive protein (CRP), wherein an increase in the level of IL-6, IL-8, IL-1RA or CRP is indicative of an increased hypersensitivity reaction.

### Claim 14 (depends on 1)

The method of claim 1, wherein:
(i) said method does not cause a detectable IRR and/or hypersensitivity reaction;
(ii) said method results in a decrease in the IRR and/or hypersensitivity reaction, which is less than 1%, 5%, 10%, 25%, 30%, 35% or 40%;
(iii) said method causes a reduced IRR and/or hypersensitivity reaction leading to a reduction in the administration of one or more of a steroid, an analgesic, or a histamine receptor antagonist;
(iv) said subject does not receive administration of a steroid within X hours of any of the initiation of administration of said first dose, wherein X is less than 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 24 hours or 48 hours; or
(v) if said first and second dose regimen is provided to a porcine subject, the subject will show a reduced IRR and/or hypersensitivity reaction, relative to a subject exposed to a bolus dose, or the subject prior to treatment.

### Claim 15 (depends on 1)

The method of claim 1, wherein the lipid is a cationic or a non-cationic lipid, or a combination thereof.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP, also referred to as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride in U.S. Pat. No. 8,158,601), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA, also referred to as N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride in U.S. Pat. No. 8,158,601), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), bis(3-pentyloctyl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, MC3, or M-C3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof; and/or
wherein the cationic lipid comprises from about 20 mol % to about 60 mol %, or about 40 mol % of the total lipid present in the formulation.

### Claim 17 (depends on 1)

The method of claim 1, wherein:
(i) the first dose is administered at a first nucleic acid dose rate between 1.5 and 2 μg/min/kg, and
the second dose is administered at a second nucleic acid dose rate between 4 and 6 μg/min/kg;
(ii) the first dose is administered at a first lipid dose rate between 15 and 25 μg/min/kg, and
the second dose is administered at a second lipid dose rate between 50 and 100 μg/min/kg; or
(iii) both (i) and (ii).

### Claim 18 (depends on 1)

The method of claim 1, wherein:
(i) the first dose is administered at between 0.5 and 1.5 mL/min, and the second dose is administered at between 2.5 and 3.5 mL/min;
(ii) the first dose is administered over a period of between 10 and 20 minutes, and the second dose is administered over a period of between 50 and 65 minutes; or
(iii) both (i) and (ii).

### Claim 19 (depends on 1)

The method of claim 1, wherein:
(i) the amount of the nucleic acid molecule administered in the first dose is between 20 and 30 μg/kg, and the amount of nucleic acid molecule administered in the second dose is between 250 and 300 μg/kg;
(ii) the amount of the lipid formulation administered in the first dose is between 250 and 400 μg/kg, and the amount of the lipid formulation administered in the second dose is between 2500 and 4000 μg/kg; or
(iii) both (i) and (ii).

### Claim 20 (depends on 1)

The method of claim 1, wherein:
(i) the total nucleic acid dose in the first and the second doses is between 0.2 and 0.4 mg/kg;
(ii) the total lipid dose in the first and the second doses is between 3.0 and 4.5 mg/kg; or
(iii) both (i) and (ii).

### Claim 21 (depends on 1)

The method of claim 1, wherein:
(i) the rate of administration of the nucleic acid molecule in the first dose is between 0.1 and 0.15 mg/min, and the rate of administration of the nucleic acid molecule in the second dose is between 0.3 and 0.4 mg/min;
(ii) the rate of administration of the lipid formulation in the first dose is between 1.0 and 1.5 mg/min, and the rate of administration of the lipid formulation in the second dose is between 3.5 and 4.5 mg/min; or
(iii) both (i) and (ii).

### Claim 22 (depends on 1)

The method of claim 1, wherein:
(i) the amount of the nucleic acid molecule administered in the first dose is between 1.5 and 2.0 mg and the amount of the nucleic acid molecule administered in the second dose is between 15 and 25 mg;
(ii) the amount of the lipid formulation administered in the first dose is between 15 and 25 mg, and the amount of the lipid formulation administered in the second dose is between 200 and 300 mg; or
(iii) both (i) and (ii).

### Claim 23 (depends on 1)

The method of claim 1, wherein the first dose is administered over a time period that is between 20% and 35% of the time period of administration of the second dose.

### Claim 24 (depends on 1)

The method of claim 1, wherein the rate of administration of said first dose is about 20% to about 40% of the rate of administration of said second dose.

### Claim 25 (depends on 1)

The method of claim 1, wherein:
(i) the first dose is administered at 1 mL/min, and the second dose is administered at between 3 mL/min;
(ii) the first dose is administered over a period of 15 minutes, and the second dose is administered over a period of 50 and 65 minutes; or
(iii) both (i) and (ii).

### Claim 26 (depends on 1)

The method of claim 1, wherein one, two, or all of a)-c) of the following conditions is met:
a) the amount of said composition administered in said first dose is no more than 1/10 of the total amount of said composition administered;
b) the amount of said composition administered in said first dose is no more than 30 or 40 μg nucleic acids per kg body weight; or
c) the amount of said composition administered in said second dose is greater than 100 or 200 μg nucleic acids per kg body weight, and the second dose is greater than said first dose.

### Claim 27 (depends on 2)

The method of claim 2, wherein one, two, or all of a)-c) of the following conditions is met:
a) the amount of said composition administered in said first dose is no more than 1/10 of the total amount of said composition administered;
b) the amount of said composition administered in said first dose is no more than 30 or 40 μg nucleic acids per kg body weight; or
c) the amount of said composition administered in said second dose is greater than 100 or 200 μg nucleic acids per kg body weight.

### Claim 28

A method of reducing an infusion-related response (IRR), or a hypersensitivity reaction, or both, in a subject, to a composition comprising a lipid formulation and a nucleic acid molecule, wherein the nucleic acid molecule comprises a double stranded RNA (dsRNA) molecule, said method comprising administering to a subject:
a first dose of said composition;
a second dose of said composition;
wherein the amount of said composition administered in said first dose is between 8% and 12% of the amount of said composition administered in said second dose;
wherein the first dose is administered over a time period that is no more than ½ of the time period over which the second dose is administered; and
wherein the rate of administration of said first dose is no more than ½ of the rate of administration of said second dose.

### Claim 29 (depends on 28)

The method of claim 28, wherein one, two, three, four, or all of a)-e) of the following conditions is met:
a) the amount of said composition administered in said first dose is no more than 1/9 or 1/10 of the total amount of said composition administered;
b) the first dose is administered over a time period that is no more than ⅓ or ¼ of the time period over which the second dose is administered;
c) the rate of administration of said first dose is no more than ⅓ of the rate of administration of said second dose;
d) the amount of said composition administered in said first dose is no more than 30 or 40 μg nucleic acids per kg body weight; or
e) the amount of said composition administered in said second dose is greater than 100 or 200 μg nucleic acids per kg body weight.

### Claim 30 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule comprises a sequence complementary to a target mRNA, wherein the target mRNA is an mRNA of: Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta, Erb-B, Src, CRK, GRB2, RAS, MEKK, JNK, RAF, Erk1/2, PCNA(p21), MYB, JUN, FOS, BCL-2, HAMP, Activated Protein C, Cyclin D, VEGF, antithrombin 3, aminolevulinate synthase 1, alpha-1-antitrypsin, tmprss6, apoa1, apoc3, bc11a, klf, angpt13, plk, PKN3, HBV, HCV, p53, angiopoietin, angiopoietin-like 3, complement component 3 (C3), or complement component 5 (C5).

### Claim 31 (depends on 2)

The method of claim 2, wherein the target gene is a gene of: Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta, Erb-B, Src, CRK, GRB2, RAS, MEKK, JNK, RAF, Erk1/2, PCNA(p21), MYB, JUN, FOS, BCL-2, HAMP, Activated Protein C, Cyclin D, VEGF, antithrombin 3, aminolevulinate synthase 1, alpha-1-antitrypsin, tmprss6, apoa1, apoc3, bc11a, klf, angpt13, plk, PKN3, HBV, HCV, p53, angiopoietin, angiopoietin-like 3, complement component 3 (C3), or complement component 5 (C5).

### Claim 32 (depends on 1)

The method of claim 1, wherein the lipid formulation comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, MC3, or M-C3).

### Claim 33 (depends on 1)

The method of claim 1, wherein the lipid formulation comprises about 50% of cationic lipid of Formula I/MC3, about 10% of a neutral lipid, about 38.5% of a sterol, and about 1.5% of PEG or a PEG-modified lipid.

### Claim 34 (depends on 1)

The method of claim 1, wherein the lipid formulation comprises about 50% of cationic lipid of Formula I/MC3, about 10% of DSPC, about 38.5% of cholesterol, and about 1.5% of PEG-DMG.