U.S. patent application number 13/370493 was filed with the patent office on 2012-06-07 for method of treatment of allergic rhinitis.
This patent application is currently assigned to GlaxoSmithKline Intellectual Property Management Limited. Invention is credited to Keith Biggadike, Ian Buxton, Steven John Coote, Rosalyn Kay Nice, Kenton Lewis Reed, Amyn Sayani, Avinash Chander Sharma.
Application Number | 20120142651 13/370493 |
Document ID | / |
Family ID | 27732225 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142651 |
Kind Code |
A1 |
Biggadike; Keith ; et
al. |
June 7, 2012 |
METHOD OF TREATMENT OF ALLERGIC RHINITIS
Abstract
There is provided a method of treatment of allergic rhinitis
which comprises administering to a patient a pharmaceutically
acceptable amount of a pharmaceutical formulation comprising an
aqueous suspension of particulate compound of formula (I)
##STR00001## or a solvate thereof, wherein the administration is
once-per-day and intranasal, and the total daily dose of the
compound (I) is 50 to 100 .mu.g.
Inventors: |
Biggadike; Keith;
(Stevenage, GB) ; Buxton; Ian; (Mississauga,
CA) ; Coote; Steven John; (Stevenage, GB) ;
Nice; Rosalyn Kay; (Stevenage, GB) ; Reed; Kenton
Lewis; (Mississauga, CA) ; Sayani; Amyn;
(Mississauga, CA) ; Sharma; Avinash Chander;
(Mississauga, CA) |
Assignee: |
GlaxoSmithKline Intellectual
Property Management Limited
Brentford
GB
|
Family ID: |
27732225 |
Appl. No.: |
13/370493 |
Filed: |
February 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12858940 |
Aug 18, 2010 |
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13370493 |
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10502902 |
Jan 28, 2005 |
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PCT/GB03/00504 |
Feb 4, 2003 |
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12858940 |
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10067020 |
Feb 4, 2002 |
6858596 |
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10502902 |
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Current U.S.
Class: |
514/172 |
Current CPC
Class: |
A61K 31/58 20130101;
A61K 31/56 20130101; A61P 11/02 20180101; A61P 5/44 20180101; A61K
9/0043 20130101; A61K 47/38 20130101; A61K 9/0078 20130101; A61P
29/00 20180101; A61P 37/08 20180101 |
Class at
Publication: |
514/172 |
International
Class: |
A61K 31/58 20060101
A61K031/58; A61P 11/02 20060101 A61P011/02 |
Claims
1. A method of treatment of allergic rhinitis which comprises
administering to a patient a pharmaceutically acceptable amount of
a pharmaceutical formulation comprising an aqueous suspension of
particulate compound of formula (I) ##STR00008## or a solvate
thereof, wherein the administration is once-per-day and intranasal,
and the total daily dose of the compound (I) is 50 to 100 .mu.g.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/858,940, filed 18 Aug. 2010; which is a
Continuation of U.S. patent application Ser. No. 10/502,902, filed
28 Jul. 2004, now abandoned; which is a 35 USC 371 U.S. National
Phase of International Patent Application No. PCT/GB03/00504 filed
4 Feb. 2003, which is a Continuation-In-Part of U.S. patent
application Ser. No. 10/067,020, filed 4 Feb. 2002, now granted as
U.S. Pat. No. 6,858,596.
FIELD OF THE INVENTION
[0002] The present invention relates to pharmaceutical formulations
containing an anti-inflammatory and anti-allergic compound of the
androstane series and to processes for their preparation. The
present invention also relates to therapeutic uses thereof,
particularly for the treatment of inflammatory and allergic
conditions.
BACKGROUND OF THE INVENTION
[0003] Glucocorticoids which have anti-inflammatory properties are
known and are widely used for the treatment of inflammatory
disorders or diseases such as asthma and rhinitis. For example,
U.S. Pat. No. 4,335,121 discloses 6.alpha.,
9.alpha.-Difluoro-17.alpha.-(1-oxopropoxy)-11.beta.-hydroxy-16.alpha.-met-
hyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester (known by the generic name of fluticasone
propionate) and derivatives thereof. The use of glucocorticoids
generally, and especially in children, has been limited in some
quarters by concerns over potential side effects. The side effects
that are feared with glucocorticoids include suppression of the
Hypothalamic-Pituitary-Adrenal (HPA) axis, effects on bone growth
in children and on bone density in the elderly, ocular
complications (cataract formation and glaucoma) and skin atrophy.
Certain glucocorticoid compounds also have complex paths of
metabolism wherein the production of active metabolites may make
the pharmacodynamics and pharmacokinetics of such compounds
difficult to understand. Whilst the modern glucocorticoids are very
much safer than those originally introduced, it remains an object
of research to produce new molecules and formulations of old and
new molecules which have excellent anti-inflammatory properties,
with predictable pharmacokinetic and pharmacodynamic properties,
with an attractive side effect profile, and with a convenient
treatment regime.
[0004] We have now identified a novel glucocorticoid compound and
formulation thereof which substantially meets these objectives, in
particular one suitable for intranasal administration.
[0005] Many millions of individuals suffer from seasonal and
perennial allergic rhinitis worldwide. Symptoms of seasonal and
perennial allergic rhinitis include nasal itch, congestion, runny
nose, sneezing and watery eyes. Seasonal allergic rhinitis is
commonly known as `hay fever`. It is caused by allergens which are
present in the air at specific times of the year, for example tree
pollen during Spring and Summer Perennial allergic rhinitis is
caused by allergens which are present in the environment during the
entire year, for example dust mites, mold, mildew and pet
dander.
[0006] To formulate an effective pharmaceutical nasal composition,
the medicament must be delivered readily to all portions of the
nasal cavities (the target tissues) where it performs its
pharmacological function. Additionally, the medicament should
remain in contact with the target tissues for relatively long
periods of time. The longer the medicament remains in contact with
the target tissues, the medicament must be capable of resisting
those forces in the nasal passages that function to remove
particles from the nose. Such forces, referred to as `mucociliary
clearance`, are recognised as being extremely effective in removing
particles from the nose in a rapid manner, for example, within
10-30 minutes from the time the particles enter the nose.
[0007] Other desired characteristics of a nasal composition are
that it must not contain ingredients which cause the user
discomfort, that it has satisfactory stability and shelf-life
properties, and that it does not include constituents that are
considered to be detrimental to the environment, for example ozone
depletors. In the case of administration of glucocorticoids, the
potential for any undesirable side effects should preferably be
minimised.
SUMMARY OF THE INVENTION
[0008] Thus, according to one aspect of the invention, there is
provided a pharmaceutical formulation comprising an aqueous
suspension of particulate compound of formula (I)
##STR00002##
or a solvate thereof.
[0009] Preferably, the formulation will contain one or more
suspending agents.
[0010] Preferably, the formulation will contain one or more
preservatives.
[0011] Preferably, the formulation will contain one or more wetting
agents.
[0012] Preferably, the formulation will contain one or more
isotonicity adjusting agents.
[0013] According to one particular aspect of the present invention
we provide a pharmaceutical formulation which comprises:
(i) an aqueous suspension of particulate compound of formula (I) or
a solvate thereof; (ii) one or more suspending agents; (iii) one or
more preservatives; (iv) one or more wetting agents; and (v) one or
more isotonicity adjusting agents.
[0014] The formulations of the present invention may be stabilised
by appropriate selection of pH using hydrochloric acid. Typically,
the pH will be adjusted to between 4.5 and 7.5, preferably between
5.0 and 7.0, especially around 6.5.
[0015] Examples of pharmaceutically acceptable materials which can
be used to adjust the pH of the formulation include hydrochloric
acid and sodium hydroxide. Preferably, the pH of the formulation
will be adjusted using hydrochloric acid.
[0016] The aqueous component is preferably a high grade quality of
water, most preferably purified water.
[0017] The active compound of formula (I) or solvate thereof will
suitably have a mass mean diameter (MMD) of less than 2 .mu.m,
preferably between 0.5-10 .mu.m, especially around 3-5 .mu.m, eg. 2
.mu.m. Particle size reduction, if necessary, may be achieved eg.
by micronisation. Preferably, the particles will be crystalline,
prepared for example by a process which comprises mixing in a
continuous flow cell in the presence of ultrasonic radiation a
flowing solution of compound of formula (I) or solvate thereof as
medicament in a liquid solvent with a flowing liquid antisolvent
for said medicament (as described in International Patent
Application PCT/GB99/04368).
[0018] A pharmaceutically effective amount of particulate compound
of formula (I) or solvate thereof is present within the
formulation, in an amount which is preferably between 0.005% and 1%
(w/w), preferably between 0.01% and 0.5% (w/w), especially
0.05-0.1% (w/w) based on the total weight of the formulation.
Typically, 50 .mu.l of suspension will contain 50 .mu.g of compound
of formula (I) or solvate thereof.
[0019] Examples of suspending agents include
carboxymethylcellulose, veegum, tragacanth, bentonite,
methylcellulose and polyethylene glycols. Preferably, the
suspending agent will be microcrystalline cellulose and carboxy
methylcellulose sodium, most preferably used as the branded product
Avicel RC591 (which typically contains 87-91% microcrystalline
cellulose and 9-13% carboxy methylcellulose sodium). Particulate
microcrystalline cellulose will preferably have a particle size in
the range 1 to 100 .mu.m. We believe that Avicel RC591 acts as a
suspending agent by imparting thixotropic properties to the
formulation, wherein the formulation may become a stable suspension
upon being stirred, shaken or otherwise disturbed.
[0020] Preferably, the thixotropic nature of the suspending agent
will ensure that the formulation assumes a gel like appearance at
rest, wherein the particulate medicament is dispersed and suspended
substantially uniformly, characterised by a high viscosity value.
Once the composition is subjected to shear forces, such as those
caused by agitation prior to spraying, the viscosity of the
formulation will preferably decrease to such a level to enable it
to flow readily through the spray device and exit as a spray of
fine particles in a mist. These particles will then be capable of
infiltrating the mucosal surfaces of the anterior regions of the
nose (frontal nasal cavities), the frontal sinus, the maxillary
sinuses and the turbinates which overlie the conchas of the nasal
cavities. Once deposited, the viscosity of the formulation will
preferably increase to a sufficient level to assume its gel-like
form and resist being cleared from the nasal passages by the
inherent mucocillary forces that are present in the nasal
cavities.
[0021] When the formulation of the present invention comprises a
suspending agent, it will be desirably added in a suitable amount
to achieve this function, preferably the suspending agent will be
present within the formulation in an amount of between 0.1 and 5%
(w/w), especially 1.5% (w/w), based on the total weight of the
formulation.
[0022] For stability purposes, the formulation of the present
invention should be protected from microbial contamination and
growth. Examples of pharmaceutically acceptable anti-microbial
agents that can be used in the formulation include quaternary
ammonium compounds (eg. benzalkonium chloride, benzethonium
chloride, cetrimide and cetylpyridinium chloride), mercurial agents
(eg. phenylmercuric nitrate, phenylmercuric acetate and
thimerosal), alcoholic agents (eg. chlorobutanol, phenylethyl
alcohol and benzyl alcohol), antibacterial esters (eg. esters of
para-hydroxybenzoic acid), chelating agents such as disodium
edetate (EDTA) and other anti-microbial agents such as
chlorhexidine, chlorocresol, sorbic acid and its salts and
polymyxin.
[0023] Preferably, the preservative will comprise disodium edetate,
which will preferably be present within the formulation in an
amount of between 0.001 and 1% (w/w), especially around 0.015%
(w/w), based on the total weight of the formulation.
[0024] Preferably the preservative will comprise potassium sorbate
which will preferably be present within the formulation in an
amount of between 0.01 and 1% (w/w), especially around 0.015% (w/w)
based on the total weight of the formulation.
[0025] Preferably, the preservative will comprise benzalkonium
chloride (BKC), which will preferably be present within the
formulation in an amount of between 0.001 and 1% (w/w), especially
around 0.015% (w/w), based on the total weight of the
formulation.
[0026] More preferably, the preservative comprises disodium edetate
and benzalkonium chloride or disodium edetate and potassium
sorbate, particularly disodium edetate and benzalkonium
chloride.
[0027] Formulations, eg nasal formulations which contain a
suspended medicament (such as a compound of formula (I) or a
solvate thereof) will preferably contain a pharmaceutically
acceptable wetting agent which functions to wet the particles of
medicament to facilitate dispersion thereof in the aqueous phase of
the composition. Preferably, the amount of wetting agent used will
not cause foaming of the dispersion during mixing.
[0028] It will be appreciated that any agent which is effective in
wetting the particles and which is pharmaceutically acceptable can
be used. Examples of wetting agents that can be used are fatty
alcohols, esters and ethers. Preferably, the wetting agent will be
a hydrophilic, non-ionic surfactant, most preferably
polyoxyethylene (20) sorbitan monooleate (supplied as the branded
product Polysorbate 80).
[0029] Wherein the formulation of the present invention comprises a
wetting agent, it will be desirably added in a sufficient quantity
to achieve this function, preferably the wetting agent will be
present within the formulation in an amount of between 0.001 and
0.05% (w/w), especially 0.025% (w/w), based on the total weight of
the formulation.
[0030] The presence of an isotonicity adjusting agent is to achieve
isotonicity with body fluids eg fluids of the nasal cavity,
resulting in reduced levels of irritancy associated with many nasal
formulations. Examples of suitable isotonicity adjusting agents are
sodium chloride, dextrose and calcium chloride. Preferably, the
isotonicity adjusting agent will be dextrose, most preferably used
as dextrose anhydrous.
[0031] When the formulation of the present invention comprises an
isotonicity adjusting agent it will be desirably added in a
sufficient quantity to achieve this function, preferably the
isotonicity adjusting agent will be present within the formulation
in an amount of between 0.1 and 10% (w/w), especially 5.0% w/w,
based on the total weight of the formulation.
[0032] The compound of formula (I) or a solvate thereof and
formulations thereof have potentially beneficial anti-inflammatory
or anti-allergic effects, particularly upon topical administration
to the nose, demonstrated by, for example, its ability to bind to
the glucocorticoid receptor and to illicit a response via that
receptor, with long acting effect. Hence, formulations according to
the invention are useful in the treatment of inflammatory and/or
allergic disorders of the nose, especially in once-per-day
therapy.
[0033] Formulations according to the invention may be prepared by
combining the ingredients in water. If necessary the pH may be
adjusted as a final step. Formulations so prepared may then be
filled into the receptacle.
[0034] Aqueous formulations of the invention may also be employed
for rectal, aural, otic, oral, topical or parenteral administration
or administration by inhalation for the treatment of other local
inflammatory conditions (eg dermatitis, asthma, chronic obstructive
pulmonary disease (COPD) and the like). For example formulations of
the invention may be administered to the lung by nebulisation. Such
formulations may employ excipients (eg preservatives, buffers and
the like) appropriate for the route of administration.
[0035] The particularly desirable biological properties of the
compound of formula (I) are now explained below:
[0036] Compound or formula (I) undergoes highly efficient hepatic
metabolism to yield the 17-.beta. carboxylic acid (X) as the sole
major metabolite in rat and human in vitro systems. This metabolite
has been synthesised and demonstrated to be >1000 fold less
active than the parent compound in in vitro functional
glucocorticoid assays.
##STR00003##
[0037] This efficient hepatic metabolism is reflected by in vivo
data in the rat, which have demonstrated plasma clearance at a rate
approaching hepatic blood flow and an oral bioavailability of
<1%, consistent with extensive first-pass metabolism.
[0038] In vitro metabolism studies in human hepatocytes have
demonstrated that compound (I) is metabolised in an identical
manner to fluticasone propionate but that conversion of (I) to the
inactive acid metabolite occurs approximately 5-fold more rapidly
than with fluticasone propionate. This very efficient hepatic
inactivation would be expected to minimise systemic exposure in man
leading to an improved safety profile.
[0039] Inhaled glucocorticoids are also absorbed through the lung
and this route of absorption makes a significant contribution to
systemic exposure. Reduced lung absorption could therefore provide
an improved safety profile. Studies with compound of formula (I)
have shown significantly lower exposure to compound of formula (I)
than with fluticasone propionate after dry powder delivery to the
lungs of anaesthetised pigs.
[0040] Examples of disease states in which the compound of formula
(I) has utility include inflammatory and/or allergic conditions of
the nasal passages such as rhinitis eg seasonal and perennial
rhinitis as well as other local inflammatory conditions such as
asthma, COPD and dermatitis.
[0041] It will be appreciated by those skilled in the art that
reference herein to treatment extends to prophylaxis as well as the
treatment of established conditions.
[0042] Preferable means for applying the formulation of the present
invention to the nasal passages is by use of a pre-compression
pump. Most preferably, the pre-compression pump will be a VP7 model
manufactured by Valois SA. Such a pump is beneficial as it will
ensure that the formulation is not released until a sufficient
force has been applied, otherwise smaller doses may be applied.
Another advantage of the pre-compression pump is that atomisation
of the spray is ensured as it will not release the formulation
until the threshold pressure for effectively atomising the spray
has been achieved. Typically, the VP7 model may be used with a
botte capable of holding 10-50 ml of a formulation. Each spray will
typically deliver 50-100 .mu.l of such a formulation, therefore,
the VP7 model is capable of providing at least 100 metered
doses.
[0043] A suitable dosing regime for the formulation of the present
invention when administered to the nose would be for the patient to
inhale deeply subsequent to the nasal cavity being cleared. During
inhalation the formulation would be applied to one nostril while
the other is manually compressed. This procedure would then be
repeated for the other nostril.
[0044] Typically, one or two inhalations per nostril would be
administered by the above procedure up to three times each day,
preferably once or twice daily, especially once daily.
[0045] It will be appreciated that the above dosing regime should
be adjusted according to the patient's age, body weight and/or
symptom severity.
[0046] As mentioned above, formulations comprising a compound of
formula (I) or solvate thereof are useful in human or veterinary
medicine, in particular as an anti-inflammatory and anti-allergic
agent.
[0047] There is thus provided as a further aspect of the invention
a formulation comprising the compound of formula (I) or solvate
thereof for use in human or veterinary medicine, particularly in
the treatment of patients with inflammatory and/or allergic
conditions.
[0048] According to another aspect of the invention, there is
provided the use of a formulation comprising the compound of
formula (I) or solvate thereof for the manufacture of a medicament
for the treatment of patients with inflammatory and/or allergic
conditions.
[0049] In a further or alternative aspect, there is provided a
method for the treatment of a human or animal subject with an
inflammatory and/or allergic condition, which method comprises
administering to said human or animal subject an effective amount
of a formulation comprising the compound of formula (I) or solvate
thereof.
[0050] The compound of formula (I) is long-acting, therefore
preferably the compound will be delivered once-per-day and the dose
will be selected so that the compound has a therapeutic effect in
the treatment of respiratory disorders (eg rhinitis) over 24 hours
or more.
[0051] The pharmaceutical compositions according to the invention
may also be used in combination with another therapeutically active
agent, for example, an anti-histamine or an anti-allergic. The
invention thus provides, in a further aspect, a combination
comprising the compound of formula (I) or solvate thereof together
with another therapeutically active agent, for example, an
anti-histamine or an anti-allergic.
[0052] Examples of anti-histamines include methapyrilene or
loratadine.
[0053] Other suitable combinations include, for example, other
anti-inflammatory agents eg NSAIDs (eg sodium cromoglycate,
nedocromil sodium, PDE4 inhibitors, leukotriene antagonists, iNOS
inhibitors, tryptase and elastase inhibitors, beta-2 integrin
antagonists and adenosine 2a agonists)) or antiinfective agents (eg
antibiotics, antivirals).
[0054] Also of particular interest is use of the compound of
formula (I) or a solvate thereof in combination with a
phosphodiesterase 4 (PDE4) inhibitor eg cilomilast or a salt
thereof.
[0055] Further, there is provided a process for the preparation of
such pharmaceutical compositions which comprises mixing the
ingredients.
[0056] The individual compounds of such combinations may be
administered either sequentially in separate pharmaceutical
compositions as well as simultaneously in combined pharmaceutical
formulations. Preferably additional therapeutically active
ingredients are suspended in the formulation together with the
compound of formula (I) Appropriate doses of known therapeutic
agents will be readily appreciated by those skilled in the art.
[0057] A process for preparing a compound of formula (I) comprises
alkylation of a thioacid of formula (II)
##STR00004##
or a salt thereof.
[0058] In this process the compound of formula (II) may be reacted
with a compound of formula FCH.sub.2L wherein L represents a
leaving group (eg a halogen atom, a mesyl or tosyl group or the
like) for example, an appropriate fluoromethyl halide under
standard conditions. Preferably, the fluoromethyl halide reagent is
bromofluoromethane. Preferably the compound of formula (II) is
employed as a salt, particularly the salt with
diisopropylethylamine.
[0059] In a preferred process for preparing the compound of formula
(I), the compound of formula (II) or a salt thereof is treated with
bromofluoromethane optionally in the presence of a phase transfer
catalyst. A preferred solvent is methylacetate, or more preferably
ethylacetate, optionally in the presence of water. The presence of
water improves solubility of both starting material and product and
the use of a phase transfer catalyst results in an increased rate
of reaction. Examples of phase transfer catalysts that may be
employed include (but are not restricted to) tetrabutylammonium
bromide, tetrabutylammonium chloride, benzyltributylammonium
bromide, benzyltributylammonium chloride, benzyltriethylammonium
bromide, methyltributylammonium chloride and methyltrioctylammonium
chloride. THF has also successfully been employed as solvent for
the reaction wherein the presence of a phase transfer catalyst
again provides a significantly faster reaction rate. Preferably the
product present in an organic phase is washed firstly with aqueous
acid eg dilute HCl in order to remove amine compounds such as
triethylamine and diisopropylethylamine and then with aqueous base
eg sodium bicarbonate in order to remove any unreacted precursor
compound of formula (II).
[0060] Compound of formula (I) in unsolvated form may be prepared
by a process comprising: [0061] (a) Crystallising the compound of
formula (I) in the presence of a non-solvating solvent such as
ethanol, methanol, water, ethyl acetate, toluene,
methylisobutylketone or mixtures thereof; or [0062] (b) Desolvating
a compound of formula (I) in solvated form (eg in the form of a
solvate with acetone, isopropanol, methylethylketone, DMF or
tetrahydrofuran) eg by heating.
[0063] In step (b) the desolvation will generally be performed at a
temperature exceeding 50.degree. C. preferably at a temperature
exceeding 100.degree. C. Generally heating will be performed under
vacuum.
[0064] Compound of formula (I) in unsolvated form has been found to
exist in 3 crystalline polymorphic forms, Forms 1, 2 and 3,
although Form 3 may be an unstable variant of Form 2. The Forms are
characterised by their X-ray diffraction (XRPD) patterns Broadly
speaking the Forms are characterised in their XRPD profiles as
follows:
Form 1: Peak at around 18.9 degrees 2Theta Form 2: Peaks at around
18.4 and 21.5 degrees 2Theta Form 3: Peaks at around 18.6 and 19.2
degrees 2Theta.
[0065] Forms 1 appears likely to be the thermodynamically most
stable form since Forms 2 and 3 are converted into Form 1 on
heating.
[0066] A process for preparing a compound of formula (I) as
unsolvated Form 1 polymorph comprises dissolving compound of
formula (I) in methylisobutylketone, ethyl acetate or methyl
acetate and producing compound of formula (I) as unsolvated Form 1
by addition of a non-solvating anti-solvent such as iso-octane or
toluene.
[0067] According to a first preferred embodiment of this process
the compound of formula (I) may be dissolved in ethyl acetate and
compound of formula (I) as unsolvated Form 1 polymorph may be
obtained by addition of toluene as anti-solvent. In order to
improve the yield, preferably the ethyl acetate solution is hot and
once the toluene has been added the mixture is distilled to reduce
the content of ethyl acetate.
[0068] According to a second preferred embodiment of this process
the compound of formula (I) may be dissolved in
methylisobutylketone and compound of formula (I) as unsolvated Form
1 polymorph may be obtained by addition of isooctane as
anti-solvent
[0069] Compound of formula (I) in solvated form may be prepared by
crystallising the compound of formula (I) from a solvating solvent
such as acetone or tetrahydrofuran (THF).
[0070] Preferably in processes for preparing formulations of the
invention, the compound of formula (I) will be employed in
unsolvated form, typically unsolvated Form 1.
[0071] Compounds of formula (II) may be prepared from the
corresponding 17.alpha.-hydroxyl derivative of formula (III):
##STR00005##
using for example, the methodology described by G. H. Phillipps et
al., (1994) Journal of Medicinal Chemistry, 37, 3717-3729. For
example the step typically comprises the addition of a reagent
suitable for performing the esterification eg an activated
derivative of 2-furoic acid such as an activated ester or
preferably a 2-furoyl halide eg 2-furoyl chloride (employed in at
least 2 times molar quantity relative to the compound of formula
(III)) in the presence of an organic base eg triethylamine. The
second mole of 2-furoyl chloride reacts with the thioacid moiety in
the compound of formula (III) and needs to be removed eg by
reaction with an amine such as diethylamine.
[0072] This method suffers disadvantages, however, in that the
resultant compound of formula (II) is not readily purified of
contamination with the by-product 2-furoyldiethylamide. We have
therefore invented several improved processes for performing this
conversion.
[0073] In a first such improved process we have discovered that by
using a more polar amine such as diethanolamine, a more water
soluble by-product is obtained (in this case
2-furoyldiethanolamide) which permits compound of formula (II) or a
salt thereof to be produced in high purity since the by-product can
efficiently be removed by water washing.
[0074] Thus we provide a process for preparing a compound of
formula (II) which comprises: [0075] (a) reacting a compound of
formula (III) with an activated derivative of 2-furoic acid as in
an amount of at least 2 moles of the activated derivative per mole
of compound of formula (III) to yield a compound of formula
(IIA)
##STR00006##
[0075] ; and [0076] (b) removal of the sulphur-linked 2-furoyl
moiety from compound of formula (IIA) by reaction of the product of
step (a) with an organic primary or secondary amine base capable of
forming a water soluble 2-furoyl amide.
[0077] In two particularly convenient embodiments of this process
we also provide methods for the efficient purification of the end
product which comprise either [0078] (c1) when the product of step
(b) is dissolved in a substantially water immiscible organic
solvent, purifying the compound of formula (II) by washing out the
amide by-product from step (b) with an aqueous wash, or [0079] (c2)
when the product of step (b) is dissolved in a water miscible
solvent, purifying the compound of formula (II) by treating the
product of step (b) with an aqueous medium so as to precipitate out
pure compound of formula (II) or a salt thereof.
[0080] In step (a) preferably the activated derivative of 2-furoic
acid may be an activated ester of 2-furoic acid, but is more
preferably a 2-furoyl halide, especially 2-furoyl chloride. A
suitable solvent for this reaction is ethylacetate or methylacetate
(preferably methylacetate) (when step (c1) may be followed) or
acetone (when step (c2) may be followed). Normally an organic base
eg triethylamine will be present. In step (b) preferably the
organic base is diethanolamine. The base may suitably be dissolved
in a solvent eg methanol. Generally steps (a) and (b) will be
performed at reduced temperature eg between 0 and 5.degree. C. In
step (c1) the aqueous wash may be water, however the use of brine
results in higher yields and is therefore preferred. In step (c2)
the aqueous medium is for example a dilute aqueous acid such as
dilute HCl.
[0081] We also provide an alternative process for preparing a
compound of formula (II) which comprises: [0082] (a) reacting a
compound of formula (III) with an activated derivative of 2-furoic
acid in an amount of at least 2 moles of activated derivative per
mole of compound of formula (III) to yield a compound of formula
(IIA); and [0083] (b) removal of the sulphur-linked 2-furoyl moiety
from compound of formula (IIA) by reaction of the product of step
(a) with a further mole of compound of formula (III) to give two
moles of compound of formula (II).
[0084] In step (a) preferably the activated derivative of 2-furoic
acid may be an activated ester of 2-furoic acid, but is more
preferably a 2-furoyl halide, especially 2-furoyl chloride. A
suitable solvent for his step is acetone. Normally an organic base
eg triethylamine will be present. In step (b) a suitable solvent is
DMF or dimethylacetamide. Normally an organic base eg triethylamine
will be present. Generally steps (a) and (b) will be performed at
reduced temperature eg between 0 and 5.degree. C. The product may
be isolated by treatment with acid and washing with water.
[0085] This aforementioned process is very efficient in that it
does not produce any furoylamide by-product (thus affording inter
alia environmental advantages) since the excess mole of furoyl
moiety is taken up by reaction with a further mole of compound of
formula (II) to form an additional mole of compound of formula
(II).
[0086] Further general conditions for the conversion of compound of
formula (III) to compound of formula (II) in the two processes just
described will be well known to persons skilled in the art.
[0087] According to a preferred set of conditions, however, we have
found that the compound of formula (II) may advantageously be
isolated in the form of a solid crystalline salt. The preferred
salt is a salt formed with a base such as triethylamine,
2,4,6-trimethylpyridine, diisopropylethylamine or
N-ethylpiperidine. Such salt forms of compound of formula (II) are
more stable, more readily filtered and dried and can be isolated in
higher purity than the free thioacid. The most preferred salt is
the salt formed with diisopropylethylamine. The triethylamine salt
is also of interest.
[0088] Compounds of formula (III) may be prepared in accordance
with procedures described in GB 2088877B. Compounds of formula
(III) may also be prepared by a process comprising the following
steps:
##STR00007##
[0089] Step (a) comprises oxidation of a solution containing the
compound of formula (V). Preferably, step (a) will be performed in
the presence of a solvent comprising methanol, water,
tetrahydrofuran, dioxan or diethylene glygol dimethylether. So as
to enhance yield and throughput, preferred solvents are methanol,
water or tetrahydrofuran, and more preferably are water or
tetrahydrofuran, especially water and tetrahydrofuran as solvent.
Dioxan and diethylene glygol dimethylether are also preferred
solvents which may optionally (and preferably) be employed together
with water. Preferably, the solvent will be present in an amount of
between 3 and 10 vol relative to the amount of the starting
material (1 wt.), more preferably between 4 and 6 vol., especially
5 vol. Preferably the oxidising agent is present in an amount of
1-9 molar equivalents relative to the amount of the starting
material. For example, when a 50% w/w aqueous solution of periodic
acid is employed, the oxidising agent may be present in an amount
of between 1.1 and 10 wt. relative to the amount of the starting
material (1 wt.), more preferably between 1.1 and 3 wt., especially
1.3 wt. Preferably, the oxidation step will comprise the use of a
chemical oxidising agent. More preferably, the oxidising agent will
be periodic acid or iodic acid or a salt thereof. Most preferably,
the oxidising agent will be periodic acid or sodium periodate,
especially periodic acid. Alternatively (or in addition), it will
also be appreciated that the oxidation step may comprise any
suitable oxidation reaction, eg one which utilises air and/or
oxygen. When the oxidation reaction utilises air and/or oxygen, the
solvent used in said reaction will preferably be methanol.
Preferably, step (a) will involve incubating the reagents at room
temperature or a little warmer, say around 25.degree. C. eg for 2
hours. The compound of formula (IV) may be isolated by
recrystallisation from the reaction mixture by addition of an
anti-solvent. A suitable anti-solvent for compound of formula (IV)
is water. Surprisingly we have discovered that it is highly
desirable to control the conditions under which the compound of
formula (IV) is precipitated by addition of anti-solvent eg water.
When the recrystallisation is performed using chilled water (eg
water/ice mixture at a temperature of 0-5.degree. C.) although
better anti-solvent properties may be expected we have found that
the crystalline product produced is very voluminous, resembles a
soft gel and is very difficult to filter. Without being limited by
theory we believe that this low density product contains a large
amount of solvated solvent within the crystal lattice. By contrast
when conditions of around 10.degree. C. or higher are used (eg
around ambient temperature) a granular product of a sand like
consistency which is very easily filtered is produced. Under these
conditions, crystallisation typically commences after around 1 hour
and is typically completed within a few hours (eg 2 hours). Without
being limited by theory we believe that this granular product
contains little or no solvated solvent within the crystal
lattice.
[0090] Step (b) will typically comprise the addition of a reagent
suitable for converting a carboxylic acid to a carbothioic acid eg
using hydrogen sulphide gas together with a suitable coupling agent
eg carbonyldiimidazole (CDI) in the presence of a suitable solvent
eg dimethylformamide.
[0091] The advantages of the formulation of the compound of formula
(I) according to the invention may include the fact that the
formulations demonstrate excellent anti-inflammatory properties,
with predictable pharmacokinetic and pharmacodynamic behaviour,
with an attractive side-effect profile, rapid onset of action, long
duration of action, and are compatible with a convenient regime of
treatment in human patients, in particular being amendable to
once-per day dosing. Further advantages may include the fact that
the formulation has desirable physical and chemical properties
which allow for ready manufacture and storage.
DETAILED DESCRIPTION OF THE INVENTION
[0092] The following non-limiting Examples illustrate the
invention:
Examples
General
[0093] .sup.1H-nmr spectra were recorded at 400 MHz and the
chemical shifts are expressed in ppm relative to tetramethylsilane.
The following abbreviations are used to describe the multiplicities
of the signals: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiples), dd (doublet of doublets), ddd (doublet of doublet of
doublets), dt (doublet of triplets) and b (broad). Biotage refers
to prepacked silica gel cartridges containing KP-Sil run on flash
12i chromatography module. LCMS was conducted on a Supelcosil
LCABZ+PLUS column (3.3 cm.times.4.6 mm ID) eluting with 0.1%
HCO.sub.2H and 0.01 M ammonium acetate in water (solvent A), and
0.05% HCO.sub.2H 5% water in acetonitrile (solvent B), using the
following elution gradient 0-0.7 min 0% B, 0.7-4.2 min 100% B,
4.2-5.3 min 0% B, 5.3-5.5 min 0% B at a flow rate of 3 ml/min. The
mass spectra were recorded on a Fisons VG Platform spectrometer
using electrospray positive and negative mode (ES+ve and
ES-ve).
Intermediates
Intermediate 1: 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
diisopropylethylamine salt
[0094] A stirred suspension of 6.alpha.,
9.alpha.-difluoro-11.beta.,17.alpha.-dihydroxy-16.alpha.-methyl-3-oxo-and-
rosta-1,4-diene-17.beta.-carbothioic acid (prepared in accordance
with the procedure described in GB 2088877B) (49.5 g) in
methylacetate (500 ml) is treated with triethylamine (35 ml)
maintaining a reaction temperature in the range 0-5.degree. C.
2-Furoyl chloride (25 ml) is added and the mixture stirred at
0-5.degree. C. for 1 hour. A solution of diethanolamine (52.8 g) in
methanol (50 ml) is added and the mixture stirred at 0-5.degree. C.
for at least 2 hours. Dilute hydrochloric acid (approx 1M, 550 ml)
is added maintaining a reaction temperature below 15.degree. C. and
the mixture stirred at 15.degree. C. The organic phase is separated
and the aqueous phase is back extracted with methyl acetate
(2.times.250 ml). All of the organic phases are combined, washed
sequentially with brine (5.times.250 ml) and treated with
di-isopropylethylamine (30 ml). The reaction mixture is
concentrated by distillation at atmospheric pressure to an
approximate volume of 250 ml and cooled to 25-30.degree. C.
(crystallisation of the desired product normally occurs during
distillation/subsequent cooling). Tertiary butyl methyl ether
(TBME) (500 ml) is added, the slurry further cooled and aged at
0-5.degree. C. for at least 10 minutes. The product is filtered
off, washed with chilled TBME (2.times.200 ml) and dried under
vacuum at approximately 40-50.degree. C. (75.3 g, 98.7%). NMR
(CDCl.sub.3) .delta.: 7.54-7.46 (1H, m), 7.20-7.12 (1H, dd),
7.07-6.99 (1H, dd), 6.48-6.41 (2H, m), 6.41-6.32 (1H, dd),
5.51-5.28 (1H, dddd .sup.2J.sub.H-F 50 Hz), 4.45-4.33 (1H, bd),
3.92-3.73 (3H, bm), 3.27-3.14 (2H, q), 2.64-2.12 (5H, m), 1.88-1.71
(2H, m), 1.58-1.15 (3H, s), 1.50-1.38 (15H, m), 1.32-1.23 (1H, m),
1.23-1.15 (3H s), 1.09-0.99 (3H, d)
Intermediate 2: 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester
Unsolvated Form 1
[0095] A mobile suspension of Intermediate 1 (12.61 g, 19.8 mmol)
in ethyl acetate (230 ml) and water (50 ml) is treated with a phase
transfer catalyst (benzyltributylammonium chloride, 10 mol %),
cooled to 3.degree. C. and treated with bromofluoromethane (1.10
ml, 19.5 mmol, 0.98 equivalents), washing in with prechilled
(0.degree. C.) ethyl acetate (EtOAc) (20 ml). The suspension is
stirred overnight, allowing to warm to 17.degree. C. The aqueous
layer is separated and the organic phase is sequentially washed
with 1M HCl (50 ml), 1% w/v NaHCO.sub.3 solution (3.times.50 ml)
and water (2.times.50 ml). The ethylacetate solution is distilled
at atmospheric pressure until the distillate reaches a temperature
of approximately 73.degree. C. at which point toluene (150 ml) is
added. Distillation is continued at atmospheric pressure until all
remaining EtOAc has been removed (approximate distillate
temperature 103.degree. C.). The resultant suspension is cooled and
aged at <10.degree. C. and filtered off. The bed is washed with
toluene (2.times.30 ml) and the product oven dried under vacuum at
60.degree. C. to constant weight to yield the title compound (8.77
g, 82%) LCMS retention time 3.66 min, m/z 539 MH.sup.+, NMR .delta.
(CDCl.sub.3) includes 7.60 (1H, m), 7.18-7.11 (2H, m), 6.52 (1H,
dd, J=4.2 Hz), 6.46 (1H, s), 6.41 (1H, dd, J 10, 2 Hz), 5.95 and
5.82 (2H dd, J 51, 9 Hz), 5.48 and 5.35 (1H, 2 m), 4.48 (1H, m),
3.48 (1H, m), 1.55 (3H, s), 1.16 (3H, s), 1.06 (3H, d, J 7 Hz).
Pharmacological Activity
In Vitro Pharmacological Activity
[0096] Pharmacological activity was assessed in a functional in
vitro assay of glucocorticoid agonist activity which is generally
predictive of anti-inflammatory or anti-allergic activity in
vivo.
[0097] For the experiments in this section, compound of formula (I)
was used as unsolvated Form 1 (Intermediate 2)
[0098] The functional assay was based on that described by K. P.
Ray et al., Biochem J. (1997), 328, 707-715. A549 cells stably
transfected with a reporter gene containing the NF-.kappa.B
responsive elements from the ELAM gene promoter coupled to sPAP
(secreted alkaline phosphatase) were treated with test compounds at
appropriate doses for 1 hour at 37.degree. C. The cells were then
stimulated with tumour necrosis factor (TNF, 10 ng/ml) for 16
hours, at which time the amount of alkaline phosphatase produced is
measured by a standard colourimetric assay. Dose response curves
were constructed from which EC.sub.50 values were estimated.
[0099] In this test the compound of formula (I) showed an EC.sub.50
value of <1 nM.
[0100] The glucocorticoid receptor (GR) can function in at least
two distinct mechanisms, by upregulating gene expression through
the direct binding of GR to specific sequences in gene promotors,
and by downregulating gene expression that is being driven by other
transcription factors (such as NF B or AP-1) through their direct
interaction with GR.
[0101] In a variant of the above method, to monitor these
functions, two reporter plasmids have been generated and introduced
separately into A549 human lung epithelial cells by transfection.
The first cell line contains the firefly luciferase reporter gene
under the control of a synthetic promoter that specifically
responds to activation of the transcription factor NF B when
stimulated with TNF. The second cell line contains the renilla
luciferase reporter gene under the control of a synthetic promotor
that comprises 3 copies of the consensus glucocorticoid response
element, and which responds to direct stimulation by
glucocorticoids. Simultaneous measurement of transactivation and
transrepression was conducted by mixing the two cell lines in a 1:1
ratio in 96 well plate (40,000 cells per well) and growing
overnight at 37.degree. C. Test compounds were dissolved in DMSO,
and added to the cells at a final DMSO concentration of 0.7%. After
incubation for 1 h 0.5 ng/ml TNF (R&D Systems) was added and
after a further 15 hours at 37.degree. C., the levels of firefly
and renilla luciferase were measured using the Packard Firelite kit
following the manufacturers' directions. Dose response curves were
constructed from which EC.sub.50 values were determined
TABLE-US-00001 Transactivation (GR) Transrepression (NF B)
ED.sub.50 (nM) ED.sub.50 (nM) Compound of 0.06 0.20 Formula (I)
Metabolite (X) >250 >1000 Fluticasone propionate 0.07
0.16
In Vivo Pharmacological Activity
[0102] Pharmacological activity in vivo was assessed in an
ovalbumin sensitised Brown Norway rat eosinophilia model. This
model is designed to mimic allergen induced lung eosinophilia, a
major component of lung inflammation in asthma.
[0103] For the experiments in this section, compound of formula (I)
was used as unsolvated Form 1.
[0104] Compound of formula (I) produced dose dependant inhibition
of lung eosinophilia in this model after dosing as an
intra-tracheal (IT) suspension in saline 30 min prior to ovalbumin
challenge. Significant inhibition is achieved after a single dose
of 30 .mu.g of compound of formula (I) and the response was
significantly (p=0.016) greater than that seen with an equivalent
dose of fluticasone propionate in the same study (69% inhibition
with compound of formula (I) vs 41% inhibition with fluticasone
propionate).
[0105] In a rat model of thymus involution 3 daily IT doses of 100
.mu.g of compound (I) induced significantly smaller reductions in
thymus weight (p=0.004) than an equivalent dose of fluticasone
propionate in the same study (67% reduction of thymus weight with
compound (I) vs 78% reduction with fluticasone propionate).
[0106] Taken together these results indicate a superior therapeutic
index for compound (I) compared to fluticasone propionate.
In Vitro Metabolism in Rat and Human Hepatocytes
[0107] Incubation of compound (I) with rat or human hepatocytes
shows the compound to be metabolised in an identical manner to
fluticasone propionate with the 17-carboxylic acid (X) being the
only significant metabolite produced. Investigation of the rate of
appearance of this metabolite on incubation of compound (I) with
human hepatocytes (37.degree. C., 10 M drug concentration,
hepatocytes from 3 subjects, 0.2 and 0.7 million cells/mL) shows
compound (I) to be metabolised ca. 5-fold more rapidly than
fluticasone propionate:
TABLE-US-00002 17-.beta. acid metabolite production Subject Cell
density (pmol/h) number (million cells/mL) Compound (I) Fluticasone
propionate 1 0.2 48.9 18.8 1 0.7 73.3 35.4 2 0.2 118 9.7 2 0.7 903
23.7 3 0.2 102 6.6 3 0.7 580 23.9
[0108] Median metabolite production 102-118 .mu.mol/h for compound
(I) and 18.8-23.0 .mu.mol/h for fluticasone propionate.
Pharmacokinetics after Intravenous (Iv) and Oral Dosing in Rats
[0109] Compound (I) was dosed orally (0.1 mg/kg) and IV (0.1 mg/kg)
to male Wistar Han rats and pharmacokinetic parameters determined
Compound (I) showed negligible oral bioavailability (0.9%) and
plasma clearance of 47.3 mL/min/kg, approaching liver blood flow
(plasma clearance of fluticasone propionate=45.2 mL/min/kg).
Pharmacokinetics after Intra-Tracheal Dry Powder Dosing in the
Pig.
[0110] Anaesthetised pigs (2) were dosed intra-tracheally with a
homogenous mixture of compound (I) (1 mg) and fluticasone
propionate (1 mg) as a dry powder blend in lactose (10% w/w).
Serial blood samples were taken for up to 8 h following dosing.
[0111] Plasma levels of compound (I) and fluticasone propionate
were determined following extraction and analysis using LC-MS/MS
methodology, the lower limits of quantitation of the methods were
10 and 20 pg/mL for compound (I) and fluticasone propionate
respectively. Using these methods compound (I) was quantifiable up
to 2 hours after dosing and fluticasone propionate was quantifiable
up to 8 hours after dosing. Maximum plasma concentrations were
observed for both compounds within 15 min after dosing. Plasma
half-life data obtained from IV dosing (0.1 mg/kg) was used to
calculate AUC(0-inf) values for compound (I). This compensates for
the plasma profile of Compound (I) only being defined up to 2 hours
after an IT dose and removes any bias due to limited data between
compound (I) and fluticasone propionate.
[0112] C.sub.max and AUC(0-inf) values show markedly reduced
systemic exposure to compound (I) compared to fluticasone
propionate:--
TABLE-US-00003 Cmax AUC (0-inf) (pg/mL) (hr pg/mL) Pig 1 Pig 2 Pig
1 Pig 2 Compound of Formula (I) 117 81 254 221 Fluticasone
propionate 277 218 455 495
[0113] The pharmacokinetic parameters for both compound (I) and
fluticasone propionate were the same in the anaesthetised pig
following intravenous administration of a mixture of the two
compounds at 0.1 mg/kg. The clearance of these two glucocorticoids
is similar is this experimental pig model.
Examples
Example 1
Nasal formulation containing 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester
[0114] A formulation for intranasal delivery was prepared with
ingredients as follows: 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester (prepared according to Intermediate 2,
micronised to MMD 3 .mu.m) 0.05% w/w
TABLE-US-00004 Polysorbate 80 0.025% w/w Avicel RC591 1.5% w/w
Dextrose 5.0% w/w BKC 0.015% w/w EDTA 0.015% w/w water to 100%
in a total amount suitable for 120 actuations and the formulation
was filled into a bottle (plastic or glass) fitted with a metering
valve adapted to dispense 50 or 100 .mu.l per actuation
[0115] The device was fitted into a nasal actuator (Valois, e.g.
VP3, VP7 or VP7D)
[0116] The formulation was prepared following the following
protocol:
Part A
[0117] 1. Dissolve dextrose in purified water 2. Dissolve EDTA in
dextrose solution 3. Add Avicel RC591 while stirring 4. Allow
suspension to hydrate
Part B (Separately)
[0118] 1. Dissolve polysorbate 80 in purified water at
50-60.degree. C. 2. Prepare slurry of drug in Polysorbate 80
solution
Part C
[0119] 1. Combine suspension of A4 with suspension of B2 and stir
2. Add solution of BKC in purified water and stir 3. Adjust pH with
1N HCl 4. Add purified water to correct weight
Example 2
Nasal formulation containing 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester
[0120] A formulation for intranasal delivery was prepared with
ingredients as follows: 6.alpha.,
9.alpha.-Difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy-16.-
alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester (prepared according to Intermediate 2,
micronised to MMD 3 .mu.m) 0.1% w/w
TABLE-US-00005 Polysorbate 80 0.025% w/w Avicel RC591 1.5% w/w
Dextrose 5.0% w/w BKC 0.015% w/w EDTA 0.015% w/w water to 100%
in a total amount suitable for 120 actuations and the formulation
was filled into a bottle fitted with a metering valve adapted to
dispense 50 or 100 .mu.l per actuation. The device was fitted into
a nasal actuator (Valois).
[0121] Stability studies on Examples 1 and 2 showed them to be
stable up to 3 months at 40.degree. C. (measurements were not taken
beyond this time).
[0122] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer or step or group of
integers but not to the exclusion of any other integer or step or
group of integers or steps.
[0123] The patents and patent applications described in this
application are herein incorporated by reference.
* * * * *