Title of Invention


Abstract POWDER PHARMACEUTICAL COMPOSITIONS, USES, AND KITS COMPRISING ANHYDROUS DEHYDROEPIANDROSTERONE PARTICLES OF RESPIRABLE OR INHALABLE SIZE This invention relates to a sealed container containing a powder formulation comprising a dehydroepiandrosterone, its analogue(s) or salt(s) by itself or with a pharmaceutically or veterinarily acceptable carrier or diluent, and having a particle size of about 0.1 urn to about 100 urn. The formulation can be used to treat or prevent asthma, chronic obstructive pulmonary disease, lung inflammation, and other respiratory diseases or conditions. The formulation may be prepared by jet milling, and may be delivered through the respiratory tract or other routes using a nebulizer. The sealed container is provided in a device and/or a therapeutic kit.
This application is a non-provisional application that claims priority to the U. S.
Provisional Patent Application Ser. No. 60/389,242, filed June 17, 2002 ; and is a nonprovisional
application that claims priority to the U. S. Provisional Patent Application
(Attorney Docket No. 02486.0077. PZUSOO), filed June 11,2003.
Field of the Invention
This invention relates to a respirable dry powder formulation comprising a
pharmaceutically or veterinarily acceptable carrier and a dehydroepiandrosterone
(DHEA), DHEA derivative, or pharmaceutically or veterinarily acceptable salt thereof,
sealed in a nebulizable form. Methods for preparation and delivering of the dry powdered
formulation, and for treating asthma, chronic obstructive pulmonary disease (COPD), or
other respiratory disease or condition, including microbial (including bacteria) or viral
caused respiratory disease, such as severe acute respiratory syndrome (SARS). The
formulation is provided in the form of a kit.
Description of the Background
Asthma and COPD and other respiratory ailments, associated with a variety of diseases
and conditions, are extremely common in the general population, and more so in certain
ethnic groups, such as African Americans. Respiratory ailments include microbial
infections or viral infections (such as SARS). In many cases they are accompanied by
inflammation, which aggravates the condition of the lungs. Asthma, for example, is one
of the most common diseases in industrialized countries. In the United States it accounts
for about 1% of all health care costs. An alarming increase in both the prevalence and
mortality of asthma over the past decade has been reported, and asthma is predicted to be
the preeminent occupational lung disease in the next decade. While the increasing
mortality of asthma in industrialized countries could be attributable to the reliance upon
beta agonists in the treatment of this disease, the underlying causes of asthma remain
poorly understood.
Asthma is a condition characterized by variable, in many instances reversible obstruction
of the airways. This process is associated with lung inflammation and in sum cases lung
allergies. Many patients have acute episodes referred to as "asthma attacks," while others
are afflicted with a chronic condition. The asthmatic process is believed to be triggered in
some cases by inhalation of antigens by hypersensitive subjects. This condition is
generally referred to as "extrinsic asthma." Other asthmatics have an intrinsic
predisposition to the condition, which is thus referred to as "intrinsic asthma," and may
be comprised of conditions of different origin, including those mediated by the adenosine
receptor(s), allergic conditions mediated by an immune IgE-mediated response, and
others. All asthmas have a group of symptoms, which are characteristic of this condition:
bronchoconstriction, lung inflammation and decreased lung surfactant. Existing
bronchodilators and anti-inflammatories are currently commercially available and are
prescribed for the treatment of asthma. The most common anti-inflarnmatories,
corticosteroids, have considerable side effects but are commonly prescribed nevertheless.
Most of the drugs available for the treatment of asthma are, more importantly, barely
effective in a small number of patients.
Chronic obstructive pulmonary disease (COPD) causes a continuing obstruction of
airflow in the airways. COPD is characterized by airflow obstruction that is generally
caused by chronic bronchitis, emphysema, or both. Commonly, the airway obstruction is
mostly irreversible. In chronic bronchitis, airway obstruction results from chronic and
excessive secretion of abnormal airway mucus, inflammation, bronchospasm, and
infection. Chronic bronchitis is also characterized by chronic cough, mucus production,
or both, for at least three months in at least two successive years where other causes of
chronic cough have been excluded. In emphysema, a structural element (elastin) in the
terminal bronchioles is destroyed leading to the collapse of the airway walls and inability
to exhale "stale" air. In emphysema there is permanent destruction of the alveoli.
Emphysema is characterized by abnormal permanent enlargement of the air spaces distal
to the terminal bronchioles, accompanied by destruction of their walls and without
obvious fibrosis. COPD can also give rise to secondary pulmonary hypertension.
Secondary pulmonary hypertension itself is a disorder in which blood pressure in the
pulmonary arteries is abnormally high. In severe cases, the right side of the heart must
work harder than usual to pump blood against the high pressure. If this continues for a
long period, the right heart enlarges and functions poorly, and fluid collects in the ankles
(edema) and belly. Eventually the left heart begins to fail. Heart failure caused by
pulmonary disease is called corpulmonale.
COPD characteristically affects middle aged and elderly people, and is one of the leading
causes of morbidity and mortality worldwide, hi the United States it affects about 14
million people and is the fourth leading cause of death, and the third leading cause for
disability in the United States. Both morbidity and mortality, however, are rising. The
estimated prevalence of this disease in the United States has risen by 41% since 1982,
and age adjusted death rates rose by 71% between 1966 and 1985. This contrasts with the
decline over the same period in age- adjusted mortality from all causes (which fell by
22%), and from cardiovascular diseases (which fell by 45%). In 1998 COPD accounted
for 112,584 deaths in the United States.
COPD, however, is preventable, since it is believed that its main cause is exposure to
cigarette smoke. Long-term smoking is the most frequent cause of COPD. It accounts for
80 to 90% of all cases. A smoker is 10 times more likely than a non-smoker to die of
COPD. The disease is rare in lifetime non-smokers, in whom exposure to environmental
tobacco smoke will explain at least some of the airways obstruction. Other proposed
etiological factors include airway hyper responsiveness or hypersensitivity, ambient air
pollution, and allergy. The airflow obstruction in COPD is usually progressive in people
who continue to smoke. This results in early disability and shortened survival time.
Stopping smoking reverts the decline in lung function to values for non-smokers. Other
risk factors include: heredity, second-hand smoke, exposure to air pollution at work and
in the environment, and a history of childhood respiratory infections. The symptoms of
COPD include: chronic coughing, chest tightness, shortness of breath, an increased effort
to breathe, increased mucus production, and frequent clearing of the throat.
There is very little currently available to alleviate symptoms of COPD, prevent
exacerbations, preserve optimal lung function, and improve daily living activities and
quality of life. Many patients will use medication chronically for the rest of their lives,
with the need for increased doses and additional drugs during exacerbations. Medications
that are currently prescribed for COPD patients include: fast-acting (B2-agonists,
anticholinergic bronchodilators, long-acting bronchodilators, antibiotics, and
expectorants. Amongst the currently available treatments for COPD, short term benefits,
but not long term effects, were found on its progression, from administration of anticholinergic
drugs, 62 adrenergic agonists, and oral steroids.
Short and long acting inhaled 62 adrenergic agonists achieve short-term bronchodilation
and provide some symptomatic relief in COPD patients, but show no meaningful
maintenance effect on the progression of the disease. Short acting 62 adrenergic agonists
improve symptoms in subjects with COPD, such as increasing exercise capacity and
produce some degree of bronchodilation, and even an increase in lung function in some
severe cases. The maximum effectiveness of the newer long acting inhaled, 62 adrenergic
agonists was found to be comparable to that of short acting 62 adrenergic agonists.
Salmeterol was found to improve symptoms and quality of life, although only producing
modest or no change in lung function. In asthmatics, however, 62 adrenergic agonists
have been linked to an increased risk of death, worsened control of asthma, and
deterioration in lung function. 62-agonists, such as albuterol, help to open narrowed
airways. The use of 62-agonists can produce paradoxical bronchospasm, which may be
life threatening to the COPD patient. In addition, the use of 62-agonists can produce
cardiovascular effects, such as altered pulse rate, blood pressure and electrocardiogram
results. In rare cases, the use of 62-agonists can produce hypersensitivity reactions, such
as urticaria, angioedema, rash and oropharyngeal edema. In these cases, the use of the 62-
agonist should be discontinued. Continuous treatment of asthmatic and COPD patients
with the bronchodilators ipratropium bromide or fenoterol resulted in a faster decline in
lung function, when compared with treatment provided on a need basis, therefore
indicating that they are not suitable for maintenance treatment. The most common
immediate adverse effect of 62 adrenergic agonists, on the other hand, is tremors, which
at high doses may cause a fall in plasma potassium, dysrhythmias, and reduced arterial
oxygen tension. The combination of a 62 adrenergic agonist with an anti-cholinergic drug
provides little additional bronchodilation compared with either drug alone. The addition
of ipratropium to a standard dose of inhaled 62 adrenergic agonists for about 90 days,
however, produces some improvement in stable COPD patients over either drug alone.
Anti-cholinergic agents were found to produce greater bronchodilation in combination
with anti-cholinergic agents than B2 adrenergic agonists, hi people with COPD. Overall,
the occurrence of adverse effects with 62 adrenergic agonists, such as tremor and
dysrhythmias, is more frequent than with anti-cholinergics. Thus, neither anticholinergic
drugs nor P2 adrenergic agonists have an effect on all people with COPD; nor
do the two agents combined.
Anti-cholinergic drugs achieve short-term bronchodilation and produce some symptom
relief in people with COPD, but no improved long-term prognosis even with inhaled
products. Most COPD patients have at least some measure of airways obstruction that is
somewhat alleviated by ipratropium bromide. "The lung health study" found in men and
women smokers spirometric signs of early COPD. Three treatments compared over a five
year period found that ipratropium bromide had no significant effect on the decline in the
functional effective volume of the patient's lungs whereas smoking cessation produced a
slowing of the decline in the functional effective volume of the lungs. Ipratropium
bromide, however, produced serious adverse effects, such as cardiac symptoms,
hypertension, skin rashes, and urinary retention. Anticholinergic bronchodilators, such as
ipratropium bromide, and theophylline derivatives, help to open narrowed airways. Longacting
bronchodilators help to relieve constriction of the airways and help prevent
bronchospasm associated with COPD. Theophyllines have a small bronchodilatory effect
in COPD patients whereas they have some common adverse effects, and they have a
small therapeutic range given that blood concentrations of 15-20 mg/1 are required for
optimal effects. Adverse effects include nausea, diarrhea, headache, irritability, seizures,
and cardiac arrhythmias, and they occur at highly variable blood concentrations and, in
many people, they occur within the therapeutic range. The theophyllines' doses must be
adjusted individually according to smoking habits, infection, and other treatments, which
is cumbersome. Although theophyllines have been claimed to have an anti-inflammatory
effect in asthma, especially at lower doses, none has been reported in COPD, although
their bronchodilating short-term effect appears to be statistically different from placebo.
The adverse effects of theophyllines and the need for frequent monitoring limit their
usefulness. There is no evidence that anti-cholinergic agents affect the decline in lung
function, and mucolytics have been shown to reduce the frequency of exacerbations but
with a possible deleterious effect on lung function. The long-term effects of B2 adrenergic
agonists, oral corticosteroids, and antibiotics have not yet been evaluated, and up to the
present time no other drug has been shown to affect the progression of the disease or
Oral corticosteroids elicit some improvement in baseline functional effective volume hi
stable COPD patients whereas systemic corticosteroids have been found to be harmful at
least producing some osteoporosis and inducing overt diabetes. The longer term
administration of oral corticosteroids may be useful in COPD, but then* usefulness must
be weighed against their substantial adverse effects. Inhaled corticosteroids have been
found to have no real short-term effect on airway hyper-responsiveness to histamine, but
a small long-term effect on lung function, e.g., hi pre-bronchodilator functional effective
volume. Fluticasone treatment of COPD patients showed a significant reduction in
moderate and severe (but not mild) exacerbations, and a small but significant
improvement in lung function and six minute walking distance. Oral prednisolone,
inhaled beclomethasone or both had no effects in COPD patients, but lung function
improved oral corticosteroids. Mucolytics have a modest beneficial effect on the
frequency and duration of exacerbations but an adverse effect on lung function. Neither
N-acetylcysteine nor other mucolytics, however, have a significant effect in people with
severe COPD (functional effective volume reductions hi frequency of exacerbation. N-acetylcysteine produced gastrointestinal side
effect. Long-term oxygen therapy administered to hypoxaemic COPD and congestive
cardiac failure, patients, had little effect on their rates of death for the first 500 days or so,
but survival rates in men increased afterwards and remained constant over the next five
years. In women, however, oxygen decreased the rates of death throughout the study.
Continuous oxygen treatment of hypoxemic COPD patients (functional effective
volume only life style changes, smoking cessation and long term treatment with oxygen (in
hypoxaemics), have been found to alter the long-term course of COPD.
Antibiotics are also often given at the first sign of a respiratory infection to prevent
further damage and infection in diseased lungs. Expectorants help loosen and expel
mucus secretions from the airways, and may help make breathing easier.
In addition, other medications may be prescribed to manage conditions associated with
COPD. These may include: diuretics (which are given as therapy to avoid excess water
retention associated right-heart failure), digitalis (which strengthens the force of the
heartbeat), painkillers cough suppressants, and sleeping pills. This latter list of
medications help alleviate symptoms associated with COPD but do not treat COPD.
Thus, there is very little currently available to alleviate symptoms of COPD, prevent
exacerbations, preserve optimal lung function, and improve daily living activities and
quality of life.
Severe acute respiratory syndrome (SARS) is a respiratory illness that has recently been
reported in Asia, North America, and Europe. In general, SARS patients initial
experience a fever of greater than 100. 4°F (>38.0°C). This may be accompanied or
followed by headache, an overall feeling of discomfort, and body aches. Certain patients
also experience respiratory symptoms. Following 2 to 7 days, SARS patients may also
develop a dry cough and experience breathing trouble. SARS appears to spread primarily
by close person-to-person contact. The majority of SARS patients appear to have been
involved people who cared for or lived with others with SARS, or had direct contact with
an infectious material (e.g., respiratory secretions) from another patient with SARS.
Potential ways in which SARS can be spread include touching the skin of other people or
objects that are contaminated with infectious droplets and then touching your eye(s),
nose, or mouth. This can happen when someone who is sick with SARS coughs or
sneezes droplets onto themselves, other people, or nearby surfaces.
Scientists at the Centers for Disease Control and Prevention (CDC) and other laboratories
have detected a previously unrecognized coronavirus in patients with SARS: SARS-CoV,
which is the leading hypothesis for the cause of SARS (see website sciencemag.org/cgi/rapidpdf/1085952vl.pdf>). The sequence of SARS-CoV has been
sequenced and all of the sequence, except for the leader sequence, was derived directly
from viral RNA. The genome of the SARS coronavirus is 29,727 nucleotides in length
and the genome organization is similar to that of other coronaviruses. Open reading
frames have been identified that correspond to the predicted polymerase protein
(polymerase la, Ib), spike protein (S), small membrane protein (E), membrane protein
(M) and nucleocapsid protein (N) (see website gov/ricidod/sars/pdf/nucleoseq. pdf).
Researchers worldwide are been working frantically to develop a treatment for SARS.
Currently no treatment has been found to be effective at stopping the SARS-CoV
coronavirus associated with SARS. The antiviral drugs currently used, or considered, for
treating SARS include ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and
glycyrrhizin. However, all these drugs have serious side effects (e.g., side effects of
glycyrrhizin include raised blood pressure and lowered potassium levels). Treatment with
the anti-inflammatory drug methylprednisolone has been shown achieve some
improvement in SARS patients (So, L. K., et al., "Development of a standard treatment
protocol for severe acute respiratory syndrome", Lancet 361(9369): 1615-7,2003).
Dehydroepiandrosterones are non-glucocorticoid steroids. DHEA, also known as 5-
androsten-3 beta-ol-17-one and DHEA sulfate (DHEA-S), a sulfated form of DHEA, are
endogenous hormones secreted by the adrenal cortex in primates and a few non-primate
species in response to the release of ACTH. DHEA is a precursor of both androgen and
estrogen steroid hormones important in several endocrine processes. Current medical use
of DHEA is limited to controlled clinical trials, and as a food supplement, and is thought
to have a role in levels of DHEA in the central nerve system (CNS), and in psychiatric,
endocrine, gynecologic, obstetric, immune, and cardiovascular functions.
DHEA-S or its pharmaceutically acceptable salts are believed to improve uterine cervix
maturation and uterine musculature sensitivity to oxytocin in late phase pregnancy.
DHEA-S and its pharmaceutically acceptable salts are thought to be effective in the
therapy for dementia, for the therapy of hyperlipemia, osteoporosis, ulcers, and for
disorders associated with high levels of, or high sensitivity to adenosine, such as steroiddependent
asthma, and other respiratory and lung diseases. Dehydroepiandrosterone itself
was administered intravenously previously, subcutaneously, percutaneously, vaginally,
topically and orally in clinical trials. In pre- formulation studies, however, the anhydrous
form of DHEA sodium sulfate (DHEA-SNa) was found to be unstable to humidity, and
its dihydrate form (DHEA-SNa) was found to be more stable under conditions of normal
As is known, various operations may be performed on medicinal agents during
pharmaceutical processing that often affect the physicochemical properties and stability
of the compounds. Prolonged grinding of the dehydroepiandrosterone sodium sulfate
dihydrate produced a decrease in crystallinity and loss of hydration water; the latter
decreasing storage stability and producing DHEA, its degradation product.
Accordingly, there is a need for a powder formulation of dehydroepiandrosterone
compounds, their analogues and salts, that will show good dispersibility and shelf
stability, as well as appropriate respirable properties. Such formulation would make it
possible to deliver the dehydroepiandrosterone compounds, analogues and salts in a
highly efficacious and cost effective manner.
U. S. Patent No. 5,527,789 discloses a method of combating cancer in a subject by
administering to the subject dehydroepiandrosterone (DHEA) or DHEA-related
compound, and ubiquinone to combat heart failure induced by the DHEA or DHEArelated
U. S. Patent No. 6,087,351 discloses an in vivo method of reducing or depleting
adenosine in a subject's tissue by administering to the subject dehydroepiandrosterone
(DHEA) or DHEA- related compound. U. S. Patent No. 6,087,351 discloses that solid
particulate compositions containing respirable dry particles of micronized active
compound may be prepared by grinding dry active compound with a mortar and pestle,
and then passing the micronized composition through a 400 mesh screen to break up or
separate out large agglomerates. Also, a solid participate composition comprised of the
active compound may optionally contain a dispersant which serves to facilitate the
formation of an aerosol; and a suitable dispersant is lactose, which may be blended with
the active compound in any suitable ratio (e.g., a 1 to 1 ratio by weight).
DHEA and DHEA-S have been described to treat COPD (U. S. Patent Application Ser.
No. 10/454, 061, filed June 3,2003, and International Application No. PCT/US02/12555,
filed April 21,2002, published October 31,2002).
The invention relates to a sealed container containing a powder pharmaceutical
composition comprising an agent and a pharmaceutically or veterinarily acceptable
carrier or diluent, wherein the agent comprises a dehydroepiandrosterone (DHEA)
compound, or analogue thereof, or hydrated form thereof, sealed in a nebulizable form
wherein said dry powder pharmaceutical composition is particles of respirable or
inhalable size. Preferably, the agent is dehydroepiandrosterone sulfate (DHEA-S),
wherein the sulfate is covalently bound to DHEA. More preferably, the agent is
dehydroepiandrosterone sulfate dihydrate. Preferably, the dry powder pharmaceutical
composition has particles of greater than about 80% of the particles about 0.1 um to
about 100 um in diameter. The dehydroepiandrosterone compound, or analogue thereof,
comprise compounds of chemical formula (I), (IT), (IB), (TV) and (V), either formulated
alone or in combination with a powder, liquid or gaseous carrier. The pharmaceutical
composition may or may not further comprise an excipient. The formulation may be
administered to a subject together with another therapeutic agent(s), either in the same
composition, or by joint administration of separate compositions.
Preferably, the agent is DHEA-S in the dihydrate form (DHEA-S-2H2O). The dihydrate
form of DHEA-S is more stable than the anhydrous form of DHEA-S. The anhydrous
form of DHEA-S is more heat labile than the dihydrate form of DHEA-S. Preferably, the
carrier is lactose. Preferably, the agent is in a powder form. Preferably, the agent is in a
crystalline form. More preferably, the agent is in a crystalline powder form.
Preferably, the sealed container is vacuumed sealed and usable for nebulizer to be
administered a patient or subject in need of prophylaxis or treatment with a
therapeutically effective amount of the powder pharmaceutical composition.
Another aspect of the present invention is a method for prophylaxis or treatment of
asthma, comprising administering to a subject in need of such prophylaxis or treatment a
therapeutically effective amount of the powder pharmaceutical composition.
Another aspect of the present invention is a method for prophylaxis or treatment of
chronic obstructive pulmonary disease, comprising administering to a subject in need of
such prophylaxis or treatment a therapeutically effective amount of the powder
pharmaceutical composition.
Another aspect of the present invention is a method of reducing or depleting adenosine in
a subject's tissue, comprising administering to a subject in need of such treatment a
therapeutically effective amount of the powder pharmaceutical composition to reduce or
deplete adenosine levels in the subject's tissue.
Another aspect of the present invention is a method for prophylaxis or treatment of a
disorder or condition associated with high levels of, or sensitivity to, adenosine in a
subject's tissue, comprising administering to a subject in need of such prophylaxis or
treatment a therapeutically effective amount of the powder pharmaceutical composition
to reduce adenosine levels in the subject's tissue and prevent or treat the disorder.
Preferably, the subject suffers from airway inflammation, allergy, asthma, impeded
respiration, cystic fibrosis, Chronic Obstructive Pulmonary Diseases (COPD), allergic
rhinitis, Acute Respiratory Distress Syndrome, microbial infection, viral infection, such
as SARS, pulmonary hypertension, lung inflammation, bronchitis, airway obstruction, or
Preferably, the dry powder formulation is prepared starting from the dry pharmaceutical
agent, altering the particle size of the agent to form a powder formulation of particles
greater than about 80% of about 0.1 to about 100 um in diameter, e. g. altered by milling,
e.g. fluid energy milling, sieving, homogenization granulation, and/or other known
The powder formulation of the invention may be delivered through the respiratory tract
by direct administration from a device, either by itself, or along with a powdered, liquid
or gaseous carrier or propellant. Preferably, the device is a nebulizer capable of
administering the powdered formulation to a patient or subject incapable of inhaling the
powdered formulation without the device. The formulation described herein is suitable
for treating any diseases; for example those associated with respiratory and lung diseases,
such as bronchoconstriction, allergy(ies), asthma, lung inflammation, chronic obstructive
pulmonary disease (COPD), allergic rhinitis, ARDS, cystic fibrosis, cancer and
inflammation, among others.
Another aspect of the present invention is an use of the dehydroepiandrosterone
compound, or analogue thereof, or hydrated form thereof, in the manufacture of a
medicament for prophylaxis or treating of asthma, COPD, lung inflammation, any
respiratory disorder or condition, or reducing or deleting adenosine in a subject's tissue.
Another aspect of the invention is a kit comprising a device for delivering the powder
pharmaceutical composition to the subject. Preferably, the device is a nebulizer or
aerosolizer, which may be pressurized, either comprising the powder formulation.
Preferably, the kit further comprises one or more capsules, cartridges or blisters with the
formulation, wherein the capsules, cartridges or blisters are to be inserted in the device
prior to use.
Figure 1 depicts fine particle fraction of neat micronized DHEA-S-2H2O delivered from
the single-dose Acu-Breathe inhaler as a function of flow rate. Results are expressed as
DHEA- S. IDL data on virtually anhydrous micronized DHEA-S are also shown in this
figure where the 30 L/min result was set to zero since no detectable mass entered the
Figure 2 depicts HPLC chromatograms of virtually anhydrous DHEA-S bulk after
storage as neat and lactose blend for 1 week at 50°C. The control was neat DHEA-S
stored at room temperature.
Figure 3 depicts HPLC chromatograms for DHEA-S -2^0 bulk after storage as neat and
lactose blend for 1 week at 50°C. The control was neat DHEA-S-2H2O stored at room
Figure 4 depicts solubility of DHEA-S as a function of NaCl concentration at two
Figure 5 depicts DHEA-S solubility as a function of the reciprocal sodium cation
concentration at 24-25 °C.
Figure 6 depicts DHEA-S solubility as a function of the reciprocal sodium cation
concentration at 7-8 °C.
Figure 7 depicts solubility of DHEA-S as a function of NaCl concentration with and
without buffer at room temperature.
Figure 8 depicts DHEA-S solubility as a function of the reciprocal of sodium cation
concentration at 24-25 °C with and without buffer.
Figure 9 depicts solution concentration of DHEA-S versus time at two storage conditions.
Figure 10 depicts solution concentration of DHEA versus time at two storage conditions.
Figure 11 depicts the schematic for nebulization experiments.
Figure 12 depicts mass of DHEA-S deposited in by-pass collector as a function of initial
solution concentration placed in the nebulizer.
Figure 13 depicts particle size by cascade impaction for DHEA-S nebulizer solutions.
The data presented are the average of all 7 nebulization experiments.
The term "agent", as used herein, means a chemical compound, a mixture of chemical
compounds, a synthesized compound, a therapeutic compound, an organic compound, an
inorganic compound, a nucleic acid, an oligonucleotide (oligo), a protein, a biological
molecule, a macromolecule, lipid, oil, fillers, solution, a cell or a tissue. Agents
comprises an active compound(s) that is a DHEA, its derivative or pharmaceutically or
veterinarily acceptable salt thereof. Agents may be added to prepare a formulation
comprising an active compound and used in a formulation or a kit in a pharmaceutical or
veterinary use.
The term "airway", as used herein, means part of or the whole respiratory system of a
subject which exposes to air. The airway includes, but not exclusively, throat, windpipes,
nasal passages, sinuses, a respiratory tract, lungs, and lung lining, among others. The
airway also includes trachea, bronchi, bronchioles, terminal bronchioles, respiratory
bronchioles, alveolar ducts, and alveolar sacs.
The term "airway inflammation", as used herein, means a disease or condition related to
inflammation on airway of subject. The airway inflammation may be caused or
accompanied by allergy(ies), asthma, impeded respiration, cystic fibrosis (CF), Chronic
Obstructive Pulmonary Diseases (COPD), allergic rhinitis (AR), Acute Respiratory
Distress Syndrome (ARDS), microbial or viral infections, pulmonary hypertension, lung
inflammation, bronchitis, airway obstruction, and bronchoconstriction.
The term "carrier", as used herein, means a biologically acceptable carrier in the form of
a gaseous, liquid, solid carriers, and mixtures thereof, which are suitable for the different
routes of administration intended. Preferably, the carrier is pharmaceutically or
veterinarily acceptable.
The composition may optionally comprise other agents such as other therapeutic
compounds known in the art for the treatment of the condition or disease, antioxidants,
flavoring agents, coloring agents, fillers, volatile oils, buffering agents, dispersants,
surfactants, RNA inactivating agents, propellants and preservatives, as well as other
agents known to be utilized in therapeutic compositions.
"Composition", as used herein, means a mixture containing a dry powdered formulation
comprising an active compound used in this invention and a carrier. The composition
may contain other agents. The composition is preferably a pharmaceutical or veterinary
"An effective amount" as used herein, means an amount which provides a therapeutic or
prophylactic benefit.
The terms "preventing" or "prevention", as used herein, mean a prophylactic treatment
made before a subject obtains a disease or ailing condition symptoms such that it can
have a subject avoid having a disease symptoms or condition related thereto.
The term "respiratory diseases", as used herein, means diseases or conditions related to
the respiratory system. Examples include, but not limited to, airway inflammation,
allergy(ies), asthma, impeded respiration, cystic fibrosis (CF), Chronic Obstructive
Pulmonary Diseases (COPD), allergic rhinitis (AR), Acute Respiratory Distress
Syndrome (ARDS), pulmonary hypertension, lung inflammation, bronchitis, airway
obstruction, bronchoconstriction, microbial infection, and viral infection, such as SARS.
"Target", as used herein, means an organ or tissue that the active compound(s) affect and
are associated with a disease or condition.
The terms "treat" or "treating", as used herein, mean a treatment which decreases the
likelihood that the subject administered such treatment will manifest symptoms of disease
or other conditions.
This invention provides a powder formulation comprising a DHEA, its derivatives,
and/or its pharmaceutically or veterinarily acceptable salts, or a hydrated form thereof,
alone, or along with a pharmaceutically or veterinarily acceptable carrier or diluent,
wherein a proportion of the formulation particles about 80% are about 0.1 to about 200,
urn in diameter, e.g., greater than about 80% particles. Examples of a DHEA, its
analogues and its salts suitable for use in this invention are represented by chemical
formulas (I), (II), (El), (IV) and (V) shown below. One group is represented by the
compound of chemical formula
(Figure Removed)
wherein R comprises H or halogen; the H at position 5 maybe present in the alpha or beta
configuration or a racemic mixture of both configurations; and RI comprises H, or a
multivalent inorganic or organic dicarboxylic acid covalently bound to the compound.
Preferably, the multivalent inorganic or organic dicarboxylic acid is SO2OM, phosphate
or carbonate. Preferably, the multivalent organic dicarboxylic acid is a succinate,
maleate, fumarate, or a suitable dicarboyxlate.
M comprises a counterion, for example, H, sodium, potassium, magnesium, aluminum,
zinc, calcium, lithium, ammonium, amine, arginine, lysine, histidine, triethylamine,
ethanolamine, choline, triethanoamine, procaine, benzathine, tromethanine, pyrrolidine,
piperazine, diethylamine, sulphatide
(Figure Removed)
wherein R2 and R3, which may be the same or different, comprise straight or branched
(Ci-Cn) alkyl or glucuronide;
(Figure Removed)
and pharmaceutically acceptable salts thereof.
RI can be an acidic or basic compound covalently bound to DHEA. If RI is an acidic
compound than the salt is formed by adding a base to the agent. Preferably, the base is
any suitable base that would result in the formation of a salt of the agent, such as sodium
hydroxide, potassium hydroxide, or the like. If RI is a basic compound than the salt is
formed by adding an acid to the agent. Preferably, the acid is any suitable acid that would
result in the formation of a salt of the agent, such as organic acids, such as rumaric acid,
maleic acid, lactic acid, or inorganic acids, such as hydrochloric acid, nitric acid, sulfuric
acid, or the like.
Preferably, the agent is DHEA-S in the dihydrate form (DHEA-S-2H2O). The dihydrate
form of DHEA-S is more stable than the anhydrous form of DHEA-S. The anhydrous
form of DHEA-S is more heat labile than the dihydrate form of DHEA-S. Preferably, the
carrier is lactose. Preferably, the agent is in a powder form. Preferably, the agent is in a
crystalline form. More preferably, the agent is in a crystalline powder form.
The present invention is the first report of using DHEA-S in the dihydrate form in
pharmaceutical composition, and that DHEA-S in the dihydrate form has the unexpected
property of a better stability, especially at higher temperatures, such as equal or greater
than 50°C, than anhydrous DHEA-S. Anhydrous DHEA-S mixed with lactose is much
less stable than crystalline dihydrate DHEA-S mixed with lactose. This discovery is
reported for the first time in this application (see Examples 3 and 5).
Compounds illustrative of formula (I) above include dehydroepiandrosterone (DHEA),
itself wherein R and RI are each H and the double bond is present; 16-alpha
bromoepiandrosterone, where R comprises Br, RI comprises H, and the double bond is
present; 16-alpha-fluoroepiandrosterone, wherein R comprises F, RI comprises H and
double bond is present; etiocholanolone, where R and RI each comprises hydrogen and
the double bond is absent; dehydroepiandrosterone sulfate, wherein R comprises H, RI
comprises SC^OM and M comprises sulphatide as defined above, and the double bond is
present; dehydroepiandrosterone sodium sulfate dihydrate, wherein R is H, RI is SOaOM
and M is a sodium group as defined above, and the double bond present, among others. In
the compound of formula (I), R preferably comprises halogen e. g. , bromo, chloro, or
fluoro, RI comprises H, and the double bond is present, more preferably the compound of
formula (1) comprises 16-alpha-fluoro epiandrosterone, the compound of formula (I),
wherein R comprises H, RI comprises SO2OM, M comprises sulphatide and the double
bond is present, and more preferably the compound of formula (I) is the dihydrate form
of dehydroepiandrosterone sodium sulfate (DHEA-S-2H2O) of chemical formula (II)
(Figure Removed)
The compounds of formula (I) and (II) may be synthesized in accordance with known
procedures or variations thereof that will be apparent to those skilled in the art. See, for
example, U. S. Patent No. 4,956, 355; UK Patent No. 2,240, 472; EPO Patent Publication
No. 429,187 ; PCT Patent Publication No. 91/04030; M. Abou-Gharbia et al., J. Pharm.
Sci. 70,1154-1157 (1981); Merck Index Monograph No. 7710; 11th Ed. (1989).
Other examples of a dehydroepiandrosterone derivative, are represented by the
compounds of chemical formulas III, FV and V shown below, and their pharmaceutically
or veterinarily acceptable salts.
(Figure Removed)
wherein R], R2, RS, R4, Re, R?, Rg, R9, RIO, Rn, Ri2,
H, OH, halogen, CMO alkyl or CMO alkoxy;
M and Ri9 are independently
RS comprises H, OH, halogen, CMO alkyl, CMO alkoxy or OSO2R2o ;
RIS comprises (1) H, halogen, CMO alkyl or CMC alkoxy when Ri6 comprises C(O)OR2i,
or (2) H, halogen, OH or CMO alkyl when Ri6 is H, halogen, OH or CMO alkyl, or (3) H,
halogen, CMO alkyl, CMO alkenyl, CMO alkynyl, formyl, CMO alkanoyl or epoxy when
Ri6 comprises OH; or RIS and Ri6 taken together comprise =O; Rn and Rig comprise
independently (1) H, OH, halogen, CMO alkyl or CMO alkoxy when Rie comprises H, OH,
halogen, CMO alkyl or -- C(O)OR2i, or (2) H, (CMO alkyl)n amino, (CMO alkyl)n amino-
CMO alkyl, CMO alkoxy, hydroxy - CMO alkyl, CMO alkoxy -CMO alkyl, (halogen)m -CMO
alkyl, CMO alkanoyl, formyl, CMO carbalkoxy or CMO alkanoyloxy when RIS and Ri6
taken together comprise =O; or Rn and Rig taken together comprise =O or taken together
with the carbon to which they are attached form a 3-6 member ring comprising 0 or 1
oxygen atoms; or
RIS and Rn taken together with the carbons to which they are attached form an epoxide
ring, R2o comprises OH, pharmaceutically acceptable ester or pharmaceutically
acceptable ether, R2i is H, (halogen)m -CMO alkyl or CMO alkyl, n is 0,1 or 2; and m is 1,2
or 3; with the proviso that
(a) R3 is not H, OH or halogen when RI, R2, R4, Re, R?, R9, RIO, Ri2, RIS, RH, Rn
and Ri9 are H and R5 is OH or CMO alkoxy and Rg is H, OH or halogen and RU is H or
OH and Rig is H, halogen or methyl and RIS is H and Ri6 is OH;

(b) R3 is not H, OH or halogen when RI, R2, R4, Re, R?, Rp, RIO, Ri2, RB, RU and
Ri9 are H and RS is OH or GI.JO alkoxy and Rg is H, OH or halogen and RH is H or OH
and Rig is H, halogen or methyl and RIS and Ri6 taken together are =O;
(c) RS is not H, halogen, CI.IQ alkoxy or OSOjRio when RI, R2, RS, R>, Re, R?, Rg,
R9, RIO, Ri2, RB, Ri4 and Rn are H and RH is H, halogen, OH or CMO alkoxy and Rig is
H or halogen and RIS and RI& taken together are =O; and
(d) Rs is not H, halogen, CMO alkoxy or OSOaRao when RI, R2, RS, R*, R Rg, R$, RIO, Ri2, Rn , RH and Rn are H and RH is H, halogen, OH or Ci-io alkoxy and
Rig is H or halogen and Ri5 is H and Rie is H, OH or halogen;
or a compound of the chemical formula
H3C p
(Figure Removed)
or pharmaceutically or cosmetically acceptable salts thereof, wherein
R is A-CH(OH)-C(O)- and A comprises H hydrogen or a (Ci-C22) alkyl or alkenyl
which may be substituted with one or more (Ci-C4) alkyl, phenyl, halogen or HO groups,
the phenyl being optionally with one or more halogen, HO or i
Compounds of general formulas (HI), (IV) and (V) may be synthesized as described in U.
S. Patent Nos. 4,898, 694; 5,001,119; 5,028,631; 5,175, 154; 6,187,767; and 6,284,750,
the relevant portions of which are incorporated herein by reference. The compounds
represented by the general formulas (III), (IV) and (V) exist as different stereoisomers
and these formulas are intended to encompass each individual stereoisomer and their
Examples of representative compounds which fall within the scope of general formulas
(III), (IV) and (V) include 5a-androstan-17-one; 16a-fluoro-5a-androstan-17-one; 3P-
methyl-Sa -androsten-17-one; 16a -fluoro-5a -androstan-17-one; 17B-bromo-5-androsten-
16-one; 17B-fliM>ro-3B-methyl-5-androsten-16-one; 17a-fluoro-5a-androstan-16-one; 36-
hydroxy-5-androsten-l 7-one; 17a -methyl-5a -androstan-16-one; 16a -methyl-5-androsten-
17-one; 17B, 16a-dimethyl-5-androsten-l 7-one; 38, 17a-dimethyl-5-androsten-16-one
16a -hydroxy-5-androsten-17-one; 16a -fluoro-16p-methyl-5-androsten-17-one; 16ct -
methyl-5a -androstan-17-one; 16-dimethylaminomethyl-5a -androstan-17-one; 16Bmethoxy-
5-androsten-17-one; 16a -fluoromethyl-5-androsten-17-one; 16-methylene-5-
androsten-17-one; 16-cyclopropyl-5a -androstan-17-one; 16-cyclobutyl-5-androsten-
one; 16-hydroxymethylene-5-androsten-l 7-one; 3a -bromo-16a -methoxy-5-androsten-17-
one; 16-oxymethylene-5-androsten-l 7-one; 3p-methyl-16.xi.-trifluoromethyl-5a -
androstan-17-one; 16-carbomethoxy-5-androsten-l 7-one; 3B-methyl-166-methoxy-5a -
androstan-17-one; 36-hydroxy-16a -dimethylamino-5-androsten-17-one; 17a -methyl-5-
androsten-17beta-ol; 17a -ethynyl-5a -androstan-17B-ol; 17fi-formyl-5a -androstan-176-ol;
20,2 l-epoxy-5a-preplan-17a-ol; 36-hydroxy-20, 21-epoxy-5a-pregnan-17a-ol; 16afluoro-
17a -ethenyl-5-androsten-l 7a -ol; 16a -hydroxy-5-androsten-l 7o -ol; 16a -methyl-5
a -androstan-17a -ol; 16a -methyl-166-fluoro-5 alpha-androstan-17a -ol; 16a -methyl-
fluoro-3-hydroxy-5-androsten-17a-ol; 3B, 16 beta-dimethyl-5-androsten-17B-ol; 36,16,
16-trimethyl-5-androsten-176-ol; 36,16,16-trimethyl-5-androsten-17-one; 36-hydroxy-4a
-methyl-5-androsten-17a -ol; 3B-hydroxy-4a -methyl-5-androsten-l 7-one; 3a -hydroxy-la
-methyl-5-androsten-l 7-one; 3a-ethoxy-5a-androstan-176-ol; 5a-pregnan-20-one; 36-
methyl-5a -pregnan-20-one; 16a -methyl-5-pregnen-20-one; 16a -methyl-36-hydroxy-5-
pregnen-20-one; 17a -fluoro-5-pregnen-20-one; 21 -fluoro-5a -pregnan-20-one; 17a -
methyl-5-pregnen-20-one; 20-acetoxy-cis-17(20)-5a -pregnene; 3a -methyl-16,17-epoxy-
5 -pregnen-20-one.
The compounds used in this invention may be administered per se or in the form of
pharmaceutically and veterinarily acceptable salts; all of these being referred to as "active
compounds". Examples of pharmaceutically or veterinarily acceptable carrier or diluent
include biologically acceptable carriers, known in the art, including lactose and other
inert or G.R.A.S. (generally regarded as safe) agents in gaseous, liquid, or solid form,
where the final form of the formulation is as a powder or a powder with a propellant and
or co-solvent that may be under pressure.
The powdered formulation may be prepared starting from a dry product comprising a
dehydroepiandrosterone, its analogue, its salt or mixtures thereof, by altering the particle
size of the agent, to form a dry formulation of particle size about 0.01 um to about 500
um in diameter; and selecting particles of the formulation comprising at least or greater
than about 80%, about 85%, about 90%, about 95%, or about 100% particles of about
0.01 p,m, 0:1 um or 0. 5 \im to about 100 urn or 200 urn in diameter. The particle size is
desirably less than about 200 (am, preferably in the range about 0.05 um, about 0.1 urn,
about 1 um, about 2 urn to about 5 (Am, about 6 um, about 8 um, about 10 urn, about 20
um, about 50 um, about 100 urn. Preferably, the selected particles of the formulation of
about 0.1 to about 200 um in diameter. More preferably, the selected particles of the
formulation of about 0.1 to about 100 um in diameter. Even more preferably, the selected
particles of the formulation of about 0.1 to about 10 um in diameter. Even much more
preferably, the selected particles of the formulation of about 0.1 to about 8 urn in
diameter. Even further much more preferably, the selected particles of the formulation of
about 0.1 to about 5 um in diameter.
The particle size of the dry agent may be then altered so as to permit the absorption of a
substantial amount of the agent into the lungs upon inhalation of the formulation. The
particle size of the medicament may be reduced by any known means, for example by
milling or micronization. Typically, the particle size for the agent is altered by milling the
dry agent either alone or in combination with a formulation ingredient to a suitable
average particle size, preferably in the about 0.05 um, about 5 jam range (inhalation) or
about 10 um, to about 50 um (nasal delivery or lung instillation). Jet milling, also known
as fluid energy milling, may be employed and are preferred among the procedures to give
the particle size of interest using known devices. Jet milling is the preferred process. It
should be understood that although a large percentage of the particles will be in the
narrow range desired, this will not generally be true for all particles. Thus, it is expected
that the overall particle range may be broader than the preferred range as stated above.
The proportion of particles within the preferred range may be greater than about 80%,
about 85%, about 90%, about 95%, and so on, depending on the needs of a specific
The particle size may be also altered by sieving, homogenization, and/or granulation,
amongst others. These techniques are used either separately or in combination with one
another. Typically, milling, homogenization and granulation are applied, followed by
sieving to obtain the dry altered particle size formulation. These procedures may be
applied separately to each ingredient, or the ingredients added together and then
Examples of the formulation ingredients that may be employed are not limited to, but
include, an excipient, preservatives, stabilizers, powder flowability improving agents, a
cohesiveness improving agent, a surfactant, other bioactive agents, a coloring agent, an
aromatic agent, anti-oxidants, fillers, volatile oils, dispersants, flavoring agents, buffering
agents, bulking agents, propellents or preservatives. One preferred formulation comprises
the active agent and an excipient(s) and/or a propellant(s).
The particle size may be altered not only in a dry atmosphere but also by placing the
active agent in solution, suspension or emulsion in inter-mediate steps. The active agent
may be placed in solution, suspension, or emulsion, either prior to, or after, altering the
particle size of the agent. An example of this embodiment that may be performed by
dissolving the agent in a suitable solvent solution, and heating to an appropriate
temperature. The temperature may be maintained in the vicinity of the appropriate
temperature for a predetermined period of time to allow for crystals to form. The solution
and the fledgling crystals then are cooled to a second lower temperature to grow the
crystals by maintaining them at the second temperature for a period of time as is known
in the art. The crystals are then allowed to reach room temperature when recrystalization
is completed and the crystals of the agent have grown sufficiently. The particle size of the
agent may also be altered by sample precipitation, which is conducted from solution,
suspension or emulsion in an adequate solvent(s).
Spray drying is useful in altering the particle size, as well. By "spray dried or spray
drying" what is meant is that the agent or composition is prepared by a process in which a
homogeneous mixture of the agent in a solvent or composition termed herein the "prespray
formulation", is introduced via an atomizer, e.g. a two-fluid nozzle, spinning disk
or an equivalent device into a heated atmosphere or a cold fluid as fine droplets. The
solution may be an aqueous solution, suspension, emulsion, slurry or the like, as long as
it is homogeneous to ensure uniform distribution of the material in the solution and,
ultimately, in the powdered formulation. When sprayed into a stream of heated gas or air,
the each droplet dries into a solid particle. Spraying of the agent into the cold fluid results
in a rapid formation of atomized droplets that form particles upon evaporation of the
solvent. The particles are collected, and then any remaining solvent may be removed,
generally through sublimation (lyophilization), in a vacuum. As discussed below, the
particles may be grown, e.g. by raising the temperature prior to drying. This produces a
fine dry powder with particles of a specified size and characteristics, that are more fully
discussed below. Suitable spray drying methodologies are also described below. See, for
example U. S. Pat. Nos. 3,963,559; 6,451,349; and, 6,458,738, the relevant portions of
which are incorporated herein by reference.
As used herein, the term "powder" means a composition that consists of finely dispersed
solid particles that are relatively free flowing and capable of being readily dispersed in an
inhalation or dry powder device and subsequently inhaled by a patient so that the
particles can reach the intended region of the lung. Thus, the powder is "respirable" and
suitable for pulmonary delivery. When the particle size of the next agent or the
formulation is above about 10 um, the particles are of such size that a good proportion of
them will deposit in the nasal cavities, and will be absorbed there through.
The term "dispersibility" means the degree to which a dry powder formulation may be
dispersed, i.e. suspended, in a current of air so that the dispersed particles may be
respired or inhaled into the lungs or absorbed through the walls of the nasal cavities of a
subject. Thus, a powder that is only 20% dispersible means that only 20% of the mass of
particles may be suspended for inhalation into the lungs. The present formulation
preferably has a dispersibility of about 1 to 99 %, although others are also suitable.
The dry powder formulation may be characterized on the basis of a number of
parameters, including, but not limited to, the average particle size, the range of particle
size, the fine powder fraction (FPF), the average particle density, and the mass median
aerodynamic diameter (MMAD), as is known in the art.
In a preferred embodiment, the agent is DHEA-S in a dihydrate crystalline form. The
DHEA-S is first crystallized into the dihydrate crystalline form. The crystals are then put
through the jet mill to produce it into a powder form. The preparation can further
comprise lactose that is separately sieved or milled and mixed with the powdered
crystalline dihydrate DHEA-S.
In a preferred embodiment, the dry powder formulation of this invention is characterized
on the basis of their average particle size that was described above. The average particle
size of the powdered agent or formulation may be measured as the mass mean diameter
(MMD) by conventional techniques. The term, "about" means the numerical values could
have an error in the range of about 10% of the numerical value. The dry powdered
formulation of this invention may also be characterized on the basis of its fine particle
fraction (FPF). The FPF is a measure of the aerosol performance of a powder, where the
higher the fraction value, the better. The FPF is defined as a powder with an aerodynamic
mass median diameter of less than 6.8 urn as determined using a multiple-stage liquid
impinger with a glass throat (MLSI, Astra, Copley Instrument, Nottingham, UK) through
a dry powder inhaler (Dryhalter™, Dura Pharmaceuticals). Accordingly, the dry powder
formulation of the invention preferably has a FPF of at least about 10%, with at least
about 20% being preferred, and at least about 30% being especially preferred. Some
systems may enable very high FPFs, of the order of 40 to 50%.
The dry powdered formulation may be characterized also on the basis of the density of
the particles containing the agent of the invention. In a preferred embodiment, the
particles have a tap density of less than about 0.8 g/cm3, with tap densities of less than
about 0.4 g/cm3 being preferred, and a tap density of less than about 0.1 g/cm3 being
especially preferred. The tap density of dry powder particles may be measured using a
GeoPyc™ (Micrometrics Instruments Corp), as is known in the art. Tap density is a
standard measure of the envelope mass density, which is defined generally as the mass of
the particle divided by the minimum sphere envelope volume within which it may be
In another preferred embodiment, the aerodynamic particle size of the dry powdered
formulation may be characterized as is generally outlined in the Examples. Similarly, the
mass median aerodynamic diameter (MMAD) of the particles may be evaluated, using
techniques well known in the art. The particles may be characterized on the basis of their
general morphology as well.
The term "dry" means that the formulation has a moisture content such that the particles
are readily dispersible in an inhalation device to form an aerosol. The dry powdered
formulation in the invention comprises preferably substantially active compound,
although some aggregation may occur, particularly upon long storage periods. As is
known for many dry powder formulation, some percentage of the material in a powder
formulation may aggregate, this resulting in some loss of activity. Accordingly, the dry
powdered formulation has at least about 70% w/w active compound, i.e. % of total
compound present, with at least about 80% w/w active compound being preferred, and at
least about 90% w/w active compound being especially preferred. More highly active
compound or agent is also contemplated, and may be prepared by the present method, i.e.
,an activity greater than about 95% and higher. The measurement of the total compound
present will depend on the compound and, generally, will be done as is known in the art,
on the basis of activity assays, etc. The measurement of the activity of the agent will be
dependent on the compound and will be done on suitable bioactivity assays as will be
appreciated by those in the art.
In spray drying, an individual stress event may arise due to atomization (shear stress and
air-liquid interfacial stress), cold or heat denaturation, optionally freezing (ice-water
interfacial stress and shear stress), and/or dehydration. Cryoprotectants and lyoprotectants
have been used during lyophilization to counter freezing destabilization, and dehydration
and long-term storage destabilization, respectively. Cryoprotectant molecules, e.g.,
sugars, amino acids, polyols, etc., have been widely used to stabilize active compounds in
highly concentrated unfrozen liquids associated with ice crystallization. These are not
required in the formulation.
The dry powdered formulations comprising an active compound may or not contain an
excipient. "Excipients" or "protectants" including cryoprotectants and lyoprotectants
generally refers to compounds or materials that are added as diluents or to ensure or
increase flowability and aerosol dispersibility of the active compounds during the spray
drying step and afterwards, and for long-term flowability of the powdered product.
Suitable excipients are generally relatively free flowing particulate solids, do not thicken
or polymerize upon contact with water, are basically innocuous when placed in the
respiratory tract of a patient and do not substantially interact with the active compound in
a manner that alters its biological activity.
Suitable excipients include, but are not limited to, proteins such as human and bovine
serum albumin, gelatin, immunoglobulins, carbohydrates including monosaccharides
(galactose, D-mannose, sorbose, etc.), disaccharides (lactose, trehalose, sucrose, etc.),
cyclodextrins, and polysaccharides (raffinose, maltodextrins, dextrans, etc.); an amino
acid such as monosodium glutamate, glycine, alanine, arginine or histidine, as well as
hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.); a lubricant
such as magnesium stearate; a methylamine such as betaine; an excipient salt such as
magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,
erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol;
polyethylene glycol; pluronics; surfactants; (lipid and non-lipid surfactants) and
combinations thereof. Preferred excipients are trehalose, sucrose, sorbitol, and lactose, as
well as mixtures thereof. When excipients are used, they are used generally in amounts
ranging from about 0.1, about 1, about 2, about 5, about 10 to about 15, about 10, about
15, about 20, about 40, about 60, about 99% w/w composition. Preferred are formulations
containing lactose, or low amounts of excipient or other ingredients.
In another preferred embodiment, the dry powdered formulation of this invention is
substantially free of excipients. "Substantially free" in this case generally means that the
formulation contains less than about 10%, w/w preferably less than about 5%, w/w more
preferably less than about 2-3% w/w, still more preferably less than about 1% w/w of any
components other than the agent. Generally, for the purposes of this invention, the
formulation may include a propellant and a co-solvent, buffers or salts, and residual
water, hi one preferred embodiment the dry powdered formulation (prior to the addition
of bulking agent, discussed below) consists of the agent and protein as a major
component, with small amounts of buffer(s), salt(s) and residual water. Generally, in this
embodiment, the spray drying process comprises a temperature raising step prior to
drying, as is more fully outlined below.
In another preferred embodiment, the pre-spray dried formulation, i.e. the solution
formulation used in the spray drying process comprises the active agent in solution, e.g.
aqueous solution, with only negligible amounts of buffers or other compounds. The prespray
dried formulation containing little or no excipient may not be highly stable over a
long period of time. It is, thus, desirable to perform the spray drying process within a
reasonable short time after the pre-spray dried formulation is produced. Although, the
pre-spray dried formulation utilizing little or no excipient may not be highly stable, the
dry powder made from it may, and generally is both surprisingly stable and highly
dispersible, as shown in the Examples.
The agents that are spray dried to form the formulations of the invention comprise the
agent and optionally a buffer, and may or may not contain additional salts. The suitable
range of the pH of the buffer in solution can be readily ascertained by those in the art.
Generally, this will be in the range of physiological pH, although the agent of the
invention may flowable at a wider range of pHs, for example acidic pH. Thus, preferred
pH ranges of the pre-spray dry formulation are about 1, about 3, about 5, about 6 to about
7, about 8, about 10, and a pH about 7 being especially preferred. As will be appreciated
by those in the art, there are a large number of suitable buffers that may be used. Suitable
buffers include, but are not limited to, sodium acetate, sodium citrate, sodium succinate,
sodium phosphate, ammonium bicarbonate and carbonate. Generally, buffers are used at
molarities from about 1 mM, about 2 mM to about 200 mM about 10 mM, about 0.5 M,
about 1 M, about 2 M, about 50 M being particularly preferred.
When water, buffers or solvents are used during the preparation process, they may
additionally contain salts as already indicated.
In addition, the dry powdered formulation of the invention is generally substantially free
of "stabilizers". The formulation may contain, however, an additional surfactant that has
its own prophylactic or therapeutic effect on the respiratory system on the lungs. These
active agents may compensate for loss of lung surfactant or generally act by other
mechanisms. The dry powdered formulations of the invention is also generally
substantially free of microsphere-forming polymers. See, e.g. WO 97/44013; U. S. Patent
No. 5,019,400. That is, the powders of the invention generally comprise the active agent
(s) and excipient, and do not require the use of polymers for structural or other purposes.
The dry powdered formulations of the invention is also preferably stable. "Stability" may
mean one of two things, retention of biological activity and retention of dispersibility
over time, with preferred embodiments showing stability hi both areas.
The dry powdered formulation of the invention generally retains biological activity over
time, e.g. physical and chemical stability and integrity upon storage. Losses of biological
activity are generally due to aggregation, and/or oxidation of agent's particles. However,
when the agent is agglomerate around particles of excipient, the resulting agglomerates
are highly stable and active. As will be appreciated by those in the art, there may be an
initial loss of biological activity as a result of spray drying, due to the extreme
temperatures used in the process. Once this has occurred, however, further loss of activity
will be negligible, as measured from the time the powder is made. Moreover, the dry
powdered formulation of the invention have been found to retain dispersibility over time,
as quantified by the retention of a high FPF over time, the minimally aggregation, caking
or clumping observed over tune.
The agent(s) of the invention is (are) made by methods known in the art. See, for
example, U. S. Patent Nos. 6,087,351; 5,175,154; and, 6,284,750. The pre-spray drying
composition may be formulated for stability as a liquid or solid formulation. For spray
drying, the liquid formulations are subjected generally to diafiltration and/or
ultrafiltration, as required, for buffer exchange (or removal) and/or concentration, as is
known in the art. The pre-spray dry formulations comprise from about 1 mg/ml, about 5
mg/ml, about 10 mg/ml, about 20 mg/ml to about 60 mg/ml, about 75 mg/ml of the agent.
Buffers and excipients, if present, are present at concentrations discussed above. The prespray
drying formulation is then spray dried by dispersing the agent into hot air or gas, or
by spraying it into a cold or freezing fluid, e.g. a liquid or gas. The pre-spray dry
formulation may be atomized as is known in the art, for example via a two-fluid or
ultrasonic nozzle using filtered pressurized air, into, for example, a fluid. Spray drying
equipment may be used (Buchi; Niro Yamato; Okawara; Kakoki). It is generally
preferable to slightly heat the nozzle, for example by wrapping the nozzle with heating
tape to prevent the nozzle head from freezing when a cold fluid is used. The pre-spray
dry formulation may be atomized into a cold fluid at a temperature of about -200°C to
about -100°C, about -80 °C. The fluid may be a liquid such as liquid nitrogen or other
inert fluids, or a gas such as air that is cooled. Dry ice in ethanol may be used as well as
super-critical fluids. In one embodiment it is preferred to stir the liquid as the atomization
process occurs, although this may not be required.
Micronization techniques involve placing bulk drug into a suitable mill. Such mills are
commercially available from, for example, DT Industries, Bristol, Pa., under the
tradename STOKES™. Briefly, the bulk drug is placed in an enclosed cavity and
subjected to mechanical forces from moving internal parts, e.g., plates, blades, hammers,
balls, pebbles, and so forth. Alternatively, or in addition to parts striking the bulk drug,
the housing enclosing the cavity may turn or rotate such that the bulk drug is forced
against the moving parts. Some mills, e.g. , fluid energy or air-jet mills, include a highpressure
air stream that forces the bulk powder into the air within the enclosed cavity for
contact against internal parts. Once the size and shape of the drug is achieved, the process
may be stopped and drug having the appropriate size and shape is recovered. Generally,
however, particles having the desired particle size range are recovered on a continuous
basis by elutriation.
There are many different types of size reduction techniques that can be used to reduce to
size of the particles. There is the cutting method employing the use of a cutter mill that
can reduce the size of particles to about 100 um. There is the compression method
employing the use of an end-runner mill that can reduce the size of particles to less than
about 50um. There is the impact method employing the use of a vibration mill that can
reduce the size of particles to about 1 um or a hammer mill that can reduce the size of
particles to about 8 um. There is the attrition method employing the use of a roller mill
that can reduce the size of particles to about 1 urn. There is the combined impact and
attrition method employing the use of a pin mill that can reduce the size of particles to
about 10 jim, a ball mill that can reduce the size of particles to about 1 urn, a fluid energy
mill (or jet mill) that can reduce the size of particles to about 1 um. One of ordinary skill
in the art is able through routine experimentation determine the particle size reduction
method and means to produce the desired particle size of the composition.
Supercritical fluid processes may be used for altering the particle size of the agent.
Supercritical fluid processes involve precipitation by rapid expansion of supercritical
solvents, gas anti-solvent processes, and precipitation from gas-saturated solvents. A
supercritical fluid is applied at a temperature and pressure that are greater than its critical
temperature (Tc) and critical pressure (Pc), or compressed fluids in a liquid state. It is
known that at near-critical temperatures, large variations in fluid density and transport
properties from gas-like to liquid-like can result from relatively moderate pressure
changes around the critical pressure (0.9-1.5 PC) While liquids are nearly incompressible
and have low diffusivity, gases have higher diffusivity and low solvent power.
Supercritical fluids can be made to possess an optimum combination of these properties.
The high compressibility of supercritical fluids (implying that large changes in fluid
density can be brought about by relatively small changes in pressure, making solvent
power highly controllable) coupled with their liquid-like solvent power and better-thanliquid
transport properties (higher diffusivity, lower viscosity and lower surface tension
compared with liquids), provide a means for controlling mass transfer (mixing) between
the solvent containing the solutes (such as a drug) and the supercritical fluid.
The two processes that use supercritical fluids for particle formation and that have
received attention in the recent past are: (1) Rapid Expansion of Supercritical Solutions
(RESS} (Tom, J. W. Debenedetti, P. G., 1991, The formation of bioerodible polymeric
microspheres and microparticles by rapid expansion of supercritical solutions.
BioTechnol. Prog. 7:403-411), and (2) Gas Anti-Solvent (GAS) Recrystallization
(Gallagher, P.M., Coffey, M.P., Krukonis, V.J., and Klasutis, N., 1989, GAS antisolvent
recrystallization: new process to recrystallize compounds in soluble and supercritical
fluids. Am. Chem. Sypm. Sen, No. 406; Yeo et al. (1993); U. S. Pat. No. 5,360,478 to
Krukonis et al.; U. S. Pat. No. 5,389,263 to Gallagher et al.). In the RESS process, a
solute (from which the particles are formed) is first solubilized in supercritical CO2 to
form a solution. The solution is then, for example, sprayed through a nozzle into a lower
pressure gaseous medium. Expansion of the solution across this nozzle at supersonic
velocities causes rapid depressurization of the solution. This rapid expansion and
reduction in CO2 density and solvent power leads to supersaturation of the solution and
subsequent recrystallization of virtually contaminant-free particles. The RESS process,
however, may not be suited for particle formation from polar compounds because such
compounds, which include drugs, exhibit little solubility in supercritical CO2 Cosolvents
(e.g. ,methanol) may be added to C(>2 to enhance solubility of polar compounds; this,
however, affects product purity and the otherwise environmentally benign nature of the
RESS process. The RESS process also suffers from operational and scale-up problems
associated with nozzle plugging due to particle accumulation in the nozzle and to
freezing of CO2 caused by the Joule-Thompson effect accompanying the large pressure
In the GAS process, a solute of interest (typically a drug) that is in solution or is
dissolved in a conventional solvent to form a solution is sprayed, typically through
conventional spray nozzles, such as an orifice or capillary tube, into supercritical CC>2
which diffuses into the spray droplets causing expansion of the solvent. Because the COi
-expanded solvent has a lower solubilizing capacity than pure solvent, the mixture can
become highly supersaturated and the solute is forced to precipitate or crystallize. The
GAS process enjoys many advantages over the RESS process. The advantages include
higher solute loading (throughput), flexibility of solvent choice, and fewer operational
problems in comparison to the RESS process. In comparison to other conventional
techniques, the GAS technique is more flexible in the setting of its process parameters,
and has the potential to recycle many components, and is therefore more environmentally
acceptable. Moreover, the high pressure used in this process (up to 2,500 psig) can also
potentially provide a sterilizing medium for processed drug particles; however, for this
process to be viable, the selected supercritical fluid should be at least partially miscible
with the organic solvent, and the solute should be preferably insoluble in the supercritical
Gallagher et al. (1989) teach the use of supercritical COa to expand a batch volume of a
solution of nitroguanadine and recrystallize particles of the dissolved solute. Subsequent
studies disclosed by Yeo et al. (1993) used laser-drilled, 25-30 jam capillary nozzles for
spraying an organic solution into COa. Use of 100 um and 151 \im capillary nozzles also
has been reported (Dixon, D. J. and Johnston, K.P.,1993, Formation of microporous
polymer fibers and oriented fibrils by precipitation with a compressed fluid antisolvent. J.
App. Polymer Sci. 50:1929-1942; Dixon, D. G., Luna-Barcenas, G., and Johnson K. P.,
1994, Microcellular microspheres and microballoons by precipitation with a vapor-liquid
compressed fluid antisolvent. Polymer 35:3998-4005).
Examples of solvents are selected from carbon dioxide (CC>2), nitrogen (N2), Helium
(He), oxygen (Ch), ethane, ethylene, ethylene, ethane, methanol, ethanol,
trifluoromethane, nitrous oxide, nitrogen dioxide, fluoroform (CHFs), dimethyl ether,
propane, butane, isobutanes, propylene, chlorotrifluormethane (CCIF3), sulfur
hexafluoride (SFe), bromotrifluoromethane (CBrFs), chlorodifluoromethane (CHC1F2),
hexafluoroethane, carbon tetrafluoride carbon dioxide, 1,1,1,2-tetrafluoroethane,
1,1,1,2,3,3,3-heptafluoropropane, xenon, acetonitrile, dimethylsulfoxide (DMSO),
dimethylformamide (DMF), and mixtures of two or more thereof.
The atomization conditions, including atomization gas flow rate, atomization gas
pressure, liquid flow rate, etc., are generally controlled to produce liquid droplets having
an average diameter of from about 0.5 urn, about 1 um, about 5 um to about 10 um,
about 30 um, about 50 um, about 100 um, with droplets of average size about 10 um and
about 5 um being preferred. Conventional spray drying equipment is generally used.
(Buchi, Niro Yamato, Okawara, Kakoki, and the like). Once the droplets are produced,
they are dried by removing the water and leaving the active agent, any excipient(s), and
residual buffers), solvent(s) or salt(s). This may be done in a variety of ways, such as by
lyophilization, as is known in the art. i.e. freezing as a cake rather than as droplets.
Generally, and preferably, vacuum is applied, e.g. at about the same temperature as
freezing occurred. However, it is possible to relieve some of the freezing stress on the
agent by raising the temperature of the frozen particles slightly prior to or during the
application of vacuum. This process, termed "annealing", reduces agent inactivation, and
may be done in one or more steps, e.g. the temperature may be increased one or more
times either before or during the drying step of the vacuum with a preferred mode
utilizing at least two thermal increases. The particles may be incubated for a period of
time, generally sufficient time for thermal equilibrium to be reached, i.e. depending on
sample size and efficiency of heat exchange 1 to several hours, prior to the application of
the vacuum, then vacuum is applied, and another annealing step is done. The particles
may be lyophilized for a period of time sufficient to remove the majority of the water not
associated with crystalline structure, the actual period of time depending on the
temperature, vacuum strength, sample size, etc.
Spheronization involves the formation of substantially spherical particles and is well
known in the art. Commercially available machines for spheronizing drugs are known
and include, for example, Marumerizer™ from LCI Corp. (Charlotte, N.C. ) and CFGranulator
from Vector Corp. (Marion, Iowa). Such machines include an enclosed cavity
with a discharge port, a circular plate and a means to turn the plate, e.g., a motor. Bulk
drug or moist granules of drug from a mixer/granulator are fed onto the spinning plate,
which forces them against the inside wall of the enclosed cavity. The process results in
particles with spherical shape. An alternative approach to Spheronization that may be
used includes the use of spray drying under controlled conditions. The conditions
necessary to spheronize particles using spray-drying techniques are known to those
skilled in the art and described in the relevant references and texts, e.g., Remington: The
Science and Practice of Pharmacy, Twentieth Edition (Easton, Pa.: Mack Publishing Co.,
In a preferred embodiment, a secondary lyophilization drying step is conducted to
remove additional water at temperatures about 0 °C, about 10 °C, to about 20 °C, to about
25 °C, with about 20 °C being preferred. The powder is collected then by using
conventional techniques, and bulking agents, if desirable, may be added although not
required. Once made, the dry powder formulation of the invention may be being readily
dispersed by a dry powder inhalation device and subsequently inhaled by a patient so that
the particles penetrate into the target regions of the lungs. The powder of the invention
may be formulated into unit dosages comprising therapeutically effective amounts of the
active agent and used for delivery to a patient, for example, for the prevention and
treatment of respiratory and lung disorders.
The dry powder formulation of this invention is formulated and dosed in a fashion
consistent with good medical practice, taking into account, for example, the type of
disorder being treated, the clinical condition of the individual patient, whether the active
agent is administered for preventative or therapeutic purposes, its concentration in the
dosage, previous therapy, the patient's clinical history and his/her response to the active
agent, the method of administration, the scheduling of administration, the discretion of
the attending physician, and other factors known to practitioners. The "effective amount"
or "therapeutically effective amount" of the active compound for purposes of this patent
include preventative and therapeutic administration, and will depend on the identity of
the active agent and is, thus, determined by such considerations and is an amount that
increases and maintains the relevant, favorable biological response of the subject being
treated. The active agent is suitably administered to a patient at one time or over a series
of treatments, preferably once a day, and may be administered to the patient at any time
from diagnosis onwards. A "unit dosage" means herein a unit dosage receptacle
containing a therapeutically effective amount of a micronized active agent. The dosage
receptacle is one that fits within a suitable inhalation device to allow for the
aerosolization of the dry powdered formulation by dispersion into a gas stream to form an
aerosol. These can be capsules, foil pouches, blisters, vials, etc. The container may be
formed from any number of different materials, including plastic, glass, foil, etc, and may
be disposable or rechargeable by insertion of a filled capsule, pouch, blister etc. The
container generally holds the dry powder formulation, and includes directions for use.
The unit dosage containers may be associated with inhalers that will deliver the powder
to the patient. These inhalers may optionally have chambers into which the powder is
dispersed, suitable for inhalation by a patient.
The dry powdered formulations of the invention may be further formulated in other ways,
e.g. as a sustained release composition, for example, for implants, patches, etc. Suitable
examples of sustained-release compositions include semi-permeable polymer matrices in
the form of shaped articles, e.g. films or microcapsules. Sustained-release matrices
include for example polylactides. See for example, U. S. Pat. No. 3,773, 919; EP 58,481.
Copolymers of L-glutamic acid and gamma-ethyl-L-glutamate are also suitable. See, e.g.
Sidman et al., Biopolymers 22: 547-556 (1983] ) as poly (2-hydroxyethyl methacrylate).
See Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech., 12:
98-105 (1982). Also suitable are ethylene vinyl acetate and poly-D-(-)-3-hydroxyburyric
acid. See, Langer et al, supra; (EP 133,988). Sustained- release compositions also include
liposomally entrapped agent, that may be prepared by known methods. See, for example,
DE 3,218,121; Epstein et al., Proc. Natl Acad. Sci. USA 82: 3688- 3692 (1985); Hwang
et al., Proc. Natl. Acad Sci. USA 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese Pat. Application 83-118008; U. S. Pat. Nos.
4,485, 045 and 4,544,545; EP 102,324. The relevant sections of all referenced techniques
are hereby incorporated by reference. Ordinarily, the liposomes are of the small
unilamellar liposomes in about 200 to 800 Angstroms which the lipid content is greater
than about 30 mol.% cholesterol, the selected proportion being adjusted for optimal
In a preferred embodiment, the dry powdered formulation in the invention may not be
inhaled but rather injected as a dry powder, using relatively new injection devices and
methodologies for injecting powders. In this embodiment, the dispersibility and
respirability of the powder is not important, and the particle size may be larger, for
example in about 10 urn, about 20 urn to about 40 um, about 50 um to about 70 um,
about 100 um. The dry powdered formulations in the invention may be reconstituted for
injection as well. As the powder of the invention shows good stability, it may be
reconstituted into liquid form using a diluent and then used in non-pulmonary routes of
administration, e.g. by injection, subcutaneously, intravenously, etc. Known diluents may
be used, including physiological saline, other buffers, salts, as well as non-aqueous
liquids etc. It is also possible to reconstitute the dry powder of the invention and use it to
form liquid aerosols for pulmonary delivery, either for nasal or intrapulmonary
administration or for inhalation. As used herein, the term "treating" refers to therapeutic
and maintenance treatment as well as prophylactic and preventative measures. Those in
need of treatment include those already diagnosed with the disorder as well as those
prone to having the disorder and those where the disorder is to be prevented. Consecutive
treatment or administration refers to treatment on at least a daily basis without
interruption in treatment for one or more days. Intermittent treatment or administration,
or treatment or administration in an intermittent fashion, refers to treatment that is not
consecutive, but rather cyclic in nature. The treatment regime herein may be consecutive
or intermittent or of any other suitable mode. The dry powdered formulation may be
obtained, for example, by sieving, lyophilization, spray-lyophilization, spray drying, and
freeze drying, etc. These methods may be combined for unproved effect. Filters may be
employed for sieving, as will be known to a skilled artisan. The alteration and selection
of the agent's particle size may be conducted in a single step, preferably, by micronizing
under conditions effective to attain the desired particle size as previously described.
The dry powdered formulation may be then stored under controlled conditions of
temperature, humidity, light, pressure etc., so long as the flowability of the agent is
preserved. The agent's stability upon the storing may be measured at a selected
temperature for a selected time period and for rapid screening a matrix of conditions are
run, e.g. at 2-8 °C, 30 °C and sometimes 40 °C, for periods of 2, 4 and 24 weeks. The
length of time and conditions under which a formulation should be stable will depend on
a number of factors, including the above, amount made per batch, storage conditions,
turnover of the product, etc. These tests are usually done at 38% (rh) relative humidity.
Under these conditions, the agent generally loses less than about 30% biological activity
over 18 months, sometimes less than about 20%, or less than about 10%. The dry powder
of the invention loses less than about 50% FPF, in some cases less than about 30%, and
in others less than about 20%.
The dry powder formulation of the invention may be combined with formulation
ingredients, such as bulking agents or carriers, which are used to reduce the concentration
of the agent in the dry powder being delivered to a patient. The addition of these
ingredients to the formulation is not required, however, in some cases it may be desirable
to have larger volumes of material per unit dose. Bulking agents may also be used to
improve the flowability and dispersibility of the powder within a dispersion device, or to
improve the handling characteristics of the powder. This is distinguishable from the use
of bulking agents or carriers during certain particle size reduction processes (e.g. spraying
drying). Suitable bulking agents or excipients are generally crystalline (to avoid water
absorption) and include, but are not limited to, lactose and mannitol. If lactose, is added,
for example, in amounts of about 99: about 1: about 5: active agent to bulking agent to
about 1: 99 being preferred, and from about 5 to about 5: and from about 1: 10 to about 1:
The dry powder formulations of the invention may contain other drugs, e.g.,
combinations of therapeutic agents may be processed together, e.g. spray dried, or they
may be processed separately and then combined, or one component may be spray dried
and the other may not, while it is processed in one of the other manners enabled herein.
The combination of drugs will depend on the disorder for which the drugs are given, as
will be appreciated by those in the art. The dry powder formulation of the invention may
also comprise, as formulation ingredients, excipients, preservatives, detergents,
surfactants, antioxidants, etc, and may be administered by any means that transports the
agent to the airways by any suitable means, but are preferably administered through the
respiratory system as a respirable formulation, more preferably in the form of an aerosol
or spray comprising the agent's particles, and optionally, other therapeutic agents and
formulation ingredients.
In another embodiment, the dry powdered formulations may comprise the dry
pharmaceutical agent of this invention and one or more surfactants. Suitable surfactants
or surfactant components for enhancing the uptake of the active compounds used in the
invention include synthetic and natural as well as full and truncated forms of surfactant
protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant
protein E, di-saturated phosphatidylcholine (other than dipalmitoyl),
dipahnitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol,
phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,
ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyllysophosphatidylcholine,
dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-
phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine,
dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline,
choline, choline phosphate; as well as natural and artificial lamelar bodies which are the
natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic
acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or
propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene,
monomeric-and polymeric-, poly (vinylamine) with dextran and/or alkanoyl side chains,
Brij 35®, Triton X-100®, and synthetic surfactants ALEC®, Exosurfo®, Survan®, and
Atovaquonee®, among others. These surfactants may be used either as single or part of a
multiple component surfactant in a formulation, or as covalently bound additions to the
active compounds.
Examples of other therapeutic agents for use in the present formulation are analgesics
such as Acetaminophen, Anilerdine, Aspirin, Buprenorphine, Butabital, Butorpphanol,
Choline Salicylate, Codeine, Dezocine, Diclofenac, Diflunisal, Dihydrocodeine,
Elcatoninin, Etodolac, Fenoprofen, Hydrocodone, Hydromorphone, Ibuprofen,
Ketoprofen, Ketorolac, Levorphanol, Magnesium Salicylate, Meclofenamate, Mefenamic
Acid, Meperidine, Methadone, Methotrimeprazine, Morphine, Nalbuphine, Naproxen,
Opium, Oxycodone, Oxymorphone, Pentazocine, Phenobarbital, Propoxyphene,
Salsalate, Sodium Salicylate, Tramadol and Narcotic analgesics in addition to those listed
above. See, Mosby's Physician's GenRx.
Anti-anxiety agents are also useful including Alprazolam, Bromazepam, Buspirone,
Chlordiazepoxide, Chlormezanone, Clorazepate, Diazepam, Halazepam, Hydroxyzine,
Ketaszolam, Lorazepam, Meprobamate, Oxazepam and Prazepam, among others. Antianxiety
agents associated with mental depression, such as Chlordiazepoxide,
Amitriptyline, Loxapine Maprotiline and Perphenazine, among others. Anti-inflammatory
agents such as non-rheumatic Aspirin, Choline Salicylate, Diclofenac, Diflunisal,
Etodolac, Fenoprofen, Floctafenine, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen,
Magnesium Salicylate, Meclofenamate, Mefenamic Acid, Nabumetone, Naproxen,
Oxaprozin, Phenylbutazone, Piroxicam, Salsalate, Sodium Salicylate, Sulindac,
Tenoxicam, Tiaprofenic Acid, Tolmetin, anti-inflammatories for ocular treatment such as
Diclofenac, Flurbiprofen, Indomethacin, Ketorolac, Rimexolone (generally for postoperative
treatment), anti-inflammatories for, non-infectious nasal applications such as
Beclomethaxone, Budesonide, Dexamethasone, Flunisolide, Triamcinolone, and the like.
Soporifics (anti-insomnia/sleep inducing agents) such as those utilized for treatment of
insomnia, including Alprazolam, Bromazepam, Diazepam, Diphenhydramine,
Doxylamine, treatments such as Tricyclic Antidepressants, including Amitriptyline HC1
(Elavil), Amitriptyline HC1, Perphenazine (Triavil) and Doxepin HC1 (Sinequan).
Examples of tranquilizers Estazolam, Flurazepam, Halazepam, Ketazolam, Lorazepam,
Nitrazepam, Prazepam Quazepam, Temazepam, Triazolam, Zolpidem and Sopiclone,
among others. Sedatives including Diphenhydramine, Hydroxyzine, Methotrimeprazine,
Promethazine, Propofol, Melatonin, Trimeprazine, and the like.
Sedatives and agents used for treatment of petit mal and tremors, among other conditions,
such as Amitriptyline HC1; Chlordiazepoxide, Amobarbital; Secobarbital, Aprobarbital,
Butabarbital, Ethchiorvynol, Glutethimide, L-Tryptophan, Mephobarbital, MethoHexital
Na, Midazolam HC1, Oxazepam, Pentobarbital Na, Phenobarbital, Secobarbital Na,
Thiamylal Na, and many others. Agents used in the treatment of head trauma (Brain
Injury/Ischemia), such as Enadoline HC1 (e.g. for treatment of severe head injury; orphan
status, Warner Lambert), cytoprotective agents, and agents for the treatment of
menopause, menopausal symptoms (treatment), e.g. Ergotamine, Belladonna Alkaloids
and Phenobarbital, for the treatment of menopausal vasomotor symptoms, e.g. Clonidine,
Conjugated Estrogens and Medroxyprogesterone, Estradiol, Estradiol Cypionate,
Estradiol Valerate, Estrogens, conjugated Estrogens, esterified Estrone, Estropipate, and
Ethinyl Estradiol. Examples of agents for treatment of pre-menstrual syndrome (PMS)
are Progesterone, Progestin, Gonadotrophic Releasing Hormone, Oral contraceptives,
Danazol, Luprolide Acetate, Vitamin B6. Examples of agents for treatment of
emotional/psychiatric, anti-depressants and anti-anxiety agents are Diazepam (Valium),
Lorazepam (Ativan), Alprazolam (Xanax), SSRI's (selective Serotonin reuptake
inhibitors), Fluoxetine HC1 (Prozac), Sertaline HC1 (Zoloft), Paroxetine HC1 (Paxil),
Fluvoxamine Maleate (Luvox), Venlafaxine HC1 (Effexor), Serotonin, Serotonin
Agonists (Fenfluramine), and other over the counter (OTC) medications.
Such combination therapeutic formulations can be manufactured using many
conventional techniques. It may be necessary to micronize the active compounds and if
appropriate (i.e. where an ordered mixture is not intended) any carrier in a suitable mill,
for example in a jet mill at some point in the process, in order to produce primary
particles in a size range appropriate for maximal deposition in the lower respiratory tract
(i.e., from about 0.1 um to about 10 um). For example, one can dry mix DHEA and
carrier, where appropriate, and then micronize the substances together; alternatively, the
substances can be micronized separately, and then mixed. Where the compounds to be
mixed have different physical properties such as hardness and brittleness, resistance to
micronization varies and they may require different pressures to be broken down to
suitable particle sizes. When micronized together, therefore, the obtained particle size of
one of the components may be unsatisfactory. In such case it would be advantageous to
first micronize the different components separately and then mix them.
It is also possible first to dissolve the active component including, where an ordered
mixture is not intended, any carrier in a suitable solvent, e. g. water, to obtain mixing on
the molecular level. This procedure also makes it possible to adjust the pH-value to a
desired level. The pharmaceutically accepted limits of pH 3.0 to 8.5 for inhalation
products must be taken into account, since products with a pH outside these limits may
induce irritation and constriction of the airways. To obtain a powder, the solvent must be
removed by a process which retains the biological activity of DHEA. Suitable drying
methods include vacuum concentration, open drying, spray drying, freeze drying and use
of supercritical fluids. Temperatures over 50°C for more than a few minutes should
generally be avoided, as some degradation of the DHEA may occur. After drying step the
solid material can, if necessary, be ground to obtain a coarse powder, and then, if
necessary, micronized.
If desired, the micronized powder can be processed to improve the flow properties, e.g.,
by dry granulation to form spherical agglomerates with superior handling characteristics,
before it is incorporated into the intended inhaler device. In such a case, the device would
be configured to ensure that the agglomerates are substantially deagglomerated prior to
exiting the device, so that the particles entering the respiratory tract of the patient are
largely within the desired size range. Where an ordered mixture is desired, the active
compound may be processed, for example by micronization, in order to obtain, if desired,
particles within a particular size range. The carrier may also be processed, for example to
obtain a desired size and desirable surface properties, such as a particular surface to
weight ratio, or a certain texture, and to ensure optimal adhesion forces in the ordered
mixture. Such physical requirements of an ordered mixture are well known, as are the
various means of obtaining an ordered mixture which fulfils the said requirements, and
may be determined easily by one skilled in the art.
The dry powder formulation of this invention may be administered into the respiratory
tract as a formulation of respirable size particles i.e. particles of a size sufficiently small
to pass through the nose, mouth, larynx or lungs upon inhalation, nasal administration or
lung instillation, to the bronchi and alveoli of the lungs, hi general, respirable particles
range from about 0.1 urn to about 100 urn, and inhalable particles are about 0.1 urn to
about 10 um, to about 5 urn in size. Mostly, when inhaled, particles of non-respirable
size that are included in the aerosol tend to deposit in the throat and be swallowed, which
reduces the quantity of non- respirable particles in the aerosol. For nasal administration, a
particle size in the range of about 10 um to about 20 um, about 50 um, about 60 urn, or
about 100 um, is preferred to ensure retention in the nasal cavity.
The size and shape of the particles may be analyzed using known techniques for
determine and ensure proper particle morphology. For example, one skilled in the art can
visually inspect the particles under a microscope and/or determine particle size by
passing them through a mesh screen. Preferred techniques for visualization of particles
include scanning electron microscopy (SEM) and transmission electron microscopy
(TEM). Particle size analysis may take place using laser diffraction methods.
Commercially available systems for carrying out particle size analysis by laser diffraction
are available from Clausthal-Zellerfeld, Germany (HELOS HI006).
The dry powdered formulation of the invention may be delivered with any device that
generates solid particulate aerosols, such as aerosol or spray generators. These devices
produce respirable particles, as explained above, and generate a volume of aerosol or
spray containing a predetermined metered dose of a medicament at a rate suitable for
human or animal administration. One illustrative type of solid particulate aerosol or spray
generator is an insufflator, which are suitable for administration of finely comminuted
powders. The latter may be taken also into the nasal cavity in the manner of a snuff. In
the insufflator, the powder, e.g. a metered dose of the agent effective to carry out the
treatments described herein, is contained in a capsule or a cartridge. These capsules or
cartridges are typically made of gelatin, foil or plastic, and may be pierced or opened in
situ, and the powder delivered by air drawn through the device upon inhalation or by
means of a manually-operated pump. The dry powder formulation employed in the
insufflator may consist either solely of the agent or of a powder blend comprising the
agent, and the agent typically comprises from 0.01 to 100 % w/w of the formulation. The
dry powdered formulation generally contains the active compound in an amount of about
0.01% w/w, about 1% w/w/, about 5% w/w, to about 20%, w/w, about 40% w/w, about
99.99% w/w. Other ingredients, and other amounts of the agent, however, are also
suitable within the confines of this invention.
In a preferred embodiment, the dry powdered formulation is delivered by a nebulizer.
This is means is especially useful for patients or subjects who are unable to inhale or
respire the powder pharmaceutical composition under their own efforts. In serious cases,
the patients or subjects are kept alive through artificial respirator. The nebulizer can use
any pharmaceutically or veterinarily acceptable carrier, such as a weak saline solution.
Preferably, the weak saline solution is less than about 1.0 or 0.5% NaCl. More
preferably, the weak saline solution is less than about 0.2% or 0.15% NaCl. Even more
preferably, the weak saline solution is less than about 0.12% NaCl. The nebulizer is the
means by which the powder pharmaceutical composition is delivered to the target of the
patients or subjects in the airways. The stability of anhydrous compounds, such as
anhydrous DHEA-S, can be maintained or increased by eliminating or reducing the water
content within the sealed container, e.g. vial, containing the compound. Preferably,
besides the compound, it is a vacuum within the sealed container.
The formulation of the invention is also provided in various forms that are tailored for
different methods of administration and routes of delivery. The formulations that are
contemplated are, for example, a transdermal formulation also containing an excipient
and other agents suitable for delivery through the skin, mouth, nose, vagina, anus, eyes,
and other body cavities, intradermally, as a sustained release formulation, intrathecally,
intravascularly, by inhalation, nasally, intrapulmonarily, into an organ, by implantation,
by suppositories, as creams, gels, and the like, all known in the art. In one embodiment,
the dry powdered formulation comprises a respirable formulation, such as an aerosol or
spray. The dry powder formulation of the invention is provided in bulk, and in unit form,
as well as in the form of an implant, a capsule, blister or cartridge, which may be
openable or piercable as is known in the art. A kit is also provided, that comprises a
delivery device, and in separate containers, the dry powdered formulation of the
invention, and optionally other excipient and therapeutic agents, and instructions for the
use of the kit components.
In one preferred embodiment, the agent is delivered using suspension metered dose
inhalation (MDI) formulation. Such a MDI formulation can be delivered using a delivery
device using a propellant such as hydrofluroaJkane (HFA). Preferably, the HFA
propellants contain 100 parts per million (PPM) or less of water. N. C. Miller (In :
Respiratory Drug Delivery, P. R. Bryon (ed.), CRC Press, Boca Raton, 1990, pp. 249-
257) reviewed the effect of water content on crystal growth in MDI suspensions. When
exposed to water, anhydrous DHEA-S will hydrate and eventually form large particles.
This hydration process can happen in a suspension of the anhydrous DHEA-S in an HFA
propellant which has a water content. This hydration process would accelerate the crystal
growth due to the formation of strong interparticle bonds and cause the formation of large
particles. In contrast, the dihydrate form is already hydrated thus more stable, and thus
more preferred, than the anhydrous form in a MDI, as the dihydrate form will not further
form larger particles. If DHEA-S forms a solvate with a HFA propellant that has a lower
energy than the dihydrate form, then this DHEA-S solvate would be the most stable, and
hence more preferred, form for an MDI.
In one preferred embodiment, the delivery device comprises a dry powder inhalator (DPI)
that delivers single or multiple doses of the formulation. The single dose inhalator may be
provided as a disposable kit which is sterilely preloaded with enough formulation for one
application. The inhalator may be provided as a pressurized inhalator, and the
formulation in a piercable or openable capsule or cartridge. The kit may optionally also
comprise in a separate container an agent such as other therapeutic compounds,
excipients, surfactants (intended as therapeutic agents as well as formulation ingredients),
antioxidants, flavoring and coloring agents, fillers, volatile oils, buffering agents,
dispersants, surfactants, antioxidants, flavoring agents, bulking agents, propellants and
preservatives, among other suitable additives for the different formulations. The dry
powdered formulation of this invention may be utilized by itself or in the form of a
composition or various formulations in the treatment and/or prevention of a disease or
condition associated with bronchoconstriction, allergy(ies), lung cancer and/or
inflammation. Examples of diseases are airway inflammation, allergy(ies), asthma,
impeded respiration, CF, COPD, AR, ARDS, pulmonary hypertension, lung
inflammation, bronchitis, airway obstruction, bronchoconstriction, microbial infection,
viral infection (such as SARS), among others. Clearly the present formulation may be
administered for treating any disease that afflicts a subject, with the above just being
examples. Typically, the dry powdered formulation may be administered in an amount
effective for the agent to reduce or improve the symptom of the disease or condition.
The dry powdered formulation may be administered directly to the lung(s), preferably as
a respirable powder, aerosol or spray. Although an artisan will know how to titrate the
amount of dry powdered formulation to be administered by the weight of the subject
being treated in accordance with the teachings of this patent, the agent is preferably
administered in an amount effective to attain an intracellular concentration of about 0.05
to about 10 uM agent, and more preferably up to about 5 uM. Propellants may be
employed under pressure, and they may also carry co-solvents. The dry powdered
formulation of the invention may be delivered in one of many ways, including a
Tansdermal or systemic route, orally, intracavitarily, intranasally, intraanally,
ntravaginally, transdermally, intrabucally, intravenously, subcutaneously,
intramuscularly ; intratumorously, into a gland, by implantation, intradermally, and many
others, including as an implant, slow release, transdermal release, sustained release
formulation and coated with one or more macromolecules to avoid destruction of the
agent prior to reaching the target tissue. Subject that may be treated by this agent include
humans and other animals in general, and in particular vertebrates, and amongst these
mammals, and more specifically and small and large, wild and domesticated, marine and
farm animals, and preferably humans and domesticated and farm animals and pets.
The following examples serve to more fully describe the manner of using the abovedescribed
invention, as well as to set forth the best modes contemplated for carrying out
various aspects of the invention. It is understood that these examples in no way serve to
limit the true scope of this invention, but rather are presented for illustrative purposes.
The relevant portions of all references cited herein are incorporated by reference in their
entirety. In these examples, uM means micromolar, mM means millimolar, ml means
milliliters, um or micron means micrometers, mm means millimeters, cm means
centimeters, °C means degrees Celsius, jig means micrograms, mg means milligrams, g
means grams, kg means kilograms, M means molar, and h means hours.
Airjet Milling of Anhydrous DHEA-S & Determination of Respirable Dose
DHEA-S is evaluated as a once-per-day asthma therapy alternative to inhaled
corticosteroid treatment that is not expected to share the safety concerns associated with
that class. The solid-state stability of DHEA-S, sodium dehydroepiandrostenone sulfate
(NaDHEA-S) or sodium prasterone sulfate, has been studied for both bulk and milled
material (Nakagawa, H., Yoshiteru, T., and Fujimoto, Y.(1981) Claim. Pharm. Bull. 29
(5) 1466-1469; Nakagawa, H., Yoshiteru, T., and Sugimoto, I. (1982) Chem. Pharm.
Bull 30 (1) 242-248). DHEA-S is most stable and crystalline as the dihydrate form. The
DHEA-S anhydrous form has low crystallinity and is very hygroscopic. The DHEA-S
anhydrous form is stable as long as it picks up no water on storage. Keeping a partially
crystalline material free of moisture requires specialized manufacturing and packing
technology. For a robust product, minimizing sensitivity to moisture is essential during
the development process.
(1) Micronization of DHEA-S
Anhydrous DHEA sulfate was micronized using a jet milling (Jet-O-Mizer Series #00,
100-120 PSI nitrogen). Approximately 1 g sample was passed through the jet mill, once,
and approximately 2 g sample were passed through the jet mill twice. The particles from
each milling run were suspended in hexane, in which DHEA-S was insoluble and Spa85
surfactant added to prevent agglomeration. The resulting solution was sonicated for 3
minutes and appeared fully dispersed. The dispersed solutions were tested on a Malvem
Mastersizer X with a small volume sampler (S VS) attachment. One sample of dispersed
material was tested 5 times. The median particle size or D(v,0.5) of unmilled material
was 52.56 um and the % RSD (relative standard deviation) was 7.61 for the 5 values. The
D(v,0.5) for a single pass through the jet mill was 3. 90 um and the % RSD was 1.27, and
the D(v, 0.5) from a double pass through the jet mill 3.25 um and the % RSD was 3.10.
This demonstrates that DHEA-S can be jet milled to particles of size suitable for
(2) HPLC Analysis
Two vials (A; single-pass; 150 mg) and (B double-pass; 600 mg) of the micronized drug
were available for determining drug degradation during jet milling micronization.
Weighed aliquots of DHEA-S from vials A and B were compared to a standard solution
of unmilled DHEA-S (10 mg/ml) in an acetonitrile-water solution (1:1). The
chromatographic peak area for the HPLC assay of the unmilled drug standard solution
(10 mg/ml) gave a value of 23,427. Weighed aliquots of micronized DHEA-S form vials
A and B, (5 mg/ml) was prepared in an acetonitrile-water solution (1:1). The
chromatographic peak areas for vials A and B were 11,979 and 11, 677, respectively.
Clearly, there was no detectable degradation of the drug during the jet milling
micronization process.
(3) Emitted Dose Studies
DHEA-S powder was collected in Nephele tubes and assayed by HPLC. Triplicate
experiments were performed at each airflow rate for each of the three dry powder inhalers
tested (Rotahaler, Diskhaler and IDL's DPI devices). A Nephele tube was fitted at one
end with a glass filter (Gelman Sciences, Type A/E, 25 um), which in turn was connected
to the airflow line to collect the emitted dose of the drug from the respective dry powder
inhaler being tested. A silicone adapter, with an opening to receive the mouthpiece of the
respective dry powder inhaler being tested at the other end of the Nephele tube was
secured. A desired airflow, of 30,60, or 90 L/min, was achieved through the Nephele
tube. Each dry powder inhaler's mouthpiece was inserted then into the silicone rubber
adapter, and the airflow was continued for about four sees after which the tube was
removed and an end-cap screwed onto the end of each tube. The end- cap of the tube not
containing the filter was removed and 10 ml of an HPLC grade water- acetonitrile
solution (1:1) added to the tube, the end-cap reattached, and the tube shaken for 1-2
minutes. The end-cap then was removed from the tube and the solution was transferred to
a 10 ml plastic syringe fitted with a filter (Cameo 13N Syringe Filter, Nylon, 0. 22um).
An aliquot of the solution was directly filtered into an HPLC vial for later drug assay via
HPLC. The emitted dose experiments were performed with micronized DHEA-S (about
12.5 or 25 mg) being placed in either a gelatin capsule (Rotahaler) or a Ventodisk blister
(Diskhaler and single-dose DPI (IDL)). When the micronized DHEA-S (only vial B
used), was weighed for placement into the gelatin capsule or blister, there appeared to be
a few aggregates of the micronized powder. The results of the emitted dose tests
conducted at an airflow rate of 30,60 and 87.8 L/min are displayed in Tables 1 through 4.
Table 1 contains the results for Rotahaler experiments at 3 different flow rates. Table 2
contains the results for Diskhaler experiments at 3 different flow rates, and Table 3
contains the results of multi-dose experiments at 3 different flow rates. Table 4
summarizes the results of the experiments.
Table 1. Emitted Dose with Rotahaler
(Table Removed)
(4) Respirable Dose Studies
The respirable dose (respirable fraction) studies were performed using a standard sampler
cascade impactor (Andersen), consisting of an inlet cone (an impactor pre-separator was
substituted here), 9 stages, 8 collection plates, and a backup filter within 8 aluminum
stages held together by 3 spring clamps and gasket O-ring seals, where each impactor
stage contains multiple precision drilled orifices. When air is drawn through the sampler,
multiple jets of air in each stage direct any airborne particles toward the surface of the
collection plate for that stage. The size of the jets is constant for each stage, but is smaller
in each succeeding stage. Whether a particle is impacted on any given stage depends
upon its aerodynamic diameter. The range of particle sizes collected on each stage
depends upon on the jet velocity of the stage, and the cut- off point of the previous stage.
Any particle not collected on the first stage follows the air stream around the edge of the
plate to the next stage, where it is either impacted or passed on to the succeeding stage,
and so on, until the velocity of the jet is sufficient for impaction. To prevent particle
bounce during the cascade impactor test, the individual impactor plates were coated with
a hexane-grease (high vacuum) solution (100:1 ratio). As noted above, the particle size
cut-off points on the impactor plates changed at different airflow rates. For example,
Stage 2 corresponds to a cut-off value greater than 6.2 um particles at 60 L/min, and
greater than 5. Sum particles at 30 L/min, and stage 3 had a particle size cut-off value at
90 L/min greater than 5.6um. Thus, similar cut-off particle values are preferentially
employed at comparable airflow rates, i.e. ranging from 5.6 to 6.2um. The set-up
recommended by the United States Phamacopeia for testing dry powder inhalers consists
of a mouthpiece adapter (silicone hi this case) attached to a glass throat (modified 50 ml
round-bottom flask) and a glass distal pharynx (induction port) leading top the preseparator
and Andersen sampler. The pre-separator sample includes washings from the
mouthpiece adaptor, glass throat, distal glass pharynx and pre-separator. 5 ml acetonitrile:
water (1:1 ratio) solvent was placed in the pre-separator before performing the cascade
impactor experiment, that were performed in duplicate with 3 different dry powder
inhaler devices and at 3 airflow rates, 30,60 and 90 L/min. The drug collected on the
cascade impactor plates were assayed by the HPLC, and a drug mass balance was
performed for each Diskhaler and multi-dose cascade impactor experiment consisting of
determining the amount of drug left in the blister, the amount of drug remaining in the
device (Diskhaler only), the non- respirable amount of the dose retained on the silicone
rubber mouth piece adaptor, glass throat, glass distal pharynx and pre-separator, all
combined into one sample, and the respirable dose, i.e. Stage 2 through filter impactor
plates for airflow rates of 30 and 60 L/min and Stages 1 through filter impactor plates for
90 L/min experiments.
Table 5. Cascade Impactor Experiments (90L/min)
(Table Removed)
*a: Multi-dose device was not washed; as solvents would attack SLA components. Multidose
device retention percentage is obtained by difference.
*b: oven dried drug for 80 minutes
*c: oven dried drug for 20 hours
The following conclusions are derived from the emitted dose and cascade impactor
experiments. The low respirable dose values achieved in the cascade impactor
experiments were due to agglomerated drug particles, which could not be separated, even
at the highest airflow rate tested. It is our opinion that agglomeration of the drug particles
is a consequence of static charge build up during the mechanical milling process used for
particles size reduction and that this situation is further compounded by subsequent
moisture absorption of the particles. A micronization method that produces less static
charge or a less hygroscopic, fully hydrated crystalline form of DHEA-S (i.e. dihydrate
form) should provide a freer flowing powder with diminished potential for
Spray Drying of Anhydrous DHEA Sulfate & Determination of Respirable Dose
(1) Micronization of the Drug
1.5 g of anhydrous DHEA sulfate were dissolved to 100 ml of 50% ethanol: water to
produce a 1.5 % solution. The solution was spray-dried with a B-191 Mini Spray-Drier
(Buchi, Flawil, Switzerland) with an inlet temperature of 55°C, outlet temperature of
40°C, at 100% aspirator, at 10% pump, nitrogen flow at 40 mbar and spray flow at 600
units. The spray-dried product was suspended in hexane and Span85 surfactant added to
reduce agglomeration. The dispersions were sonicated with cooling for 3-5 minutes for
complete dispersion and the dispersed solutions tested on a Malvern Mastersizer X with a
Small Volume Sampler (SVS) attachment.
The two batches of spray dried material were found to have mean particle sizes of 5.07
±0.70 urn and 6.66 ±0.9lum. Visual examination by light microscope of the dispersions
of each batch confirmed that spray drying produced small respirable size particles. The
mean particle size was 2.4 jam and 2.0 um for each batch, respectively. This demonstrates
that DHEA-S can be spray dried to a particle size suitable for inhalation.
(2) Respirable Dose Studies
The cascade impactor experiments were conducted as described in Example 1. Four
cascade impactor experiments were done, three with a IDL multi-dose device and one
with a Diskhaler, all at 90 L/min. The results of the cascade impactor experiments are
presented in Table 6 below.
Table 6: Cascade Impactor Results with Spray-Dried Drug Product
(Table Removed)
The spray-dried anhydrous material in these experiments produced a two-fold increase in
the respirable dose compared to micronized anhydrous DHEA-S. While it does appear
that increased respirable doses were obtained with spray drying as compared to jetmilling,
the % respirable dose was still low. This was due to agglomeration likely the
result of moisture absorption of the anhydrous form.

Air Jet Milling of DHEA-S Dihydrate (DHEA-S-2H20) & Determination of
Respirable Dose
(1) Recrystallization of DHEA-S dihydrate. Anhydrous DHEA-S is dissolved in a
boiling mixture of 90% ethanol/water. This solution is rapidly chilled in a dry
ice/methanol bath to recrystallize the DHEA-S. The crystals are filtered, washed twice
with cold ethanol, than placed in a vacuum desiccator at room temperature for a total of
36h. During the drying process, the material is periodically mixed with a spatula to break
large agglomerates. After drying, the material is passed through a 500 (urn sieve.
(2) Micronization and physiochecmical testing. DHEA-S dihydrate is micronized with
nitrogen gas in a jet mill at a venturi pressure of 40 PSI, a mill pressure of 80 PSI, feed
setting of 25 and a product feed rate of about 120 to 175 g/hour. Surface area is
determined using five point BET analyses are performed with nitrogen as the adsorbing
gas (P/Po = 0.05 to 0.30) using a Micromeritics TriStar surface area analyzer. Particle size
distributions are measured by laser diffraction using a Micromeritics Saturn Digisizer
where the particles are suspended in mineral oil with sodium dioctyl sodium
sulfosuccinate as a dispersing agent. Drug substance water content is measured by Karl
Fischer titration (Schott Titroline KF). Pure water is used as the standard and all relative
standard deviations for triplicates are less than 1%. Powder is added directly to the
titration media. The physicochemical properties of DHEA-S-dihydrate before and after
micronization are summarized in Table 7.
Table 7. Physicochemical properties of DHEA-S-dihydrate before and
after micronization.
(Table Removed)
The only significant change measured is in the particle size. There is no significant loss
of water or increase in impurities. The surface area of the micronized material is in
agreement with an irregularly shaped particle having a median size of 3 to 4 microns. The
micronization successfully reduces the particle size to a range suitable for inhalation with
no measured changes in the solid-state chemistry.
(3) Aerosolization of DHEA-S'dihydrate. The single-dose Acu-Breathe device is used
for evaluating DHEA-S-dihydrate. Approximately 10 mg of neat DHEA-S-dihydrate
powder is filled and sealed into foil blisters. These blisters are actuated into the Andersen
8-stage cascade impactor at flow rates ranging from 30 to 75 L/min with a glass twinimpinger
throat Stages 1-5 of the Andersen impactor are rinsed together to obtain an
estimate of the fine particle fraction. Pooling the drug collected from multiple stages into
one assay make the method much more sensitive. The results for this series of
experiments is shown in Figure 1.
At all flow rates, the dihydrate yields a higher fine particle fraction than the virtually
anhydrous material. Since the dihydrate powder is aerosolized using the single-dose
inhaler, it is very reasonable to conclude that its aerosol properties are significantly better
than the virtually anhydrous material. Higher crystallinity and stable moisture content are
the most likely factors contributing the dihydrate's superior aerosol properties. This
unique feature of DHEA- S-dihydrate has not been reported in any previous literature.
While the improvement in DHEA-S's aerosol performance with the dihydrate form is
significant, neat drug substance may not be the optimal formulation. Using a carrier with
a larger particle size typically improves the aerosol properties of micronized drug
Anhydrous DHEA-S and DHEA-S Dihydrate Stability with and without Lactose
The initial purity (Time-0) was determined for anhydrous DHEA and for DHEA-S
dihydrate by high pressure liquid chromatography (HPLC). Both forms of DHEA-S were
then either blended with lactose at a ratio of 50:50, or used as a neat powder and placed
in open glass vials, and held at SOT for up to 4 weeks. These conditions were used to
stress the formulation in order to predict its long-term stability results. Control vials
containing only DHEA-S (anhydrous or dihydrate) were sealed and held 25°C for up to 4
weeks. Samples were taken and analyzed by HPLC also at 0,1,2, and 4 weeks to
determine the amount of degradation, as determined by formation of DHEA.
After one week, virtually anhydrous DHEA-S blended with lactose (50% w/w,
nominally) stored at 50°C in sealed glass vials acquires a brown tinge that is darker for
the lactose blend. This color change is accompanied by a significant change in the
chromatogram as shown in Figure 1. The primary degradant is dehydroepiandrosterone or
DHEA. Qualitatively from Figure 2, the amount of DHEA in the blend is higher than the
other two samples. To quantitatively estimate the % DHEA in the samples, the area for
the DHEA peak is divided by the total area for the DHEA-S and DHEA peaks (see Table
8 for results). The higher rate of decomposition for the blend indicates a specific
interaction between lactose and the virtually anhydrous DHEA-S. In parallel with the
increase in DHEA, the brown color of the powders on accelerated storage increased over
time. The materials on accelerated storage become more cohesive with time as evidenced
by clumping during sample weighing for chemical analysis. Based on these results, it is
not possible to formulate virtually anhydrous DHEA-S with lactose. This is a
considerable disadvantage since lactose is the most commonly used inhalation excipient
for dry powder formulations. Continuing with the virtually anhydrous form would mean
limiting formulations to neat powder or undertaking more comprehensive safety studies
to use a novel excipient.
Table 8: DHEA % formed from Anhydrous DHEA-S at 50°C
(Table Removed)
In contrast to Figure 2, there is virtually no DHEA generated after storage for 1 week at
50 °C (see Figure 3). Furthermore, the materials show no change in color. The moisture
content of DHEA-S-dihydrate remains virtually unchanged after one week at 50°C. The
water content after accelerated storage is 8.66% versus a starting value of 8.8%. The %
DHEA measured during the course of this stability program is shown in Table 9.
Table 9: Percent DHEA formed from DHEA-S Dihydrate at 50°C
(Table Removed)
By comparing Figures 1 and 2 and Tables 8 and 9, one can see that the dihydrate form of
DHEA-S is the more stable form for progression into further studies. The superior
compatibility of DHEA-S-dihydrate with lactose over that of the virtually anhydrous
material has not been reported in the patent or research literature. The solubility of this
substance is reported in the next section as a portion of the development work for a
nebulizer solution.
DHEA-S Dihydrate/Lacotse blends, Determination of Respirable Dose & Stability
(1) DHEA-S Dihydrate/Lactose blend. Equal weights of DHEA-S and inhalation grade
lactose (Foremost Aero Flo 95) are mixed by hand then passed through a 500 urn screen
to prepare a pre- blend. The pre-blend is then placed in a BelArt Micro-Mill with the
remaining lactose to yield a 10% w/w blend of DHEA-S. The blender is wired to a

variable voltage source to regulate the impeller speed. The blender voltage is cycled
through 30%, 40%, 45% and 30% of full voltage for 1, 3, 1.5, and 1.5 minutes,
respectively. The content uniformity of the blend was determined by HPLC analysis.
Table 10 shows the result of content uniformity samples for this blend. The target value is
10% w/w DHEA-S. The blend content is satisfactory for proximity to the target value and
content uniformity.
Table 10. Content uniformity for a blend of DHEA-S-dihydrate with lactose.
(Table Removed)
(2) Aerosolization of DHEA-S-dihydrate/Lactose blend. Approximately 25 mg of this
powder is filled and sealed in foil blisters and aerosolized using the single-dose device at
60 L/min. Two blisters are used for each test and the results for fine particle fraction
(material on stages 1-5) are shown in Table 11.
Table 11. Fine particle fraction for lactose blend in two different experiments
(Table Removed)
The aerosol results for this preliminary powder blend are satisfactory for a respiratory
drug delivery system. Higher fine particle fractions are possible with optimization of the
powder blend and blister/device configuration. The entire particle size distribution of Test
2 is shown in Table 12.
Table 12. Particle size distribution of aerosolized DHEA-S dihydrate/Lactose Blend
(Table Removed)
This median diameter for DHEA-S for this aerosol is ~2. 5 um. This diameter is smaller
than the median diameter measured for micronized DHEA-S-dihydrate by laser
diffraction. Irregularly shaped particles can behave aerodynamically as smaller particles
since their longest dimension tends to align with the air flow field. Therefore, it is
common to see a difference between the two methods. Diffraction measurements are a
quality control test for the input material while cascade impaction is a quality control test
for the finished product.
(3) Stability of DHEA-S Dihydrate/Lactose Blend. This lactose formulation is also
placed on an accelerated stability program at 50°C. The results for DHEA-S content are
in Table 13. The control is the blend stored at room temperature.
Table 13. Stressed stability data on DHEA-S-dihydrate/lactose blend at 50°C.
(Table Removed)
There is no trend in the DHEA-S content over time for either condition and all the results
are within the range of samples collected for content uniformity testing (see Table 13).
Furthermore, there are no color changes or irregularities observed in the chromatograms.
The blend appears to be chemically stable.
Nebulizer Formulation of DHEA-S
Solubility of DHEA-S. An excess of DHEA-S dihydrate, prepared according to
"Recrystallization of DHEA-S-Dihydrate (Example 4)", is added to the solvent medium
and allowed to equilibrate for at least 14 hours with some periodic shaking. The
suspensions are then filtered through a 0.2 micron syringe filter and immediately diluted
for HPLC analysis. To prepare refrigerated samples, the syringes and filters are stored in
the refrigerator for at least one hour before use.
Inhalation of pure water can produce a cough stimulus. Therefore, it is important to add
halide ions to a nebulizer formulation with NaCl being the most commonly used salt.
Since DHEA-S is a sodium salt, NaCl could decrease solubility due to the common ion
effect. The solubility of DHEA-S at room temperature (24-26 °C) and refrigerated (7-8
°C) as a function of NaCl concentration is shown in Figure 4.
DHEA-S's solubility decrease with NAC1 concentration. Lowering the storage
temperature decrease the solubility at all NAC1 concentrations. The temperature effect is
weaker at high NAC1 concentrations. For triplicates, the solubility at -25 °C and 0%
NAC1 range from 16.5-17.4 mg/mL with a relative standard deviation of 2.7%. At 0.9%
NAC1 refrigerated, the range for triplicates is 1.1-1.3 mg/mL with a relative standard
deviation of 8.3%.
The equilibrium between DHEA-S in the solid and solution states is:
NaDHEA-Ssolid *->DHEA-S'+ Na+
K= [DHEA-S*] [Na*]/[NaDHEA-Sjso,id
Since the concentration of DHEA-S in the solid is constant (i.e., physically stable
dihydrate), the equilibrium expression is simplified:
Ksp = [DHEA-Sl [Na+]
Based on this presumption, a plot of DHEA-S solubility versus the reciprocal of the total
sodium cation concentration is linear with a slope equal to Ksp. This is shown in Figures
5 and 6 for equilibrium at room temperature and refrigerated, respectively.
Based on the correlation coefficients, the model is a reasonable fit to the data at both
room and refrigerated temperatures where the equilibrium constants were 2236 and 665
mM2, respectively. To maximize solubility, the NAC1 level needs to be as low as
possible. The minimum halide ion content for a nebulizer solution should be 20 mM or
To estimate a DHEA-S concentration for the solution, a 10°C temperature drop in the
nebulizer during use is assumed (i.e., 15 °C). Interpolating between the equilibrium
constants versus the reciprocal of absolute temperature, the Ksp at 15 °C would be ~
1316 mM2. Each mole of DHEA-S contributes a mole of sodium cation to the solution,
Ksp = [DHEA-S'][Na+] = [DHEA-S'] [Na+ + DHEA-S*]
= [DHEA-Sl2 + [Na^fDHEA-Sl
which is solve for [DHEA-S"] using the quadratic formula. The solution for 20 mM Na+
with a Ksp of 1316 mM2 is 27.5 mM DHEA-S' or 10.7 mg/mL. Therefore a 10 mg/mL
DHEA-S solution in 0.12% NaCl is selected as a good candidate formulation to progress
into additional testing. The estimate for this formula does not account for any
concentration effects due to water evaporation from the nebulizer.
The pH of a 10 mg/mL DHEA-S solution with 0.12% NACl range from 4.7 to 5.6. While
this would be an acceptable pH level for an inhalation formulation, the effect of using a
20 mM phosphate buffer is evaluated. The solubility results at room temperature for
buffered and unbuffered solutions are shown in Figure 7.
The presence of buffer in the formulation suppress the solubility, especially at low NACl
levels. As shown in Figure 8, the solublity data for the buffered solution falls on the same
equilibrium line as for the unbuffered solution. The decrease in solubility with the buffer
is due to the additional sodium cation content.
Maximizing solubility is an important goal and buffering the formulation reduces
solubility. Furthermore, Ishihora and Sugimoto ((1979) Drug Dev. Indust. Pharm. 5(3)
263-275) did not show a significant improvement in NaDHEA-S stability at neutral pH.
Stability Studies. A 10 mg/mL DHEA-S formulation is prepared in 0.12% NACl for a
short- term solution stability program. Aliquots of this solution are filled into clear glass
vials and stored at room temperature (24-26 °C) and at 40 °C. The samples are checked
daily for DHEA-S content, DHEA content, and appearance. For each time point,
duplicate samples are withdrawn and diluted from each vial. The DHEA-S content over
the length of this study is shown in Figures 9 and 10.
At the accelerated condition, the solution show a faster decomposition rate and became
cloudy after two days of storage. The solution stored at room temperature is more stable
and a slight precipitate is observed on the third day. The study is stopped on day three.
DHEA-S decomposition is accompanied by an increase in DHEA content as shown in
Figure 10.
Since DHEA is insoluble in water, it only takes a small quantity in the formulation to
create a cloudy solution (accelerated storage) or a crystalline precipitate (room storage).
This explains why earlier visual evaluations of DHEA-S solubility severely
underestimate the compound's solubility: small quantities of DHEA would lead the
experimenter to conclude the solubility limit of DHEA-S had been exceeded. While this
is not a promising commercial formulation, the solution should easily be stable for the
day of reconstitution in a clinical trial. The following section describes the aerosol
properties of this formulation.
Nebulizer Studies. DHEA-S solutions are nebulized using a Pari ProNeb Ultra
compressor and LC Plus nebulizer. The schematic for the experiment set-up is shown in
Figure 11. The nebulizer is filled with 5 mL of solution and nebulization is continued
until the output became visually insignificant (4l/2 to 5 min.). Nebulizer solutions are
tested using a California Instruments AS-6 6-stage impactor with a USP throat. The
impactor is run at 30 L/min for 8 seconds to collect a sample following one minute of
nebulization time. At all other times during the experiment, the aerosol is drawn through
the by-pass collector at approximately 33 L/min. The collection apparatus, nebulizer, and
impactor are rinsed with mobile phase and assayed by HPLC. 5 mL of DHEA-S in 0.12%
NaCl is used in the nebulizer. This volume is selected as the practical upper limit for use
in a clinical study. The results for the first 5 nebulization experiments are shown below:
Table 14. Results for nebulization studies with DHEA-S
(Table Removed)
* Only assayed liquid poured from nebulizer; did not weigh before and after
aerosolization or rinse entire unit
Nebulizer #1 runs to dryness in about 5 minutes while Nebulizer #2 takes slightly less
than 4.5 minutes. In each case, the liquid volume remaining in the nebulizer is
approximately 2 mL. This liquid is cloudy initially after removal from the nebulizer then

clears within 3-5 minutes. Even after this time, the 10 mg/mL solutions appear to have a
small amount of coarse precipitate in them. Fine air bubbles in the liquid appear to cause
the initial cloudiness. DHEA-S appears to be surface active (i.e., promoting foam) and
this stabilizes air bubbles within the liquid. The precipitate in 10 mg/mL solutions
indicates that the drug substance's solubility is exceeded in the nebulizer environment.
Therefore, the additional nebulization experiments in Table 15 are run at lower
Table 15 presents additional data of "dose" linearity versus solution concentration.
Table 15. Results from additional nebulizer experiments with DHEA-S.
(Table Removed)
Nebulizer #3 takes slightly less than 4.5 minutes to reach dryness. The mass in the bypass
collector is plotted versus the initial solution concentration in Figure 12.
Semi-quantitatively, there is good linearity from 0 to 7.5 mg/mL then the amount
collected appears to start leveling-off. While the solubility reduction by cooling is
included in the calculation of the 10 mg/mL solution, any concentration effects on drug
and NaCl content were neglected. Therefore, it is possible for a precipitate to form via
supersaturation of the nebulizer liquid. The data in Figure 12 and the observation of some
particulates in the 10 mg/mL solution following nebulization indicate that the highest
solution concentration for a proof of concept clinical trial formulation is approximately
7.5 mg/mL.
An aerosol sample is drawn into a cascade impactor for particle size analysis. There is no
detectable trend in particle size distribution with solution concentration or nebulizer
number. The average particle size distribution for all nebulization experiments is shown
in Figure 13. The aerosol particle size measurements are in agreement with
published/advertised results for this nebulizer (i.e., median diameter ~2um).
While the in vitro experiments demonstrate that a nebulizer formulation can deliver
respirable DHEA-S aerosols, the formulation is unstable and takes 4-5 minutes of
continuous nebulization. Therefore, a stable DPI formulation has significant advantages.
DHEA-S-dihydrate is identified as the most stable solid state for a DPI formulation. The
anhydrous form is also suitable for administration with the nebulizer if its stability is
maintained by eliminating its contact with water prior to nebulization.
An optimal nebulizer formulation is 7.5 mg/mL of DHEA-S in 0.12% NAC1 for clinical
trials for DHEA-S. The pH of the formulation is acceptable without a buffer system. The
aqueous solubility of DHEA-S is maximized by minimizing the sodium cation
concentration. Minimal sodium chloride levels without buffer achieve this goal. This is
the highest drug concentration with 20 mM of Cl" that will not precipitate during
nebulization. This formulation is stable for at least one day at room temperature.
Although the invention has been described with reference to the presently preferred
embodiments, it should be understood that various modifications can be made without
departing from the spirit of the invention.
All publications, patents, and patent applications, and web sites are herein incorporated
by reference in their entirety to the same extent as if each individual publication, patent,
or patent application, was specifically and individually indicated to be incorporated by
reference in its entirety.

We Claim:
1. A pharmaceutical composition comprising
(i) dehydroepiandrosterone (DHEA) or dehydroepiandrosterone sulfate (DHEAS)
and/or a pharmaceutically or veterinarily acceptable salt thereof, in an anhydrous
form wherein the dry powdered composition has at least 0.01 to 100 % w/w of the
composition of active compound and
(ii) a pharmaceutically or veterinarily acceptable excipient wherein excipients are
used in amounts ranging from 0.1, to 99% w/w composition,
of particles of respirable or inhalable size, wherein greater than 80%) of the particles
are about 0.1 urn to about 100 µm in diameter.
2. The pharmaceutical composition as claimed in claim 1, wherein said excipient is one selected from human protein, bovine serum albumin, gelatin, immunoglobulins, galactose, D-mannose, sorbose, trehalose, sucrose, cyclodextrins, raffinose, maltodextrins, dextrans, monosodium glutamate, glysine, alanine, arginine or histidine, tryptophan, tyrosine, leucine, phenylalanine, betaine, magnesium sulfate, magnesium stearate, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, propylene glycol, polyethylene glycol, pluronics, surfactants, and a mixture thereof.
3. The pharmaceutical composition as claimed in claim 1, wherein said powder pharmaceutical composition is deliverable using a nebulizer, a dry powder inhaler, an insufflator, or an aerosol or spray generator.
4. The pharmaceutical composition as claimed in claim 1, wherein said powder pharmaceutical composition is produced by jet-milling.
5. The pharmaceutical composition as claimed in claim 1, wherein greater than 80% of the particles are about 0.1 µm to about 50 µm.

6. The pharmaceutical composition as claimed in claim 5, wherein greater than 80% of the particles are preferably about 0.1 µm to about 10 µm.
7. The pharmaceutical composition as claimed in claim 6, wherein greater than 90% of the particles are most preferably about 0.1 µm to about 5 µm.

8. The pharmaceutical composition as claimed in claim 1 for use in the manufacture of a medicament and a kit comprising said composition ,pharmaceutically acceptable propellant for the said composition and a nebulizer in a vaccum sealed container for treatment of asthma, for treatment of chronic obstructive pulmonary disease, for reducing or depleting adenosine in a subject's tissue, for treatment of airway inflammation, allergy, asthma, impeded respiration, cystic fibrosis, Chronic Obstructive Pulmonary Diseases, allergy rhinitis, Acute Respiratory Disease Syndrome, microbial infection, SARS, pulmonary hypertension, lung inflammation, bronchitis, airway obstruction, or bronchoconstriction , for treatment of a disorder or condition associated with high levels of, or sensitive to, adenosine in a subject's tissue, wherein the medicament comprises a therapeutically effective amount of the powder pharmaceutical composition.
9. Pharmaceutical compositions, comprising anhydrous dehydroepiandrosterone particles of respirable or inhalable size substantially as described in the specification and illustrated in the accompanying examples and drawings.

















3700-delnp-2004-description (complete).pdf























Patent Number 236147
Indian Patent Application Number 3700/DELNP/2004
PG Journal Number 41/2009
Publication Date 09-Oct-2009
Grant Date 30-Sep-2009
Date of Filing 24-Nov-2004
Applicant Address 7 CLARK DRIVE, CRANBURRY, NJ 08512 (US).
# Inventor's Name Inventor's Address
PCT International Classification Number A61K 31/704
PCT International Application Number PCT/US2003/018944
PCT International Filing date 2003-06-17
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/477,987 2003-06-11 U.S.A.
2 60/389,242 2002-06-17 U.S.A.