Note: Descriptions are shown in the official language in which they were submitted.
CA 02863377 2014-09-15
CYANOCOBALAMIN LOW VISCOSITY AQUEOUS FORMULATIONS
FOR INTRANASAL DELIVERY
This application is a division of application number 2,656,823, filed in
Canada on June 23, 2006.
BACKGROUND OF THE INVENTION
10011 Vitamin 1312 is a dietary essential, a deficiency
of which results in
defective synthesis of DNA in any cell in which chromosomal replication and
division
= 10 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
B12
deficiencies. An early sign of B12 deficiency is a megaloblastic anemia.
Dietary B12,
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 812-
15 intrinsic factor complex reaches the ileum, it interacts
with a receptor on the mucosal
cell surface and is actively transported into circulation. Adequate intrinsic
factor, bile
and sodium bicarbonate (suitable pH) all are required for ileal transport of
vitamin B12.
Vitamin B12 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.
20 Achlorhydria and decreased secretion of intrinsic factor by
parietal cells secondary to
gastric atrophy or gastric surgery is a common cause of vitamin B12 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 B12 malabsorption is seen with pancreatic disorders (loss
of
25 pancreatic protease secretion), bacterial overgrowth,
intestinal parasites, sprue, and
localized damage to Beal mucosa' cells by disease or as a result of surgery.
The
recommended daily intake of vitamin 812 in adults is 2.4 }Lg.
CA 02863377 2014-09-15
[002] There are four main forms of vitamin B12: cyanocobalamin:
hydroxocobalamin, methylcobalamin and adenosylcobahunin. Methylcobalamin and
adenosylcobalamin are unstable and damaged by light. They are therefore
unsuitable
for use in dietary supplements or pharmaceuticals and are not essential since
they can
be formed from cyanocobalamin or hydroxocobalamin within the body. The main
form
of vitamin B12 found in food is hydroxocobalamin. The main fonn used
therapeutically
and in nutritional supplements is cyanocobalamin, chosen because it is the
most stable
form and therefore easiest to synthesize and formulate.
[003] 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
B12-intrinsic factor complex transport mechanism, vitamin B12 must therefore
be
administered systemically. Currently, therapeutic amounts of cyanocobalamin
are
administered by intramuscular or deep subcutaneous injection of
cyanocobalamin.
However, patients must return to the physician's office periodically to
receive
additional injections to maintain their levels of vitamin B12. However, an
intranasal
gel cyanocobalamin preparation, NASCOBAL is currently being marketed in which
cyanocobalamin is administered intranasally as maintenance vitamin B12
therapy.
However, many patients find the consistency of the intranasal gel unpleasant
and
would prefer to have administered intranasally a low viscosity spray
containing
cyanocobalamin.
[004] The prior art suggests that for vitamin B12 to be absorbed
intranasally in
therapeutically beneficial amounts, the concentration of the B12 in solution
must
either be greater that 1% by weight, see Merkus, U.S. Patent No. 5,801,161
(hereafter
"Merkus") or be administered intranasally in a viscous gel, Wenig, U.S. Patent
No.
4,724,231 (hereafter "Wenig") so that the gel remains in the nostril for an
extended
period of time. In fact Wenig states that B12 administered intranasally in a
low
viscosity solution is not in contact with the nasal mucosa long enough for a
sufficient
period of time to permit useful absorption. Wenig claims that most of the B12
is
wasted if the solution has a low viscosity. Merkus developed intranasal
formulations
of hydroxocobalamin having a concentration of hydroxocobalamin greater than
1%,
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however hydroxocobalamin is not very stable and thus has a short shelve-life.
Merlcus chose hydroxocobalamin because cyanocobalamin is not soluble in an
aqueous solution at concentrations greater than 1%.
[0051 The teachings of Wenig are clear in that the subject disclosure
fails to
teach a cyanocobalamin formulation in an aqueous form, suitable and effective
for
intranasal administration as described herein below. Wenig unambiguously
teaches
away from any cyanocobalamin formulation having a viscosity below the
expressly-
stated, critical range taught by Wenig of 2500-6000 cPs. In particular, Wenig
teaches
that an intranasal cyanocobalamin formulation "will contain a sufficient
amount of a
thickening agent so that the viscosity is from about 2500 to 6500 cps,
although more
viscous compositions even up to 10,000 cps may be employed." (Col. 2, lines 37-
39, ,
emphasis added). Nowhere does Wenig teach or suggest adjustment or selection
to
lower viscosity of an intranasal cyanocobalamin formulation as disclosed by
Applicant. On the contrary, Wenig's disclosure teaches directly away from
intranasal
cyanocobalamin formulations having a viscosity less than about 1000 cPs. The
cyanocobalamin gel compositions described by Wenig have a vastly higher
viscosity
than this, in all embodiments contemplated. Thus, Wenig teaches that "[a]
typical
composition of this invention" has a viscosity of "about 4500 cps" (Col. 3,
lines 41-
51). Each of the reported working embodiments provided by Wenig (for which
viscosity values are provided, and which are reportedly validated in terms of
bioavailability by measurement of blood plasma cyanocobalamin levels), have
respective viscosities of 4000 cps, 3500-4000 cps, and 4000 cps (see, e.g.,
Example 1;
formulations A, B, and C). As noted above, Wenig further emphasizes that "more
viscous compositions even up to 10,000 cps may be employed."
[006] Accordingly, there is clearly no teaching nor suggestion
provided by
Wenig to substitute an aqueous liquid (spray or drop) cyanocobalamin
formulation for
the disclosed intranasal gel formulation of Wenig. In addition, Wenig
expressly
teaches in the background section of the disclosure that non-gel (powder and
aqueous)
formulations of cyanocobalamin are ineffective for intranasal administration
to treat
vitamin B12 deficiency. With regard to aqueous formulations, Wenig cites,
e.g., a
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proposed aqueous isotonic sodium chloride solution of vitamin B12 reported by
Monto et al. (Am. J. Med. Sci. 223:113, 1953; Arch. Int. Med. 93:219, 1954).
Wenig
states that this solution, along with powdered cyanocobalamin formulations, is
ineffective for intranasal use to treat vitamin B12 deficiency, because:
[M]ost of the B12 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 B12 so administered is, in fact wasted. (Col. 1, lines
63-68).
[007] Thus, 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. In particular, Wenig teaches that effective intranasal
cyanocobalamin
formulations must have "a sufficient amount of a thickening agent so that the
viscosity
is from about 2500 to 6500 cps" (supra)¨which Wenig describes as a critical
parameter
to render the formulations "sufficiently viscous to maintain themselves in the
nasal
passages for a period of time which is long enough so that most of the B12 is
absorbed." (Col. 2, lines 24-29). These properties are directly contrasted by
Wenig to
the properties of non-gel (liquid and powder) cyanocobalamin compositions,
which,
according to Wenig's express teachings, fail to exhibit sufficient nasal
mucosal
residence time to achieve effective intranasal delivery/bioavailability of
cyanocobalamin.
[008] In other attempts to develop useful cyanocobalamin fommlations for
nasal
administration, Garcia-Arieta et al., Biol. Pharm. Bull. 24:1411-1416, 2001
(hereafter,
"Garcia-Arieta et al.") describes "Spray-Dried Powders as Nasal Absorption
Enhancers
of Cyanocobalamin" (Title). The only disclosure by Garcia-Arieta et al. with
regard to
any cyanocobalamin spray and drop formulation is derived from a comparative
experiment, from which the authors expressly report that liquid (spray and
drop)
cyanocobalamin compositions are inoperable for intranasal use. More
specifically,
Garcia-Arieta et al. tested bioavailability of three spray-dried, intranasal
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cyanocobalamin formulations alongside two experimental cyanocobalamin nasal
solutions (drops and spray containing 0.1% cyanocobalamin; no other
formulation
parameters specified) (p. 1412, right colunm). Garcia-Arieta et al. expressly
report that
the experimental nasal spray and drop formulations of cyanocobalamin yielded
no
detectable bioavailability whatsoever. As described at page 1415, left column,
Garcia-
Arieta et al. found from their experiments that:
"[N]either the nasal solution in drops nor in spray were able
to increase the basal level of serum cobalamin in rabbits to
a statistically significant level. This means that either the
cyanocobalamin is hardly absorbed by the nasal route when
it is administered without absorption enhancers or that due
to the lack of any viscosity-enhancing agent these
formulations were not retained in the nasal cavity for long
enough to allow their absorption. (emphasis supplied)."
[0091 These reports expressly describe inoperability of an aqueous,
low viscosity
cyanocobalamin formulation, which accords closely with the conclusions by
Wenig
noted above. Accordingly, both Wenig and Garcia-Arieta et al., teach that non-
gel,
liquid intranasal formulations of cyanocobalamin were understood to be
ineffective to
achieve useful therapeutic results. Wenig expressly describes a critical
threshold
viscosity above 2500 cps, and a much higher range of 3500-4500 cps for all
demonstrated working embodiments, of a useful intranasal gel formulation of
cyanocobalamin. Garcia-Arieta et al. further provides direct experimental
evidence that
simple 1% aqueous cyanocobalamin spray and drop formulations yielded no
detectable
bioavailability.
[00101 In other attempts to provide useful intranasal formulations to
treat vitamin
B12 deficiency, Slot et al., Gastroenterology 113:430-433, 1997 (hereafter
"Slot et al.),
report an intranasal formulation of hydroxocobalamin. Notably, Slot et al.
expressly
teach that hydroxocobalamin is the preferred form of vitamin B12 for treating
vitamin
B12 deficiency, and that cyanocobalamin is not an effective or practical
treatment agent
for use in liquid, intranasal delivery formulations or methods. Like Wenig
discussed
above, Slot et al. comment on a previously-described, isotonic saline solution
of
cyanocobalamin, stating that "[N]one of these proposals found a follow-up in
clinical
practice. Apparently the results were not very practical." (page 432, right
column).
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Slot et al. further teach away from intranasal cyanocobalamin formulations and
methods
for treating vitamin B12 deficiency, by disclosing that:
"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 B12 deficiencies. (page 432, right
column)."
[0011] Another prior art reference that teaches away from the
selection of
cyanocobalamin as a useful form of cobalamin in intranasal formulations and
methods
is Merkus, USPN 5,801,161, discussed above. Similar to the teachings of Slot
et al.,
Merkus expressly states that hydroxocobalamin is a preferred treatment agent
for
vitamin B12 deficiency in comparison to cyanocobalamin. In the specific
context of
nasal formulations, Merkus emphasizes that:
"[T]he most effective concentrations of vitamin B12 in the
formulations for nasal administration are higher than 1%.
The maximal concentration that can be reached with
cyanocobalamin is about 1%. 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 B12
per unit of volume can be administered and subsequently
absorbed nasally, when hydroxocobalamin is used. (Col. 3,
lines 43-53)"
[0012] Based on the foregoing teachings, persons of ordinary skill in the art
would not have found practical motivation to develop a low viscosity
cyanocobalamin
formulations and methods, particularly as an effective treatment tool for
intranasal
administration to treat vitamin B12 deficiency. Considering the prior art as a
whole,
there was simply no reasonable expectation that cyanocobalamin formulations
and
methods as described herein, below could be developed and employed to
successfully
achieve therapeutically effective delivery/bioavailability of cyanocobalamin
sufficient
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to alleviate vitamin 812 deficiency, as presently disclosed. Both Wenig and
Garcia-
Arieta et al. teach directly away from low viscosity liquid cyanocobalarnin
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.
Similarly
with regard to the selection and concentration of cyanocobalamin as an active
form of
cobalamin for use in an intranasal liquid formulation, Slot et al. and Merkus
collectively teach that cyanocobalamin would be expected to be ineffective, or
at best
strongly disfavored in comparison to hydroxocobalamin, in an intranasal
formulation
for treating vitamin 812 deficiency (independent from viscosity
considerations).
Exemplifying these teachings, Merlcus emphasizes that:
"A high and efficient intranasal absorption of vitamin B12
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 B12 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.
[0014] International Patent Application No. PCT/US86/00665, publication no.
WO 86/05987, discloses nasal spray composition containing vitamin 1312 as
cyanocobalamin. However, the specific spray formulations all contained a
mercury
compound as a preservative, however the disclosure did require the presence of
mercury compounds. Other preservatives were also mentioned including
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benzalkonium chloride and chlorobutanol. As was stated above, an iniranasal
gel
containing cyanocobalamin, NASCOBAL , is currently being produced and
marketed by Nastech Pharmaceutical Company Inc. of Bothell, Washington. It is
very effective in maintaining levels of vitamin B12 for patients who have been
deficient in the past but have recovered their levels of B12 through
intramuscular
injections. However, a number of patients find the consistency of the gel
unpleasant
in their nose, and would prefer an intranasal formulation that has a lower
viscosity
and is free of mercury compounds. Thus, there is a need to produce a
pharmaceutically stable aqueous solution of cyanocobalamin that has a low
viscosity,
is optionally free of mercury compounds, and has sufficient bioavailability to
be used
as a maintenance therapy for vitamin B12.
SUMMARY OF THE INVENTION
[0015] 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.
[0016] In alternate embodiments, 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. In certain embodiments 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. In certain embodiments, the solution is essentially free of
mercury
and mercury-containing compounds.
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[0017] Certain compositions within the scope of this invention will contain a
humectant to inhibit drying of the 1111100.118 membranes and to prevent
irritation. Any
of a variety of humectants can be used including but not limited to sorbitol,
propylene
glycol or glycerol. An illustrative, useful humectant is glycerin.
[0018] A preservative is generally employed to increase the shelf life of the
compositions. Examples of preservative include but are not limited to benzyl
alcohol,
chlorobutanol and benzalkonium chloride. In illustrative embodiments, the
preservative is benzalkonhun 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.
[0019] An exemplary formulation 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 0.02% and 96.79% water. In other detailed
embodiments, a nasal spray solution of Cyanocobalamin is provided in a spray
applicator containing 2.3 mL of a 500 mcg/0.1 mL 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. After
initial
priming, an exemplary spray pump spray delivers an average of 500 mcg of
cyanocobalamin and the 2.3 mL of spray solution contained in the bottle will
deliver 8
doses of the nasal spray.
[0020] 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.
In
certain embodiments 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. In more detailed embodiments,
the
cyanocobalamin solution administered according to the methods of the invention
is
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essentially free of mercury and mercury-containing compounds. In this context,
"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.
[0021] 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 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 cyanocobalamin. In a more
detailed
embodiment the B12 CSF levels are increased so that the ratio of B12 in the
CSF to
the levels in the blood serum is at least 1.9.
[0022] 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 gm 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 gm or less in size; 90% of the
droplets
produced by the spray are 55.3 gm or less in size; or 10% of the droplets
produced by
the spray are 12.5 gm or less in size.
CA 02863377 2014-09-15
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. lA shows a nasal spray pump kit containing the cyanocobalamin solution of
the present invention having an actuator that is not engaged.
FIG. 1B 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.
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DETAILED DESCRIPTION OF THE INVENTION
[0023] The following definitions may aid in the understanding of the present
invention.
[0024] "About": is taken to be a relative term denoting an approximation of
plus
or minus 20% of the nominal value it refers to. For the field of pharmacology
and
clinical medicine and analogous arts that are the subject of this disclosure,
this level
of approximation is appropriate unless the value is specifically stated to be
critical or
to require a tighter range.
[0025] "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.
[0026] "Aqueous": refers to a solution formed in water, but may contain lesser
amounts of other co-solvents.
[0027] "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)].
[0028] "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 pig of cyanocobalamin would have a
100% bioavailability, if an intranasal dose of cyanocobalamin contains 100 pig
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
pig, 6 pig, or
7 pig of cyanocobalamin would be taken up into the blood vasculature.
Likewise, if
the intranasal dose of cyanocobalamin contained 500 pig, at least 35 pig of
cyanocobalamin would be taken up into the blood vasculature, if the intranasal
formulation had a bioavailability of at least 7%.
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[0029] "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. In a
similar vein,
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.
[0030] "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.
[0031] "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. This includes the superior, middle and inferior
nasal
turbinates and the nasal pharynx. Note that the olfactory region is
concentrated in the
superior (upper 1/3) of the nasal turbinates. Cilial action pushes material
back toward
the oropharynx, so material deposited in the nasal vestibule encounters the
nasal
mucosa before entering the throat.
[0033] "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
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preferably less than 1%, by weight based on the total weight of active
ingredients in
the composition.
[0034] 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.
[0035] As used herein "peak concentration (C..õ) 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) of vitamin in a blood
plasma" are pharmacolcinetic parameters known to one skilled in the art.
[Laursen et
al., Eur. J. Endocrinology, 135: 309-315, (1996)]. The "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.," is the
maximum
concentration of cyanocobalamin in the blood serum of a subject following a
single
dosage of cyanocobalamin to the subject. The term "tmax" 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.
[0036] As used herein, "area under concentration vs. time curve (AUC) of
cyanocobalamin in a blood plasma" is calculated according to the linear
trapezoidal
rule and with addition of the residual areas. A decrease of 23% or an increase
of 30%
between two dosages would be detected with a probability of 90% (type II error
f3 =
10%). The "delivery rate" or "rate of absorption" is estimated by comparison
of the
time (tm..) to reach the maximum concentration (C.O. Both Cõ,.õ and tinax are
analyzed using non-parametric methods. Comparisons of the pharmacokinetics of
subcutaneous, intravenous and intranasal cyanocobalamin administrations were
performed by analysis of variance (ANOVA). For pair wise comparisons a
Bonferroni-Holmes sequential procedure was used to evaluate significance. The
dose-response relationship between the three nasal doses was estimated by
regression
14
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analysis. P <0.05 was considered significant. Results are given as mean values
+/-
SEM. (Laursen et al., 1996.)
[0037] The above-described cyanocobalamin solutions are designed to be
administered to the nasal mucosa either in drop or in spray form. However, 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
#
63385 produced by Pfeiffer GmbH, Radolfzell, Germany.
' Nasal Administration of Cvanocobalamin
[0038] Cyanocobalamin is administered intranasally using a nasal spray
according
to the present invention. In this area the following definitions are useful.
[0039] Aerosol ¨ A product that is packaged under pressure and contains
therapeutically active ingredients that are released upon activation of an
appropriate
valve system.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] Spray ¨ A liquid minutely divided as by a jet of air or steam.
[0044] Metered spray ¨ A non-pressurized dosage form consisting of valves that
allow the dispensing of a specified quantity of spray upon each activation.
[0045] 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.
CA 02863377 2014-09-15
[0046] The fluid dynamic characterization of the aerosol spray emitted by
metered
nasal spray pumps as a drug delivery device ("DDD"). Spray characterization is
an
integral part of the regulatory submissions necessary for Food and Drug
Administration ("FDA") approval of research and development, quality assurance
and
stability testing procedures for new and existing nasal spray pumps.
[0047] Thorough characterization of the spray's geometry has been found to be
the best indicator of the overall performance of nasal spray pumps. In
particular,
measurements of the spray's divergence angle (plume geometry) as it exits the
device;
the spray's cross-sectional ellipticity, uniformity and particle/droplet
distribution
(spray pattern); and the time evolution of the developing spray have been
found to be
the most representative performance quantities in the characterization of a
nasal spray
pump. During quality assurance and stability testing, plume geometry and spray
pattern measurements are key identifiers for verifying consistency and
conformity
with the approved data criteria for the nasal spray Pumps.
Definitions
[0048] Plume Height¨the measurement from the actuator tip to the point at
which the plume angle becomes non-linear because of the breakdown k of linear
flow.
Based on a visual examination of digital images, and to establish a
measurement point
for width that is consistent with the farthest measurement point of spray
pattern, a
height of 30 mm is defined for this study
[0049] Major Axis ¨ the largest chord that can be drawn within the fitted
spray
pattern that crosses the COMw in base units (mm)
[0050] Minor Axis ¨ the smallest chord that can be drawn within the fitted
spray
pattern that crosses the COMw in base units (mm)
[0051] Ellipticity Ratio ¨ the ratio of the major axis to the minor axis
[0052] D10¨ the diameter of droplet for which 10% of the total liquid volume
of
sample consists of droplets of a smaller diameter (um)
[0053] D50 - the diameter of droplet for which 50% of the total liquid volume
of
sample consists of droplets of a smaller diameter (im), also known as the mass
median diameter
16
CA 02863377 2014-09-15
[0054] D90- the diameter of droplet for which 90% of the total liquid volume
of
sample consists of droplets of a smaller diameter (gm)
[0055] Span ¨ measurement of the width of the distribution, The smaller the
value, the narrower the distribution. Span is calculated as (Doo -Dio)
Dso
[0056] % RSD ¨ percent relative standard deviation, the standard deviation
divided by the mean of the series and multiplied by 100, also known as % CV.
[0057] Cyanocobalamin nasal spray kit.
[0058] The present invention is further comprised of a cyanocobalamin nasal
spray kit and method of administering the cyanocobalamin solution using the
nasal
spray kit.
[0059] The nasal spray kit is exemplified by Figures 1A, 1B and Figure 2.
Figure
1A and 1B show a nasal spray device 10 before engagement (FIG. 1A) and after
engagement (FIG.1B). 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. 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, 22, pattern of Figure 2, is elliptical and has a major
axis, 24, and a
minor axis 26.
[0060] In one exemplary embodiment, 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. In
certain
embodiments less than 5% of the droplets of the cyanocobalamin solution are
less
than 10 minp size, the spray pattern has a major axis and minor axis of 25
and 40
mm, respectively, 50% of the droplets are 26.9 11M or less in size, 90% of the
droplets
are 55.3 gm or less in size, and 10% of the droplets are 12.5 gm or less in
size.
17
CA 02863377 2014-09-15
[00611 As noted above, 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.
Examples
of 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.
[0062] In exemplary therapy methods, 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 B12 therapy. For
example, an initial therapy regimen may involve the patient receiving daily
intramuscular injections of 100 ug 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 lig as doses in excess of 100 lig are rapidly
cleared
from the plasma into the urine, and administration of larger amounts of
vitamin B12
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.
[0063] Instead of a once a month injection of 100 p.g of cyanocobalamin, using
the cyanocobalamin spray described herein the patient self-administers a dose
of the
nasal spray, e.g., containing 500 ug 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 B12 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. Maintenance
cyanocobalamin therapy using the cyanocobalamin solution of the present
invention is
also indicated for those afflicted with malabsorption of vitamin B12 resulting
from
structural or functional damage to the stomach, where intrinsic factor is
secreted or to
the ileum, where intrinsic factor facilitates B12 absorption. These conditions
include
18
CA 02863377 2014-09-15
tropical sprue and nontropical sprue (Idiopathic steatorrhea, gluten-induced
enteropathy).
[0064] Maintenance cyanocobalamin therapy using the cyanocobalamin solution
of the present invention is also indicated for those afflicted with
malabsorption of
vitamin B12 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 B12 deficiency include ileitis, ileal resections, Crohn's disease
and
malignancies. Vitamin B12 deficiencies may also be the result of competition
by
intestinal parasites, and inadequate utilization of vitamin B12 occurring if
antimetabolites for the vitamin are employed in the treatment of neoplasia.
[0065] The intranasal cyanocobalamin solution of the present invention can
also
be used for individuals who require above normal levels of vitamin B12, due
to, for
example, pregnancy, thyrotoxicosis, hemolytic anemia, hemorrhage, malignancy,
hepatic and renal disease.
[0066] As noted above, 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.
[0067] In certain exemplary embodiments, 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
19
CA 02863377 2014-09-15
embodiments at least about 8%, 9%, 10%, 11%, or 12% or more, of the
bioavailability of an intramuscular injection of cyanocobalamin.
[0068] In certain embodiments, compositions according to the invention will
contain 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.
[0069] A preservative is generally employed to increase the shelf life of the
compositions. Examples of 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
0.002% to 2% based upon the total weight, although there may be appreciable
variation depending upon the agent selected.
[0070] In certain exemplary embodiments, 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.
[0071] Other 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.
[0072] 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. In certain embodiments, 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.
CA 02863377 2014-09-15
[0073] 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 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. In
certain
embodiments the B12 CSF levels are increased so that the ratio of B12 in the
CSF to
the levels in the blood serum is at least 1:9.
[0074] This is a significant embodiment of the present invention because
vitamin
B12 deficiency can result in irreversible damage to the nervous system.
Progressive
swelling of myelinated neurons, derayelination, and neuronal cell death are
seen in the
spinal colnum 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.
Since the neurological damage can be dissociated from the changes in the
hematopoietic, vitamin B12 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 B12 in the CSF can have tremendous benefit for neurological patients.
Thus,
intranasal administration of vitamin B12 according to the invention can be
used to
treat such diseases as Alzheimer's disease, dementia, and multiple sclerosis.
21
CA 02863377 2014-09-15
[0075] Exemplary formulations of the invention include the following:
Cyanocobalmin Nasal Spray 500 mcg/0.1 mL
Formulation:
Component Nasal Solution
Quantity (% w/w)
Cyanocobalamin, USP 0.50
Citric acid anhydrous, USP 0.12
Sodium citrate dihydrate, USP 0.32
Glycerin, USP 2.23
Benzalkonium chloride (50%), 0.04
NF
Purified water q.s. 100.0
[0076] Alternative buffer systems and amounts that can be used for
Cyanocobalamin Nasal Spray
Quantity (% w/w)
1) Citric Acid-Phosphate buffer
Citric Acid anhydrous, USP 0.240
Dibasic Sodium Phosphate anhydrous 0.357
2) Acetate buffer
Sodium Acetate anhydrous, USP 0.220
Acetic Acid, glacial, USP 0.064
3) Phosphate buffer
Monobasic Potassium Phosphate anhydrous, 0.483
NF
Dibasic Sodium Phosphate anhydrous 0.004 =
22
CA 02863377 2014-09-15
[0077] 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 0.1mL per
squirt and has a diptube length of 36.05 mm. It can be purchased from Pfeiffer
of
America of Princeton, NJ.
100781 The following examples are provided by way of illustration, not
limitation.
EXAMPLE 1
Comparison of Intranasal Cyanocobalamin Solution of the Present Invention with
NASCOBALD and Intramuscular Injections of Cyanocobalamin
Introduction
[0079] Nascobal (Cyanocobalamin, USP) is a synthetic form of vitamin 1312
with
equivalent vitamin B12 activity. The chemical name is 5,6-dimethyl-
benzimidazoly1
cyanocobamide. Currently, Nascobal (Cyanocobalamin, US?) is marketed as a
self-
administered nasal gel. The recommended dose of Nascobal (Cyanocobalamin,
USP) in subjects with vitamin B12 malabsorption who are in remission following
inject able vitamin B12 therapy is 500- g administered intranasally once
weekly.
[00801 Vitamin B12 deficiency has a number of causes, including malabsorption
of vitamin B12 resulting from structural or functional damage to the
gastrointestinal
system and dietary deficiency of vitamin B12.
[0081] The purposes of this study are to compare the bioequivalence of vitamin
B12 nasal gel versus the nasal spray, and to evaluate the relative
bioavailability of
three preparations of vitamin B12 in a fasted state in normal healthy male and
female
subjects.
100821 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.
23
CA 02863377 2014-09-15
Study Objectives
[0083] To compare the phannacokinetic profile of a single
intranasally-administered spray, single intranasally-administered gel
(Nascobal6), and
single intramuscular-administered vitamin B12 in a fasted state in normal
healthy male
and female subjects.
24
CA 02863377 2014-09-15
Investigational plan
Overall Study Design and Plan.
[0084] This study was a single-site, open-label, 3-way (3-treatment, 6-
sequence)
crossover, pharmacoldnetic study of vitamin B12 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:
[0085] Treatment A: One IN spray administration of 500- g vitamin B12. 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.
[0086] Treatment B: One rx gel administration of 500- g vitamin B12
(Nascobal6).
[0087] Treatment C: One IM administration of 100- g vitamin B12.
[0088] Subjects were on a Vitamin B12-free diet throughout each confinement
period. Subsequent treatments will be dosed no sooner than 14 days following
the
preceding treatment dose administration.
Treatments
Treatments Administered
[0089] On Day 1 of Periods I, II, and III after an 8 hour fast, subjects
received a
single IN spray of 500- g vitamin B12 (Treatment A), a single IN gel of 500- g
vitamin B12 (Nascobal ) (Treatment B), or a single IM administration of 100- g
vitamin B12 (Treatment C) based upon a randomization generated by the PPD
Development Biostatistician in one of six sequences. Following all periods,
all
subjects were to have received each treatment in a crossover manner. A washout
period of 14 days separated the three dosing periods.
[0090] On the morning of Day 1, subjects assigned to Treatment A received a
single IN spray administration of 500 g of vitamin B12. Subjects assigned to
CA 02863377 2014-09-15
Treatment B received a single IN gel administration of 500 pg of vitamin B12
(Nascobal ). Subjects assigned to Treatment C received a single IM
administration of
100 pg of vitamin B12. Doses were preceded by an overnight fast (i.e., at
least 8
hours) from food (not including water) and were followed by a fast from food
(not
including water) for at least 4 hours post-dose.
[0091] While confined at the clinical site, subjects received a
standardized
vitamin B12-deficient diet at scheduled times which did not conflict with
other study-
related activities. A registered dietician set up the diet, and the food staff
maintained a
diet diary. No dietary supplements were permitted during the study. Subjects
abstained
from consuming alcohol-containing, grapefruit-containing, or caffeine-
containing foods
or beverages for 72 hours prior to Check-in.
Study Variables
[0092] For each subject, the following pharrnacokinetic parameters
were
calculated whenever possible, based on the serum concentrations of vitamin B12
from
Treatments A, B, and C according to the model independent approach: Cmax,
Tmax, and
AUCo-i
Pharmacoldnetie Measurements
[0093] Blood samples for PI( analysis of vitamin B12 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
, 25 of vitamin B12 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
[0094] The phannacolcinetic parameters used in this study were those
typically
used to assess bioequivalence. All assessments of bioequivalence were based on
comparisons of AUC0-i, Tmax, and Cmax (test versus reference treatments).
[0095] AUC is a measure of the extent of drug bioavail ability and
reflects the
total amount of drug that reaches the systemic circulation.
26
CA 02863377 2014-09-15
[0096] Cm. represents the maximum senun concentration obtained after drug
administration and provides an indication that sufficient drug has reached the
systemic
circulation to provide a therapeutic response. In addition, Cõ,,õ provides
warning of
possible toxic drug levels.
[0097] T,õõ, was calculated and presented as median range.
Phannacoldnetic Variables
[0098] For each subject, the following phannacolcinetic parameters
were
calculated, whenever possible, based on the serum concentrations of vitamin
B12 from
Treatments A, B, and C, according to the model independent approach (Ref. 1):
[0099] Cõ,õõ Maximum observed concentration.
[00100] tmax Time to maximum concentration.
[00101] AUCo..t Area under the concentration-time curve from time 0 to the
time of
last measurable concentration, calculated by the linear trapezoidal rule.
[00102] Pharmacolcinetic calculations were performed, using SAS (SAS Inst.,
Version 8.02).
Statistical Methods Planned in the Protocol and Determination of Sample Size
Statistical and Analytical Plans/Pharmacokinetic Analysis
[00103] Levels of vitamin B12 in serum samples were measured as pg/mL. Serum
concentration values below the quantifiable limits of detection were treated
as zero.
Actual sampling times, rather than scheduled sampling times, were used in all
computations of the pharmacokinetic parameters. For ease of presentation,
however,
scheduled sampling times were used to present results in tables, listings, and
figures.
[00104] From the concentration data, non-compartmental pharmacokinetic
parameters (AUCo_t, Cmax,Tmax,) were calculated as described in Section 8.4.3.
Statistical Analysis
[00105] All statistical tests were conducted at the 0.05 significance level,
unless
otherwise specifically identified. Summary statistics of continuous parameters
consisted of number (N), mean, median, SD, and range.
27
CA 02863377 2014-09-15
[00106] Descriptive statistics were obtained and tabulated by treatment for
levels
of vitamin B12 at each time point and for the pharinacokinetic parameters
calculated.
[00107] 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. Cmax and AUCo.t were natural log (loge) transformed prior to
analysis.
The corresponding 90% confidence intervals for the geometric mean ratio were
obtained by taking the antilog of the 90% confidence intervals for the
difference
between the means on the log scale.
[00108] It was assumed that the test (Treatment A) is non-inferior (with
respect to
the reference (Treatment B) if the lower bound of the 90% confidence intervals
from
loge-transformed C., and AUCo_t were greater than or equal to 80%. If the
lower
bound of the 90% confidence intervals from loge-transformed C. and AUC04 were
less than 80%, it was assumed that non-inferiority could not be established.
[00109] The sequence effect was tested using the mean square error (MSE) for
subject within sequence as the error term. All other main effects were tested
against the
MSE from the ANOVA model.
[00110] 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.
[00111] For Tn,õõ, the analyses were run using Wilcoxon's matched pairs method
to
determine if differences exist between the test group and each reference
group.
28
CA 02863377 2014-09-15
SUMMARY - CONCLUSIONS
PHARMACOKINETIC RESULTS:
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.=
(1480 pg/mL, 1670 pg/mL, respectively) and AUCo.t (92000 pg*hrimL, 97000
pehrimL, respectively).
Additionally, the median difference for T.õ, between the spray and gel IN
formulation was less than 15
minutes (-0.24). The C. value for the IM formulation was significantly higher
than the Cm values for
the two IN formulations (p<0.0001).
Bioequivalence was established for. the Vitamin Bp IN spray with regard to the
gel data based on C.
and AUCo.t. The 90% confidence intervals for the loge-transformed C. and AUC01
for the spray and
gel formulations fell within the range of 80% to 125%. Additionally, non-
inferiority can be assumed
when comparing the two IN formulations because the lower bounds of the
confidence intervals are
greater than 80% for both AUC0.1 and Cm..
CONCLUSIONS:
= The relative bioavailability for the two IN formulations was 0.9715.
Bioavailability for
Treatment A (spray) -versus Treatment C (IM) was 0.6105, and 0.6284 when
comparing Treatment
B (gel) versus Treatment C (IM).
= The pharrnacokinetic profiles of the spray formulation and the gel
formulation are similar for
Cm. (1480 pgimL, 1670 pg/mL, respectively) and AUCo.t (92000 pehr/mL, 97000
pehr/mL,
respectively). Additionally, the median difference for T. between the spray
and gel IN
formulation was less than 15 minutes (-0.24). The C. value for the IM
formulation was
significantly higher than the C. values for the two IN formulations
(p<0.0001).
= Bioequivalence between the Vitamin 1312 spray formulation and the Vitamin
B12 gel formulation
was established using loge-transformed 90% confidence intervals for AUCo.t and
Cmõõ. The 90%
confidence intervals for the loge-transformed Cm. and AUC04 for the spray and
gel formulations fell
within the range of 0.80 to 1.25. Noninferiority can be assumed for the two IN
formulations
(Treatment A versus Treatment B).
All Vitamin Bp formulations were safe and well tolerated by healthy male and
female volunteers.
- ¨
29
CA 02863377 2014-09-15
[00112] As noted above, in certain embodiments of the invention 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) of 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.
[00113] The present disclosure provides detailed comparative bioavailability
studies and results to evince these unexpected performance characteristics of
the
methods and compositions of the invention. As described above, the relative
bioavailability for two exemplary intranasal (IN) formulations was 0.9715.
Relative
bioavailability when comparing treatment A (Spray) versus treatment C
(intramuscular = IM) was 0.6105, and 0.6284 when comparing Treatment B (gel)
versus Treatment C (IM). The pharmacokinetic profiles of the compared
cyanocobalamin spray formulation and gel formulation were similar for Cmax
(1480
pg/mL, 1670p, respectively) and AUCO-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).
[00114] While these data are not expressed directly in the form of comparative
AUC values for IN versus IM biovailability of cyanocobalamin, the relative AUC
values are readily and accurately derivable from the data presented above. In
particular, the comparative bioavailability study results presented above
demonstrate
that the "relative bioavailability" ratio of an exemplary spray cyanocobalmin
formulation compared to IM cyanocobalamin bioavailability, and of an exemplary
gel
formulation compared to IM bioavailability, is 0.6105, and 0.6284,
respectively.
These values represent ratios of the natural log of geometric means of the AUC
based
CA 02863377 2014-09-15
on nominal doses. These data were dosed normalized according to conventional
practice to the appropriate dose multiple based on a dose of 500 lig given
intranasal
and 100 lig given by IM. The skilled artisan will readily comprehend these
data and
fully appreciate that the dose normalized data yield a ratio of
bioavailability between
an IN cyanocobalamin solution of the invention and IM-administration that
reasonably corresponds to the disclosed relative minimum relative
bioavailability of at
least about 5%, 6%, or 7%. This determination can be made by a standard
mathematical operation to derive the dose normalized relative AUC values for
an IN
spray and IM injection. In the example provided above, this standard
operation/result
is 0.6105 X 100 pg,/500 g X100 = 12%; or a ratio of the AUC between the IN
spray
and IM injection of 0.12. In addition, the actual arithmetic AUC are provided
above
for an exemplary IN cyanocobalamin spray and gel, as 92000 and 97000
pg*Iir/mL,
respectively. These data likewise evince the corresponding AUC for the IM
injected
study comparator, according to the instant disclosure. For example, the
arithmetic
mean of the AUC for IM is calculated as 147155 pg*hr/mL (as readily derived by
90 reverse mathematical operation from the ratios given above--for example
for the spray
92000/147155 = 0.62 ratio). When dose normalized these data correspond
directly to
an exemplary relative bioavailability value within the presently-described
range of at
least 7%, and in other embodiments at least 9%, 10%, 11% or 12% or greater for
an
IN cyanocobalamin formulation of the invention compared to IM cyanocobalamin
bioavailability.
EXAMPLE 2
[00115] Also provided by the instant disclosure are results of a non-blinded,
single
dose, parallel group study to compare the uptake of Vitamin B12 into the
cerebrospinal
fluid (CSF) after intranasal and intramuscular administration in healthy male
and non-
pregnant female volunteers. This study compared CSF levels to plasma levels
produced by both formulations.
[00116] Thirty-six healthy male and non-pregnant female subjects, age 18 and
over, were enrolled in the study. Eighteen
subjects received a single
31
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intranasal dose of 500 mcg delivered as a 0.1 mL spray and eighteen subjects
received
a single intramuscular dose of 100 mcg delivered intramuscularly. Each subject
visited the clinical site three times in a one-month period. These visits
consisted of a
screening visit, one dosing visit and a final visit.
[00117] 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). 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
[00118] In addition to the above, on the day of dosing 7 mL blood samples were
drawn before dosing and post dosing at 5, 10, 15 and 20 minutes, and at 0.5,
1, 11/2, 2,
3, 4, 6, and 8 hours post-dose (prior to discharge).
[00119] The cerebrospinal fluid was evaluated for total Vitamin B12 content.
It was
the objective of the study described herein to measure the amount of Vitamin
B12
present in the blood and CSF following intramuscular (IM) and nasal
administration.
Reference and Test Products
[00120] Reference Product: Cyanocobalamin 100mcg intramuscular injection.
[00121] Cyanocobalamin Injection, USP is a sterile solution of cyanocobalamin
(Vitamin B12) for intramuscular or subcutaneous injection. Each mL contains
1,000 mcg
cyanocobalamin.
[00122] Test Product: Vitamin B12 Nasal Spray = 500 mcg/0.1 mL spray. 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.
[00123] Vitamin 1312 Nasal Spray was supplied as a 2.3 mL bottle to deliver
one
dose: 500 mcg/0.1 mL per dose.
[00124] Before intranasal dosing, all subjects were given an orientation of
the
proper dosing technique and general conduct of the study.
32
CA 02863377 2014-09-15
[00125] The subjects were instructed to gently blow his/her nose. The subjects
remained in a seated position, and the primed IN applicator was inserted into
the nostril
by the subject, under the direction of the study staff. During dosing, the
contralateral
nostril was closed with the forefinger. Subjects were instructed to tilt their
heads
slightly back for dosing and to return their heads to an upright position
while sniffing in
gently immediately following dosing. According to this protocol, a 0.1 mL dose
of
vitamin B12 spray was released into the nasal cavity of each subject (a dose
is a single
application to one nostril). Subjects were instructed to refrain from blowing
their nose
for 1 hour following IN treatment.
[00126] After dosing, each subject underwent lumbar puncture, involving the
retrieval of 4.0 mL of 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. At the appropriate time after
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
(1% xylocaine, 1-5 mL). Upon reaching a state of adequate anesthesia, 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.
Upon completion of CSF collection, the spinal needle was removed.
[00127] The levels of vitamin B12 were determined in both the CSF and blood
serum using Vitamin B12 concentrations in the CSF will be analyzed for
determination
of Vitamin B12 using a validated TOSOH Nex. lA procedure.
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CA 02863377 2014-09-15
Results and Conclusion
[00128] The data showed that the ratio of vitamin B12 to serum was higher in
those
individuals receiving intranasal administration of vitamin B12 than those
receiving
intramuscular injections of vitamin B12.
[00129] The average ratio (B12 CSF/I312 Serum x 100) ranged from 1.1 to 1.9
for
those individuals receiving intranasal administration of vitamin B12 while
those who
received intramuscular injections of vitamin B12 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 B12. This indicates that intranasal administration of vitamin B12
reaches the
CSF much more effectively than by intramuscular injection.
34
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EXAMPLE 3
Production of a Cyanocobalatnin Solution
1001301 A 4000 g batch of a cyanocobalamin solution of the present invention
was
prepared, which had a concentration of 500 mcg/0,1g of solution.
Starting Materials
I. Formula Record
Ingredient Name Theoretical Weight (Groins)
Cyanocobalamin, USP 20.0
Citric Acid, USP (Anhydrous) 4.8
Sodium Citrate, USP (Dihydrate) 12.8
Glycerin, USP 89.2
Benzalkonitun Chloride Solution, NF (50%) 1.6
Purified Water, USP 3871.6*
1001311 The 3871.6 grams of water was placed in a stainless steel container,
which
had been placed on a hot plate. The water was heated to about 30 C and
stirred. Into
the heated water was added 12.8 g of sodium citrate while the water was being
stirred
at 300 rpm for 5 minutes. The 4.8 g of citric acid was then added and stirred
for 10
minutes. Into this mixture was added 20.0 g of cyanocobalamin and stirred for
30
minutes at 30 Cat 300 rpm. The hot plate was then turned off. The 89.2 g of
glycerin was added and stirred for 5 minutes at 300 rpm. 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.
CA 02863377 2014-09-15
EXAMPLE 4
[00132] 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.
[00133] The volume of the aerosol can be between about 5 microliters and 1.0
ml,
preferably between 20 and 200 microliters.
[00134] This test method describes the procedure for characterizing plume
geometry of the cyanocobalamin nasal solution formulations using the SprayView
NSP system. The plume geometry is characterized using a SprayView High Speed
Optical Spray Characterization System (SprayView NSP) with Integrated
SprayView
NSx actuation station (Image Therm 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.
[00135] Using the formulation of table 1 the spray characterization and
droplet size
of the formulation in both a 3 mL bottle both having a nasal Spray Pump w/
Safety
Clip, Pfeiffer SAP #65550, which delivers a dose of 0.1mL per squirt and has a
diptube length of 36.05 mm.
[00136] The droplet size data are shown in the following table.
Droplet Size for Nasal Spray Bottle and Pfeiffer SAP # 60548
% < 10
D10 D50 Dgo Span micromet
lmL Salmon cyanocobalamin 12.5 26.9 55.3 1.6 5.1
36
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Below are listed the spray pattern results:
Spray Pattern MajorAxis MinorAxis EllipticityRatio
Active 3mL 35.3nun 30.8 min 1.14
=
=
37
