JP2013227319A - Cyanocobalamin low viscosity aqueous formulation for intranasal delivery - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stable pharmaceutical aqueous solution of cyanocobalamin comprising cyanocobalamin and water, and a method for elevating a vitamin B12 level in a cerebral spinal fluid (CSF).SOLUTION: A pharmaceutical aqueous solution of cyanocobalamin is provided, which is suitable for intranasal administration, has a viscosity of less than about 1000 cPs, and yields a bioavailability of cyanocobalamin of at least about 5% when administered intranasally, relative to an intramuscular injection of cyanocobalamin. A method for elevating a vitamin B12 level in a cerebral spinal fluid (CSF) is provided, which comprises intranasally administering an aqueous solution of cyanocobalamin having a pH of 4 to 6, including citric acid, a sodium citrate swelling agent and an antiseptic, and results in at least 5% bioavailability relative to an intramuscular injection of cyanocobalamin.

Description

(Background of the invention)
Vitamin B12 is a food essential element, and its deficiency results in poor synthesis of DNA in any cell that undergoes chromosomal replication and division. The hematopoietic system is particularly sensitive to vitamin B12 deficiency because the tissues with the greatest cell rotation show the most dramatic changes. An early sign of B12 deficiency is megaloblastic anemia. Food B12 is released from food and saliva-binding protein in the presence of gastric acid and pancreatic proteases and binds to gastric factors. When the vitamin B12 endogenous factor complex reaches the ileum, it reacts with receptors on the mucosal cell surface and is actively transported into the circulation. Sufficient endogenous factors, bile and sodium bicarbonate (preferred pH) are all required for ileal transport of vitamin B12. Vitamin B12 deficiency in adults is rarely the result of an inadequate diet, but rather reflects defects in one or another aspect of this complex absorption chain. Hydrochloric deficiency and reduced endogenous factor secretion by mural cells associated with gastric atrophy or gastric surgery are common causes of vitamin B12 deficiency in adults. Antibodies against mural cells or endogenous factor complexes can also play a significant role in causing deficiency. Some bowel diseases can interfere with absorption. Vitamin B12 malabsorption results in pancreatic disorders (decreased pancreatic protease secretion
) Found in bacterial overgrowth, intestinal parasites, sprue, and local damage to ileal mucosal cells due to disease or as a result of surgery. The recommended daily intake of vitamin B12 in adults is 2.4μ
g.

Vitamin B _12, cyanocobalamin, hydroxocobalamin, there are four main forms of methylcobalamin and adenosyl cobalamin. Methylcobalamin and adenosylcobalamin are unstable and are damaged by light. Therefore, they are not essential because they are unsuitable for use in dietary supplements or pharmaceuticals and can be formed from cyanocobalamin or hydroxocobalamin in the body. The main form of vitamin B ₁₂ found in food is hydroxocobalamin. The main form used for therapeutic and dietary supplements is cyanocobalamin, which was chosen because it is the most stable form and therefore the easiest to synthesize and formulate.

In general, if vitamin B12 deficiency occurs because of a disruption in the transport mechanism of vitamin B12 endogenous factor complex that prevents vitamins from being absorbed into the small intestine, vitamin B12 must be absorbed systemically. Currently, therapeutic amounts of cyanocobalamin are administered by intramuscular or deep subcutaneous injection of cyanocobalamin. However, patients must visit the clinic regularly to receive further injections to maintain vitamin B12 levels. However, NASCOBAL®, a nasal gel cyanocobalamin formulation, is currently marketed and cyanocobalamin is administered intranasally as maintenance vitamin B12 therapy. However, many patients feel uncomfortable with the viscosity of the intranasal gel and prefer to administer a low viscosity spray containing cyanocobalamin intranasally.

To absorb a therapeutically effective amount of vitamin B12 in the nose, the concentration of B12 in the solution is greater than 1% by weight (Merkus, U.S. Pat.No. 5,801,161 (hereinafter referred to as `` Merkus '')) Must be administered intranasally with a viscous gel to remain (Wenig, U.S. Pat.No. 4,724,23
The prior art suggests (No. 1) (hereinafter “Wenig”). In fact, B12 administered intranasally in a low-viscosity solution must be in contact with the nasal mucosa for a long enough time to allow beneficial absorption.
enig says. Wenig argues that if the solution is low viscosity, most of B12 is wasted. Merkus has developed an intranasal formulation of hydroxocobalamin with a hydroxocobalamin concentration exceeding 1%, but hydroxocobalamin is not very stable and has a short shelf life. Hydroxocobalamin was chosen because cyanocobalamin is not soluble in aqueous solutions at concentrations above 1%.

Wenig's teaching is clear in that the subject disclosure does not teach an aqueous form of a cyanocobalamin formulation suitable and effective for intranasal administration as described below. Wenig clearly teaches the elimination of cyanocobalamin formulations with viscosities below the stated critical range of 2500-6000 cPs taught by Wenig. In particular, the nasal cyanocobalamin formulation “We can use a higher viscosity composition up to 10,000 cps, but will contain a sufficient amount of thickener to reach a viscosity of about 2500 to 6500 cps”. Teaches (column 2, lines 37-39, emphasis added)
. Wenig does not teach or disclose adjustments or selections for the lower viscosity nasal cyanocobalamin formulations disclosed by Applicants. In contrast, Wenig's disclosure teaches completely eliminating intranasal cyanocobalamin formulations with viscosities less than about 1000 cPs. The cyanocobalamin gel composition described in Wenig has a much higher viscosity in all possible embodiments. Thus, Wenig states that “an exemplary composition of the invention” is “about 450
0 cps "(column 3, lines 41-51). Each of the reported working embodiments presented by Wenig, whose viscosity values are shown and reported to be verified in terms of bioavailability by measuring plasma cyanocobalamin values, are 4000 cps and 3500-4000 cps, respectively. And 400
Having 0 cps (see, eg, Example 1; Formulations A, B, and C). As mentioned above, Wenig said, “Furthermore
It further emphasizes that higher viscosity compositions up to 10,000 cps can be employed.

Thus, there is no teaching or suggestion by Wenig to use an aqueous liquid (spray or drop) cyanocobalamin formulation instead of the intranasal gel formulation disclosed by Wenig. In addition, W
enig clearly teaches in the background section of the disclosure that non-gel (powder and aqueous) formulations of cyanocobalamin are not effective for intranasal administration to treat vitamin B12.
For aqueous formulations, Wenig cites, for example, the proposed isotonic sodium chloride aqueous solution of vitamin B12 reported by Monto et al. (Am. J. Med. Sci. 223: 113, 1953; Arch. In
t. Med. 93: 219, 1954). Wenig said that this solution, together with the powdered cyanocobalamin formulation,
He states that it is not effective for intranasal use to treat vitamin B12 deficiency and gives the following reasons:
Most of B12 goes directly into the throat. No contact with the nasal mucosa for a time sufficient to allow beneficial and uniform absorption. The majority of B12 so administered is actually wasted (first
Column, lines 63-68).

Thus, Wenig not only teaches to eliminate reducing the viscosity of nasal cyanocobalamin formulations to below the critical minimum range of 2500-6500 cps, but instead of the nasal gel formulation described by Wenig, an aqueous composition The possibility of using (spray or drops) is clearly denied. Wenig presents clear evidence and reasons that teach us to completely eliminate the proposed modification. In particular, an effective intranasal cyanocobalamin formulation has a critical parameter and Wenig that gives the formulation a “sufficient viscosity to maintain the formulation in the nasal passage for a long enough time that most of B12 is absorbed”. “We must have a sufficient amount of thickener to be about 2500 to 6500 cps (supra),” Wenig teaches (column 2, lines 24-29).
These properties are attributed to non-gels (liquids and liquids) that, according to Wenig, according to Wenig's clear teachings, cannot exert sufficient nasal mucosal residence time to achieve effective intranasal delivery / bioavailability of cyanocobalamin. Powder) directly contrasted with the properties of the cyanocobalamin composition.

In another attempt to develop a useful cyanocobalamin formulation for nasal administration, Garcia-Ari
eta et al., Biol. Pharm. Bull. 24: 1411-1416, 2001 (hereinafter “Garcia-Arieta et al.”) describes “Spray-Dried Powders as nasal absorption enhancers of cyanocobalamin.
as Nasal Absorption Enhancers of Cyanocobalamin) ”(title). The only disclosure by Garcia-Arieta et al. About optional cyanocobalamin sprays and drops is derived from a comparative experiment, which allows the authors to find that liquid (spray and drops) cyanocobalamin compositions are effective for intranasal use. It is reported clearly that there is no. More specifically, Garcia-A
rieta et al. demonstrated the bioavailability of three spray-dried nasal cyanocobalamin formulations along with two experimental cyanocobalamin nasal solutions (drops and sprays containing 0.1% cyanocobalamin; other formulation parameters not specified). Tested (page 1412, right column). Garcia showed that experimental nasal sprays and drops of cyanocobalamin did not show any detectable bioavailability.
-Arieta et al. Report clearly. As described in the left column on page 1415, Garcia-Arieta et al. Found the following from their experiments.
“Neither the nasal solution in the drops nor the nasal solution in the spray was able to increase the basal value of serum cobalamin to a statistically significant value in rabbits. This is because cyanocobalamin was administered without an absorption enhancer. This means that these formulations were not retained in the nostril for long enough to allow their absorption, either due to poor absorption in the nasal passages or lack of viscosity improvers. Emphasis added) ''

These reports describe the ineffectiveness of aqueous low-viscosity cyanocobalamin formulations that are in strict agreement with the above conclusion by Wenig. Thus, Wenig and Garcia-Arieta et al. Teach that a non-gel liquid intranasal formulation of cyanocobalamin was understood to be ineffective in achieving beneficial therapeutic results. Wenig describes a critical threshold viscosity above 2500 cps and a larger range of 3500-4500 cps for all proven working embodiments of useful nasal gel formulations of cyanocobalamin. Garcia-Arieta et al. Further show that simple 1% aqueous cyanocobalamin sprays and drops did not show detectable bioavailability.

In another attempt to provide a useful intranasal formulation for treating vitamin B12 deficiency, Slot et al., Gastroenterology 113: 430-433, 1997 (hereinafter “Slot et al.”) Includes a hydroxocobalamin intranasal formulation. Has been reported. In particular, Slot et al. Show that hydroxocobalamin is a preferred form of vitamin B12 for treating vitamin B12 deficiency, and cyanocobalamin is effective or practical for use in liquid nasal delivery formulations or methods. It is taught that it is not a therapeutic agent. Similar to Wenig described above, Slot et al. Expressed their opinions on the isotonic saline solution of cyanocobalamin already mentioned, “Neither of these proposals has been followed in clinical practice. It was extremely impractical. ”(Page 432, right column). Slot et al. Further teach eliminating the nasal cyanocobalamin formulations and methods for treating vitamin B12 by disclosing the following.
“Hydrocobalamin binds to plasma proteins more strongly than cyanocobalamin and has a longer half-life in the body. As a result, hydroxocobalamin is better retained in the body and therefore less frequently administered. Hydroxocobalamin is the best drug to ameliorate vitamin B12 deficiency because it is contraindicated in patients with tropical amblyopia and concurrent cigarette use, and in patients with pernicious anemia with optic neuropathy (page 432, right column). )

Another prior art document that teaches the elimination of the selection of cyanocobalamin as a useful form of cobalamin in intranasal formulations and methods is Merkus, USPN 5,801,161, supra. Similar to the teachings of Slot et al., Merkus has a hydroxocobalamin, a vitamin compared to cyanocobalamin.
It is specified that this is the preferred treatment for B12 deficiency. In the specific context of nasal preparations, Merkus emphasizes:
“The most effective concentration of vitamin B12 in formulations for nasal administration is higher than 1%. The maximum concentration that can be achieved with cyanocobalamin is about 1%. Concentrations above about 1% have good water solubility It can only be obtained with hydroxocobalamin for some reason, the solubility of the hydroxocobalamin substrate can reach 10%, which is up to about 10 times per unit volume when using hydroxocobalamin. It means that vitamin B12 can be administered and absorbed nasally (column 3, lines 43-53) "

Based on the foregoing teachings, those skilled in the art have found no practical motivation to develop low-viscosity cyanocobalamin formulations and methods, particularly as an effective therapeutic means for intranasal treatment to treat vitamin B12 deficiency. Become. In view of the prior art as a whole, the following cyanocobalamin formulations and methods have been developed and adopted to provide therapeutically effective delivery / biological utilization of cyanocobalamin sufficient to ameliorate vitamin B12 deficiency as disclosed herein There was simply no reasonable expectation that performance could be achieved smoothly. Wenig and Garcia-Arie
all of ta et al. teach the complete elimination of low viscosity liquid cyanocobalamin formulations. Wenig clearly teaches that the critical minimum viscosity for effective intranasal cyanocobalamin is 2500-4000 cps. Both Wenig and Garcia-Arieta et al teach that a non-gel liquid cyanocobalamin formulation will not be retained for a time sufficient to allow for nasal absorption. Garcia-Arieta et al. Specifically reported experimental results that no significant bioavailability was detected after nasal administration of a 1% simple aqueous cyanocobalamin formulation. Similarly with respect to the selection and concentration of cyanocobalamin as the active form of cobalamin used in intranasal liquid formulations, Slot et al. And Merkus also indicate that cyanocobalamin is not effective in intranasal formulations for treating vitamin B12 deficiency. It is taught collectively (independent of viscosity) that it is highly undesirable compared to hydroxocobalamin, or at best. Illustrating these teachings, Merkus emphasizes that:
“High and efficient nasal absorption of vitamin B12 is beneficial in medical treatment and can only be obtained by using hydroxocobalamin, which exhibits significantly higher water solubility than cyanocobalamin. Only by hydroxocobalamin is much higher. An excellent nasal composition in an aqueous medium can be produced that has a concentration of vitamin B12 and, consequently, nasal absorption of vitamin B12 is much more efficient. Less frequent and easier and cheaper to treat (column 2, lines 27-37) "

In other prior art relating to the general field of the invention, Deihl U.S. Pat.No. 4,525,341 discloses administering vitamins intranasally, but specific formulations containing only cyanocobalamin are described. Is not present or enabled.

International Patent Application No. PCT / US86 / 00665, in the Publication No. WO86 / 05987, nasal spray composition comprising vitamin B ₁₂ is disclosed as cyanocobalamin. However, although all of the specific spray formulations contained mercury as a preservative, the disclosure required the presence of mercury compounds. Other preservatives have also been mentioned including benzalkonium chloride and chlorobutanol. As noted above, NASCOBAL®, a nasal gel containing cyanocobalamin, is currently manufactured and marketed by Nastech Pharmaceutical Company Inc., Bothell, Washington. It is very effective in maintaining vitamin B12 levels for patients who have been deficient in the past but have recovered B12 levels through intramuscular injection. However, some patients feel uncomfortable with the consistency of the gel in the nose and prefer a nasal formulation that is less viscous and free of mercury compounds. Accordingly, there is a need to produce a pharmaceutically stable aqueous solution of cyanocobalamin having a low viscosity, optionally free of mercury compounds and sufficient bioavailability for use as a vitamin B12 maintenance therapy.

(Summary of the Invention)
The present invention is a stable pharmaceutical solution of cyanocobalamin suitable for intranasal administration having a viscosity of less than about 1000 cPs, said nasal solution of cyanocobalamin being at least 5 of the bioavailability of intramuscular injection of cyanocobalamin. %, In some embodiments, this need is met by providing a pharmaceutical solution having at least 6% or 7% bioavailability.

In an alternative embodiment, the therapeutically or pharmaceutically effective formulation of the present invention may consist of cyanocobalamin, citric acid, sodium citrate and water, with a viscosity of 1000 cPs.
And the solution of cyanocobalamin has a bioavailability of at least about 5%, in some embodiments, at least about 6% or 7% of the bioavailability of intramuscular injection of cyanocobalamin. In some embodiments, the solution of cyanocobalamin according to the present invention has a bioavailability of at least about 8%, 9%, 10%, 11% or 12% of the bioavailability of intramuscular injection of cyanocobalamin. Have. In some embodiments, the solution is substantially free of mercury and mercury-containing compounds.

Some compositions within the scope of the present invention will contain a wetting agent to inhibit mucosal drying and prevent irritation. Any of a variety of wetting agents can be used, including but not limited to sorbitol, propylene glycol or glycerol. An exemplary useful wetting agent is glycerin.

Preservatives are widely employed to extend the shelf life of the composition. Examples of preservatives include, but are not limited to, benzyl alcohol, chlorobutanol and benzalkonium chloride. In an exemplary embodiment, the preservative is benzalkonium chloride. A suitable concentration of preservative is 0.002% to 2.0% based on the total weight, but can vary considerably depending on the drug selected.

Typical formulation is 0.5% cyanocobalamin (percentage of total weight), 0.12% citric acid
Sodium citrate has a concentration of 0.32%, glycerin 2.23%, benzalkonium chloride 0.02% and water 96.79%. In another detailed embodiment, a nasal spray solution of cyanocobalamin in a spray applicator comprising 2.3 mL of 500 mcg / 0.1 mL solution of cyanocobalamin with sodium citrate, citric acid, glycerin and benzalkonium chloride in purified water I will provide a. A typical spray solution in this context has a pH of 4.5 to 5.5. After initial priming, a typical spray pump spray will deliver an average of 500 mcg of cyanocobalamin, and 2.3 mL of the spray solution contained in the bottle will deliver eight doses of nasal spray.

Another embodiment of the present invention is a method of administering cyanocobalamin comprising injecting an aqueous solution of cyanocobalamin into the nose, wherein the cyanocobalamin solution has a viscosity of less than 1000 cPs, said cyanocobalamin solution being a muscle of cyanocobalamin A method having a bioavailability of at least about 5% of bioavailability of internal injection, and in some embodiments, at least about 6% or 7%. In some embodiments, the solution of cyanocobalamin according to the present invention has a bioavailability of at least about 8%, 9%, 10%, 11% or 12% of the bioavailability of intramuscular injection of cyanocobalamin. Have. In a more detailed embodiment, the cyanocobalamin solution administered according to the method of the present invention is substantially free of mercury and mercury-containing compounds. In this context, “substantially free” generally refers to a solution in which a particular substance is less than 2% by weight, while in other embodiments the solution is less than 1% by weight, 0.05% by weight of the substance. Less than 0.01% by weight,
Or, for example, mercury or mercury-containing compounds are not completely present so that they cannot be detected in solution using conventional detection methods.

Furthermore, the present invention relates to the ratio of vitamin B12 in CSF to vitamin B12 in serum (B12C
A method to increase vitamin B12 levels in cerebral spinal fluid (CSF), including intranasally administering a solution of cyanocobalamin to raise the mean value of SF / B12 serum x 100) to at least about 1.1 The solution of cyanocobalamin relates to a method having a bioavailability of at least 7% for intramuscular injection of cyanocobalamin. In a more detailed embodiment, B1 such that the ratio of B12 in CSF to B12 value in serum is at least 1.9.
Increase 2CSF level.

The method of the present invention further comprises administering a cyanocobalamin solution as a spray to an individual's nose via an actuator chip, the spray having, in some embodiments, one or more of the following characteristics: The spray pattern ellipticity when measured at a height of 3.0 cm from the actuator chip is about 1.0 to about 1.4; or the spray produces droplets and less than about 5% of the droplets are less than 10 μm in size. The spray has a spray pattern major axis of about 35.3 mm and a minor axis of about 30.8 mm; 50% of the droplets produced by the spray are 26.9 μ
have a size of m or less; 90% of the droplets produced by spray have a size of 55.3 μm or less; or 10% of the droplets produced by spray have a size of 12.5 μm or less.

(Brief description of the drawings)
It is a figure which shows the nasal spray pump kit which has the actuator which contains the cyanocobalamin solution of this invention and does not interlock | cooperate. It is a figure which shows the nasal spray pump kit which has the actuator which interlock | cooperates including the cyanocobalamin solution of this invention and ejects the spray plume of the cyanocobalamin solution of this invention. It is a figure which shows the spray pattern produced | generated by the actuator of a spray pump kit.

(Detailed description of the invention)
The following definitions may be helpful in understanding the present invention.

“About” is taken to be a relative term that represents an approximate value of plus or minus 20% of the nominal value it points to. In the fields of pharmacology and clinical medicine and similar technologies that are the subject of this disclosure,
This approximate level is appropriate unless the value is a critical value or specifically specified to require a tighter range.

“Nasal mucosa”; the nasal mucosa is considered to be the lining of the nasal entrance that is vascularized and extends inward toward the boundary between the central pharynx and the cavity.

“Aqueous” refers to a solution formed with water, but may contain smaller amounts of other co-solvents.

“Bioavailability” is defined as the rate and extent to which an active ingredient or active moiety is absorbed from a drug product and made available at the active site [21 CFR §
320.1 (a)].

“Bioavailability of intranasal spray for intramuscular injection of cyanocobalamin” means the percentage of the amount of intranasal dose absorbed into the systemic vasculature compared to the same amount of cyanocobalamin injected. For example, assuming that an intramuscular injection of a cyanocobalamin solution containing 100 μg cyanocobalamin has 100% bioavailability, an intranasal cyanocobalamin dose of 100
containing 5 μg, 6 μg, or at least about 6% or 7% of the bioavailability of intramuscular injection of cyanocobalamin, in some embodiments,
7 μg of cyanocobalamin will be absorbed by the vascular system. Similarly, if the intranasal administration of cyanocobalamin contains 500 μg, at least 35 μg of cyanocobalamin will be absorbed into the vasculature if the intranasal formulation has at least 7% bioavailability. .

`` Stability '': measured at a parameter during storage, examples of which include concentration, degradation, viscosity, pH or particle size that may significantly affect the quality attributes of the product over time, including Any non-limiting composition change refers to instability. Similarly, changes that are not expected to significantly affect the quality attributes of the product refer to stability. The time to measure stability is relative depending on the intended use of the composition. Accelerated stability at higher temperatures may be viewed as a faster method of inferring stability over a longer time than is actually measured.

“Pharmaceutically acceptable”: refers to a composition that, when administered to a human or mammal by the designated route of administration, does not cause adverse reactions disproportionate to the benefits obtained by administration of the compound.

“Mammal” includes any of the warm-blooded, more advanced vertebrates that nourish the pups with milk secreted by the mammary glands and usually have some hair-covered skin, including humans and non-human primates For both male and female neonates and young adults, including domestic animals such as horses, cattle, sheep and goats, and research and domestic animal species including dogs, cats, mice, rats, guinea pigs and rabbits Includes non-exclusively. As used herein, “patient” or “subject”
Used interchangeably with "mammal".

“Intranasal administration” refers to the delivery of a drug primarily through the mucosa of the nasal cavity. This is the top,
Includes the middle and lower turbinates and nasopharynx. Note that the olfactory region is concentrated in the upper part of the nasal concha (upper 1/3). The ciliary action pushes the substance back to the center of the pharynx so that the substance deposited at the nasal entrance enters the throat after encountering the nasal mucosa.

“Substantially free” refers to the amount of a particular active ingredient in the composition of the invention, wherein the particular active ingredient is less than 20% by weight, preferably 10% by weight relative to the total amount of active ingredient in the composition. %, More preferably less than 5% by weight, most preferably less than 1% by weight.

Delivery vehicles herein that are considered useful include actuator dispensers widely used for nasal solutions and gels. Embodiments of this technology include multiple dispensers, single dose dispensers, metered dose dispensers, pediatric safety dispensers, disposable dispensers and kits thereof.

As used herein, “maximum concentration of cyanocobalamin in plasma (C _max )”,
“Cyanocobalamin concentration in plasma versus area under time curve (AUC)”, “Time of vitamin in plasma vs maximum plasma concentration (t _max )” are pharmacokinetic parameters known to those skilled in the art [[Laursen Et al., Eur. J. Endocrinology, 135: 309-315, (1996)]. A “concentration versus time curve” is a measure of the concentration of cyanocobalamin in the serum of a subject after administration to the subject of a cyanocobalamin administration by intranasal, subcutaneous or other parenteral administration route. "
“C _max ” is the maximum concentration of cyanocobalamin in the serum of a subject after a single dose of cyanocobalamin to the subject. The term “t _max ” is the time to reach the maximum concentration of cyanocobalamin in the subject's serum after administration of a single dose of cyanocobalamin to the subject.

As used herein, “Cyanocobalamin concentration in plasma versus area under time curve (AUC)” is calculated according to the linear trapezoidal rule and by adding the remaining areas. A 23% decrease or 30% increase between the two doses will be detected with a 90% probability (type II error β = 10%). By comparing the time (t _max ) to reach the maximum concentration (C _max ), the “delivery rate” or “
Estimate the "absorption rate". Analyze both C _max and t _max using non-parametric methods. subcutaneous,
Comparison of pharmacokinetics of intravenous and intranasal cyanocobalamin administration was performed by difference analysis (ANOVA). For one-to-one comparisons, Bonferroni-Holmes sequential treatment was used to assess significance. The dose response relationship of the three nasal doses was estimated by regression analysis. P <0.05 was considered significant.
Results are shown as mean ± SEM (Laursen et al., 1996).

The cyanocobalamin solution is designed to be administered to the nasal mucosa in the form of drops or sprays. However, the preferred mode of administration is in spray form, i.e. in the form of fine droplets. An example of a suitable spray pump is Pfeiffer Spray manufactured by Pfeiffer GmbH (Radolfzell, Germany)
Pump Model # 63385.

(Nasal administration of cyanocobalamin)
Cyanocobalamin is administered intranasally using a nasal spray according to the present invention. The following definitions are useful in this field.

Aerosols-Products containing therapeutically active ingredients that are packaged under pressure and released upon activation of the appropriate valve system.

Metered aerosol-A pressurized dosage form consisting of a metered dose valve that allows delivery of a uniform amount of spray per activation.

Powder aerosol-A product containing a therapeutically active ingredient in the form of a powder that is packaged under pressure and released upon activation of a suitable valve system.

Spray aerosol-An aerosol product that utilizes a compressed gas as a propellant that provides the necessary force to deliver the product as a wet spray. The present invention can be widely applied to a solution in which a medicine is dissolved in an aqueous solvent.

  Spray-A liquid that is subdivided by a jet of air or water vapor.

Metered spray-a non-pressurized dosage form consisting of a valve that allows a specified amount of spray to be dispensed with each activation.

Suspension spray-a liquid formulation containing solid particles dispersed in a liquid medium and in the form of coarse droplets or as a fine solid.

Rheological characterization of aerosol sprays delivered by metered nasal spray pumps as drug delivery devices (“DDD”). Spray characterization determines the legal compliance required for US Food and Drug Administration (`` FDA '') approval for research and development, quality assurance and stability testing procedures for new and existing nasal spray pumps. It is an important part.

Full characterization of the spray geometry has been found to be the best indicator of the overall performance of a nasal spray pump. In particular, measurements of spray opening as it exits the device (plume geometry); cross-sectional ellipticity of spray, uniformity and particle / droplet distribution (spray pattern); It turned out to be the most representative performance value in characterization of nasal spray pumps. Through quality assurance and stability testing, plume geometry and spray pattern measurements are key identifiers for verifying consistency and conformance with approved data standards for nasal spray pumps.

(Definition)
Plume height—Measured from the actuator tip to the point at which the plume angle becomes nonlinear due to the breaking of the linear flow. Based on visual inspection of the digital image and to establish a measurement point with a width that coincides with the farthest measurement point of the spray pattern, this test defines a height of 30 mm.

Long axis-the largest string that can be drawn in a fixed spray pattern that intersects COMw in basic units (mm).

Short axis-the smallest string that can be drawn in a fixed spray pattern that intersects COMw in basic units (mm).

  Ellipticity-Ratio of major axis to minor axis.

D _10- Droplet diameter (μm), where 10% of the total liquid volume of the sample consists of smaller diameter droplets.

D _50- droplet diameter (μm), where 50% of the total liquid volume of the sample consists of droplets of smaller diameter,
Also known as the mass center diameter.

D ₉₀ —Drop diameter (μm), where 90% of the total liquid volume of the sample consists of smaller diameter droplets.

Span-a measure of the distribution width. The smaller the value, the narrower the distribution. The total length is calculated by the following formula.

% RSD-percent relative standard deviation. Divide the standard deviation by the average of the sequence and multiply by 100, also known as% CV.

  Cyanocobalamin nasal spray kit

The present invention further comprises a cyanocobalamin nasal spray kit and a method of administering a cyanocobalamin solution using the nasal spray kit.

The nasal spray kit is illustrated in FIGS. 1A, 1B and FIG. Figures 1A and 1B are before production (Figure 1A)
And shows the nasal spray device 10 after operation (FIG. 1B). Cyanocobalamin nasal spray kit 10 comprises a container, in this case a bottle 12 in which a cyanocobalamin preparation is placed, as well as bottle 12
And an actuator 14 that fluidly connects with the solution of cyanocobalamin in the bottle 12. Actuator 14, when activated or activated, passes cyanocobalamin spray plume 16 through actuator chip 15. The spray plume is composed of droplets of a solution of cyanocobalamin. The spray pattern is measured by taking a picture of a cross section of the spray plume 16 that exceeds the predetermined height of the plume. Spray plume actuator
When exiting 14, it has an emission angle of 20. The spray pattern of the spray plume 16 is shown in FIG. The spray pattern of FIG. 2 is elliptical and has a major axis 24 and a minor axis 26.

In one exemplary embodiment, the actuator is 3.0 cm from the actuator chip.
Produces a spray of cyanocobalamin solution having a spray pattern ellipticity of about 1.0 to about 1.4 when measured at a height of. In some embodiments, less than 5% of the droplets of the cyanocobalamin solution are less than 10 μm in size and the spray pattern is 25 mm and 40 mm, respectively.
With 50% of the droplets being 26.9μm or less, 90% of the droplets being 55.3μm or less, and 10% of the droplets being 12.5μm or less is there.

As noted above, the present invention provides improved methods and compositions for intranasal administration of cyanocobalamin to mammalian subjects for the treatment or prevention of various diseases, disorders and conditions. Examples of suitable mammalian subjects for treatment and prevention according to the methods of the present invention include human and non-human primates, domestic animal species such as horses, cows, sheep and goats, and dogs, cats, mice, rats, This includes, but is not limited to, research and domestic animal species, including guinea pigs and rabbits.

In an exemplary treatment method, the cyanocobalamin nasal spray of the present invention can be directed to maintaining the blood state of a sedated patient following intramuscular vitamin B12 treatment. For example, initial therapeutic dosing can include receiving daily intramuscular injections of 100 μg cyanocobalamin over a period of about 1 to 2 weeks with 1 to 5 mg folic acid. Intramuscular injections of cyanocobalamin do not exceed 100 μg because doses exceeding 100 μg are rapidly excreted from plasma into the urine, and larger doses of vitamin B12 do not retain longer amounts of vitamins And Treatment with the methods and compositions of the present invention can be performed after the initial treatment process as follows.

Instead of injecting 100 μg cyanocobalamin once a month, using the cyanocobalamin spray described herein, a patient may receive a nasal spray dose containing, for example, 500 μg cyanocobalamin once a week or Self-administer twice. The maintenance treatment of nasal cyanocobalamin addresses any patient diagnosed with vitamin B12 deficiency, but especially the diet of vitamin B12 that occurs in pernicious anemia, thorough vegetarians, so-called vegetarians who do not eat so-called animal products Respond to patients being treated for sexual deficiency. The maintenance cyanocobalamin treatment using the cyanocobalamin solution of the present invention is for patients suffering from vitamin B12 malabsorption due to structural or functional damage of the stomach where endogenous factor is secreted or endogenous factor promotes B12 absorption Is also suggested. These conditions include tropical and non-tropical sprue (pediatric steatosis,
Gluten-induced enteropathy).

Maintenance cyanocobalamin treatment using the cyanocobalamin solution of the present invention can result in inadequate secretion of endogenous factors, lesions that destroy the gastric mucosa (ingestion of corrosives, large tumor formation), and variable degrees of gastric atrophy. Several conditions involved (multiple sclerosis, human immunodeficiency virus (HIV) infection,
It is also suggested for patients suffering from vitamin B12 malabsorption due to certain endocrine disorders, iron deficiency and partial gastrectomy. Structural lesions that lead to B12 deficiency include ileitis, ileectomy, Crohn's disease and malignancy. Vitamin B12 deficiency can also be the result of intestinal parasite competition and insufficient utilization of vitamin B12 that occurs when antimetabolites to vitamins are employed to treat neoplasms.

The intranasal cyanocobalamin solution of the present invention is used for individuals who need a higher amount of vitamin B12 than usual due to, for example, pregnancy, hyperthyroidism, hemolytic anemia, massive bleeding, malignancy, liver disease and kidney disease You can also

As described above, the present invention is suitable for intranasal administration and is a stable pharmaceutical solution of cyanocobalamin having a viscosity of less than about 1000 cPs, wherein the intranasal cyanocobalamin solution comprises:
When administered intranasally, at least 5%, 6% of the bioavailability of intramuscular injection of cyanocobalamin
Alternatively, a cyanocobalamin solution having 7% bioavailability is provided. Intranasal formulations often consist of water and cyanocobalamin plus a buffer to maintain the pH between 4 and 6, preferably about 5, an optional wetting agent to prevent mucosal drying, and an optional preservative. Will be.

In some exemplary embodiments, the composition according to the invention consists of cyanocobalamin, citric acid, sodium citrate and water, the viscosity is less than 1000 cPs, and the solution of cyanocobalamin is for intramuscular injection of cyanocobalamin. At least 5%, 6% of bioavailability or
7%, in some embodiments, at least about 8%, 9%, 10%, 11%, or 12% bioavailability.

In some embodiments, the compositions of the present invention will contain a wetting agent to inhibit mucosal drying and prevent irritation. Any of a variety of wetting agents can be used including sorbitol, propylene glycol or glycerol. A typical wetting agent is glycerol.

Preservatives are widely used to extend the shelf life of the composition. Examples of the preservative include benzyl alcohol, parabenztimerosal, chlorobutanol, benzethonium chloride and benzalkonium chloride. An exemplary preservative useful within the formulations and methods of the present invention is benzalkonium chloride. A suitable concentration of preservative is 0.002% to 2% based on the total weight, but there may be considerable differences depending on the drug selected.

In some exemplary embodiments, a formulation according to the invention comprises 0.5% cyanocobalamin (percentage of total weight), 0.12% citric acid, 0.32% sodium citrate, 2.2% glycerin.
It has a concentration of 3%, benzalkonium chloride solution 0.02% and water 96.79%.

Other buffer combinations useful within the scope of the present invention include, but are not limited to, monopotassium phosphate and disodium phosphate; potassium hydrogen phthalate and sodium hydroxide; and sodium acetate and acetic acid. .

Another embodiment of the present invention is a method of administering sialobalacin comprising injecting an aqueous solution of cyanocobalamin into the nose, said cyanocobalamin solution having a viscosity of less than 1000 cPs, said cyanocobalamin solution being intramuscular of cyanocobalamin. At least about compared to injection
A method with 5%, 6% or 7% bioavailability. In some embodiments, the bioavailability of the cyanocobalamin solutions of the present invention is at least about 8%, 9%, 10%, 11% and 12 compared to the bioavailability of intramuscular injection of cyanocobalamin. Up to% or more.

Furthermore, the present invention relates to the ratio of vitamin B12 in CSF to vitamin B12 in serum (B12C
A method to increase vitamin B12 levels in cerebral spinal fluid (CSF), including intranasally administering a solution of cyanocobalamin to raise the mean value of SF / B12 serum x 100) to at least about 1.1 The solution of cyanocobalamin relates to a method having a bioavailability of at least 7% for intramuscular injection of cyanocobalamin. In some embodiments, B12CSF has a ratio of B12 in CSF to B12 value in serum of at least 1: 9.
Increase value.

Since vitamin B12 deficiency can result in irreversible damage to the nervous system, this is a significant embodiment of the present invention. Progressive swelling of myelinated neurons, demyelination, and neuronal cell death are found in the spinal column and cerebral cortex. This includes a wide range of neurological signs and symptoms, including paralysis of the limbs, reduced vibrations and positional sensations, reduced deep tendon reflexes, and confusion, moodiness, memory loss, and even central vision loss in later stages. cause. Patients can exhibit delusions, hallucinations or even overt mental illness. Because neurological disorders can be separated from hematopoietic changes, vitamin B12
Deficiency should be seen as one possibility in older patients with dementia and mental disorders, even if they are not anemic. Thus, embodiments of the invention relating to increasing vitamin B12 levels in CSF can have significant advantages for neurological patients. Therefore, intranasal administration of vitamin B12 according to the present invention can be used to treat diseases such as Alzheimer's disease, dementia and multiple sclerosis.

Exemplary formulations of the present invention include:
Cyanocobalamin nasal spray 500mcg / 0.1mL
Formulation:

Alternative buffer systems and amounts that can be used in cyanocobalamin nasal sprays

Any spray bottle or syringe can be used to administer the intranasal formulation of the present invention. A preferred nasal spray bottle delivers a 0.1 mL dose per jet and has a dip tube length of 36.05 mm “nasal spray pump w / safety clip, Pfeiffer SAP # 605
48 ”. It can be purchased from Pfeiffer of America in Princeton, New Jersey.

The following examples are given for purposes of illustration and not limitation.
(Example 1)
Comparison of intranasal cyanocobalamin solution of the present invention with intramuscular injection of NASCOBAL® and cyanocobalamin
(Introduction)

Nascobal® (cyanocobalamin, USP) is a synthetic form of vitamin B ₁₂ that has comparable vitamin B ₁₂ activity. The chemical name is 5,6-dimethyl-benzimidazolyl cyanocobamide. Currently, Nascobal® (cyanocobalamin, USP) is marketed as a self-administered nasal gel. The recommended dose of Nascobal® (cyanocobalamin, USP) is 5 once a week for subjects with poor absorption of vitamin B ₁₂ who are sedated after injectable vitamin B ₁₂ treatment.
00 μg is administered intranasally.

Vitamin B ₁₂ deficiency, there are several causes, including dietary deficiency malabsorption and vitamin B ₁₂ Vitamin B ₁₂ due to structural or functional damage to the gastrointestinal system.

The purpose of this study was to compare the bioequivalence of vitamin B12 nasal gels and nasal sprays, as well as the relative organisms of the three formulations of vitamin B12 in the fasted state of normal healthy male and female subjects. Is to evaluate the availability.

Intranasal cyanocobalamin gel is approved for a dose of 500 μg. Current studies also utilize the same 500 μg dose of cyanocobalamin nasal spray and 100 μg intramuscular dose.

(Purpose of the test)
Comparing a single intranasal administration sprays, single intranasal gel (Nascobal (R)) and pharmacokinetic profile of single intramuscular administration of vitamin B ₁₂ in the fasting state of a subject of normal healthy male and female .

(Survey plan)
Overall study design and designThis study included the following: Intranasal (IN) spray (500 μg), IN gel (Nascobal®) (500 μg) and fasting normal healthy men and women single site of vitamin B ₁₂ is administered via intramuscular (IM) injection (100 [mu] g), open label, three-way (3 therapeutics, 6 series) was crossed pharmacokinetic studies.

Treatment A: 1 single IN spray administration of vitamin B ₁₂ of 500 [mu] g. The intranasal formulation is comprised of an exemplary embodiment of the present invention and is composed of cyanocobalamin, citric acid at a concentration of 0.5% (percentage of total weight).
0.12%, sodium citrate 0.32%, glycerin 2.23%, 50% benzalkonium chloride solution 0.04
% And 96.79% water.

Therapeutic B: 500 μg vitamin B ₁₂ once in an IN gel (Nascobal®)

Treatment C: 1 single IM administration of vitamin B ₁₂ of 100μg.

Subjects were given a diet without vitamin B ₁₂ throughout each restraint period. Administer next treatment 14 days after administration of previous treatment.

(Therapeutic)
The drug to administer
In one of the 6 series, a single IN spray of 500 μg vitamin B ₁₂ (therapeutic agent) on the first day of periods I, II and III after an 8-hour fast, based on the randomization generated by PPD Development Biostastician A), 500 μg vitamin B ₁₂ single IN gel (Nascobal) ® (therapeutic agent B) or 100
Subjects were given a single IM dose of μg vitamin B ₁₂ (therapeutic agent C). After all periods, all subjects were given each treatment in a cross. A 14-day washout period divided the three dosing periods.

To 1 day in the morning, the subject therapeutic agent A is assigned a single IN spray administration of vitamin B ₁₂ of 500μg was given. Subjects to whom therapeutic B is assigned are 500 μg vitamin B ₁₂ single IN
A gel dose (Nascobal®) was given. Subjects who are assigned therapeutic C are 100μ
A single IM dose of g vitamin B ₁₂ was given. Overnight (ie at least 8 hours) prior to administration
Fasted (excluding water) and fasted for at least 4 hours after administration (excluding water).

While restrained in the clinical setting, subjects were given a standardized vitamin B ₁₂ deficient diet at scheduled times consistent with other study-related activities. A registered dietitian set up a meal and the food staff kept a meal diary. Dietary supplements were not allowed during the study. Subjects refrained from eating alcohol-containing, grapefruit-containing or caffeine-containing foods or beverages for 72 hours prior to check-in.

(Research variables)
For each subject, as far as possible, model-independent approaches, i.e. C _max , T _max and AUQ _{0
According to -t} , the following pharmacokinetic parameters were calculated based on vitamin B ₁₂ serum concentrations of therapeutic agents A, B and C.

(Pharmacokinetic measurements)
Blood samples for PK analysis of vitamin B ₁₂ values are collected via an indwelling catheter and / or direct venipuncture using a 5 mL yellow top Vacutainer® Hemogard® vacuum serum separator collection tube did. Blood sample for PK analysis of vitamin B ₁₂ levels 0, 6 on day 1
And 12 hours and 0 hours after the first day (i.e. before administration); 30 minutes after; 1, 1.5, 2 after administration for each period
4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84 and 96 hours later.

(Relevance of measured values)
The pharmacokinetic parameters used in this study were typically those used to assess bioequivalence. All assessments of bioequivalence are AUC _0-t , T _max and C _m
Based on comparison of _ax (study and baseline treatment).

AUC measures the range of drug bioavailability and reflects the total amount of drug that reaches the systemic circulation.

C _max represents the maximum serum concentration obtained after drug administration, indicating that sufficient drug has reached the systemic circulation to give a therapeutic response. In addition, C _max indicates a warning of the expected toxic drug amount.

T _max was calculated and expressed as median ± range.

(Pharmacokinetic variables)
For each subject, whenever possible, follow the model-independent approach (criteria 1)
The following pharmacokinetic parameters were calculated based on vitamin B ₁₂ serum concentrations of B, C and C:

C _max maximum observed concentration

t _max Time vs. maximum concentration

Area under the concentration-time curve from time 0 to the final measurable concentration, calculated using the AUC _0-t linear trapezoidal rule

  Pharmacokinetic calculations were performed using SAS (SAS Inst., Version 8.02).

(Measurement of statistical methods and sample sizes planned in the protocol)
Vitamin B ₁₂ value in statistical and analytical plans / pharmacokinetic analysis serum samples were measured in pg / mL. Serum concentration values below the quantifiable detection limit were treated as zero. The actual sampling time was used instead of the planned sampling time in all calculations of pharmacokinetic parameters. However, for ease of display, the results were represented in tables, lists and figures using planned sampling times.

Sectional non-compartmental pharmacokinetic parameters (AUC _0-t , C _max and T _max ) from concentration data
Calculated as described in 8.4.3.

(Statistical analysis)
All statistical tests were performed at the 0.05 significance level, unless otherwise specified. Summary statistics for continuous parameters consisted of number (N), mean, median, SD and range.

Descriptive statistics were obtained and summarized in a table by treatment for vitamin B ₁₂ levels and calculated pharmacokinetic parameters at each time point.

Bioequivalence was evaluated for the test (therapeutic agent A-nasal spray) versus the criterion (therapeutic agent B-gel). A difference analysis (ANOVA, Criterion 2) was performed to generate a 90% confidence interval for the test / reference ratio. C _m
ax and AUC _0-t were natural logarithms (log _e ) converted before analysis. The corresponding 90% confidence interval for the geometric mean ratio was determined by taking the true value of the 90% confidence interval for the difference between the logarithmic mean values.

log _{e -} if the lower limit of the 90% confidence interval from conversion C _max and AUC _0-t is 80% or higher
Was assumed not to be inferior to the standard (therapeutic agent B). log _e -from conversion C _max and AUC _0-t
It was assumed that non-inferiority could not be determined if the lower limit of the 90% confidence interval was less than 80%.

The sequence effect was tested using the root mean error (MSE) for the object in the sequence as the error term.
All other main effects were tested against MSE from ANOVA model.

Bioavailability was evaluated for the study (therapeutic agents A and B—nasal spray and gel, respectively) and the reference (therapeutic agents C-IM) groups. Standard (therapeutic agent C) group average test (therapeutic agent
A and B) Relative bioavailability was assessed by examining the 90% confidence interval for the ratio of group means.

To determine if there was a difference between the test group and each reference group, T _max was analyzed using the Wilcoxon paired pair method.
Summary-Conclusion Pharmacokinetic study results:
The relative bioavailability for the two IN formulations was 0.9715. Bioavailability is
When comparing therapeutic agent A (spray) and therapeutic agent C (IM), it was 0.6105, and when comparing therapeutic agent B (gel) and therapeutic agent C (IM), it was 0.6284.
The pharmacokinetic profiles of spray and gel formulations are C _max (1480 pg / mL, 1670 respectively
pg / mL) and AUC _0-t (92000 pg * hr / mL, 97000 pg * hr / mL, respectively).
The median difference in T _max between the spray and gel IN formulations was less than 15 minutes (-0.24). C _max values for the IM formulation was significantly greater than the C _max value of the two IN formulations (p <0.0001).
For gel data based on C _max and AUC _0-t , bioequivalence for vitamin B ₁₂ IN spray was established. Log _e -conversion C _max and AUC _0-t for spray and gel formulations
The 90% confidence interval was in the range of 80% to 125%. In addition, the lower bound of the confidence interval is AUC _0-t and C _m
Since it exceeds 80% for both _ax , non-inferiority can be assumed when comparing the two IN formulations.
Conclusion:
The relative bioavailability for the two IN formulations was 0.9715. Bioavailability is
It is 0.6105 for therapeutic agent A (spray) versus therapeutic agent C (IM), therapeutic agent B (gel) and therapeutic agent C (IM)
Was 0.6284.
The pharmacokinetic profiles of spray and gel formulations are C _max (1480 pg / mL, 1670 respectively
pg / mL) and AUC _0-t (92000 pg * hr / mL, 97000 pg * hr / mL, respectively).
The median difference in T _max between the spray and gel IN formulations was less than 15 minutes (-0.24). C _max values for the IM formulation was significantly greater than the C _max value of the two IN formulations (p <0.0001).
The log _e -conversion 90% confidence interval for AUC _0-t and C _max was used to establish bioequivalence between the vitamin B ₁₂ spray formulation and the vitamin B ₁₂ gel formulation. The 90% confidence interval for log _e -converted C _max and AUC _0-t for spray and gel formulations was in the range of 0.8 to 1.25. Non-inferiority can be assumed for the two IN formulations (therapeutic agent A and therapeutic agent B).
Both vitamin B ₁₂ formulations were safe and well tolerated by healthy male and female partners.

As noted above, in some embodiments of the present invention, the minimum relative or comparative bioavailability of the nasal cyanocobalamin formulations of the present invention (e.g., suitable where a placebo or placebo formulation is administered) (In parallel test subjects administered equivalent doses of intramuscular cyanocobalamin or nasal cyanocobalamin), tested for plasma or CSF concentrations of cyanocobalamin relative to the control subject) is a bioavailability achieved by intramuscular injection. At least 5%, 6% or 7%,
In some cases 8%, 9%, 10%, 11% or 12% or more.

The present disclosure provides detailed comparative bioavailability studies and results to demonstrate these unexpected performance characteristics of the methods and compositions of the present invention. As mentioned above, two typical intranasal (
The bioavailability for the IN) formulation was 0.9715. Relative bioavailability is therapeutic A
It was 0.6105 when comparing (spray) and therapeutic agent C (intramuscular = IM), and 0.6284 when comparing therapeutic agent B (gel) and therapeutic agent C (IM). The pharmacokinetic profiles of the cyanocobalamin spray and gel formulations compared are C _max (1480 pg / mL and 1670 pg / mL, respectively) and AUC _0-t (
9200 pg * hr / mL and 9700 pg * hr / mL), respectively. The median difference in T _max between the spray and gel IN formulations was less than 15 minutes (-0.24). The C _max value for the IM formulation is 2
It was significantly greater than the C _max value of the IN formulation (p <0.0001).

These data show a comparative AUC for cyanocobalamin IN vs. IM bioavailability.
Although not directly expressed in the form of values, the relative AUC values can be easily and accurately derived from the data shown above. In particular, the results of the comparative bioavailability studies shown above are representative spray cyanocobalamin formulations for the bioavailability of IM cyanocobalamin, and
The `` relative bioavailability '' of a typical gel formulation for IM bioavailability is 0, respectively.
Proving that it is 6105 and 0.6284. These values represent the ratio of the natural logarithm of the geometric mean value of AUC based on the nominal dose. These data were normalized for dose according to conventional methods for appropriate multiple doses based on doses of 500 μg administered intranasally and 100 μg administered by IM. One of ordinary skill in the art can easily comprehend these data and the dose normalization data reasonably corresponds to at least about 5%, 6% or 7% of the disclosed relative minimum relative bioavailability. It will be appreciated that the bioavailability ratio between the IN cyanocobalamin solution of the present invention and IM administration is derived. This measurement can be made by standard mathematical processing to derive a relative AUC value normalized for dose to IN spray and IM injection.
In the examples shown above, this standard treatment / result is 0.6105 × 100 μg / 500 μg × 100 = 12%;
Or the ratio of AUC between IN spray and IM injection is 0.12. In addition, the actual arithmetic AUC is shown above as 92000 and 97000 pg * hr / mL for representative IN cyanocobalamin sprays and gels, respectively. These data similarly prove the corresponding AUC for the IM injection study comparator according to the present disclosure. For example, the arithmetic average of AUC for IM is 1471
Calculated as 55 pg * hr / mL (easily derived from inverse ratio processing from the ratios shown above-for example, 92000/147155 = 0.62 ratio for spray). When normalized for dose, these data indicate that the IN of the present invention as compared to the bioavailability of IM cyanocobalamin.
Directly that a typical relative bioavailability value for a cyanocobalamin formulation is at least 7%, and in other embodiments at least 9%, 10%, 11% or 12% or more within the ranges described herein. Correspond.

(Example 2)
After intranasal and intramuscular administration in women co-workers that are not healthy male and pregnancy vitamin B ₁
The results of an open-label single-dose parallel group study to compare _two cerebrospinal fluid (CSF) uptake are also shown by the present disclosure. In this study, CSF values were compared with plasma values produced by both formulations.

Thirty-six healthy men and women who were not pregnant were enrolled in the study. 18
One human subject received a 500 mcg single intranasal dose delivered as a 0.1 mL spray and 18 subjects received a 100 mcg single intramuscular dose delivered intramuscularly. Each subject visited the clinical site three times during a one month period. These visits consisted of screening visits, single-dose visits, and final visits.

After each administration, each subject received only one lumbar puncture and a total of 4.0 mL CSF (4 tubes, 1.0 mL per tube) was collected. One third of subjects collected CSF samples 60 minutes after administration, one third of subjects collected CSF samples 90 minutes after administration, and one third of subjects received CSF 120 minutes after administration.
A sample was collected.

In addition to the above, on the day of administration, before administration, 5, 10, 15 and 20 minutes after administration, and 0.5
1, 1.5, 2, 3, 4, 6 and 8 hours (before drainage), 7 mL blood samples were collected.

The evaluation of the cerebrospinal fluid was carried out for all the vitamin B ₁₂ content. The purpose of the studies described herein was to determine the amount of vitamin B ₁₂ present in the blood and CSF after intramuscular (IM) and intranasal administration.

(Standards and test products)
Reference product: Cyanocobalamin 100mcg intramuscular injection

Cyanocobalamin injection, USP, is a sterile solution of cyanocobalamin (vitamin B ₁₂ ) for intramuscular or subcutaneous injection. Every 1mL contains 1000mcg cyanocobalamin.

Test product: Vitamin B ₁₂ nasal spray = 500mcg / 0.1mL spray. The nasal aqueous solution of cyanocobalamin in this study is 0.5% (percentage of total weight) of cyanocobalamin, citric acid 0.12%, sodium citrate 0.32%, glycerin 2.23%, 50% benzalkonium chloride solution
It contained 0.04% and 96.79% water.

One dose: Vitamin B ₁₂ nasal spray was supplied as a 2.3 mL bottle to deliver 500 mcg / 0.1 mL per dose.

Prior to intranasal administration, all subjects were instructed on proper dosing techniques and the overall conduct of the study.

Subject was instructed to bite his nose gently. The subject remained seated and a primed IN applicator was inserted into the nostril by the subject under the direction of the research staff. During dosing, the contralateral nostril was blocked with an index finger. Subjects were instructed to tilt their head slightly backwards for dosing and return their heads to an upright position while breathing in immediately after dosing. According to this protocol, and release vitamin B ₁₂ spray dose of 0.1mL in nasal cavity of each object (administration thereof is a single coated product for one nostril). Subjects were instructed not to blow their nose for 1 hour after IN treatment.

After administration, each subject received a lumbar puncture and 4.0 mL CSF (4 tubes, 1.0 mL per tube)
Was recovered. One-third of the subjects in each group collected CSF samples 60 minutes after dosing, one-third of subjects collected CSF samples 90 minutes after dosing, and one-third of subjects received 120 minutes Later CSF
A sample was collected. At the appropriate time after administration, the examiner placed the patient in a position suitable for lumbar puncture. The waist was prepared and covered with a normal sterile cloth. Local anesthesia was utilized (1% xylocaine, 1-5 mL). When sufficient anesthesia was reached, the spinal needle (20 or 22G) was introduced into the spinal canal at a level deemed appropriate by the examiner. CSF samples were collected 60, 90 or 120 minutes after administration. A total of 4.0 mL of CSF was collected from each patient and distributed into four separate collection tubes. The tube was appropriately labeled with the patient identifier and subjected to bioanalysis. When CSF recovery was complete, the spinal needle was removed.

With vitamin B ₁₂ concentrations were measured vitamin B12 values in both the CSF and serum in CSF, it is analyzed to determine the vitamin B ₁₂ using a validated for TOSOH Nex.1A procedures.

(Results and conclusions)
The ratio of vitamin B12 to serum was higher in individuals who received nasal vitamin B12.
Data showed that it was larger than individuals who received an intramuscular injection of B12.

The average ratio (B12 CSF / B12 serum x 100) for individuals who received intranasal vitamin B12 was 1.1.
Individuals who received an intramuscular injection of vitamin B12 had an average ratio of 0.17 to 0.24, compared to 1.9 to 1.9. This is a surprising result in that intranasal administration has only about 7-12% bioavailability in serum relative to intramuscular injection of vitamin B12. This shows that intranasal administration of vitamin B12 reaches CSF much more efficiently than intramuscular injection.

(Example 3)
Preparation of cyanocobalamin solution A 4000 g batch of the cyanocobalamin solution of the present invention having a concentration of 500 mcg per 0.1 g of solution was prepared.
Starting material

3871.6 grams of water was placed in a stainless steel container placed on a hot plate. Water was heated to about 30 ° C. and stirred. Add 12.8 g of sodium citrate to the heated water and add 3
Stir at 00 rpm for 5 minutes. 4.8 g of citric acid was then added and stirred for 10 minutes. To this mixture, 20.0 g of cyanocobalamin was added and stirred at 30 ° C. and 300 rpm for 30 minutes. The hot plate was then turned off. 89.2 g of glycerin was added and stirred at 300 rpm for 5 minutes. Next, 1.6 g of an aqueous solution containing 50% by weight of benzalkonium chloride was added to the cyanocobalamin solution and stirred at 300 rpm for 5 minutes. The pH was then measured and adjusted if the pH was not in the range of 4.5-5.5. Additional water was added to bring the solution weight to 4000 g.

(Example 4)
In this example, a representative pharmaceutical composition of the invention comprising an aqueous solution of salmon cyanocobalamin at a concentration sufficient to provide a therapeutically effective plasma concentration and delivered via an actuator to produce an aerosol of said solution A pharmaceutical composition is described wherein the aerosol spray pattern ellipticity is from 1.00 to 1.40 when measured at a height of 30 cm from the actuator chip.

The volume of the aerosol can be about 5 microliters to 1.0 ml, preferably 20 to 200 microliters.

This test method describes a procedure for characterizing the plume geometry of a cyanocobalamin nasal solution formulation using the SprayView NSP system. In accordance with the methods described in U.S. Patent No. 6,665,421 and U.S. Patent Application Publication No. 20030018416 published January 23, 2003, an integrated SprayView NSx actuation station (Image Therm Engineering, Inc.
SprayView high speed light spray characterization system (SprayV) with Sudbury, Massachusetts
iew NSP) to characterize the plume geometry.

Use the formulations in Table 1 to deliver a dose of 0.1 mL per jet and have both “nasal spray pump w / safety clip, Pfeiffer SAP # 60550” with a dip tube length of 36.05 mm Spray characterization and droplet size of the formulation in 3 mL bottles.

以下にスプレーパターン結果を示す。

The droplet size data is shown in the table below.

The spray pattern results are shown below.

Claims (73)

A stable aqueous pharmaceutical solution of cyanocobalamin composed of cyanocobalamin and water, wherein said cyanocobalamin solution is suitable for intranasal administration and has a viscosity of less than about 1000 cPs, said cyanocobalamin solution administered intranasally The aqueous pharmaceutical solution wherein the bioavailability of cyanocobalamin is at least about 5% for intramuscular injection of cyanocobalamin. The aqueous solution of cyanocobalamin according to claim 1, which is composed of citric acid, sodium citrate and water and has a pH of about 4-6.   The aqueous solution of claim 2, wherein the pH is about 5.   The aqueous solution of claim 2, further comprising a wetting agent. 5. The aqueous solution according to claim 4, wherein the wetting agent is selected from the group consisting of sorbitol, propylene glycol and glycerin.   6. The aqueous solution according to claim 5, wherein the wetting agent is glycerin.   The aqueous solution of claim 6, wherein the glycerin is present at a concentration of about 2.23%.   The aqueous solution according to claim 2, wherein the solution further comprises a preservative. 9. The aqueous solution according to claim 8, wherein the preservative is selected from the group consisting of benzyl alcohol, chlorobutanol and benzalkonium chloride.   10. The aqueous solution according to claim 9, wherein the preservative is benzalkonium chloride. 11. The aqueous solution of claim 10, wherein the benzalkonium chloride is present in the solution at a concentration of about 0.02%. The aqueous solution of claim 2, wherein cyanocobalamin is present at a concentration of about 0.5% of the total weight, citric acid is present at a concentration of about 0.12%, and sodium citrate is present at a concentration of about 0.32%.   13. The aqueous solution according to claim 12, wherein the pH of the solution.   13. The aqueous solution of claim 12, further comprising a wetting agent. 15. The aqueous solution according to claim 14, wherein the wetting agent is selected from the group consisting of sorbitol, propylene glycol and glycerin.   16. The aqueous solution according to claim 15, wherein the wetting agent is glycerin.   17. The aqueous solution of claim 16, wherein the glycerin is present in the solution at a concentration of about 2.23%.   13. The aqueous solution according to claim 12, further comprising a preservative. 19. The aqueous solution according to claim 18, wherein the preservative is selected from the group consisting of benzyl alcohol, chlorobutanol and benzalkonium chloride.   20. The aqueous solution according to claim 19, wherein the preservative is benzalkonium chloride. 21. The aqueous solution of claim 20, wherein the benzalkonium chloride is present in the solution at a concentration of about 0.02%. The cyanocobalamin solution is at least about an intramuscular injection of cyanocobalamin.
The aqueous solution of claim 1 having a bioavailability of 7%. Cyanocobalamin at a concentration of about 0.5% of the total weight of the solution, citric acid at a concentration of about 0.12%, about 0.32%
A stable pharmaceutical aqueous solution of cyanocobalamin composed of sodium citrate at a concentration of approximately 2.23%, glycerin at a concentration of approximately 2.23%, benzalkonium chloride at a concentration of approximately 0.02%, and water, wherein the cyanocobalamin solution is suitable for intranasal administration. Preferably having a viscosity of less than about 1000 cPs, wherein the cyanocobalamin solution has a bioavailability of cyanocobalamin when administered intranasally of at least about 5% relative to an intramuscular injection of cyanocobalamin. The pharmaceutical aqueous solution. A method of administering cyanocobalamin comprising injecting an aqueous solution of cyanocobalamin into the nose, wherein the cyanocobalamin solution has a viscosity of less than 1000 cPs, and the cyanocobalamin solution has a cyanocobalamin bioavailability that is The method, which is about 5% for internal injection. 25. The method of claim 24, wherein the cyanocobalamin solution is further comprised of citric acid and sodium citrate, and the solution has a pH of about 4-6.   26. The method of claim 25, wherein the solution has a pH of about 5.   26. The method of claim 25, wherein the cyanocobalamin is present in the solution at a concentration of 0.5-1% by weight.   28. The method of claim 27, wherein the concentration of cyanocobalamin in solution is about 0.5%. The citric acid is present in the aqueous solution at a concentration of about 0.12% and the sodium citrate is about 0.32%
29. The method of claim 28, wherein the method is present in the aqueous solution at a concentration of A method of administering cyanocobalamin comprising injecting an aqueous solution of cyanocobalamin into the nose, wherein the aqueous solution of cyanocobalamin comprises cyanocobalamin at a concentration of about 0.5% of the total weight of the solution, citric acid at a concentration of about 0.12%, about 0.32% Composed of sodium citrate at a concentration of about 2.23%, glycerin at a concentration of about 2.23%, benzalkonium chloride at a concentration of about 0.02%, and water, the cyanocobalamin solution is suitable for intranasal administration and has a viscosity of less than about 1000 cPs And the solution of cyanocobalamin when administered intranasally has a bioavailability of cyanocobalamin of at least about 5% for intramuscular injection of cyanocobalamin. A method for increasing vitamin B12 levels in cerebrospinal fluid (CSF), sufficient to increase the average ratio of vitamin B12 in CSF to vitamin B12 in serum (B12 CSF / B12 serum x 100) to at least about 1.1 Nasally administering a solution of a sufficient amount of cyanocobalamin,
The aqueous solution of cyanocobalamin has a concentration of about 0.5% cyanocobalamin, about the total weight of the solution.
Consisting of citric acid at a concentration of 0.12%, sodium citrate at a concentration of about 0.32%, glycerin at a concentration of about 2.23%, benzalkonium chloride at a concentration of about 0.02% and water, the cyanocobalamin solution Suitable for administration and having a viscosity of less than about 1000 cPs, said cyanocobalamin solution has a bioavailability of cyanocobalamin when administered intranasally of at least about 5% relative to intramuscular injection of cyanocobalamin. Said method. An aqueous solution of cyanocobalamin and excipients in a container;
A nasal drug delivery pharmaceutical kit comprising a droplet generating actuator mounted on the container and fluidly connected to the cyanocobalamin in the container, wherein the actuator is a tip of the actuator when the actuator acts Generating a spray of the cyanocobalamin solution, wherein the spray of cyanocobalamin solution has a spray pattern ellipticity of about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator chip. The spray includes droplets, and less than 5% of the droplets are less than 10 μm in size.
The described kit. 35. The kit of claim 32, wherein the aqueous solution of cyanocobalamin has a viscosity of less than 1000 cPs and the solution of cyanocobalamin has about 5% cyanocobalamin bioavailability for intramuscular injection of cyanocobalamin. The spray is composed of droplets of the cyanocobalamin solution, and less than 5% of the droplets are 10 μm.
35. The kit of claim 34, wherein the kit is less than m. The kit according to claim 3, wherein the spray has a spray pattern major axis and minor axis of 25 to 40 mm, respectively. The cyanocobalamin solution further comprises citric acid and sodium citrate;
35. The kit of claim 34, wherein the solution has a pH of about 4-6.   38. The kit of claim 37, wherein the pH of the solution is about 5. 35. The kit of claim 34, wherein the cyanocobalamin is present in the solution at a concentration of 0.5-1% by weight.   40. The kit of claim 39, wherein the concentration of cyanocobalamin in the solution is about 0.5% by weight. The citric acid is present in the aqueous solution at a concentration of about 0.12% and the sodium citrate is about 0.32%
38. The kit of claim 37, wherein the kit is present in an aqueous solution at a concentration of 35. The kit of claim 34, wherein the cyanocobalamin spray is composed of droplets of the cyanocobalamin solution, and 50% of the droplets are 26.9 μm or less in size. 35. The kit of claim 34, wherein the cyanocobalamin spray is composed of droplets of the cyanocobalamin solution, and 90% of the droplets have a size of 55.3 μm or less. 35. The kit of claim 34, wherein the cyanocobalamin spray produces droplets of the solution, wherein 10% of the droplets are 12.5 μm or less in size. A kit for intranasally administering a cyanocobalamin solution, the kit comprising a container, a solution of cyanocobalamin in the container, and an actuator attached to the container, and spraying the cyanocobalamin solution acts by the actuator Sometimes released through the tip of the actuator, the aqueous solution of cyanocobalamin is about 0.5% concentration of cyanocobalamin, about 0.12% concentration of citric acid, about 0.32% concentration of sodium citrate, Consisting of glycerin at a concentration of about 2.23%, benzalkonium chloride at a concentration of about 0.02% and water, the cyanocobalamin solution is suitable for intranasal administration and has a viscosity of less than about 1000 cPs, When the solution is administered intranasally, the bioavailability of cyanocobalamin has increased At least about 5% for internal injection, and the spray is about 1.0 to about when measured at a height of 3.0 cm from the actuator chip.
The kit having a spray pattern ellipticity of 1.4. 46. The kit of claim 45, wherein the cyanocobalamin spray is composed of droplets of a cyanocobalamin solution, and less than 5% of the droplets of the cyanocobalamin spray are less than 10 μm in size. 46. The kit according to claim 45, wherein the cyanocobalamin spray is composed of droplets of a cyanocobalamin solution, and 50% of the droplets of the cyanocobalamin spray have a size of 26.9 μm or less. 46. The kit of claim 45, wherein the cyanocobalamin spray produces droplets of the solution, wherein 90% of the droplets are 55.3 μm or less in size. 46. The kit of claim 45, wherein the cyanocobalamin spray produces droplets of the solution, wherein 10% of the droplets are 12.5 μm or smaller in size. The spray has a spray pattern major axis of about 35.3 mm and a minor axis of about 30.8 mm.
45 kit. A method for intranasal administration of cyanocobalamin comprising providing an aqueous solution of cyanocobalamin, said cyanocobalamin solution having a viscosity of less than 1000 cPs, said cyanocobalamin solution being about 5% for intramuscular injection of cyanocobalamin The cyanocobalamin bioavailability is administered as a spray to an individual's nose through an actuator tip, and the spray is about 3.0 cm when measured at a height of 3.0 cm from the actuator tip. The method having a spray pattern ellipticity of 1.0 to about 1.4. The spray produces droplets, and less than 5% of the droplets are less than 10 μm in size.
The method according to 1. 52. The method of claim 51, wherein the spray has a spray pattern major axis and minor axis of 25 to 40 mm, respectively. 52. The method of claim 51, wherein the cyanocobalamin solution is further comprised of citric acid and sodium citrate, and the solution has a pH of about 4-6.   52. The method of claim 51, wherein the solution has a pH of about 5.   52. The method of claim 51, wherein the cyanocobalamin is present in the solution at a concentration of 0.5-1% by weight.   57. The method of claim 56, wherein the concentration of cyanocobalamin in the solution is about 0.5%. The citric acid is present in the aqueous solution at a concentration of about 0.12% and the sodium citrate is about 0.32%
52. The method of claim 51, wherein the method is present in an aqueous solution at a concentration of 52. The method of claim 51, wherein the cyanocobalamin spray produces droplets of the solution, and 50% of the droplets are 26.9 μm or less in size. 52. The method of claim 51, wherein the cyanocobalamin spray produces droplets of the solution, wherein 90% of the droplets are 55.3 μm or less in size. 52. The method of claim 51, wherein the cyanocobalamin spray produces droplets of the solution, wherein 10% of the droplets are 12.5 μm or less in size. A method of administering cyanocobalamin comprising providing an aqueous solution of cyanocobalamin, wherein the aqueous solution of cyanocobalamin comprises about 0.5% concentration of cyanocobalamin, about 0.12% concentration of citric acid, about 0.32% concentration. Sodium citrate, glycerin at a concentration of about 2.23%, benzalkonium chloride at a concentration of about 0.02% and water, and the cyanocobalamin solution is suitable for intranasal administration and has a viscosity of less than about 1000 cPs. The cyanocobalamin solution, when administered intranasally, has a bioavailability of cyanocobalamin of at least about 5% for intramuscular injection of cyanocobalamin, and the cyanocobalamin solution is sprayed through the actuator chip. Administered to an individual's nose and the spray is measured at a height of 3.0 cm from the actuator tip. About 1.0 with about 1.4 spray pattern ellipticity from the method. The cyanocobalamin spray produces droplets of the solution, and less than 5% of the droplets are 10 μm
64. The method of claim 62, wherein the method is less than size. 64. The method of claim 62, wherein the cyanocobalamin spray produces droplets of the solution, and 50% of the droplets are 26.9 μm or less in size. 64. The method of claim 62, wherein the cyanocobalamin spray produces droplets of the solution, wherein 90% of the droplets are 55.3 [mu] m or less in size. 63. The method of claim 62, wherein the cyanocobalamin spray produces droplets of the solution, wherein 10% of the droplets are 12.5 μm or less in size. 64. The method of claim 62, wherein the spray has a spray pattern major axis and minor axis of about 25-40 mm each. A method for increasing vitamin B12 levels in cerebrospinal fluid (CSF), sufficient to increase the average ratio of vitamin B12 in CSF to vitamin B12 in serum (E12 CSF / B12 serum x 100) to at least about 1.1 Nasally administering a solution of a sufficient amount of cyanocobalamin,
The aqueous solution of cyanocobalamin has a concentration of about 0.5% cyanocobalamin, about the total weight of the solution.
Consisting of citric acid at a concentration of 0.12%, sodium citrate at a concentration of about 0.32%, glycerin at a concentration of about 2.23%, benzalkonium chloride at a concentration of about 0.02% and water, the cyanocobalamin solution Suitable for administration and having a viscosity of less than about 1000 cPs, said cyanocobalamin solution has a bioavailability of cyanocobalamin when administered intranasally of at least about 5% relative to intramuscular injection of cyanocobalamin. The cyanocobalamin solution is administered as a spray to an individual's nose through an actuator tip, and the spray has a spray pattern ellipticity of about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator tip. Said method. The cyanocobalamin spray produces droplets of the solution, and less than 5% of the droplets are 10 μm
69. The method of claim 68, wherein the method is less than a size. 69. The method of claim 68, wherein the cyanocobalamin spray produces droplets of the solution, wherein 50% of the droplets are 26.9 μm or less in size. 69. The method of claim 68, wherein the cyanocobalamin spray produces droplets of the solution, wherein 90% of the droplets are 55.3 μm or less in size. 69. The method of claim 68, wherein the cyanocobalamin spray produces droplets of the solution, wherein 10% of the droplets are 12.5 μm or less in size. 69. The method of claim 68, wherein the spray has a spray pattern major axis and minor axis of about 25-40 mm each.

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