Vitamin b12 formulation for intranasal administration

Abstract

The invention relates to an aqueous vitamin B12 formulation for intranasal administration, comprising: · 3-100 mg / mL of a vitamin B12 component selected from hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, pharmaceutically acceptable salts of these cobalamins and combinations thereof · 30-300 mg / mL of sorbitol; wherein the vitamin B12 component and sorbitol are present in a weight ratio of vitamin B12 component : sorbitol of at least 0.2 and wherein the formulation has a pH in the range of 3.0 to 5.5.

Description

[0001] VITAMIN B12 FORMULATION FOR INTRANASAL ADMINISTRATION

[0002] Technical field of the invention

[0003] This invention relates to an aqueous vitamin B12 formulation for intranasal administration. The aqueous vitamin B12 formulation of the present invention is concentrated, very stable and has a surprisingly high intranasal bioavailability.

[0004] The invention also provides a metered-dose pump spray device for intranasal delivery of the vitamin B12 formulation.

[0005] The aqueous vitamin B12 formulation of the present invention can suitably be used in the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis.

[0006] Background of the invention

[0007] Vitamin B12, also called cobalamin, is a vitamin that is involved in the metabolism of every cell of the human body: it is a cofactor in DNA synthesis, and in both fatty acid and amino acid metabolism. Vitamin B12 is particularly important in the normal functioning of the nervous system via its role in the synthesis of myelin, and in the maturation of developing red blood cells in the bone marrow.

[0008] Vitamin B12 is one of eight B vitamins; it is the largest and structurally most complex vitamin. It contains the biochemically rare element cobalt positioned in the centre of a corrin ring. Four of the six coordination sites of vitamin B12 are provided by the corrin ring, and a fifth by the dimethylbenzimidazole group. The sixth coordination site, the reactive centre, is variable, being a cyano group (cyanocobalamin), a hydroxyl group (hydroxocobalamin), a methyl group (methylcobalamin) or a 5'-deoxyadenosyl group (adenosylcobalamin).

[0009] There are no naturally-occurring vegetable sources of vitamin B12, so vegans and vegetarians are advised to take a vitamin B12 containing supplement or vitamin B12 fortified foods. Most omnivorous people in developed countries obtain enough vitamin B12 from consuming animal products including meat, milk, eggs, and fish. A serum B12 concentration above 300 pg / mL is interpreted as normal. B12 levels between 200 and 300 pg / mL are considered borderline. Subjects with B12 levels below 200 pg / mL are considered deficient.

[0010] The most common cause of vitamin B12 deficiency in developed countries is impaired absorption due to a loss of gastric intrinsic factor, which must be bound to food-source B12 in order for absorption to occur. Another group affected are those on long term antacid therapy, using proton pump inhibitors, H2 blockers or other antacids.

[0011] Vitamin B12 supplements are available in single agent or multivitamin tablets; and pharmaceutical preparations may be given by intramuscular injection. The nasal administration route is recognized for its ability to facilitate rapid and efficient absorption of substances. In the US, a nasal spray containing an aqueous solution of cyanocobalamin is marketed by Questcor Pharmaceuticals, Inc. under the brand name Nascobal®. In the Netherlands, a nasal spray containing an aqueous solution of vitamin B12 s marketed by Pronova Laboratories under the brand name Pronofit®.

[0012] US 2020 / 0268651 describes a vitamin B12 nasal spray composition that comprises (i) a therapeutic dose of vitamin B12; (ii) synergistic amounts of a polymer, carrageenan and potassium acetate; (iii) optionally a buffer to adjust pH of the formulation between 4.5 and 7.5; (iv) a preservative; (v) a humectant; (vi) purified water; (vii) optionally hydrochloric acid and / or sodium hydroxide to adjust the pH of the formulation between 4.5 and 7.5, and (viii) optionally a drug(s) known to impair absorption of vitamin B12. Used therapeutic dose of vitamin B12 ranges from 0.01 to 10 mg / mL.

[0013] ES 2074396 concerns a composition for the nasal administration of cyanocobalamin. The composition comprises cyanocobalamin, sorbitol, pantothenol, methylparaben, acetic acid, sodium acetate, methyl cellulose, glycerine, sodium chloride, water and a combination of polyglycerol esters of fatty acids.

[0014] WO 86 / 05988 describes a composition for nasal administration comprising vitamin B12 in an isotonic aqueous buffer at a pH of 4 to 6 and having viscosity of 2500 to 6500 cps.

[0015] WO 2018 / 142358 describes an intranasal composition comprising methylcobalamin and at least one gelling agent. WO 2022 / 043526 relates to a liquid aqueous pharmaceutical formulation containing hydroxocobalamin and / or hydroxocobalamin salts dissolved in a concentration above 0.1% (w / v) in water containing at least 3% (w / v) and less than 12% (w / v) mannitol.

[0016] CA1300014C describes an intranasal formulation containing vitamin B12 in an isotonic aqueous buffer having a pH of 4 to 6 and a viscosity of 2500-6500 cps.

[0017] CA 2 656 823 describes a pharmaceutical aqueous solution for intranasal administration, comprising cyanocobalamin; citric acid; sodium citrate; a humectant selected from the group consisting of sorbitol, propylene glycol, and glycerin; a preservative and water.

[0018] Hadinata Lie et al. (Sorbitol enhances the physicochemical stability of B12 vitamers , Int J Vitam Nutr Res (2019), 1-9) report the outcome of a study in which they compared the degradation losses in cyanocobalamin, hydroxocobalamin and methylcobalamin due to physicochemical exposure before and after the addition of sorbitol.

[0019] Summary of the invention

[0020] The inventors have developed a concentrated aqueous vitamin B12 formulation for intranasal administration that is highly stable and that has a surprisingly high intranasal bioavailability The formulation of the invention can suitably be used in the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis.

[0021] The aqueous vitamin B12 formulation for intranasal administration according to the present invention comprises:

[0022] • 3-100 mg / mL of a vitamin B12 component selected from hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, pharmaceutically acceptable salts of these cobalamins and combinations thereof

[0023] • 30-300 mg / mL of sorbitol; wherein the vitamin B12 component and sorbitol are present in a weight ratio of vitamin B12 component : sorbitol of at least 0.2 and wherein the formulation has a pH in the range of 3.0 to 5.5.

[0024] The formulations of the present invention can achieve a surprisingly high intranasal bioavailability relative to the intramuscular route of about 20%. A stability at ambient temperature of 1 year or more can also be realised by the present formulations. The presence of sorbitol in the intranasal formulation is deemed essential for achieving these benefits. A second aspect of the invention relates to a metered-dose pump spray device comprising

[0025] • a container holding the aqueous vitamin B12 formulation according to the invention,

[0026] • a nozzle, and

[0027] • a manual pump connecting the nozzle and the container, said pump being configured to deliver one spray-unit upon actuation by manual force.

[0028] Another aspect of the invention relates to the use of the aforementioned aqueous vitamin B12 formulation in the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis, said use comprising intranasal administration of the vitamin B12 formulation according to the invention.

[0029] Detailed description of the invention

[0030] Accordingly, a first aspect of the invention relates to an aqueous vitamin B12 formulation for intranasal administration comprising:

[0031] • 3-100 mg / mL of a vitamin B12 component selected from hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, pharmaceutically acceptable salts of these cobalamins and combinations thereof;

[0032] • 30-300 mg / mL of sorbitol; wherein the vitamin B12 component and sorbitol are present in a weight ratio of vitamin B12 component : sorbitol of at least 0.2 and wherein the formulation has a pH in the range of 3.0 to 5.5.

[0033] According to a particularly preferred embodiment, the aqueous B12 formulation of the present in invention is an aqueous solution, and not an emulsion (e.g. a micro-emulsion or nanoemulsion.

[0034] The aqueous vitamin B12 formulation for intranasal administration preferably comprises IQ- 90 mg / mL, more preferably 20-80 mg / mL, even more preferably 24-70 mg / mL of the vitamin B12 component.

[0035] According to a particularly preferred embodiment, the vitamin B12 component that is employed in accordance with the present invention is selected from hydroxocobalamin, pharmaceutically acceptable salts of hydroxocobalamin and combinations thereof. The inventors have found that when hydroxocobalamin is used, a particularly concentrated vitamin B12 formulation can be prepared. According to a particularly preferred embodiment, the aqueous vitamin B12 formulation comprises 15-90 mg / mL, more preferably 20-80 mg / mL, even more preferably 30-70 mg / mL of hydroxocobalamin and / or a pharmaceutically acceptable salt thereof.

[0036] The aqueous vitamin B12 formulation preferably comprises 50-250 mg / mL, more preferably 70-160 mg / mL and most preferably 80-150 mg / mL sorbitol.

[0037] Preferably, vitamin B12 component and sorbitol are contained in the formulation in a weight ratio of 0.25 to 2.0, more preferably of 0.28 to 1 .2 and most preferably of 0.30 to 0.70.

[0038] According to a particularly preferred embodiment, the vitamin B12 formulation contains a thickening agent. The thickening agent is preferably contained in the aqueous vitamin B12 formulation in a concentration of 2-50 mg / mL, more preferably in a concentration of 2.5-30 mg / mL, most preferably in a concentration of 4-20 mg / mL.

[0039] Examples of thickening agents that may employed in aqueous vitamin B12 formulation include cellulose ethers, xanthan gum, gellan gum, alginate, tragacanth, gum Arabic, guar gum, starch, dextran, gelatin, pectin, chitosan, polyacrylic acid polymers, poly hydroxyethyl methylacrylate, polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, carbomer and combinations thereof. More preferably, the thickening agent is selected from cellulose ethers, xanthan gum, gellan gum, alginate, carbomer and combinations thereof

[0040] According to a particularly preferred embodiment, the aqueous vitamin B12 formulation comprises 2-40 mg / mL, more preferably 4-20 mg / mL of the cellulose ether.

[0041] The cellulose ether is preferably selected from hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxyethylcellulose (HEC), ethyl hydroxyethylcellulose (EHEC), methyl ethyl hydroxyethylcellulose (MEHEC), hydroxypropyl cellulose (HPC), dihydroxypropylcellulose (DHPC), methylcellulose (MC), ethyl cellulose (EC) and combinations thereof. More preferably, the cellulose ether is selected from hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxyethylcellulose (HEC), ethyl hydroxyethylcellulose (EHEC), methyl ethyl hydroxyethylcellulose (MEHEC), hydroxypropyl cellulose (HPC), dihydroxypropylcellulose (DHPC) and combinations thereof.

[0042] According to a particularly preferred embodiment, the cellulose ether in the aqueous vitamin B12 formulation is HPMC. The aqueous vitamin B12 formulation preferably has a pH in the range of 3.2 to 5.0, more preferably of 3.5 to 4.8.

[0043] The pH of the vitamin B12 formulation is preferably set using an acetate buffer and / or a citrate buffer.

[0044] In one embodiment, the formulation comprises 3-20 pmol / mL of acetate, more preferably 5- 12 pmol / mL of acetate.

[0045] Tin another embodiment, the formulation comprises 2-15 pmol / m of citrate, more preferably 3-8 pmol / mLof citrate.

[0046] Preferably, the aqueous vitamin B12 formulation contains at least 70 wt.% water, more preferably at least 75 wt.% water, most preferably 78-90 wt.% water.

[0047] According to a preferred embodiment, the combination of the vitamin B12 component, sorbitol, the thickening agent and water constitutes at least 90 wt.%, more preferably at least 95 wt.% of the aqueous vitamin B12 formulation.

[0048] The aqueous vitamin B12 formulation preferably has a dissolved oxygen content of less than 8 ppm, more preferably of less than 1.5 ppm.

[0049] The aqueous vitamin B12 formulation preferably has an osmolarity of between 150 and 2000 mOsm / L, more preferably between 300 and 800 mOsm / L.

[0050] As explained herein before, the aqueous vitamin B12 formulation preferably is an aqueous and not an emulsion. Accordingly, the aqueous vitamin B12 formulation preferably contains less than 1 mg / mL of polyglycerol ester of fatty acids, more preferably the formulation does not contain polyglycerol ester of fatty acids.

[0051] According to a particularly preferred embodiment, the aqueous vitamin B12 formulation contains less than 1 mg / mL of emulsifier having an HLB of 10 or more. Most preferably, the formulation does not contain an emulsifier having an HLB of 10 or more.

[0052] The aqueous vitamin B12 formulation of the present invention may suitably contain additional ingredients, such as preservatives, penetration enhancers, anti-oxidants, flavouring. Examples of preservatives that may be employed include benzalkonium chloride, potassium sorbate and sorbic acid.

[0053] According to a particularly preferred embodiment, the aqueous vitamin B12 formulation of the present invention is a viscous liquid having a viscosity in the range of 5-100 mPa.s, more preferably in the range of 10-70 mPa.s and most preferably in the range of 20-50 mPa.s at a shear rate of 100 rpm and a temperature of 20°C. The viscosity may suitably be determined using a Brookfield™ DV-II viscometer that is equipped with spindle 3.

[0054] According to a second aspect of the present invention there is provided a metered-dose pump spray device comprising

[0055] • a container holding the aqueous vitamin B12 formulation according to any one of the preceding claims,

[0056] • a nozzle, and

[0057] • a manual pump connecting the nozzle and the container, said pump being configured to deliver one spray-unit upon actuation by manual force.

[0058] Preferably, the metered-dose pump spray device of the invention is adapted to deliver a dose of 10-250 pl, more preferably of 15-200 pl, even more preferably of 20-150 pl, most preferably 25-125 pL.

[0059] According to a preferred embodiment, the metered-dose pump spray device can be operated to produce a spray without allowing subsequent ingress of air.

[0060] According to a particularly preferred embodiment, the metered-dose pump spray device comprises a filter membrane that prevents ingress of micro-organisms with venting air. The aqueous vitamin B12 formulation that is contained in this pump spray device preferably is preservative-free.

[0061] A third aspect of the present invention relates to the aqueous vitamin B12 formulation for use in the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis, said use comprising intranasally administering the formulation as described herein before.

[0062] The aqueous vitamin B12 formulation of the present invention can suitably be used in the treatment of Parkinson’s disease or Alzheimers disease. Potential mechanisms are via interaction with alfa-synuclein and amyloid beta synthesis, entanglement and disentanglement. Another contributing pathway is via homocysteine induced neurotoxicity that develops at low B12 levels.

[0063] According to the MS-AdoCbl hypothesis, multiple sclerosis (MS) is caused by autoimmune attack of ATP:cob(l)alamin adeno-syltransferase (ATR) that converts cobalamins to the bioactive form 50-deoxy-50-adenosyl cobamide (adenosylcobalamin, co-enzyme B12 or AdoCbl). When the concentration of ATR is reduced, production of AdoCbl is limited resulting in metabolic changes that are pathological to the nervous system by causing myelin to become antigenic. Thus, aspects of MS pathogenesis are related to deficiency of AdoCbl.

[0064] The use of the present aqueous vitamin B12 formulation in the prevention and treatment of Parkinson’s disease, Alzheimers disease or multiple sclerosis preferably achieves serum B12 concentrations above normal physiological levels. Preferably, said use achieves an elevated serum B12 concentration of at least 500 pmol / L, more preferably of at least 1000 pmol / l. The latter may be achieved by nasal administration of 2,500 ug hydroxocobalamin HCI every 3 days.

[0065] In accordance with a particularly preferred embodiment, the formulation is intranasally administered by the metered-dose pump spray according to the invention.

[0066] Preferably, the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis in accordance with the present invention comprises intranasally administering the aqueous vitamin B12 formulation in a dosage of 10-250 pl, more preferably of 15-200 pl, even more preferably of 20 to 150 pl, most preferably 30-120 pL.

[0067] Preferably, the aqueous vitamin B12 formulation is intranasally administered to provide a dosage of 0.025-10 mg of the vitamin B12 component, more preferably a dosage of 0.1 -8,0 mg of the vitamin B12 component and most preferably a dosage of 0.5-5.0 mg of the vitamin B12 component.

[0068] The present treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis preferably comprises at least once daily intranasal administration of the aqueous vitamin B12 formulation. Most preferably, it comprises once daily intranasal administration of the aqueous vitamin B12 formulation.

[0069] The invention is further illustrated by the following non-limiting examples. Examples

[0070] Example 1

[0071] An aqueous vitamin B12 formulation according to the invention was prepared on the basis of the recipe that is shown in Table 1.

[0072] Table 1

[0073] The formulation was prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH was adjusted to 4.5 using acetic acid.

[0074] The vitamin B12 formulation had a viscosity of 32 mPa.s at 100 rpm and 20 °C (Brookfield DV-II, spindle 3).

[0075] The vitamin B12 formulation is stable for up to 3 years when stored at 5°C.

[0076] Example 2

[0077] Using a pump spray device, the aqueous vitamin B12 formulation of Example 1 was administered intranasally to 2 healthy volunteers in a dose of 53 pL (2.65 mg hydroxocobalamin). Plasma concentration of B12 was determined immediately before as well as 1 , 2, 7 and 24 hours after administration. The results are summarised in Table 2.

[0078] Table 2 Example 3

[0079] An aqueous vitamin B12 formulation according to the invention was prepared on the basis of the recipe that is shown in Example 1, except that the hydroxocobalamin content was reduced to 25 mg / mL.

[0080] Using a pump spray device, the aqueous vitamin B12 formulation was administered intranasally to 2 healthy volunteers in a dose of 50 pL (1.25 mg hydroxocobalamin). Plasma concentration of B12 was determined immediately before as well as 2 and 4 hours after administration. The results are summarised in Table 3.

[0081] Table 3

[0082] Van der Kuy et al. (Bioavailability of oral hydroxocobalamin, J. Clin. Pharmacol., (2009), 49,395-396) performed a cross-over randomised open study in six healthy volunteers to compare absorption of orally (10 mg) and intramuscularly (1 mg) administered hydroxocobalamin. Hydroxocobalamin was orally administered as 200 mL of an aqueous solution containing 10 mg hydroxocobalamin. The results of this study are summarised in Table 4.

[0083] Table 4

[0084] Van der Kuy et al. conclude that oral absorption of hydroxocobalamin was negligible. The pharmacokinetic results obtained for the intranasal formulations of the present invention indicate that these formulations had an intranasal bioavailability relative to the intramuscular route of about 20%. Example 4

[0085] An aqueous vitamin B12 formulation according to the invention was prepared on the basis of the recipe that is shown in Table 5.

[0086] Table 5

[0087] The formulation was prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH was adjusted to 5.0 using acetic acid.

[0088] Using a pump spray device, delivering 50 pL, the aqueous vitamin B12 formulation was administered intranasally to two healthy volunteers to deliver 0.5 mg of adenosylcobalamin. Plasma concentration of B12 was determined immediately before as well as 2 and 4 hours after administration. The results are summarised in Table 6.

[0089] Table 6

[0090] Example 5

[0091] An aqueous vitamin B12 formulation according to the invention was prepared on the basis of the recipe that is shown in Table 7.

[0092] Table 7 The formulation was prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH was adjusted to 4.5 using acetic acid. Using a pump spray device, the aqueous vitamin B12 formulation was administered intranasally to two healthy volunteers in a dose of 50 pL to deliver 0.5 mg of methylcobalamin. Plasma concentration of B12 was determined immediately before as well as 2 and 4 hours after administration. The results are summarised in Table 8.

[0093] Table 8

[0094] Example 6

[0095] An aqueous vitamin B12 formulation according to the invention is prepared on the basis of the recipe that is shown in Table 9.

[0096] Table 9

[0097] The formulation is prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH is adjusted to 5.0 using acetic acid.

[0098] Example 7

[0099] An aqueous vitamin B12 formulation according to the invention is prepared on the basis of the recipe that is shown in Table 10. Table 10

[0100] The formulation is prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH is adjusted to 5.0 using acetic acid.

[0101] Example 8

[0102] An aqueous vitamin B12 formulation according to the invention is prepared on the basis of the recipe that is shown in Table 11.

[0103] Table 11

[0104] The formulation is prepared by adding all components to a beaker and stirring until a homogeneous solution is obtained. The pH is adjusted to 5.0 using acetic acid.

[0105] Comparative Example

[0106] An aqueous vitamin B12 formulation was prepared on the basis of the recipe that is shown in Table 1, except that sorbitol was replaced by mannitol.

[0107] Using a pump spray device, the aqueous vitamin B12 formulation was administered intranasally to a healthy volunteers in a dose of 53 pL (2.65 mg hydroxocobalamin). Plasma concentration of B12 was determined immediately before as well 3 hours after administration. The results are summarised in Table 12. Table 12

Claims

CLAIMS1 . An aqueous vitamin B12 formulation for intranasal administration comprising:• 3-100 mg / mL of a vitamin B12 component selected from hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, pharmaceutically acceptable salts of these cobalamins and combinations thereof;• 15-300 mg / mL of sorbitol; wherein the vitamin B12 component and sorbitol are present in a weight ratio of vitamin B12 component : sorbitol of at least 0.2 and wherein the formulation has a pH in the range of 3.0 to 5.5.

2. Formulation according to claim 1 , wherein the vitamin B12 component is selected from hydroxocobalamin, pharmaceutically acceptable salts of hydroxocobalamin and combinations thereof.

3. Formulation according to any one of the preceding claims, wherein the formulation comprises 10-90 mg / mL of vitamin B12.

4. Formulation according to any one of the preceding claims, wherein the formulation comprises 25-250 mg / mL of sorbitol.

5. Formulation according to any one of the preceding claims, wherein the vitamin B12 component and sorbitol are contained in the formulation in a weight ratio of 0.25 to 1 .0.

6. Formulation according to any one of the preceding claims, wherein the formulation contains 2-50 mg / mL of thickening agent.

7. Formulation according to claim 5, wherein the thickening agent is selected from cellulose ethers, xanthan gum, gellan gum, alginate, tragacanth, araya gum, guar gum, starch, dextran, gelatin, pectin, chitosan, polyacrylic acid polymers, poly hydroxyethyl methylacrylate, polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, carbomer and combinations thereof.

8. Formulation according to any one of the preceding claims, wherein the formulation contains at least 70 wt.% water.

9. Formulation according to any one of the preceding claims, wherein the combination of the vitamin B12 component, sorbitol, the thickening agent and water constitutes at least 90 wt.%.

10. Formulation according to any one of the preceding claims, wherein the formulation has a pH in the range of 3.2 to 5.0.

11. Formulation according to any one of the preceding claims, wherein the formulation comprises 3-20 pmol / mL of organic acid selected from acetate, citrate and combinations thereof.

12. Formulation according to any one of the preceding claims, wherein the formulation has a dissolved oxygen content of less than 8 ppm, more preferably of less than 1.5 ppm.

13. A metered-dose pump spray device comprising• a container holding the aqueous vitamin B12 formulation according to any one of the preceding claims,• a nozzle, and• a manual pump connecting the nozzle and the container, said pump being configured to deliver one spray-unit upon actuation by manual force.

14. Metered-dose pump spray according to claim 13, wherein the device is adapted to deliver a dose of 10-250 pL.

15. Formulation for use in the treatment or prevention of vitamin B12 deficiency, Parkinson’s disease, Alzheimer’s disease or multiple sclerosis, said use comprising intranasally administering the formulation according to any one of claims 1-12.

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