Note: Descriptions are shown in the official language in which they were submitted.
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o S
STABILIZED STEROID COMPOSITIONS
Field of the Invention
1 5The present invention relates to stabilized liquid compositions of
steroidal compounds, particularly adrenocorticosteroids. More particularly, the
present invention relates to stabilized aqueous steroidal compositions.
Background of the Invention
Many of the adrenocorticosteroids share a common structural feature,
namely, the dihydroxy acetone side chain at C-17. A number of studies has
demonstrated that the dihydroxy acetone side chain is prone to oxidative and
hydrolytic degradation in aqueous solutions. Kinetic studies germane to this
discussion include degradation in aqueous solutions of prednisolone,
described by Guttman, D.E. and Meister, P.D., '~he kinetics of the base-
catalyzed degradation of prednisolone," J. Am. Pharm. Assoc. 47 (1958) 773-
778 and Oesterling, T.O. and Guttman, D.E., "Factors influencing stability of
prednisolone in aqueous solution," J. Pharm. Sci. 53 (1964) 1189-1192,
hydrocortisone, described by Bundgard, H. and Hansen, J., "Studies on the
stability of corticosteroids. IV. Formation and degradation kinetics of 21-
3 0 dehydrocorticosteroids, key intermediates in the oxidative decomposition of 21-
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dehydrocorticosteroids, key intermediates in the oxidative decomposition of 21-
hydroxy corticosteroids," Arch. Pharm. Chem.. Sci. F~n. 8 (1980) 187-206,
Hansen, J. and Bundgard, H., "Studies on the stability of corticosteroids. 1.
Kinetics of degradation of hydrocortisone in aqueous solution," Arch. Pharm.
Chem.. Sci. Fdn. 7 (1979) 135-146, Hansen, J. and Bundgard, H., "Studies on
the stability of corticosteroids. Il. Kinetics and mechanism of the acid-catalyzed
degradation of corticosteroid," Arch. Pharm. Chem.. Sci. Edn. 8:5-14 (1980),
Hansen, J. and Bundgard, H., "Studies on the stability of corticosteroids. V. The
degradation pattern of hydrocortisone in aqueous solution," Int. J. Pharm. 6
(1980) 307-319 and Pitman, I.H., Higuchi, T., Alton, M. and Wiley, R.,
"Deuterium isotope effects on degradation of hydrocortisone in aqueous
solution," J. Pharm. Sci. 61 (1972) 918-920, cloprednol, described by Johnson,
D.M., "Degradation of cloprednol in aqueous solution. The enolization step," J.
Qr~ Chem. 47 (1982) 198-201, and triamcinolone acetonide, described by
15 Gupta, V.D., "Stability of triamcinolone acetonide solutions as determined by
high-performance liquid chromatography," J. Pharm. Sci. 72 (1983) 1453-1456
and Timmins, P. and Gray, E.A., "The degradation of triamcinolone acetonide in
aqueous solution: influence of the cyclic ketal function," J. Pharm. Pharmacol.
35 (1982) 175-177. Autoxidation has been reported to be the primary
20 degradation pathway under aerobic conditions in neutral and alkaline aqueous
solutions.
The autoxidation is strongly catalyzed by trace metal ions especially
copper and the incorporation of a sequestering agent eliminates the metal
catalysis. The oxidative degradation products have been characterized for
25 hydrocortisone and flurandrenolide in cream base. The steroidal glyoxals (21-
dehydro steroid derivatives) were found to be the key intermediates in the
oxidative decomposition of the steroids.
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Triamcinolone acetonide is a known pharmaceutically active ingredient
used for the treatment of a variety of topical, nasal, bronchial and other
inflammation conditions as described in US Patent Nos. 3,897,779 and
4,767,612, the disclosures of which are incorporated herein by reference.
Brief Summery of the Invention
The present invention relates to a pharmaceutical composition
comprising a therapeutically effective amount of triamcinolone acetonide in
admixture with an aqueous pharmaceutical acceptable carrier providing said
composition with properties resistant to triamcinolone acetonide degradation in
the presence of contaminants. A special embodiment of the invention relates to
a pharmaceutical composition comprising a therapeutically effective amount of
triamcinolone acetonide in admixture with an aqueous pharmaceutically
acceptable carrier providing said composition with properties resistant to
triamcinolone acetonide degradation in the presence of contaminants, wherein
the pH of the composition is between about 4.9 and 5.1, comprises an effective
degradation inhibiting amount of EDTA.
Brief Description of the Drawings
Figure 1 shows HPLC chromatograms of degraded triamcinolone
acetonide in aqueous solutions at 70~C for 22 hours. (a) pH 4.0, (b) pH 6.1, (c)pH 7.4, (d) pH 8.6
Figure 2 shows time-courses for triamcinolone acetonide, I (o), the
glyoxal hydrate, IV (o), the glycolic acid, V (~) and the etianic acid, Vl (-) during
the oxidative degradation of I in borate buffer of pH 8.9 at 70~C. Buffer
concenlr~lio", 0.032 M.
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Figure 3 shows the effect of borate buffer concentration on the rate of
degradation of I in the presence (o) and absence (o) of EDTA at 70~C. pH, 9.2.
Ionic strength, 0.1. EDTA concentration, 5X10 4 M.
Figure 4 shows the effect of EDTA concentration on the rate of
degradation of I in carbonate buffer of pH 9.4 at 70~C.
Figure 5 shows the effect of CuS04 concentration on the rate of
degradation of I in borate buffer of pH 8.9 at 70~C.
Figure 6 shows the log k-pH profiles for the degradation of I in aqueous
solutions at 70~C in the absence (o) and in the presence of 1 x10-5 M CuSO4 (~)
or 5x10 4 M EDTA (o).
Figure 7 shows a scheme of degradation products of triamcinolone
acetonide (I).
ne~Ailed l)escription of Preferred F~nbod~ments
Reference is made to the following non-limiting examples. These
examples utilize the following materials, equipment and analytical procedures.
~teri~
Triamcinolone acetonide was obtained from Upjohn (Kalamazoo, Ml).
The purity of the drug substance was greater than 99 % as determined by
HPLC analysis. Cupric acetate (Fisher, Pittsburgh, PA), periodic acid (Fisher),
EDTA disodium salt (Fisher) and all other chemicals were of ACS reagent
grade and used as received. Acetonitrile was HPLC grade.
HPi C An~lysis
The chromatography system consisted of a pump (Perkin Elmer 410), an
automatic injector (Perkin Elmer ISS 100), a photo diode array detector (Perkin
Elmer 480), and a networking computer data acquisition system (Waters 860).
The HPLC method employed a 250 mmx 4.6 mm i.d., 5 um particle size, octyl-
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bonded silica stationary phase column which is sterically protected (Zorbax Rx-
C8) and a mobile phase consisting of acetonitrile:water:trifluoroacetic acid
(320:680:0.68, vlvlv). The flow rate was 1.5 mUminute and the detector
wavelength for UV absorbance detection was 238 nm.
Kinetic method
Stock solutions of triamcinolone acetonide (4 mg/mL) in methanol and
buffers (0.2 M) in deionized water were prepared. An aliquot (0.5 mL) of the
triamcinolone acetonide stock solution, an appropriate amount of buffer stock
solution, hydrochloric acid (pH 1.1-2.0), chloro~cet~te (pH 3.0), ~cet~te (pH
10 4.0-5.2), phosphate (pH 6.1-7.4), borate (pH 8.6-8.9) or carbonate (pH 9.0-
10.0) buffer stock solution and an appropriate amount of 1 M NaCI to maintain
an ionic strength of 0.1 were transferred to a 100 mL volumetric flask and filled
to volume with water. A low buffer conce"lr~lion (0.02 M) was used to minimize
possible catalysis by buffer species. To study the influence of cupric ion or
15 EDTA on the oxidative degradation rate, an appro~riate amount of CuS04 (5 x
10 4 M) or Na2 EDTA (1.1 x 10-2M) stock solution was added to the flask. No
attempts were made to control the oxygen concenl,dlion in the system.
SpectrosC~DY
The 1H and 13C NMR spectra were recorded on a Varian VXRS 200 NMR
20 spectrometer using CDCI3 or DMSO-d6 as the solvent. The electron impact (El)
mass spectra were obtained using a Finnigan 4500 mass spectrometer via
direct inlet. The electron energy was 70 eV. The FAB mass spectra were
obtained using a VG 70 SE mass spectrometer and nitrobenzyl alcohol as a
matrix.
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Example 1
De~radation product in acidic solution.
Triamcinolone acetonide (200 mg) was suspended in 200 mL of 0.1 N
HCI and the suspension was refluxed for 24 hours. At the end of this time
5 period, the solution became clear. Upon cooling the solution, a white solid
material precipitated from the solution. The solid was filtered and the product
was recrystallized from 20% methanol in water. The crystalline material was
dried under vacuum at 60~C for two hours.
The El mass spectrum of the isolated product (ll in Fig. 7) showed a
molecular ion at m/z 394 (C2, H27FO6) and a peak at m/z 374(M+-HF). The
carbon NMR spectrum showed the absence of peaks at 25,26 and 110 ppm
which correspond to the carbons of the cyclic ketal group of triamcinolone
acetonide. Likewise, the proton NMR spectrum showed the absence of peaks
at 1.0 and 1.3 ppm corresponding to the methyl protons of the ketal group. The
l S mass and NMR spectra were ide"lical to those of an authentic sample of
triamcinolone (Il).
F~-~mDle 2
The steroidal ~Iyoxal hydrate (IV in Fi~. 7)
To a solution of 1 9 of triamcinolone acetonide in 125 ml of methanol
20 was added a solution of 250 mg of cupric acetate in an equal volume of
methanol. The solution was stirred at room temperature for one hour. HPLC
analysis of the solution showed that the reaction was complete with only one
product. The methanol was removed under vacuum using a rotary evaporator.
The residue was suspended in 500 ml of water and the product was extracted
25 with 200 ml of ethyl ~cet~te The ethyl acetate layer was washed with water
and evaporated to dryness under vacuum. The residue was dissolved in a
minimum amount of acetone. To the acetone solution, water was added
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carefully until the solution became slightly turbid. The solution was kept in a
refrigerator overnight. The crystallization from aqueous acetone gave fine
needles. The material was filtered and dried at 60~C under vacuum for 2 hours.
The fast atom bombardment (FAB) mass spectrum of the compound
S showed a protonated molecular ion (M+H)+ at m/z 451 and a peak at 431
(M+H-HF)+. The El mass spectrum did not show the molecular ion but
contained peaks at 432 (M-H2O)+ and 412 (432-HF)+. The carbon NMR
spectrum showed a resonance of C-21 at 85 ppm (doublet) in place of 66 ppm
(triplet) in 1. The theoretical elemental analysis values calculated for C24H3,FO7
1 0 are C 63.98, H 6.94; found, C 62.70, H 7.06. The spectral information agreed
with the structure IV in Fig. 7.
Example 3
The steroidal ~Iycolic acid (V in Fig. 7)
The glyoxal hydrate (IV) prepared from 1 9 of triamcinolone acetonide
1 5 was suspended in 250 mL of 0.1 N NaOH. The suspension was stirred at room
temperature for two hours. HPLC analysis showed that the glyoxal hydrate was
completely converted to the glycolic acid (V). The solution was filtered and the
filtrate was acidified by adding 1 N HCI dropwise until the pH of the solution
was approximately 3. The product was extracted with 250 mL of ethyl acetate
and the ethyl acetate layer was washed with water. The ethyl acetate was
removed under vacuum using a rotary evaporator. The residue was dissolved
in a minimum amount of methanol. To the solution was added water slowly
until no further prec;~ilation occured. The solid material was filtered and dried
under vacuum at 60~C for two hours.
The FAB mass spectrum showed a protonated molecular ion (M+H)+ at
- m/z 451. The El mass spectrum also showed (M+H)~ at 451 and peaks at 435
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(M-CH3)+ and 430 (M-HF)+. The carbon NMR spectrum of the compound
showed C-20 and C-21 at 71 (doublet) and 173 ppm(singlet), respectively. The
proton NMR spectrum showed an acid proton at 12.4 ppm and C-20 non-
exchangeable proton at 4.3 ppm. The mass and NMR spectra agreed with the
5 structure V.
Example 4
The eti~nic acid derivative (Vl)
To a solution of 2 g of triamcinolone acetonide in 300 mL of methanol
was added a solution of 4 9 of periodic acid in 400 mL of water. The aqueous
10 methanolic solution was left at room temperature for two days in the dark. The
methanol was removed under vacuum using a rotary evaporator and the
residue was suspended in 200 mL of water. To the aqueous solution was
added 1 N NaOH dropwise until the pH of the solution was 8-9. The solution
was filtered and the filtrate was shaken with ethyl acetate (2x30 mL). The ethyl
15 acetate layer was discarded. The aqueous layer was acidified by dropwise
addition of 1 N HCI until the pH of the solution was 2-3. The product was
extracted with ethyl acetate (3x100 mL). The ethyl acetate layer was dried over
200 mg of anhydrous Na2SO4 and removed under vacuum using a rotary
evaporator. The residue was recrystallized from methanol. The white solid was
20 dried under vacuum at 60~C for two hours.
The El mass spectrum of the compound showed a molecular ion atm/z
420 and peaks at 405 (M-CH3)+ and 400 (M-HF)'. The carbon NMR spectrum
showed a resonance of C-20 at 174 ppm in place of 210 ppmin I and loss of C-
21 at 66 ppm in 1. The proton NMR spectrum showed an acid proton at 12.8
25 ppm and loss of C-21 protons in 1. The mass and NMR spectra agreed with the
structure Vl.
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The stability-specific HPLC method was used to follow the extent of the
degradation of triamcinolone acetonide in aqueous solutions (Fig. 1). Because
of the low solubility of the drug in aqueous solutions, the conce,ll.alions of the
degradation products were not enough for isolation and identification for most
5 of the degradates. Therefore the approach taken in elucidating the degradationprofile of the steroid had two stages. The first stage involved the partial
identification of the degradates in the degraded sample solutions by molecular
weight determination using an LC-MS technique. The second stage required
the synthesis and characterization of potential degradation products followed
l O by identification of such compounds in the degraded solutions by comparing
their molecular weights and HPLC retention times.
The glyoxal synthesized from I was characterized as a hydrate(lV) by
elemental analysis, NMR and FAB mass spectra. However, the El mass
spectrum of the compound yielded the highest m/z peak corresponding to the
15 non-hydrated aldehyde, due to the loss of water during ionization of the
sample. The co-injection of a degraded sample of I and the synthetic
compound displayed one peak at 8.3 minutes. The ion-spray mass spectra of
the synthetic and degraded samples produced identical peaks at m/z 451
(M+H)+ and 492 (MH++CH3CN) in the mobile phase. Thus the degradate was
20 identified as the steroidal glyoxal. It exits in the hydrated form (IV) in aqueous
solutions as well as in the solid state. The glyoxal hydrate peak appears first in
degrading solutions of the drug in neutral and alkaline pH regions (Fig. 1 b, c,d)-
In neutral and basic solutions, the primary degradation pathway is
25 autoxidation of the primary alcoholic group at C-21 as in other corticosteroids.
The major degradation product is the steroid glyoxal hydrate (IV) as shown
before (Fig. 2). The product further degrades to V in alkaline solutions. As the
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pH of the solution decreases beiow 4, this oxidative degradation pathway is
absent (Fig. 1 a). Instead, the cyclic ketal of triamcinolone acetonide is cleaved
yielding triamcinolone (Il).
The rate of disappearance of triamcinolone acetonide exhibited a
S dependency on the buffer concentration at constant pH and ionic strength (Fig.
3). In the absence of EDTA, a plot of the rate constant against the buffer
concenlralion is curved and the rate constant levels off at high buffer
concentrations. In the presence of EDTA, the rate constant is independent of
the buffer concentration. The results strongly indicate that the buffer
10 components themselves have no catalytic influence, but that the rate increase
is due to the catalytic effect of trace-metal contaminants present in buffer
components. Similar observations were made in the degradation of
prednisolone (Oesterling and Guttman, 1964) and hydrocortisone (Hansen and
Bundgard, 1979).
The effect of EDTA concenlr~lion on the degradation rate constant is
shown in Fig. 4. The results show that EDTA even in a very low concentration
has a profound inhibitory effect, reaching the maximum inhibition level at the
concer,l,ation of approximately 1x10-5 M.
Cupric ion has been known to catalyze the oxidative degradation of 21-
20 hydroxy corticosteroids. The addition of cupric salt to a borate buffer increasedthe degradation rate (Fig. 5), with the maximum rate at the concenlralio" of
5x10-6 M CuS O4. Ferric and nickel ions exhibited negligible catalytic effects.
The degradation of triamcinolone acetonide was studied in aqueous
solutions over the pH range of 1-10 at 70~C and ionic strength of 0.1. At
25 constant pH and temperature, the degradation followed an apparent first-order
process under all experimental conditions. The results are seen as the plot of
logarithm of the rate constant versus pH (Fig. 6).
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In the pH region below 3, the log k-pH profile shows a straight line with a
slope of approximately -1 indicating that the degradation appears to be a
specific-acid-catalyzed process. The same straight line was observed when the
degradation proceeded in the presence of cupric ion or EDTA. In this pH
S region, the cleavage of the cyclic ketal is the dominant reaction yielding
triamcinolone(ll). The non-oxidative cleavage reaction is not dependent on
metal catalysis. Therefore, it is expected that incorporation of cupric ion or
EDTA into the solutions wouid have no effect on the degradation rate as shown
in Fig. 6.
At pH above 4, the predominant degradation product was the oxidation
product (IV). Between pH 4 and 7, the profile shows a straight line with a slope
of approximately +1 indicating specific-base catalysis. In this pH region,
incorporation of 1 x10-5 M of CuS04 into the solutions did not have any effect
on the degradation rate, whereas 5 x 10 4 M EDTA decreased the degradation
rate two orders of magnitude. This observation indicates that the trace metal
ions present in the buffer components catalyze the degradation to the
maximum and, therefore, additional cupric ion has no further catalyzing effect.
In the pH region above 7, a pH-independent plateau is reached,
followed by a straight line portion with a slope of approximately +1 between pH
8 and 10. Between pH 7 and 10, the experimental points are more scattered.
Fig. 3 shows that the rate constant (1.2x10-5 sec~') extrapolated to zero buffer
concentration coincide with that obtained in the presence of EDTA. Thus, on
- eliminating the buffer catalysis (trace-metal catalysis in buffer components),the
log k-pH profile would be superimposable on that determined in the presence
2 S of EDTA.
The log k-pH profile in the present study shows no plateau when CuS04
or EDTA is incorporated into the solutions and the log k increases with
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increasing pH with a slope of +1. Cupric ion enhances the rate, whereas EDTA
retards the rate. This observation strongly indicates that the plateau is not due
to the ionization of steroid molecules but to a different degree of catalysis bytrace-metal-ion contaminants present in the buffer components.
The rate expression for the copper-catalyzed and the metal-sequestered
reactions is given by
k=kH[H+]+ko+koH~OH-]
where k is the observed rate constant, kH and koH are the respective second-
order rate constants and ko is the water-catalyzed or spontaneous reaction rate
constant. The values of kH, koH and ko were estimated from Fig. 6 to be 3.0x104
sec~1 M-1, 15.9 sec 1 M-1 and 4.6x1 o~8 sec~', respectively, for the copper-
catalyzed degradation reaction, and 3.0x10-4 sec~' M-', 0.11 sec ' M-' and
2.6x1 o~8 sec~~, respectively, for the metal-sequestered reaction. It is noteworthy
that the cupric-ion-catalyzed degradation is 150 times as fast as that of the
1 5 metal-sequestered degradation in neutral and alkaline pH regions.
The steroidal glyoxal (Ill) undergoes further degradation to the
corresponding glycolic acid (V) in alkaline solutions. It is seen that the
formation of V goes through an induction period (Fig. 2).A small amount of the
corresponding etianic acid (Vl) was observed in the degradation of I in alkaline2 0 solutions (Fig. 2). This result indicates that a small amount of the steroid
undergoes cleavage between C-20 and C-21 during the oxidation. It is likely
that Vl could have been formed by oxidative cleavage of the glyoxal (Ill).
The experimental data described above and shown in the figures
demonstrate the stability and degradation-resistant properties of embodiments
2 5 and preferred embodiments according to the present invention under
accelerated laboratory conditions. These properties provide long term stability
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to the aqueous triamcinolone acetonide compositions of the present invention
under normal use, at ambient temperature for storage times awaiting use by
the distributor, pharmacist and patients. It is expected that the shelf life,
required for the commercial acceptability of the present compositions, will be at
S least 6 months to one or more years, at room temperature, that is about 25
degrees C.
The compositions of this invention are useful in the treatment of patients
suffering from certain medical disorders. For example, compounds within the
present invention are useful as bronchodilators and asthma-prophylactic
10 agents, e.g. for the treatment of inflammatory airways disease, especially
reversible airway obstruction or asthma, and for the treatment of other diseases
and conditions characterized by, or having an etiology involving, morbid
eosinophil accumulation. As further examples of conditions which can be
ameliorated may be mentioned inflammatory diseases, allergic rhinitis, adult
15 respiratory distress syndrome. A special embodiment of the therapeutic
methods of the present invention is the treating of asthma.
In practice compositions of the present invention may generally be
administered by inhalation and may be presented in forms permitting
administration suitable for use in human or veterinary medicine. These
20 compositions may be prepared according to the customar,v methods, using one
or more pharmaceutically acceptable adjuvants or excipients. The adjuvants
comprise, inter alia, diluents, sterile aqueous media and the various non-toxic
organic solvents. The compositions may be presented in the form of aqueous
solutions or suspensions, and can contain one or more agents chosen from the
25 group co"".risi"g surfactants, flavorings, colorings, or preservatives in order to
obtain pharmaceutically acceptable preparations.
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Suitable compositions containing the compounds of the invention may
be prepared by conventional means. For example, compounds of the invention
may be dissolved or suspended in a suitable carrier for use in a nebulizer or a
suspension or solution aerosol.
The percentage of active ingredient in the compositions of the invention
may be varied, it being necessary that it should constitute a proportion such
that a suitable dosage shall be obtained. Obviously, several unit dosage forms
may be administered at about the same time. The dose employed will be
determined by the physician, and depends upon the desired therapeutic effect,
l 0 the route of administration and the duration of the treatment, and the condition
of the patient. In the adult, the doses are generally from about 0.001 to about
50, preferably about 0.001 to about 5, mg/kg body weight per day by inhalation
In each particular case, the doses will be determined in accordance with the
factors distinctive to the subject to be treated, such as age, weight, general
state of health and other characteristics which can influence the efficacy of the
medicinal product.
The products according to the invention may be administered as
frequently as necessary in order to obtain the desired therapeutic effect. Some
patients may respond rapidly to a higher or lower dose and may find much
weaker maintenance doses adequate. For other patients, it may be necessary
to have long-term treatments at the rate of 1 to 4 doses per day, in accordance
with the physiological requirements of each particular patient. Generally, the
active product may be administered orally 1 to 4 times per day. It goes without
saying that, for other patients, it will be necessary to prescribe not more thanone or two doses per day.
The present invention may be embodied in other specific forms without
departing from the spirit or essential attributes thereof and, accordingly,
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reference should be made to the appended claims, rather than the
specification, as indicating the scope of the invention.
