Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7135—Compounds containing heavy metals
  • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics

Definitions

• Vitamin B 12 is a dietary essential, a deficiency of which results in defective synthesis of DNA in any cell in which chromosomal replication and division are taking place. Since tissues with the greatest rate of cell turnover show the most dramatic changes, the hematopoietic system is especially sensitive to vitamin B 12 deficiencies.
• An early sign of Bl 2 deficiency is a megaloblastic anemia. Dietary B 12, in the presence of gastric acid and pancreatic proteases, is released from food and salivary binding protein and bound to gastric intrinsic factor. When the vitamin B 12- intrinsic factor complex reaches the ileum, it interacts with a receptor on the mucosal cell surface and is actively transported into circulation.
• Vitamin B 12 deficiency in adults is rarely the result of a deficient diet; rather, it usually reflects a defect in one or another aspect of this complex sequence of absorption.
• Acblorhydria and decreased secretion of intrinsic factor by parietal cells secondary to gastric atrophy or gastric surgery is a common cause of vitamin B 12 deficiency in adults.
• Antibodies to parietal cells or intrinsic factor complex also can play a prominent role in producing deficiency. A number of intestinal diseases can interfere with absorption.
• Vitamin Bl 2 malabsorption is seen with pancreatic disorders (loss of pancreatic protease secretion), bacterial overgrowth, intestinal parasites, sprue, and localized damage to ileal mucosal cells by disease or as a result of surgery.
• the recommended daily intake of vitamin B 12 in adults is 2.4 ⁇ g.
• cyanocobalamin hydroxocobalamin, methylcobalamin and adenosylcobalamin. Methylcobalamin and adenosylcobalamin are unstable and damaged by light.
• vitamin B 12 Because deficiencies of vitamin B12 are generally caused by the inability of the vitamin to be absorbed in the small intestine due to a breakdown in the vitamin Bl 2-intrinsic factor complex transport mechanism, vitamin B 12 must therefore be administered systemically.
• therapeutic amounts of cyanocobalamin are administered by intramuscular or deep subcutaneous injection of cyanocobalamin.
• patients must return to the physician's office periodically to receive additional injections to maintain their levels of vitamin B 12.
• an intranasal gel cyanocobalamin preparation, NASCOBAL® is currently being marketed in which cyanocobalamin is administered intranasally as maintenance vitamin B 12 therapy.
• Merkus chose hydroxocobalamin because cyanocobalamin is not soluble in an aqueous solution at concentrations greater than 1%.
• [M]ost of the B 12 passes immediately into the throat. It is not in contact with the nasal mucosa for a sufficient period of time to permit useful and uniform absorption. Most of the B 12 so administered is, in fact wasted. (Col. 1, lines 63-68).
• Wenig not only teaches away from decreasing viscosity of an intranasal cyanocobalamin formulation below a critical, minimum range of 2500-6500 cps, but also expressly negates the prospect of using an aqueous (spray or drops) composition to substitute for the intranasal gel formulation described by Wenig.
• Wenig provides clear evidence and reasoning that teaches directly away from such a proposed modification.
• Hydroxocobalamin binds more extensively to plasma proteins and has a longer half time in the body than cyanocobalamin. As a result, hydroxocobalamin is better retained in the body and, therefore, requires less frequent dosing. Moreover, cyanocobalamin is contraindicated in patients with tropical amblyopia and simultaneous tobacco usage and in patients with pernicious anemia with optic neuropathy; hence, hydroxocobalamin is the drug of choice in restoring vitamin B 12 deficiencies, (page 432, right column)."
• hydroxocobalamin Concentrations above 1% can only be obtained with hydroxocobalamin, because its good solubility in water.
• the solubility of hydroxocobalamin substances can be as high as 10%, which means that up to about 10 times more vitamin B 12 per unit of volume can be administered and subsequently absorbed nasally, when hydroxocobalamin is used. (Col. 3, lines 43-53)"
• Arieta et al. teach directly away from low viscosity liquid cyanocobalamin formulations. Wenig expressly teaches a critical, minimum viscosity for an effective, intranasal cyanocobalamin between 2500-4000 cps. Both Wenig and Garcia-Arieta et al. teach that non-gel, liquid cyanocobalamin formulations would not be retained for a sufficient time to allow for intranasal absorption. Garcia-Arieta et al. specifically report experimental results that no significant bioavailability was detected following nasal administration of a 1% simple aqueous cyanocobalamin formulation.
• a high and efficient intranasal absoiption of vitamin B 12 is advantageous in medical therapy and can be obtained only by using hydroxocobalamin, which shows a significant higher solubility in water than cyanocobalamin. Only with hydroxocobalamin a superior nasal composition in an aqueous medium can be produced with by far the highest concentration of vitamin B 12 and consequently a much more efficient nasal absorption of vitamin B12. Such a nasal formulation can be taken less frequently by patients, making the therapy much easier and less expensive. (Col. 2, lines 27-37)." [0013] In other prior art pertaining to the general field of the invention, U.S.
• Patent No. 4,525,341, Deihl discloses a method of administering vitamins intranasally but does not describe or enable a specific formulation containing only cyanocobalamin.
• the present invention fills this need by providing a stable pharmaceutical solution of cyanocobalamin suitable for intranasal administration, having a viscosity less than about 1000 cPs, wherein said intranasal solution of cyanocobalamin has a bioavailability of at least 5%, and in certain embodiments at least 6% or 7%, of the bioavailability of an intramuscular injection of cyanocobalamin.
• a therapeutic or pharmaceutically effective formulation of the invention may be comprised of cyanocobalamin, citric acid, sodium citrate, and water wherein the viscosity is less than 1000 cPs, and wherein the solution of cyanocobalamin has a bioavailability of at least about 5% of, and in certain embodiments at least about 6% or 7% of, the bioavailability of an intramuscular injection of cyanocobalamin.
• the solution of cyanocobalamin according to the invention has a bioavailability of at least about 8%, 9%, 10%, 11%, or 12% of, the bioavailability of an intramuscular injection of cyanocobalamin.
• the solution is essentially free of mercury and mercury-containing compounds.
• compositions within the scope of this invention will contain a humectant to inhibit drying of the mucous membranes and to prevent irritation.
• humectants can be used including but not limited to sorbitol, propylene glycol or glycerol.
• An illustrative, useful humectant is glycerin.
• a preservative is generally employed to increase the shelf life of the compositions.
• preservative include but are not limited to benzyl alcohol, chlorobutanol and benzalkonium chloride.
• the preservative is benzalkonium chloride.
• a suitable concentration of the preservative will be from 0.002% to 2.0% based upon the total weight, although there may be appreciable variation depending upon the agent selected.
• An exemplary formulation has the concentration of cyanocobalamin at
• a nasal spray solution of Cyanocobalamin is provided in a spray applicator containing 2.3 mL of a 500 mcg/0.1 niL solution of cyanocobalamin with sodium citrate, citric acid, glycerin and benzalkonium chloride in purified water.
• An exemplary spray solution in this context has a pH between 4.5 and 5.5.
• an exemplary spray pump spray delivers an average of 500 meg of cyanocobalamin and the 2.3 mL of spray solution contained in the bottle will deliver 8 doses of the nasal spray.
• Another embodiment of the present invention is a method for administering cyanocobalamin comprised of infusing the nose with an aqueous solution of cyanocobalamin, wherein the solution of cyanocobalamin has a viscosity of less than 1000 cPs, and wherein said solution of cyanocobalamin has a bioavailability of at least about 5% of, and in certain embodiments at least about 6% or 7% of, the bioavailability of an intramuscular injection of cyanocobalamin.
• the solution of cyanocobalamin according to the invention has a bioavailability of at least about 8%, 9%, 10%, 11%, or 12% of, the bioavailability of an intramuscular injection of cyanocobalamin.
• the cyanocobalamin solution administered according to the methods of the invention is
• essentially free generally means a solution having less than 2% by weight of a particular substance, while in other aspects the solution will have less that 1 % by weight, 0.05% by weight, 0.01% by weight, or be completely free of the substance so that, e.g., a mercury or mercury-containing compound is not detectable in the solution using conventional detection methods.
• the present invention is further directed towards a method for elevating the vitamin B12 levels in the cerebral spinal fluid (CSF) comprising intranasally administering a solution of cyanocobalamin so as to increase the average ratio of ' vitamin B 12 in the CSF to that in the blood serum (B 12 CSF/B 12 Serum x 100) to at least about 1.1, wherein said solution of cyanocobalamin has a bioavailability of at least 7% relative to an intramuscular injection of cyanocobalamin.
• the B 12 CSF levels are increased so that the ratio of B 12 in the CSF to the levels in the blood serum is at least 1.9.
• the methods of present invention are further comprised of the cyanocobalamin solutions being administered into a nose of an individual through an actuator tip as a spray, wherein the spray in certain embodiments has one or more of the following properties: a spray pattern ellipticity ratio of from about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator tip; or the spray produces droplets, less than about 5% of the droplets are less than 10 ⁇ m 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 ⁇ m or less in size; 90% of the droplets produced by the spray are 55.3 ⁇ m or less in size; or 10% of the droplets produced by the spray are 12.5 ⁇ m or less in size.
• a spray pattern ellipticity ratio of from about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator tip
• the spray produces droplets, less than about 5%
• FIG. IA shows a nasal spray pump kit containing the cyanocobalamin solution of the present invention having an actuator that is not engaged.
• FIG. IB shows the nasal spray pump kit containing the cyanocobalamin solution of the present invention having an actuator that is engaged and expelling a spray plume of the cyanocobalamin solution of the present invention.
• FIG. 2 shows the spray pattern produced by the actuator of the spray pump kit.
• nasal mucosa the nasal mucosa is taken to be the lining of the vestibule of the nose, where vascularized, and extending interiorly to the boundaries of the oropharynx and sinuses.
• Aqueous refers to a solution formed in water, but may contain lesser amounts of other co-solvents.
• Bioavailability is defined as the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action, [21 CFR ⁇ 320.1 (a)].
• Bioavailability of the intranasal spray relative to an intramuscular injection of cyanocobalamin means the percent amount a dose of the intranasal taken up by the systemic vascular system in comparison to the same amount of cyanocobalamin injected. For example, assuming an intramuscular injection of a solution of cyanocobalamin containing 100 ⁇ g of cyanocobalamin would have a
• an intranasal dose of cyanocobalamin contains 100 ⁇ g and has at least 5% of, and in certain embodiments at least about 6% or 7% of, the bioavailability of an intramuscular injection of cyanocobalamin, at least 5 ⁇ g, 6 ⁇ g, or 7 ⁇ g of cyanocobalamin would be taken up into the blood vasculature.
• the intranasal dose of cyanocobalamin contained 500 ⁇ g, at least 35 ⁇ g of cyanocobalamin would be taken up into the blood vasculature, if the intranasal formulation had a bioavailability of at least 7%.
• Stability during storage, any compositional change measured in a parameter, examples of which include but are not limited to concentration, degradation, viscosity, pH, or particle size, that is considered to significantly affect the quality attributes of the product over time, denotes instability.
• changes that are not considered to significantly affect the quality attributes of the product connote stability.
• the time period over which stability is measured is relative depending on the intended utility of the composition. Accelerated stability at higher temperature is sometimes taken as a more speedy way of extrapolating stability over longer periods of time than are actually measured.
• “Pharmaceutically acceptable” refers to a composition which when administered to a human or a mammal by the indicated route of administration, provokes no adverse reaction which is disproportionate to the benefit gained by administration of said compound.
• “Mammal” shall include any of a class of warm-blooded higher vertebrates that nourish their young with milk secreted by mammary glands and have skin usually more or less covered with hair, and non-exclusively includes humans and non-human primates, their children, including neonates and adolescents, both male and female, livestock species, such as horses, cattle, sheep, and goats, and research and domestic species, including dogs, cats, mice, rats, guinea pigs, and rabbits. "Patient” or “subject” is used herein interchangeably with “mammal.” [0032] "Intranasal delivery” shall mean delivery of a drug primarily via the mucosa of the nasal cavity.
• substantially free refers to the level of a particular active ingredient in the compositions of the invention, wherein the particular active ingredient constitutes less than 20%, preferably less than 10%, more preferably less than 5%, and most
• Delivery vehicles herein found useful include actuator dispensers commonly used for nasal solutions and gels.
• Embodiments of this technology include multiple, single-dose, metered dose, child resistant, and disposable dispensers, and their kits.
• peak concentration (C max ) of cyanocobalamin in a blood plasma "area under concentration vs. time curve (AUC) of cyanocobalamin in a blood plasma”, “time to maximal plasma concentration (t max ) of vitamin in a blood plasma” are pharmacokinetic parameters known to one skilled in the art. [Laursen et al, Eur. J. Endocrinology, 135: 309-315, (1996)].
• concentration vs. time curve measures the concentration of cyanocobalamin in a blood serum of a subject vs. time after administration of a dosage of cyanocobalamin to the subject either by intranasal, subcutaneous, or other parenteral route of administration.
• C max is the maximum concentration of cyanocobalamin in the blood serum of a subject following a single dosage of cyanocobalamin to the subject.
• t max is the time to reach maximum concentration of cyanocobalamin in a blood serum of a subject following administration of a single dosage of cyanocobalamin to the subject.
• AUC area under concentration vs. time curve
• cyanocobalamin solutions are designed to be administered to the nasal mucosa either in drop or in spray form.
• the preferred mode of administration is in spray form, i.e., in the form of finely divided droplets.
• An example of a suitable spray pump is the Pfeiffer Spray Pump Model #
• Cyanocobalamin is administered intranasally using a nasal spray according to the present invention.
• the following definitions are useful.
• Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released upon activation of an appropriate valve system.
• Metered aerosol A pressurized dosage form comprised of metered dose valves, which allow for the delivery of a uniform quantity of spray upon each activation.
• Powder aerosol A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which are released upon activation of an appropriate valve system.
• Spray aerosol An aerosol product that utilizes a compressed gas as the propellant to provide the force necessary to expel the product as a wet spray; it generally applicable to solutions of medicinal agents in aqueous solvents.
• Spray - A liquid minutely divided as by a jet of air or steam.
• Metered spray - A non-pressurized dosage form consisting of valves that allow the dispensing of a specified quantity of spray upon each activation.
• Suspension spray - A liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of course droplets or as finely divided solids.
• Plume Height the measurement from the actuator tip to the point at which the plume angle becomes non-linear because of the breakdown 1 of linear flow.
• Minor Axis the smallest chord that can be drawn within the fitted spray pattern that crosses the COMw in base units (mm)
• Ellipticity Ratio the ratio of the major axis to the minor axis
• D 10 the diameter of droplet for which 10% of the total liquid volume of sample consists of droplets of a smaller diameter ( ⁇ m)
• D 50 the diameter of droplet for which 50% of the total liquid volume of sample consists of droplets of a smaller diameter ( ⁇ m), also known as the mass median diameter
• Span - measurement of the width of the distribution The smaller the value, the narrower the distribution. Span is calculated as ⁇ 90 " 10 * . y Dso
• % RSD - percent relative standard deviation the standard deviation divided by the mean of the series and multiplied by 100, also known as % CV.
• the present invention is further comprised of a cyanocobalamin nasal spray kit and method of administering the cyanocobalamin solution using the nasal spray kit.
• the nasal spray kit is exemplified by Figures IA, IB and Figure 2.
• the cyanocobalamin nasal spray kit, 10 is comprised of a container, in this case a bottle 12 into which the cyanocobalamin formulation is placed, and an actuator 14, attached to bottle 12 and in fluid connection with the solution of cyanocobalamin in bottle 12.
• the actuator, 14 When the actuator, 14 is actuated or engaged, it forces a spray plume, 16 of cyanocobalamin through tip 15 of the actuator.
• the spray plume is comprised of droplets of the solution of cyanocobalamin.
• a spray pattern is determined by taking a photograph of a cross-section of the spray plume 16 above a predetermined height, of the plume.
• the spray plume also has angle of ejection, 20, as it leaves actuator, 14.
• a spray pattern of spray plume 16 is shown on
• FIG. 2 The Spray pattern of Figure 2, is elliptical and has a major axis, 24, and a minor axis 26.
• the actuator produces spray of the cyanocobalamin solution having a spray pattern ellipticity ratio of from about 1.0 to about 1.4 when measured at a height of 3.0 cm from the actuator tip.
• less than 5% of the droplets of the cyanocobalamin solution are less than 10 ⁇ m in size
• the spray pattern has a major axis and minor axis of 25 and 40 mm, respectively, 50% of the droplets are 26.9 ⁇ m or less in size, 90% of the droplets are 55.3 ⁇ m or less in size, and 10% of the droplets are 12.5 ⁇ m or less in size.
• the present invention provides improved methods and compositions for intranasal delivery cyanocobalamin to mammalian subjects for treatment or prevention of a variety of diseases, disorders and conditions.
• appropriate mammalian subjects for treatment and prophylaxis according to the methods of the invention include, but are not restricted to, humans and non-human primates, livestock species, such as horses, cattle, sheep, and goats, and research and domestic species, including dogs, cats, mice, rats, guinea pigs, and rabbits.
• the cyanocobalamin nasal spray of the present invention may be directed towards maintenance of the hematological status of patients who are in remission following intramuscular vitamin B 12 therapy.
• an initial therapy regimen may involve the patient receiving daily intramuscular injections of 100 ⁇ g of cyanocobalamin for about 1 to 2 weeks, together with 1 to 5 mg of folic acid.
• Intramuscular injections of cyanocobalamin should not be greater than 100 ⁇ g as doses in excess of 100 ⁇ g are rapidly cleared from the plasma into the urine, and administration of larger amounts of vitamin B 12 will not result in greater retention of larger amounts of the vitamin.
• Treatment using the methods and compositions of the invention may be enlisted following such a course of initial therapy, as follows.
• the patient self-administers a dose of the nasal spray, e.g., containing 500 ⁇ g of cyanocobalamin, once or twice a week.
• a dose of the nasal spray e.g., containing 500 ⁇ g of cyanocobalamin, once or twice a week.
• the maintenance therapy of the intranasal cyanocobalamin is for any patient that had been diagnosed with a vitamin B 12 deficiency, but especially for those treated for pernicious anemia and dietary deficiency of vitamin B12 occurring in strict vegetarians, the so-called vegans who eat no animal products.
• cyanocobalamin therapy using the cyanocobalamin solution of the present invention is also indicated for those afflicted with malabsorption of vitamin B 12 resulting from structural or functional damage to the stomach, where intrinsic factor is secreted or to the ileum, where intrinsic factor facilitates B 12 absorption.
• Maintenance cyanocobalamin therapy using the cyanocobalamin solution of the present invention is also indicated for those afflicted with malabsorption of vitamin B 12 resulting from inadequate secretion of intrinsic factor, resulting from lesion that destroys the gastric mucosa (ingestion of corrosives, extensive neoplasia), and a number of conditions associated with a variable degree of gastric atrophy (such as multiple sclerosis, human immunodeficiency viral (HIV) infection certain endocrine disorders, iron deficiency, and subtotal gastrectomy).
• Structural lesions that lead to B 12 deficiency include ileitis, ileal resections, Crohn's disease and malignancies.
• Vitamin B 12 deficiencies may also be the result of competition by intestinal parasites, and inadequate utilization of vitamin B 12 occurring if antimetabolites for the vitamin are employed in the treatment of neoplasia.
• the intranasal cyanocobalamin solution of the present invention can also be used for individuals who require above normal levels of vitamin B 12, due to, for example, pregnancy, thyrotoxicosis, hemolytic anemia, hemorrhage, malignancy, hepatic and renal disease.
• the present invention provides for a stable pharmaceutical solution of cyanocobalamin suitable for intranasal administration, having a viscosity less than about 1000 cPs, wherein said intranasal solution of cyanocobalamin has when administered intranasally a bioavailability of at least 5%, 6%, or 7% of the bioavailability of an intramuscular injection of cyanocobalamin.
• the intranasal formulation will often be comprised of, in addition to water and cyanocobalamin, a buffering agent to maintain the pH between 4 and 6 preferably about 5, an optional humectanct to inhibit drying of the mucous membranes, and an optional preservative.
• a composition according to the invention is comprised of cyanocobalamin, citric acid, sodium citrate, and water wherein the viscosity is less than 1000 cPs, and wherein the solution of cyanocobalamin has a bioavailability of at least 5%, 6%, or 7%, and in certain
• Customer No. 25,315 embodiments at least about 8%, 9%, 10%, 11%, or 12% or more, of the bioavailability of an intramuscular injection of cyanocobalamin.
• compositions according to the invention will contain a humectant to inhibit drying of the mucous membranes and to prevent irritation.
• a humectant to inhibit drying of the mucous membranes and to prevent irritation.
• Any of a variety of humectants can be used including, for example sorbitol, propylene glycol or glycerol.
• An exemplary humectant is glycerin.
• a preservative is generally employed to increase the shelf life of the compositions.
• preservative include benzyl alcohol, parabens thimerosal, chlorobutanol, benzethonium chloride and benzalkonium chloride.
• An exemplary preservative useful within the formulations and methods of the invention is benzalkonium chloride.
• a suitable concentration of the preservative will be from
• a formulation according to the invention has the concentration of cyanocobalamin at 0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%, glycerin 2.23%, benzalkonium chloride solution 0.02% and 96.79% water.
• buffering agent combinations useful within the invention include but are not limited to: Monopotassium phosphate and disodium phosphate; Potassium biphthalate and sodium hydroxide; and Sodium acetate and acetic acid.
• Another embodiment of the present invention is a method for administering cyanocobalamin comprised of infusing the nose with an aqueous solution of cyanocobalamin, wherein the solution of cyanocobalamin has a viscosity of less than 1000 cPs, and wherein said solution of cyanocobalamin has a bioavailability of at least about 5%, 6%, or 7% relative to an intramuscular injection of cyanocobalamin.
• the bioavailability of the cyanocobalamin solution of the invention is at least about 8%, 9%, 10%, 11%, and up to 12% or greater compared to bioavailability of an intramuscular injection of cyanocobalamin.
• the present invention is further directed towards a method for elevating the vitamin B 12 levels in the cerebral spinal fluid (CSF) comprising intranasally administering a solution of cyanocobalamin so as to increase the average ratio of vitamin B12 in the CSF to that in the blood serum (B12 CSF/B12 Serum x 100) to at least about 1.1, wherein said solution of cyanocobalamin has a bioavailability of at least 7% relative to an intramuscular injection of a cyanocobalamin.
• the B 12 CSF levels are increased so that the ratio of B 12 in the CSF to the levels in the blood serum is at least 1:9.
• vitamin B 12 deficiency can result in irreversible damage to the nervous system. Progressive swelling of myelinated neurons, demyelination, and neuronal cell death are seen in the spinal column and cerebral cortex. This causes a wide range of neurological signs and symptoms, including paresthesias of the hands and feet, diminution of vibration and position senses with resultant unsteadiness, decreased deep tendon reflexes, and, in the later stages, confusion, moodiness, loss of memory, and even a loss of central vision. The patient may exhibit delusions, hallucinations, or even an overt psychosis.
• vitamin B 12 deficiency Since the neurological damage can be dissociated from the changes in the hematopoietic, vitamin B 12 deficiency must be considered as a possibility in elderly patients with dementia and psychiatric disorders, even if they are not anemic. Thus, the embodiment of the present invention directed towards increasing the level of vitamin B 12 in the CSF can have tremendous benefit for neurological patients.
• intranasal administration of vitamin B 12 according to the invention can be used to treat such diseases as Alzheimer's disease, dementia, and multiple sclerosis.
• the intranasal formulations of the present invention can be administered using any spray bottle or syringe.
• a preferred nasal spray bottle is the, "Nasal Spray Pump w/ Safety Clip, Pfeiffer SAP # 60548, which delivers a dose of O.lmL per t squirt and has a diptube length of 36.05 mm. It can be purchased from Pfeiffer of America of Princeton, NJ.
• the following examples are provided by way of illustration, not limitation.
• Nascobal ® (Cyanocobalamin, USP) is a synthetic form of vitamin B i 2 with equivalent vitamin B 12 activity.
• the chemical name is 5,6-dimethyl-benzimidazolyl cyanocobamide.
• Nascobal ® (Cyanocobalamin, USP) is marketed as a self- administered nasal gel.
• the recommended dose of Nascobal ® (Cyanocobalamin, USP) in subjects with vitamin B 12 malabsorption who are in remission following inject able vitamin B 12 therapy is 500- ⁇ g administered intranasally once weekly.
• Vitamin B 12 deficiency has a number of causes, including malabsorption of vitamin B 12 resulting from structural or functional damage to the gastrointestinal system and dietary deficiency of vitamin B 12 .
• Intranasal cyanocobalamin gel is approved for a dose of 500 ⁇ g.
• the current study also utilizes a cyanocobalamin nasal spray at the same 500 ⁇ g dose and an intramuscular dose of 100 ⁇ g.
• This study was a single-site, open-label, 3 -way (3 -treatment, 6-sequence) crossover, pharmacokinetic study of vitamin B 12 administered via intranasal (IN) spray (500- ⁇ g), IN gel (Nascobal ® ) (500- ⁇ g), and intramuscular (IM) injection (100- ⁇ g) in fasted normal healthy male and female subjects, as follows:
• Treatment A One IN spray administration of 500- ⁇ g vitamin B 12 .
• the intranasal formulation was comprised of an exemplary embodiment of the present invention and contained cyanocobalamin at a concentration of 0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%, glycerin 2.23%, 50% benzalkonium chloride solution 0.04% and 96.79% water.
• Treatment B One IN gel administration of 500- ⁇ g vitamin B 12 (Nascobal ® ).
• Treatment C One IM administration of 100- ⁇ g vitamin B 12 .
• Blood samples for PK analysis of vitamin Bi 2 levels were collected via an indwelling catheter and/or via direct venipuncture using 5-mL yellow-top Vacutainer ® HemogardTM evacuated serum separator collection tube. Blood samples for PK analysis of vitamin Bi 2 levels were collected on Day-1 at 0, 6, and 12 hours and Day 1 at 0 hour (i.e., pre-dose); 30 minutes; 1, 1.5, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84 and 96 hours post-dose during each period. Appropriateness of Measurements
• AUC is a measure of the extent 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 and provides an indication that sufficient drag has reached the systemic circulation to provide a therapeutic response.
• C nmx provides warning of possible toxic drug levels.
• T max was calculated and presented as median ⁇ range.
• Bioequivalence was evaluated for the test (Treatment A - Nasal Spray) versus the reference (Treatment B - Gel).
• An analysis of variance (ANOVA, Ref. 2) was performed and the 90% confidence intervals were generated for the ratio of test/reference.
• C max and AUC 0- t were natural log (log e ) transformed prior to analysis.
• the corresponding 90% confidence intervals for the geometric mean ratio were obtained by talcing the antilog of the 90% confidence intervals for the difference between the means on the log scale.
• test (Treatment A) is non-inferior (with respect to the reference (Treatment B) if the lower bound of the 90% confidence intervals from log e -transformed C max , and AUCo- t were greater than or equal to 80%. If the lower bound of the 90% confidence intervals from log e -transformed C raax and AUCo- t were less than 80%, it was assumed that non-inferiority could not be established.
• Bioavailability was evaluated for the test (Treatments A and B - Nasal Spray and Gel, respectively) and the reference (Treatment C - IM) groups. Relative bioavailability was assessed by examining the 90% confidence intervals for the ratio of the test (Treatments A and B) group means relative to the reference (Treatment C) group mean.
• the relative bioavailability for the two IN formulations was 0.9715. Bioavailability when comparing Treatment A (spray) versus Treatment C (IM) was 0.6105, and 0.6284 when comparing Treatment B (gel) versus Treatment C (IM).
• the pharmacokinetic profiles of the spray formulation and the gel formulation were similar for C max (1480 ⁇ g/mL, 1670 pg/mL, respectively) and AUC 04 (92000 pg*hr/mL, 97000 pg*hr/mL, respectively). Additionally, the median difference for T max between the spray and gel IN formulation was less than 15 minutes (-0.24). The C max value for the IM formulation was significantly higher than the C max values for the two IN formulations (p ⁇ 0.0001).
• Vitamin B i 2 formulations were safe and well tolerated by healthy male and female volunteers.
• a minimum relative or comparative bioavailability e.g., in side-by-side test subjects administered comparable doses of intramuscular cyanocobalamin, or intranasal cyanocobalamin, tested for plasma or CSF concentration of cyanocobalamin against suitable control subjects administered sham or placebo preparations
• the intranasal cyanocobalamin formulations of the invention will be at least 5%, 6%, or 7% of the bioavailability achieved by intramuscular injection, in some cases at least 8%, 9%, 10%, 11%, or 12% or greater.
• the present disclosure provides detailed comparative bioavailability studies and results to evince these unexpected performance characteristics of the methods and compositions of the invention.
• the relative bioavailability for two exemplary intranasal (IN) formulations was 0.9715.
• the pharmacokinetic profiles of the compared cyanocobalamin spray formulation and gel formulation were similar for Cmax (1480 ⁇ g/mL, 1670 pg/mL, respectively) and AUC0-t (9200 pg*hr/mL, 9700 pg*hr/mL, respectively). Additionally, the median difference for Tmax between the spray and gel IN formulation was less than 15 minutes (-0.24). The Cmax value for the IM formulation was significantly higher than the Cmax values for the two IN formulations (p ⁇ 0.0001).
• the actual arithmetic AUC are provided above for an exemplary IN cyanocobalamin spray and gel, as 92000 and 97000 pg*hr/mL, respectively. These data likewise evince the corresponding AUC for the IM injected study comparator, according to the instant disclosure.
• the arithmetic mean of the AUC for IM is calculated as 147155 pg*hr/mL (as readily derived by reverse mathematical operation from the ratios given above-for example for the spray
• CSF cerebrospinal fluid
• each subject After each dosing, each subject underwent lumbar puncture only once, with the retrieval of a total 4.0 mL of CSF (4 tubes, 1.0 mL per tube).
• CSF 4 tubes, 1.0 mL per tube.
• One third of the subjects had a CSF sample collected at 60 minutes post dosing, one third of subjects had a CSF sample collected at 90 minutes post dosing, and one third of subjects had a CSF sample collected at 120 minutes post dosing
• the cerebrospinal fluid was evaluated for total Vitamin B 12 content. It was the objective of the study described herein to measure the amount of Vitamin B 12 present in the blood and CSF following intramuscular (IM) and nasal administration. Reference and Test Products
• Reference Product Cyanocobalamin lOOmcg intramuscular injection.
• Cyanocobalamin Injection USP is a sterile solution of cyanocobalamin (Vitamin B 12 ) for intramuscular or subcutaneous injection. Each mL contains 1,000 meg cyanocobalamin.
• the cyanocobalamin intranasal aqueous solution in this study contained cyanocobalamin at a concentration of 0.5% (percent of total weight), citric acid 0.12%, sodium citrate 0.32%, glycerin 2.23%, 50% benzalkonium chloride solution 0.04% and 96.79% water.
• Vitamin Bi 2 Nasal Spray was supplied as a 2.3 mL bottle to deliver one dose: 500 mcg/0.1 mL per dose.
• each subject underwent lumbar puncture, involving the retrieval of 4.0 mL of CSF (4 tubes, 1.0 mL per tube).
• CSF 4 tubes, 1.0 mL per tube.
• One third of subjects from each group had a CSF sample collected at 60 minutes post dosing, one third of subjects had a CSF sample collected at 90 minutes post dosing, and one third of subjects had a CSF sample collected at 120 minutes post dosing.
• the investigator positioned the patient appropriately for lumbar puncture. The lumbar area was prepared and draped in the usual aseptic fashion. Local anesthesia was utilized
• a spinal needle (20 or 22G) was introduced into the spinal canal, at the level deemed appropriate by the Investigator.
• the CSF samples were collected 60, 90 or 120 minutes after administration.
• a total of 4.0 mL of CSF were collected from each patient, and distributed into 4 separate collection tubes.
• the tubes were appropriately labeled with a patient identifier and submitted for bioanalytical analysis.
• the spinal needle was removed.
• Vitamin B 12 The levels of vitamin B 12 were determined in both the CSF and blood serum using Vitamin Bi 2 concentrations in the CSF will be analyzed for determination of Vitamin B 12 using a validated TOSOH Nex. IA procedure.
• the average ratio (B12 CSF/B12 Serum x 100) ranged from 1.1 to 1.9 for those individuals receiving intranasal administration of vitamin B 12 while those who received intramuscular injections of vitamin B 12 had an average ratio ranging from 0.17 to 0.24. This is a surprising result in that intranasal administration only has about a 7-12% bioavailability in the blood serum relative to intramuscular injection of vitamin B 12. This indicates that intranasal administration of vitamin B 12 reaches the CSF much more effectively than by intramuscular inj ection.
• a 4000 g batch of a cyanocobalamin solution of the present invention was prepared, which had a concentration of 500 mcg/O.lg of solution.
• cyanocobalamin solution Into the cyanocobalamin solution was then added 1.6 g of an aqueous solution containing 50% by weight of Benzalkonium Chloride was added to the solution and stirred for 5 minutes at 300 rpm. The pH was then measured and adjusted if the pH was not with the 4.5-5.5 range. Additional water was added to bring the weight of the solution to 4000 g.
• This example describes an exemplary pharmaceutical composition of the invention comprising an aqueous solution of salmon cyanocobalamin at a concentration sufficient to produce therapeutically effective plasma concentrations, delivered via an actuator to produce an aerosol of said solution, wherein the spray pattern ellipticity ratio of said aerosol is between 1.00 and 1.40 when measured at a height of 30 cm distance from the actuator tip.
• the volume of the aerosol can be between about 5 microliters and 1.0 ml, preferably between 20 and 200 microliters.
• This test method describes the procedure for characterizing plume geometry of the cyanocobalamin nasal solution formulations using the Spray View
• the plume geometry is characterized using a SprayView High Speed Optical Spray Characterization System (SprayView NSP) with Integrated SprayView NSx actuation station (Image The ⁇ n Engineering, Inc., Sudbury, MA) according to the methods described in U.S. Patent No. 6,665,421 and U.S. Patent Application Publication No. 20030018416 published January 23, 2003.
• SprayView NSP SprayView High Speed Optical Spray Characterization System
• Integrated SprayView NSx actuation station Image The ⁇ n Engineering, Inc., Sudbury, MA

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