Note: Descriptions are shown in the official language in which they were submitted.
CA 02379160 2002-O1-14
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SAMPLE ARRAYS AND HIGH-THROUGHPUT TESTING THEREOF TO DETECT
INTERACTIONS
This application in a continuation-in-part of Ser. No. 09/540,462 filed March
31,
2000, which claims the benefit of U.S. Provisional Application Nos.
60/146,019, filed July
28, 1999 and 60/127,755, filed April 5, 1999, the entire contents of which
continuation-in-
part application and provisional applications are incorporated herein by
reference. This
application also claims the priority benefit of U.S. Provisional Application
No. 60/146,019,
filed July 28, 1999.
Field of the Invention
This invention is directed to the generation and analysis of data concerning
multi-
component chemical compositions, in particular, pharmaceutical or other
formulations.
More specifically, the invention is directed to methods, systems, and devices
for high
throughput measuring and testing of samples for optimization of sample
properties and
discovery of new compositions.
Backeround of the Invention
Discovery of pharmaceutical formulations that optimize bioavailability and
duration
of action of the pharmaceutical and minimize undesirable properties is an
important part of
pharmaceutical development and research. Pharmaceuticals are rarely
distributed as pure
compounds because of problems with, among others, stability, solubility, and
bioavailability. In most cases, pharmaceuticals are administered in a
pharmaceutical
formulation comprising the active ingredients and excipients. It is well
documented that
physical and chemical properties, such as stability, solubility, dissolution,
permeability, and
partitioning of most pharmaceuticals are directly related to the medium in
which they are
administered. This is because the medium affects the physical and chemical and
chemical
environment of the active ingredient, e.g., a pharmaceutical. The physical and
chemical
properties of drug-in-formulation mixtures are directly related to
pharmacological and
pharmacokinetic properties, such as absorption, bioavailability, metabolic
profile, toxicity,
and potency. Such effects are caused by physical and chemical interactions
between the
excipients and the pharmaceutical and/or physical and chemical interactions
between the
excipients themselves. Other properties influenced by the formulation in which
a
pharmaceutical is administered include mechanical properties, such as
compressibility,
WO 01/09391 CA 02379160 2002-O1-14 ~CT/[JSO~n~717
compactability, and flow characteristics and sensory properties, such as
taste, smell, and
color.
Thus the goal of formulation development is to discover formulations that
optimize
desired characteristics of the pharmaceutical, such as stability, solubility,
and bioavailability
of a pharmaceutical under the conditions that it is administered. This is
normally a tedious
process, where each variable is separately assessed, at several points over a
range of
conditions or combinations. For example, if the formulation contains a
pharmaceutical
characterized by poor solubility, the solubility of the pharmaceutical in a
range of salt
concentrations; pHs; excipients; and pharmaceutical concentrations must be
prepared and
tested to find interactions between the pharmaceutical and excipients or
interactions
between excipients that affect the pharmaceutical's solubility. While some
general rules
exist, the effect of excipients and combinations of excipients on the physical
and chemical
properties of the pharmaceutical are not easily predicted. Moreover, there are
over 3,000
excipients to choose from when designing pharmaceutical formulations, each
having
differing degrees and types of interactions with each other and with the
pharmaceutical.
Because of the many variables involved, industry does not have the time or
resources to
identify, measure, or exploit interactions between excipients and
pharmaceuticals and thus
cannot provide optimized pharmaceutical formulations tailored to the
particular
pharmaceutical. Such work would require testing hundreds to thousands of
samples a day.
Assuming three hundred substances are to be tested for efficacy as excipients
in a
pharmaceutical formulation, even with no variations in concentrations and no
physical or
chemical property variations, the number of possible combinations is enormous:
when two
of the substances are selected, there are 44,850 possible combinations, for
three components
there are 4.455,100 combinations, and for four components, there are
330,791,175 possible
combinations. The complexity is increased when the relative ratio of each
component is
considered. Unfortunately, technologies that can make many pharmaceutical-
excipient
combinations at the same time, then automatically feed each combination into a
system for
identifying the combinations that have optimized properties are not known.
Today, since it
is more cost effective, most pharmaceuticals are distributed and administered
in the
standard, un-optimized formulations, see e.g., Allen's Compounded
Formulations: U.S.
Pharmacists Collection 1995 to 1998, ed. Loyd Allen.
Unfortunately, present day pharmaceutical formulation research and
development,
still relies on a select few excipients and retro-fits the active ingredients
into well-known
oral or parenteral formulation systems. This invention resists the traditional
approach.
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The need to provide optimized formulations is not limited to formulations
wherein
the active component is a pharmaceutical. Similar problems are encountered for
administering dietary supplements, alternative medicines, nutraceuticals,
sensory
compounds, agrochemicals, and consumer and industrial product formulations.
For
example, similar to a pharmaceutical formulation, a vitamin formulation can be
characterized by poor stability, solubility, bioavailability, taste, or smell.
In another
example, industrial product formulations, such as bleaching agents for paper
mills can
benefit by reformulation for higher stability so that the activity is not
diminished during
shipment.
Summary of the Invention
The invention relates to high-throughput methods to prepare a large number of
component combinations, at varying concentrations and identities, at the same
time, and
high-throughput methods to test each combination. Such methods allow detection
or
1 S measurement of interactions between inactive components and active
components; between
multiple inactive components; or between multiple active components. Such
methods also
allow detection of lack of interactions between inactive components and active
components;
between multiple inactive components; or between multiple active components.
The
invention is particularly suited for making a large number of pharmaceutical-
excipient
combinations, then rapidly testing each combination to detect or measure
interactions or to
detect lack of interactions between excipients and the pharmaceutical; between
excipients;
or between multiple pharmaceuticals. Once such interactions or lack of
interactions are
identified, they can be exploited to develop optimized formulations for
pharmaceutical
administration.
The invention thus encompasses the high-throughput testing of pharmaceutical
formulations in order to determine the overall optimal formulation for any
particular
ingredient, or to optimize any particular desired property or results, e.g.,
bioavailability,
potency, release, stability, and the like; or both. To applicant's knowledge,
a systematic,
high-throughput method for formulation generation, screening, testing, and
analysis, has not
been published prior to this invention. Moreover, an automated system for the
generation,
screening, and testing of such formulations is encompassed by this invention.
Finally,
computerized analysis of such data is encompassed by this invention. Specific
embodiments of this invention are described in detail below.
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In one embodiment, the invention concerns an array of samples, each sample
comprising a component-in-common and at least one additional component,
wherein each
sample differs from any other sample with respect to at least one of
(i) the identity of the additional component,
S (ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common.
Component-in-common means that the component is present in every sample.
Preferably,
the component-in-common is an active component, more preferably, a
pharmaceutical,
dietary supplement, alternative medicine, nutraceutical, sensory compound,
agrochemical,
the active component of a consumer product formulation, or the active
component of an
industrial product formulation. The samples and components can be in the form
of solids,
liquids, gels, foams, pastes, ointments, triturates, suspensions, or
emulsions.
In another embodiment, the invention concerns a method to measure or detect an
interaction between components, comprising:
(a) preparing an array of samples, each sample comprising a component-in-
common and at least one additional component, wherein each sample differs
from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component,
or
(iii) the physical state of the component-in-common; and
(b) testing each sample for one or more properties.
In yet another embodiment, the invention concerns a method to measure or
detect an
interaction between components, comprising:
(a) preparing an array of samples, each sample comprising at least two
components, wherein each sample differs from any other sample with respect
to the identity of the components; the samples may further differ with respect
to the ratio of the components, or the physical state of the components; and
(b) testing each sample for one or more properties.
In this embodiment, the samples do not have a component-in-common. The method
is useful for testing mixtures of components, such as chemical fragrances,
where each
component has a different identity or chemical formula.
Preferably the samples are prepared, tested, and analyzed automatically and
the data
stored and/or analyzed by a computer. Preferably, the component-in-common is
an active
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component, more preferably, a pharmaceutical, dietary supplement, alternative
medicine,
nutraceutical, sensory compound, agrochemical, the active component of a
consumer
product formulation, or the active component of an industrial product
formulation. Most
preferably, the component-in-common is a pharmaceutical. The samples and
components
can be in the form of solids, liquids, gels, foams, pastes, ointments,
triturates, suspensions,
or emulsions.
In another embodiment, the invention concerns a method for testing or
optimizing
one or more properties of a formulation of an active-component, comprising:
(a) preparing an array of samples, each sample comprising the active component
and at least one additional component, wherein each sample differs from any
other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the active component to the additional component, or
(iii) the physical state of the active component;
(b) testing each sample for at least one property to generate a property-
result for
each sample; and
(c) comparing the property-result generated for each sample to a baseline or a
control for said property to generate a comparison result for the sample.
Preferably the samples are prepared, tested, and analyzed automatically and
the data
stored and/or analyzed by a computer. Preferably, the active component is a
pharmaceutical, dietary supplement, alternative medicine, nutraceutical,
sensory compound,
agrochemical, the active component of a consumer product formulation, or the
active
component of an industrial product formulation. Most preferably, the active
component is a
pharmaceutical. The samples and components can be in the form of solids,
liquids, gels,
foams, pastes, ointments, triturates, suspensions, or emulsions.
In still another embodiment, the invention concerns a system to measure or
detect an
interaction between components, comprising:
(a) an array of samples, each sample comprising a component-in-common and at
least one additional component, wherein each sample differs from any other
sample
with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common; and
(b) a sample tester to test each sample for one or more properties
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Preferably, the component-in-common is a pharmaceutical, dietary supplement,
alternative medicine, nutraceutical, sensory compound, agrochemical, the
active component
of a consumer product formulation, or the active component of an industrial
product
formulation. Most preferably, the component-in-common is a pharmaceutical. The
samples
and components can be in the form of solids, liquids, gels, foams, pastes,
ointments,
triturates, suspensions, or emulsions.
These and other features, aspects, and advantages of the invention will become
better understood with reference to the following detailed description,
examples, and
appended claims.
Brief Description of the Drawings
Figure 1 is a schematic of the method for preparing an array of samples,
processing
the samples, analyzing the samples for one or more properties, collecting and
storing the
data, analyzing the data, and detecting or measuring an interaction or
detecting a lack of an
interaction.
Figures 2A and 2B is a more detailed schematic of a process to formulate and
analyze multiple samples, for properties such as solubility (UV-VIS HPLC) and
estimated
oral absorbance, wherein Figure 2A is a schematic of the process wherein
solids are
deposited in sample wells in the array, then reconstituted and tested; and
Figure 2B is a
schematic of the process wherein liquids are deposited into an array, dried,
reconstituted and
then separated into liquids and solids and tested.
Figure 3A is a graph of the solubility (absorbance) of 3,500 unique
formulations
containing griseofulvin with various excipients in water. Figure 3B is a graph
of the data in
Figure 3A plotted to show standard deviations for each of the unique
formulations.
Figure 4 is a graph comparing solubility (absorbance) of the commercially
available
pharmaceutical with five lead formulations (TPI-1 to TPI-5).
Figure 5 is a graph of the ratio of the solubility of various reformulations
of one of
the lead formulations, TPI-3, reformulated with only one or two of the three
excipients
shown in relative ratios in the accompanying "pie". Figure 6 is a graph of the
ratio of the
solubility of various reformulations of a lead formulation, TPI-1, comparing
the effect of
reformulating the formulation with one or two of the three excipients in the
lead
formulation, demonstrating that some excipients actually decrease solubility.
Figure 7 is a graph of the ratio of the solubility of various reformulations
of one lead
formulation, TPI-2, showing the effect of reformulating the formulation with
one or two of
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WO 01/09391 CA 02379160 2002-O1-14 pCT/US00/20717
the three excipients in the lead formulation, demonstrating that some
excipients have a
synergistic effect on solubility.
Figure 8 is a graph comparing rates of dissolution and equilibrium
solubilities for
TPI-2 and griseofulvin, showing TPI-2 having a higher rate of dissolution as
well as a
higher equilibrium solubility compared to the griseofulvin.
Detailed Descrption of the Invention
As used herein, the term "array" means a plurality of samples associated under
a
common experiment, wherein each of the samples comprises at least two
components, one
of the components being a component-in-common. The term "component-in-common"
simply means a particular component that is present in every sample of the
array, with the
exception of negative controls. The array is designed to provide a data set,
analysis of
which allows detection or measurement of interactions (including lack of
interactions)
between the component-in-common and the other component. Each sample in the
array
differs from any other sample in the array with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common.
An array can comprise 24, 36, 48, 96, or more samples, preferably 1000 or more
samples, more preferably, 10,000 or more samples. An array is typically
comprised of one
or more sub-arrays. For example, a sub-array can be a 96-well plate of sample
wells.
As used herein, the term "sample" means a mixture of a component-in-common and
one or more additional components. Preferably a sample comprises 2 or more
additional
components, more preferably, 3 or more additional components. In general, a
sample will
comprise one component-in-common but can comprise multiple components in
common.
A sample can be present in any container or holder or in or on any material or
surface, the
only requirement is that the samples be located at separate sites. Preferably,
samples are
contained in sample wells, for example, a 24, 36, 48, or 96 well plates (or
filter plates) of
volume 250 u1 available from Millipore, Bedford, MA. The sample can comprise
less than
about 100 milligrams of the component-in-common, preferably, less than about 1
milligram,
more preferably, less than about100 micrograms, and even more preferably, less
than 100
nanograms. Preferably, the sample has a total volume of 150-200 u1. Preferably
a sample
CA 02379160 2002-O1-14
WO 01/09391 PCT/US00/20717
comprises 2 or more additional components, more preferably, 3 or more
additional
components.
According to the invention described herein, the "physical state" of a
component is
initially defined by whether the component is a liquid or a solid. If the
component is a
solid, the physical state is further defined by the particle size and whether
the component is
crystalline or amorphous. If the component is crystalline, the physical state
is further
divided into: ( 1 ) whether the crystal matrix includes a co-adduct or whether
the crystal
matrix originally included a co-adduct, but the co-adduct was removed leaving
behind a
vacancy; (2) crystal habit; (3) morphology, i.e., crystal habit and size
distribution; and (4)
internal structure (polymorphism). In a co-adduct, the crystal matrix can
include either a
stoichiometric or non-stoichiometric amount of the adduct, for example, a
crystallization
solvent or water, i.e., a solvate or a hydrate. Non-stoichiometric solvates
and hydrates
include inclusions or clathrates, that is, where a solvent or water is trapped
at random
intervals within the crystal matrix, for example, in channels. A
stoichiometric solvate or
hydrate is where a crystal matrix includes a solvent or water at specific
sites in a specific
ratio. That is, the solvent or water molecule is part of the crystal matrix in
a defined
arrangement. Additionally, the physical state of a crystal matrix can change
by removing a
co-adduct, originally present in the crystal matrix. For example, if a solvent
or water is
removed from a solvate or a hydrate, a hole will be formed within the crystal
matrix,
thereby forming a new physical state. The crystal habit is the description of
the outer
appearance of an individual crystal, for example, a crystal may have a cubic,
tetragonal,
orthorhombic, monoclinic, triclinic, rhomboidal, or hexagonal shape. The
processing
characteristics are affected by crystal habit. The internal structure of a
crystal refers to the
crystalline form or polymorphism. A given compound may exist as different
polymorphs,
that is, distinct crystalline species. In general, different polymorphs of a
given compound
are as different in structure and properties as the crystals of two different
compounds.
Solubility, melting point, density, hardness, crystal shape, optical and
electrical properties,
vapor pressure, and stability, etc. all vary with the polymorphic form.
When refernng to an interaction between components, an "interaction" means
that
the components as a mixture display a property (e.g., the ability to
solubilize a specific
pharmaceutical) of a different magnitude or value than the same property
displayed by each
component in isolation. Interactions between components will affect the
properties of
samples. Merely for example, a particular combination and ratio of excipients
can interact
such that the combination has a high solubilizing power for a particular
pharmaceutical.
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Once such an interaction is detected, it can be exploited to develop enhanced
formulations
for the pharmaceutical.
As used herein, the term "component" means any substance. A component can be
active or inactive. As used herein, the term "active component" means a
substance that
S imparts the primary utility to a formulation when the formulation is used
for its intended
purpose. Examples of active components include pharmaceuticals, dietary
supplements,
alternative medicines, nutraceuticals, sensory compounds, agrochemicals, the
active
component of a consumer product formulation, and the active component of an
industrial
product formulation. As used herein, an "inactive component" means a component
that is
useful or potentially useful to serve in a formulation for administration of
an active
component, but does not significantly share in the active properties of the
active component.
Examples of suitable inactive components include, but are not limited to,
excipients,
solvents, diluents, stabilizers and combinations thereof.
Preferably, the samples of an array comprise an active component-in-common and
inactive components. A number of permutations are available to the skilled
artisan, for
example, when the active component is a pharmaceutical, dietary supplement,
alternative
medicine, or nutraceutical, the preferred inactive components are excipients.
When the
active component is a sensory compound, such as a fragrance or flavor, the
inactive
components are preferably those inactive (non-sensory) substances or
ingredients known in
the art for administration of sensory compounds. When the active component is
an
agrochemical, preferably the inactive components are those inactive substances
routinely
used in the art to administer agrochemicals. When the active component is
associated with
a consumer or an industrial product formulation, the inactive components are
preferably
inactive substances known in the art to deliver such active components.
As used herein, the term "assaying agent" means a method, biological material,
or
reagent, such as an enzyme or cell line, useful for measuring the properties
of a sample.
As used herein, the term "formulation" means a composition comprising a
predefined ratio of one or more active components and one or more inactive
components. A
formulation is used as directed to administer the active component.
As used herein, the term "administer" means the act of delivering or applying
an
active component for its intended use via a formulation. For example,
administration of
pharmaceuticals, dietary supplements, alternative medicines, and
nutraceuticals includes,
but is not limited to, oral consumption, intravenous injection, and topical
application.
Administration of agrochemical includes, but is not limited to, crop spraying
and dusting.
Administration of sensory compounds includes, but is not limited to, applying
perfumes and
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deodorants to the human body or eating a food or candy that has been
supplemented by a
flavor material. Administration of consumer and industrial product
formulations means
applying or simply using the product as directed. For example, administering a
paint means
applying the paint to a surface with a paint brush and administering a
lubricant means
applying the lubricant to a surface where lubrication is desired.
According to the invention, the ratio of the component-in-common and to a
particular additional component will differ between samples when the ratios
thereof are
intentionally varied to induce a measurable change in the sample's properties.
As used herein, the term "property" means a physical or chemical
characteristic of a
sample. Preferred properties are those that relate to the efficacy, safety,
stability, or utility
of formulations before or after administration. For example, regarding
pharmaceutical,
dietary supplement, alternative medicine, and nutraceutical formulations,
properties include
physical properties, such as rheology, friability, stability, solubility,
dissolution,
permeability, and partitioning; mechanical properties, such as
compressibility,
compactability, and flow characteristics; sensory properties, such as mouth
feel, appearance,
texture, color, taste, and smell; and properties that affect the utility, such
as absorption,
bioavailability, toxicity, metabolic profile, potency, rate-of release, and
rate-of dispersion.
Optimizing physical, sensory, and utility properties can result in a lowered
required dose for
the same therapeutic effect potentially with fewer side reactions, thereby
improving patient
compliance.
An array comprises at least two samples and preferably comprises 24, 36, 48,
72 or
more samples, more preferably 96 or more, still more preferably 1000 or more,
most
preferably 10,000 or more samples. The samples are located at separate sites
and can be
confined in any container or holder, absorbed into a suitable material, or
present at separate
sites on a flat surface. Preferably, the samples are contained in sample
wells. The sample
wells can be of any dimensions or volume. Preferably, the sample wells have a
volume with
a range of 200 to 300 p1, more preferably, 250 p1. Arrays can be prepared by
adding the
component-in-common and the additional components) to the sample wells. The
component-in-common is chosen by the skilled artisan according to the
interaction to be
measured or identified. Preferably, the component-in-common is an active
component (i. c. ,
an active component-in-common), more preferably, a pharmaceutical (i.e., a
pharmaceutical-in-common). But in some cases, the component-in-common is an
inactive
component, for example, where an array is designed to detect or measure an
interaction
between a particular excipient (an excipient-in-common) and other excipients.
Active
components and inactive components can be chosen from known lists of
substances
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published in the literature or a component can be a newly discovered
substance. Preferably,
the inactive components are already known in the art for administration of the
class of
active component under study. For example, when the component-in-common is a
pharmaceutical, the inactive components can be chosen from lists of known
excipients.
For each sample in an array, the component-in-common and each component is in
a
particular ratio. The ratios of the component-in-common to the additional
components are
readily varied from sample to sample by the skilled artisan according to the
component-in-
common's identity and the information that the array is designed to provide.
Preferably, the sample components are mixed to obtain a homogeneous solution
or
suspension. One of skill in the art will know how to add and mix the
components of each
sample based on the particular study. The components can be added and mixed
either
manually or via automation.
Preferably the components are added to the sample wells and mixed
automatically.
"Automated" refers to samples prepared by using software and robotics to add
and mix the
components. After adding and mixing the components to the sample wells, the
samples
may be processed by well known techniques, such as heating, filtration, and
lyophilization.
One of skill in the art will know how to process the sample according to the
properties
being tested. The samples can be processed individually or as a group,
preferably, as a
group.
According to the methods of the invention, each sample in the array is tested
or
assayed for a particular property to provide a data set. The samples can be
tested by a
variety of known methods depending on the components and the property in
question. The
property can just be detected or the property can be measured to provide a
value or
magnitude for it. The value or magnitude of the property can be compared to a
control or
standard value to assess whether the sample's components are interacting or if
the sample
has potential to serve as a formulation for administering an active component.
For example,
when testing an array of samples-each sample comprising the same known drug
but
different excipients- to find the combination of excipients in which the drug
displays the
highest solubility, the solubility of the drug in each formulation may be
compared to a
control or standard value for the solubility. For instance, the solubility of
the drug in each
sample can be compared to the solubility of the drug in a commercial
formulation or to the
solubility of the drug in isolation.
Pharmaceuticals Dietary Supplements. Alternative Medicines, and Nutraceuticals
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The invention described herein is useful to detect or measure interactions
between
pharmaceuticals, dietary supplements, alternative medicines, or nutraceuticals
and
excipients thereby allowing development of formulations thereof with optimal
properties.
In a one embodiment, the invention concerns an array of samples, each sample
comprising a component-in-common is a pharmaceutical, a dietary supplement, an
alternative medicine, or a nutraceutical and at least one additional
component, wherein each
sample differs from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common.
As used herein, the term "pharmaceutical" means any substance that has a
therapeutic, disease preventive, diagnostic, or prophylactic effect when
administered to an
animal or a human. The term pharmaceutical includes prescription drugs and
over the
counter drugs. Pharmaceuticals suitable for use in the invention include all
those known or
to be developed.
Examples of suitable pharmaceuticals include, but are not limited to,
cardiovascular
pharmaceuticals, such as amlodipine besylate, losartan potassium, irbesartan,
diltiazem
hydrochloride, clopidogrel bisulfate, digoxin, abciximab, furosemide,
amiodarone
hydrochloride, beraprost, tocopheryl nicotinate; anti-infective components,
such as
amoxicillin, clavulanate potassium, azithromycin, itraconazole, acyclovir,
fluconazole,
terbinafine hydrochloride, erythromycin ethylsuccinate, and acetyl
sulfisoxazole; '
psychotherapeutic components, such as sertaline hydrochloride, vanlafaxine,
bupropion
hydrochloride, olanzapine, buspirone hydrochloride, alprazolam,
methylphenidate
hydrochloride, fluvoxamine maleate, and ergoloid mesylates; gastrointestinal
products, such
as lansoprazole, ranitidine hydrochloride, famotidine, ondansetron
hydrochloride,
granisetron hydrochloride, sulfasalazine, and infliximab; respiratory
therapies, such as
loratadine, fexofenadine hydrochloride, cetirizine hydrochloride, fluticasone
propionate,
salmeterol xinafoate, and budesonide; cholesterol reducers, such as
atorvastatin calcium,
lovastatin, bezafibrate, ciprofibrate, and gemfibrozil; cancer and cancer-
related therapies,
such as paclitaxel, carboplatin, tamoxifen citrate, docetaxel, epirubicin
hydrochloride,
leuprolide acetate, bicalutamide, goserelin acetate implant, irinotecan
hydrochloride,
gemcitabine hydrochloride, and sargramostim; blood modifiers, such as epoetin
alfa,
enoxaparin sodium, and antihemophilic factor; antiarthritic components, such
as celecoxib,
nabumetone, misoprostol, and rofecoxib; AIDS and AIDS-related pharmaceuticals,
such as
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lamivudine, indinavir sulfate, stavudine, and lamivudine; diabetes and
diabetes-related
therapies, such as metformin hydrochloride, troglitazone, and acarbose;
biologicals, such as
hepatitis B vaccine, and hepatitis A vaccine; hormones, such as estradiol,
mycophenolate
mofetil, and methylprednisolone; analgesics, such as tramadol hydrochloride,
fentanyl,
metamizole, ketoprofen, morphine sulfate, lysine acetylsalicylate, ketoralac
tromethamine,
morphine, loxoprofen sodium, and ibuprofen; dermatological products, such as
isotretinoin
and clindamycin phosphate; anesthetics, such as propofol, midazolam
hydrochloride, and
Iidocaine hydrochloride; migraine therapies, such as sumatriptan succinate,
zolmitriptan,
and rizatriptan benzoate; sedatives and hypnotics, such as zolpidem, zolpidem
tartrate,
triazolam, and hycosine butylbromide; imaging components, such as iohexol,
technetium,
TC99M, sestamibi, iomeprol, gadodiamide, ioversol, and iopromide; and
diagnostic and
contrast components, such as alsactide, americium, betazole, histamine,
mannitol,
metyrapone, petagastrin, phentolamine, radioactive B,2, gadodiamide,
gadopentetic acid,
gadoteridol, and perflubron. Other pharmaceuticals for use in the invention
include those
listed in Table 1 below, which suffer from problems that could be mitigated by
developing
new administration formulations according to the arrays and methods of the
invention.
TABLE 1: Exemplary Pharmaceuticals
Bred Name Chemical Properties
SANDIMMUNE cyclosporin Poor absorption due to its low water solubility.
TAXOL paclitaxel Poor absorption due to its low water solubility.
VIAGRA sildenafil citrate Poor absorption due to its low water solubility.
NORVIR ritonavir Can undergo a polymorphic shift during shipping
and storage.
FULVICIN griseofulvin Poor absorption due to its low water solubility.
FORTOVASE saquinavir Poor absorption due to its low water solubility.
Still other examples of suitable pharmaceuticals are listed in 2000 Med Ad
Ne~~s
19:56-60 and The Physicians Desk Reference, 53rd edition, 792-796, Medical
Economics
Company (1999), both of which are incorporated herein by reference.
Examples of suitable veterinary pharmaceuticals include, but are not limited
to,
vaccines, antibiotics, growth enhancing components, and dewormers. Other
examples of
suitable veterinary pharmaceuticals are listed in The Merck Veterinary Manual,
8th ed.,
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Merck and Co., Inc., Rahway, NJ, 1998; (1997); The Encyclopedia of Chemical
Technology, 24 Kirk-Othomer (4'" ed. at 826); and Veterinary Drugs in ECT 2nd
ed., Vol
21, by A.L. Shore and R.J. Magee, American Cyanamid Co.
As used herein, the term "dietary supplement" means a non-caloric or
insignificant-
caloric substance administered to an animal or a human to provide a
nutritional benefit or a
non-caloric or insignificant-caloric substance administered in a food to
impart the food with
an aesthetic, textural, stabilizing, or nutritional benefit. Dietary
supplements include, but
are not limited to, fat binders, such as caducean; fish oils; plant extracts,
such as garlic and
pepper extracts; vitamins and minerals; food additives, such as preservatives,
acidulents,
anticaking components, antifoaming components, antioxidants, bulking
components,
coloring components, curing components, dietary fibers, emulsifiers, enzymes,
firming
components, humectants, leavening components, lubricants, non-nutritive
sweeteners, food-
grade solvents, thickeners; fat substitutes, and flavor enhancers; and dietary
aids, such as
appetite suppressants. Examples of suitable dietary supplements are listed in
(1994) The
1 S Encyclopedia of Chemical Technology, 11 Kirk-Othomer (4'" ed. at 805-833).
Examples of
suitable vitamins are listed in (1998) The Encyclopedia of Chemical
Technology, 25 Kirk-
Othomer (4'" ed. at 1 ) and Goodman & Gilman's: The Pharmacological Basis of
Therapeutics, 9th Edition, eds. Joel G. Harman and Lee E. Limbird, MeGraw-
Hill, 1996
p.1547, both of which are incorporated by reference herein. Examples of
suitable minerals
are listed in The Encyclopedia of Chemical Technology, 16 Kirk-Othomer (4'"
ed. at 746)
and "Mineral Nutrients" in ECT 3rd ed., Vol 15, pp. 570-603, by C.L. Rollinson
and M.G.
Enig, University of Maryland, both of which are incorporated herein by
reference
As used herein, the term "alternative medicine" means a substance, preferably
a
natural substance, such as a herb or an herb extract or concentrate,
administered to a subject
or a patient for the treatment of disease or for general health or well being,
wherein the
substance does not require approval by the FDA. Examples of suitable
alternative
medicines include, but are not limited to, ginkgo biloba, ginseng root,
valerian root, oak
bark, kava kava, echinacea, harpagophyti radix, others are listed in The
Complete German
Commission E Monographs: Therapeutic Guide to Herbal Medicine, Mark Blumenthal
et
al. eds., Integrative Medicine Communications 1998, incorporated by reference
herein.
As used herein the term "nutraceutical" means a food or food product having
both
caloric value and pharmaceutical or therapeutic properties. Example of
nutraceuticals
include garlic, pepper, brans and fibers, and health drinks Examples of
suitable
Nutraceuticals are listed in M.C. Linden ed. Nutritional Biochemistry and
Metabolism with
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Clinical Applications, Elsevier, New York, 1985; Pszczola et al., 1998 Food
technology
52:30-37 and Shukla et al., 1992 Cereal Foods World 37:665-666.
Preferably, when the component-in-common is a pharmaceutical, a dietary
supplement, an alternative medicine, or a nutraceutical, the additional
components) are
excipients. As used herein, the term "excipient" means the inactive substances
used to
formulate pharmaceuticals as a result of processing or manufacture or used by
those of skill
in the art to formulate pharmaceuticals, dietary supplements, alternative
medicines, and
nutraceuticals for administration to animals or humans. Preferably, excipients
are approved
for or considered to be safe for human and animal administration. Examples of
suitable
excipients include, but are not limited to, acidulents, such as lactic acid,
hydrochloric acid,
and tartaric acid; solubilizing components, such as non-ionic, cationic, and
anionc
surfactants; absorbents, such as bentonite, cellulose, and kaolin; alkalizing
components,
such as diethanolamine, potassium citrate, and sodium bicarbonate; anticaking
components,
such as calcium phosphate tribasic, magnesium trisilicate, and talc;
antimicrobial
components, such as benzoic acid, sorbic acid, benzyl alcohol, benzethonium
chloride,
bronopol, alkyl parabens, cetrimide, phenol, phenylmercuric acetate,
thimerosol, and
phenoxyethanol; antioxidants, such as ascorbic acid, alpha tocopherol, propyl
gallate, and
sodium metabisulfite; binders, such as acacia, alginic acid, carboxymethyl
cellulose,
hydroxyethyl cellulose; dextrin, gelatin, guar gum, magnesium aluminum
silicate,
maltodextrin, povidone, starch, vegetable oil, and zero; buffering components,
such as
sodium phosphate, malic acid, and potassium citrate; chelating components,
such as EDTA,
malic acid, and maltol; coating components, such as adjunct sugar, cetyl
alcohol, polyvinyl
alcohol, carnauba wax, lactose maltitol, titanium dioxide; controlled release
vehicles, such
as microcrystalline wax, white wax, and yellow wax; desiccants, such as
calcium sulfate;
detergents, such as sodium lauryl sulfate; diluents, such as calcium
phosphate, sorbitol,
starch, talc, lactitol, polymethacrylates, sodium chloride, and glyceryl
palmitostearate;
disintegrants, such as collodial silicon dioxide, croscarmellose sodium,
magnesium
aluminum silicate, potassium polacrilin, and sodium starch glycolate;
dispersing
components, such as poloxamer 386, and polyoxyethylene fatty esters
(polysorbates);
emollients, such as cetearyl alcohol, lanolin, mineral oil, petrolatum,
cholesterol, isopropyl
myristate, and lecithin; emulsifying components, such as anionic emulsifying
wax,
monoethanolamine, and medium chain triglycerides; flavoring components, such
as ethyl
maltol, ethyl vanillin, fumaric acid, malic acid, maltol, and menthol;
humectants, such as
glycerin, propylene glycol, sorbitol, and triacetin; lubricants, such as
calcium stearate,
canola oil, glyceryl palmitosterate, magnesium oxide, poloxymer, sodium
benzoate, stearic
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acid, and zinc stearate; solvents, such as alcohols, benzyl phenylformate,
vegetable oils,
diethyl phthalate, ethyl oleate, glycerol, glycofurol, for indigo carmine,
polyethylene glycol,
for sunset yellow, for tartazine, triacetin; stabilizing components, such as
cyclodextrins,
albumin, xanthan gum; and tonicity components, such as glycerol, dextrose,
potassium
chloride, and sodium chloride; and mixture thereof. Excipients include those
alter rate of
absorption, bioavailability, or other pharmacokinetic properties of
pharmaceuticals, dietary
supplements, alternative medicines, or nutraceuticals. Other examples of
suitable
excipients, such as binders and fillers are listed in Remington's
Pharmaceutical Sciences,
18th Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995 and
Handbook
of Pharmaceutical Excipients, 3rd Edition, ed. Arthur H. Kibbe, American
Pharmaceutical
Association, Washington D.C. 2000, both of which are incorporated herein by
reference.
The definition of the term "excipient", as used herein, also includes
solvents.
Aqueous solvents can be used to make mixtures, suspensions, and matrices
formed of water
soluble polymers. Organic solvents will typically be used to dissolve
hydrophobic and
some hydrophilic polymers. Preferred organic solvents are volatile or have a
relatively low
boiling point or can be removed under vacuum and which are non-toxic or
acceptable for
administration to humans in trace amounts, such as methylene chloride. Other
solvents,
such as ethyl acetate, ethanol, methanol, dimethyl formamide, acetone,
acetonitrile,
tetrahydrofuran, acetic acid, dimethyl sulfoxide, and chloroform, and mixture
thereof, also
may be used. Preferred solvents are those rated as class 3 residual solvents
by the Food and
Drug Administration, as published in the Federal Register vol. 62, number 85,
pp. 24301-
24309 (May 1997). Solvents for drugs that are administered parenterally or as
a solution or
suspension will more typically be distilled water, buffered saline, Lactated
Ringer's or some
other pharmaceutically acceptable carrier.
In one aspect of the embodiment concerning arrays of samples, wherein the
component-in-common is selected from the group of pharmaceuticals, dietary
supplements,
alternative medicines. and nutraceuticals, the additional component is also
selected from the
group of pharmaceuticals, dietary supplements, alternative medicines, and
nutraceuticals.
That is, each sample can comprise multiple active components, wherein a
particular active
component is present in all the samples (i.e., the component-in-common). An
array of such
samples can be used to detect favorable or synergistic interactions between
pharmaceuticals,
dietary supplements, alternative medicines, and nutraceuticals and other
pharmaceuticals,
dietary supplements, alternative medicines, and nutraceuticals. For instance,
an array of
samples, wherein each sample comprises the same pharmaceutical-in-common and
each
sample also comprises a different, additional component selected from the
group of a
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pharmaceutical, a dietary supplement, an alternative medicine, or a
nutraceuticals can be
used to identify particularly advantageous combinations of active components.
Such
advantageous combinations may be unexpected based on the properties of the
components
in isolation. An array of such multiple-active-component samples can also be
used to find
suitable excipient combinations to co-formulate two or more active component,
e.g.,
formulate two or more pharmaceuticals into a multiple dosage form. Such
multiple-dosage
forms can obviate the need for the patient to take multiple medications
(polypharmacy),
since the prescribed pharmaceuticals are conveniently contained in one
formulation.
In another embodiment, the invention relates to a method to measure or detect
an
interaction between components, comprising:
(a) preparing an array of samples, each sample comprising a component-in-
common is a pharmaceutical, a dietary supplement, an alternative medicine,
or a nutraceutical and at least one additional component, wherein each
sample differs from any other sample with respect to at least one of:
I S (i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common;
(b) testing each sample for a property to generate a data set; and
(c) analyzing the data set to measure or detect the interaction.
Preferably, the samples are tested for properties that relate to
administration and
pharmacokinetics of pharmaceuticals, dietary supplements, alternative
medicines, or
nutraceuticals. Preferred properties include, but are not limited to, physical
properties, such
as rheology, friability, stability, solubility, dissolution, permeability, and
partitioning;
mechanical properties, such as compressibility, compactability, and flow
characteristics;
sensory properties, such as mouth feel, appearance, texture, color, taste, and
smell; and
properties that affect the utility, such as absorption, bioavailability,
toxicity, metabolic
profile, potency, rate-of release, and rate-of dispersion. While some
biological properties
relate to in vivo performance of an active component, such as a pharmaceutical
formulation,
they can be measured by in vitro tests that can be extrapolated to in vivo
performance.
Toxicity is the pharmaceutical formulations propensity to cause detrimental
side
effects when administered to a subject or patient. Toxicity includes
hypersensitivity and
allergic reactions. Potency is the activity that the formulation has for its
intended purpose.
Both of these biological properties can be measured by in vitro techniques,
such as
microbial assays. For a discussion of in vitro biological testing methods see
Remington's
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WO 01/09391 PCT/US00/20717
Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing
Co. Easton,
PA, 1995, pp.499-500, all three of which are incorporated herein by reference.
Absorption is the process of movement of an active component, such as a
pharmaceutical from the site of application past the physiological barrier -
for example,
S crossing through the gastrointestinal tract in the case of oral dosage;
crossing through the
skin and into the blood stream in the case of transdermal dosage; or crossing
through the
stratum corneum and into the dermis in the case of an intadermal dosage. Many
factors
determine the ease with which a pharmaceutical is absorbed, for example, the
pharmaceutical's concentration, solubility, and permeation ability. While
techniques exist
to test each of these properties, some general tests are available to estimate
absorption
directly, for example, an Ussing chamber containing HT Caco-2/MS engineered
cells as
reported in Lennernas, H. 1998 J. Pharm. Sci. 87:403-410. For a detailed
discussion of the
theory of and methods for measuring absorption of pharmaceuticals see
Remington's
Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing
Co. Easton,
PA, 1995, pp. 710-714, incorporated herein by reference.
Solubility refers to the equilibrium solubility or steady state and is
measured as
weight component/volume solvent. When an active component, such as a
pharmaceutical
substance has an aqueous solubility of less than about 1 milligram/milliliter
in the
physiological pH range of 1-7, a potential bioavailability problem exists.
Descriptive terms
used to describe solubility given in parts of solvent for 1 part of solvent
are: very soluble
(<1 part); freely soluble (from 1 to 10 parts); soluble (from 10 to 30 parts);
sparingly soluble
(from 30 to 100 parts); slightly soluble (from 100 to 1,000 parts); very
slightly soluble (from
1,000 to 10,000 parts); and insoluble (> 10,000 parts). For a discussion of
solution and
phase equilibria see Remington's Pharmaceutical Sciences, 18th Edition, ed.
Alfonso
Gennaro, Mack Publishing Co. Easton, PA, 1995, Ch. 16, incorporated herein by
reference.
The solubility can be tested by mixing the sample with a test solvent and
agitating
the sample at a constant temperature until equilibrium is achieved.
Equilibrium usually
occurs upon agitating the samples for 6 to 24 hours. If the component is
acidic or basic, its
solubility can be influenced by pH and one of skill in the art will take such
factors into
consideration when testing the solubility properties of a sample. Once
equilibrium has
occurred, the sample can be tested to determine the amount of component
dissolved using
standard technology, such as mass spectroscopy, HPLC, UV spectroscopy,
fluorescence
spectroscopy, gas chromatography, optical density, or by colorimetery. For a
discussion of
the theory and methods of measuring solubility see Streng et al., 1984 J.
Pharm. Sci.
63:605; Kaplan 1972 Drug Metab. Rev. 1:15; and Remington's Pharmaceutical
Sciences,
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WO 01/09391 PCT/US00/20717
18th Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995,
pp.1456-1457,
all three of which are incorporated herein by reference. For a discussion of
heat of
dissolution, pKa, and pH solubility profile effects and techniques for
measurement thereof
see Fiese et al., in The Theory and Practice of Industrial Pharmacy, 3rd ed.,
Lachman L.;
Lieberman, H.A.; and Kanig, J.L. Eds., Lea and Febiger, Philadelphia, 1986 pp.
185-188,
incorporated herein by reference.
Dissolution refers to the process by which a solid of only fair solubility
enters into
solution. Several factors affect dissolution such as solubility, particle
size, crystalline state,
and the presence of diluents, disintegrants, or other excipients. For a
discussion of the
theory and methods of measuring dissolution see Remington's Pharmaceutical
Sciences,
18th Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995,
Chapter 34,
incorporated herein by reference.
Stability refers to the chemical and physical stability of the component
during
manufacturing, packaging, distribution, storage, and administration of the
active-
component(s), to the formulation as a whole, or components thereof.
Chemical stability refers to resistance of the formulation to chemical
reactions
induced, for example, by heat, ultraviolet radiation, moisture, chemical
reactions between
components, or oxygen. Chemical stability also refers to a compound's ability
to maintain a
particular stereoisomeric form without conversion to another stereoisomeric
form, e.g.,
optical activity. Well known methods for measuring chemical stability include
mass
spectroscopy, UV-VIS spectroscopy, polarimetry, chiral and non-chiral HPLC,
chiral and
non-chiral gas chromatography, and liquid chromatography-mass spectroscopy (LC-
MS).
In the case of surface discoloration due to oxidation or due to reaction of
the active
component with excipients or with itself, surface reflectance measurements can
be more
sensitive than other assays. For a discussion of the theory and methods of
measuring
chemical stability see Xu et al., Stability-Indicating HPLC Methods for Drug
Analysis
American Pharmaceutical Association, Washington D.C. 1999 and Remington's
Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing
Co. Easton,
PA, 1995, pp. 1458-1460, both of which are incorporated herein by reference.
Physical stability refers to a formulation's ability to maintain its physical
form, for
example maintaining particle size; maintaining crystal or amorphous form;
maintaining
complexed form, such as hydrates and solvates; resistance to absorption of
ambient
moisture, i.e., hydroscopicity; and maintaining of mechanical properties, such
as
compressibility and flow characteristics. Methods for measuring physical
stability include
spectroscopy, sieving or testing, microscopy, sedimentation, stream scanning,
and light
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scattering. Polymorphic changes, for example, are usually detected by
differential scanning
calorimetry or quantitative infrared analysis. For a discussion of the theory
and methods of
measuring physical stability see Fiese et al., in The Theory and Practice of
Industrial
Pharmacy, 3rd ed., Lachman L.; Lieberman, H.A.; and Kanig, J.L. Eds., Lea and
Febiger,
Philadelphia, 1986 pp. 193-194 and Remington's Pharmaceutical Sciences, 18th
Edition,
ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995, pp. 1448-1451, both
of which
are incorporated herein by reference.
Permeability refers to the propensity of a component to pass across biological
membranes. Biological membranes act as lipid barriers to most pharmaceuticals
and permit
the absorption of lipid-soluble substances by passive diffusion. Lipid-
insoluble substances
can pass the barrier only with considerable difficulty. An in vitro procedure
for measuring
permeability characteristics consists of an aqueous/organic-lipid
layer/aqueous system.
Another in vitro procedure is the everted sac technique using segments of rat
small
intestines. For a discussion of the theory and methods of measuring
permeability see
Remington's Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack
Publishing Co. Easton, PA, 1995, pp. 1460-1461, incorporated herein by
reference.
Partitioning refers to how a component distributes itself between two phases
so that
each phase becomes saturated. If the component is added to the immiscible
solvent system
in an amount insufficient to saturate the solutions, it will distribute
between the solvents in a
definite ratio. Understanding the partitioning effect enables one to estimate
the site of
absorption, for example, whether a component is absorbed in the stomach or
small
intestines. For a discussion of the theory and methods of measuring
partitioning see Hansch
et al., 1972 J. Pharm. Sci. 61:1; Dressman et al., 1984 J. Pharm. Sci.
73:1274; Suzuki et al.,
1970 J. Pharm. Sci. 59:644; and Remington's Pharmaceutical Sciences, 18th
Edition, ed.
Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995, pp. 1451-1452, all of
which are
incorporated herein by reference.
Metabolic profile or metabolism refers to conversion of a pharmaceutical,
dietary
supplement, alternative medicine, or nutraceutical to another chemical within
a human or
animal after administration. As soon as a pharmaceutical enters the body it
becomes
susceptible to a variety of enzymatic or chemical metabolic processes in the
stomach,
intestine, and liver as well as other areas of the body. Metabolism can occur
at different
sites in the body, formulations are especially important in optimizing, for
example,
transdermal, dermal, and gastric metabolism. Metabolism can occur by
functional group
changes, such as ring or side chain hydroxylation, nitro-group reduction,
aldehyde
oxidation, dealkylation, deamination, etc. or by conjugation, wherein the
pharmaceutical
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combines with a solulizable group, such as glucuronic acid or glycine to form
an excretable
compound. Metabolism of an active component, such as a pharmaceutical, can be
enhanced
or retarded depending on the formulation in which the active component is
administered.
Although metabolism occurs within the body, it can be measured using a variety
of in vitro
assays. For example, certain oral pharmaceuticals, such as the penicillins,
are undesirably
metabolized through acid hydrolysis in the digestive tract. To estimate
efficacy of a
formulation for mitigating such metabolism and allowing the pharmaceutical to
pass into
the blood stream, a formulation comprising the pharmaceutical in question may
be placed in
a medium approximating the digestive medium and the rate or extent of
hydrolysis
measured by an appropriate analytical technique, such as HPLC. Similarly, for
pharmaceuticals known to be metabolized by certain enzymes, the efficacy of a
formulation
for promoting or retarding metabolism can be tested by exposing the
pharmaceutical
formulation to the enzyme or a similar enzyme under appropriate conditions and
measuring
the rate of degradation. For a discussion of the theory and methods of
measuring
metabolism see Remington's Pharmaceutical Sciences, 18th Edition, ed. Alfonso
Gennaro,
Mack Publishing Co. Easton, PA, 1995, pp. 431, 742, 1665, 1753, and 1831,
incorporated
herein by reference.
Compressibility refers to a powder's capacity to decrease in volume under
pressure,
while compactability refers to its capacity to be compressed into a tablet of
particular
strength or hardness. When a powder undergoes compression, the powder
particles adopt a
more efficient packing order and upon further pressure undergo elastic or
reversible
deformation. If the force were to be removed during this phase, the powder
would recover
to the efficiently packed state. Further application of pressure results in
compaction, i.e.,
irreversible deformation of the powder. The compaction stage is very important
in the
manufacture of tablets. For tableting, the pharmaceutical-in-formulation
mixture must be
amenable to irreversible deformation (compaction) upon pressure application to
a tablet
hard enough to resist erosion and disintegration and strong enough to resist
brittle fracture.
See Remington's Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro,
Mack
Publishing Co. Easton, PA, 1995, pp. 1457-1458 and chapter 92 and Jones et
al., 1977
Pharmazeutische Industrie, 39:469, both of which are incorporated herein by
reference
herein. Numerous techniques to measure to evaluate compressibility and
compactability are
published, e.g., see Rees et al., 1987 J. Pharm. Pharmacol. 30:601 and Jones
in Polermand
ed: Formulation and Preparation of Dosage Forms, Elsevier, North Holland, 29,
1977.
Power flow characteristics refer to a bulk powder's ability to flow. Powders
can be
broadly classified as free flowing or cohesive. Flow characteristics can be
influenced by
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particle size, shape, and morphology as well as other factor such as density,
electrostatic
charge, the presence of absorbed air or moisture. Free flowing powders can be
characterized by a simple flow-rate apparatus consisting of grounded metal
tube from which
the pharmaceutical flows through the orifice onto an electronic balance. For a
discussion of
measuring properties affecting flow characteristics see Kaye Chemical
analysis: Direct
Characterization ofFine Particles. Vol. 61, John Wiley and Sons, New York
1981; Sutton
et al., Characterization ofPowder Surfaces, Academic Press, London, 1976, pp.
1,7, and
158; Hiestand, et al., 1973 J. Pharm. Sci. 62:1513; and Hiestand, et al., 1974
J. Pharm. Sci.
63:605, all of which are incorporated by reference herein.
Friability is an indicator of cohesiveness and hardness of a powdered solid-
dosages
forms, e.g., tablets, of pharmaceuticals, dietary supplements, alternative
medicines, and
nutraceuticals. Friability can be measured by well-known methods, such as with
a Roche
friabilitor. For a discussion of friability and methods for measuring see
Remington's
Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing
Co. Easton,
PA, 1995, pp. 1639-1640, incorporated herein by reference.
Rheology concerns deformation and flow of matter. For pharmaceuticals, dietary
supplements, alternative medicines, nutraceuticals, consumer product
formulation and
industrial formulations, rheology relates to mechanical properties such
adhesive strength,
tackiness, and viscosity. Rheology is especially import in pharmaceutical
applications of
biopolymers and mucoadhesives. For example, in transdermal and dermal
applications
where a bioadhesive must adhere to the skin and later be removed without
causing pain.
Rheology is also important in industrial and consumer product formulations,
such as
polymers, adhesives, glues, gels, rubbers, inks, and plastics. Rheology can be
measured by
well-known methods, for a discussion see Remington 's Pharmaceutical Sciences,
18th
Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995, Ch. 22,
incorporated
herein by reference.
Dispersion relates to the mechanical disintegration properties of solids and
liquids
into small particles and their distribution and dissolution in a fluid
vehicle. Dispersion
properties are especially important in pharmaceutical, dietary supplement,
alternative
medicine, and nutraceutical formulations. Dispersion properties of
formulations can be
enhanced by additives, such as disintegrants and lubricants. For a discussion
of dispersion
and its relationship to dissolution and disintegration and tests therefor see
Remington :s
Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing
Co. Easton,
PA, 1995, pp. 595-596, incorporated herein by reference.
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Rate-of release refers to the release of a pharmaceutical from a
pharmaceutical
formulation, thus presenting the pharmaceutical for absorption. Control of
rate-of release
allows timed delivery of a dosage over an extended period or immediate
surrender of the
total dosage. Rate-of release is intimately related to the formulation's
properties, such as
friability, dispersion, disintegration, pharmaceutical concentration,
pharmaceutical-carrier
interactions, particle size and type, pH, polarity, surface tension, and
rheology, to name but
a few. For a discussion of and assays to measure rate-of release see, see
Chemical Aspects
of Drug Delivery Systems, eds. D.R. Karsa and R.A. Stephenson, The Royal
Society of
Chemistry, Cambridge, UK, 1996, incorporated herein by reference
Sensory Materials
In a another embodiment, the invention relates to an array of samples, each
sample
comprising a sensory-material-in-common and at least one additional component,
wherein
each sample differs from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the sensory-material-in-common to the additional
component; or
(iii) the physical state of the sensory-material-in-common.
As used herein, the term "sensory-material" means any chemical or substance,
known or to be developed, that is used to provide an olfactory or taste effect
in a human or
an animal, preferably, a fragrance material, a flavor material, or a spice. A
sensory-material
also includes any chemical or substance used to mask an odor or taste.
Examples of
suitable fragrances materials include, but are not limited to, musk materials,
such as
civetone, ambrettolide, ethylene brassylate, musk xylene, TonalideC~, and
Glaxolide~;
amber materials, such as ambrox, ambreinolide, and ambrinol; sandalwood
materials, such
as a-santalol, ~3-santalol, Sandalore~, and Bacdanol~; patchouli and woody
materials, such
as patchouli oil, patchouli alcohol, Timberol~ and Polywood~; materials with
floral odors,
such as Givescone~, damascone, irones, linalool, Lilial~, Lilestralis~, and
dihydrojasmonate. Other examples of suitable fragrance materials for use in
the invention
are listed in Perfumes: Art, Science, Technology, P.M. Muller ed. Elsevier,
New York,
1991, incorporated herein by reference. Examples of suitable flavor materials
include, but
are not limited to, benzaldehyde, anethole, dimethyl sulfide, vanillin, methyl
anthranilate,
nootkatone, and cinnamyl acetate. Examples of suitable spices include but are
not limited
to allspice, tarrogon, clove, pepper, sage, thyme, and coriander. Other
examples of suitable
flavor materials and spices are listed in Flavor and Fragrance Materials-1989,
Allured
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Publishing Corp. Wheaton, IL, 1989; Bauer and Garbe Common Flavor and
Fragrance
Materials, VCFI Verlagsgesellschaft, Weinheim, 1985; and (1994) The
Encyclopedia of
Chemical Technology, 11 Kirk-Othomer (4'" ed. at 1-61), all of which are
incorporated by
reference herein.
S Preferred components for use with sensory-materials are those known in the
art of
fragrance and flavor compounding, for example, components used to make
functional
products, such as those used to formulate colognes and perfumes, skin
cleansers, shower
products, skin-care products, gels, sun screens, deodorants and
antiperspirants, hair-care
products and shampoos, and cosmetics. Other examples of suitable components
are the
excipients listed above for use with pharmaceuticals as they can also be used
to formulate
formulations comprising sensory-materials. Other examples of suitable
components for
samples comprising sensory-materials are listed in Perfumes: Art, Science,
Technology,
P.M. Muller ed. Elsevier, New York, 1991 p. 338-345, 347-362, 40-42 and (1996)
The
Encyclopedia of Chemical Technology, 18 Kirk-Othomer (4'" ed. at 171-201 ),
both of which
1 S are incorporated by reference herein. In one aspect of the embodiment
concerning arrays of
samples, wherein the component-in-common is a sensory-material, the additional
components) are other sensory-materials. That is, each sample can comprise
multiple
sensory-materials, wherein a particular sensory-material is present in all the
samples
(sensory-material-in-common). An array of such samples can be used to detect
favorable or
synergistic interactions between sensory-materials, for example,
identification of a sensory-
material combination that is particularly advantageous due to their
interaction with each
other. Such advantageous combinations can be unexpected based on the
properties of the
sensory-materials in isolation.
In another embodiment, the invention relates to a method to measure or detect
an
interaction between a sensory-material and another component, comprising:
(a) preparing an array of samples, each sample comprising a sensory-material-
in-common and at least one additional component, wherein each sample
differs from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the sensory-material-in-common to the additional
component; or
(iii) the physical state of the sensory-material-in-common;
(b) testing each sample for a property to generate a data set; and
(c) analyzing the data set to measure or detect the interaction.
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Preferably, samples comprising sensory-materials are tested for properties
related to
their use in functional products, such as perfumes, air fresheners,
deodorants, colognes,
foods, candy, or fragranced or flavored pharmaceuticals. Preferred properties
for sensory
materials include strength and quality of the odor, substantivity (how long
the odor lasts),
S rate of evaporation, solubility, partitioning, biodegradability, odor,
taste, mouth feel,
appearance, lack of odor, lack of taste, toxicity, potency, texture, color,
appearance, or
partitioning and physical and chemical stability. One of skill in the art can
readily develop
assays to measure such properties, for instance, the methods discussed above
for measuring
pharmaceutical formulation properties. For example, strength and quality of
odor can be
measured by simply smelling the sample or by neurosensory techniques, such as
electronic
noses, see e.g., Matzger et al., 2000 J. Comb. Chem. 2:301-304; Gibson et al.,
2000 Chem.
Ind. (London) 8:287-289; Ormancey et al., 1998 Semin. Food Anal. 3:77-84;
Bain, H.,
Measurement of Consumer Perceptions and Evaluation of odor as an Aid to
Perfume
Selection, In ESOMAR Seminar on Research for Flavors and Fragrances, Lyon
1989,
I S Esomar, Amsterdam, 1989, all four of which are incorporated herein by
reference.
Properties such as substantivity, rate of evaporation, solubility,
partitioning, and physical
and chemical stability can be measured by adaptation of the methods discussed
above for
pharmaceuticals.
A~rochemicals
In still another embodiment, the invention relates to an array of samples,
each
sample comprising an agrochemical-in-common and at least one additional
component,
wherein each sample differs from any other sample with respect to at least one
of:
(i) the identity of the additional component,
(ii) the ratio of the agrochemical-in-common to the additional
component; or
(iii) the physical state of the agrochemical-in-common.
As used herein, the term "agrochemical" means any substance known or to be
developed that is used on the farm, yard, or in the house or living area to
benefit gardens,
crops, ornamental plants, shrubs, or vegetables or kill insects, plants, or
fungi. Examples of
suitable agrochemicals for use in the invention include pesticides,
herbicides, fungicides,
insect repellants, fertilizers, and growth enhancers. For a discussion of
agrochemicals see
The Agrochemicals Handbook ( 1987) 2nd Edition, Hartley and Kidd; editors: The
Royal
Society of Chemistry, Nottingham, England.
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Pesticides include chemicals, compounds, and substances administered to kill
vermin such as bugs, mice, and rats and to repel garden pests such as deer and
woodchucks.
Examples of suitable pesticides that can be used according to the invention
include, but are
not limited to, abamectin (acaricide), bifenthrin (acaricide), cyphenothrin
(insecticide),
imidacloprid (insecticide), and prallethrin (insectide). Other examples of
suitable pesticides
for use in the invention are listed in Crop Protection Chemicals Reference,
6th ed.,
Chemical and Pharmaceutical Press, John Wiley & Sons Inc., New York, 1990; (
1996) The
Encyclopedia ofChemical Technology, 18 Kirk-Othomer (4'" ed. at 311-341); and
Hayes et
al., Handbook of Pesticide Toxicology, Academic Press, Inc., San Diego, CA,
1990, all of
which are incorporated by reference herein.
Herbicides include selective and non-selective chemicals, compounds, and
substances administered to kill plants or inhibit plant growth. Examples of
suitable
herbicides include, but are not limited to, photosystem I inhibitors, such as
actifluorfen;
photosystem II inhibitors, such as atrazine; bleaching herbicides, such as
fluridone and
difunon; chlorophyll biosynthesis inhibitors, such as DTP, clethodim,
sethoxydim, methyl
haloxyfop, tralkoxydim, and alacholor; inducers of damage to antioxidative
system, such as
paraquat; amino-acid and nucleotide biosynthesis inhibitors, such as
phaseolotoxin and
imazapyr; cell division inhibitors, such as pronamide; and plant growth
regulator synthesis
and function inhibitors, such as dicamba, chloramben, dichlofop, and
ancymidol. Other
examples of suitable herbicides are listed in Herbicide Handbook, 6th ed.,
Weed Science
Society of America, Champaign, Il 1989; ( 1995) The Encyclopedia of Chemical
Technology, 13 Kirk-Othomer (4'" ed. at 73-136); and Duke, Handbook of
Biologically
Active Phytochemicals and Their Activities, CRC Press, Boca Raton, FL, 1992,
all of which
are incorporated herein by reference.
Fungicides include chemicals, compounds, and substances administered to plants
and crops that selectively or non-selectively kill fungi. For use in the
invention, a fungicide
can be systemic or non-systemic. Examples of suitable non-systemic fungicides
include,
but are not limited to, thiocarbamate and thiurame derivatives, such as
ferbam, ziram,
thiram, and nabam; imides, such as captan, folpet, captafol, and
dichlofluanid; aromatic
hydrocarbons, such as quintozene, dinocap, and chloroneb; dicarboximides, such
as
vinclozolin, chlozolinate, and iprodione. Example of systemic fungicides
include, but are
not limited to, mitochondiral respiration inhibitors, such as carboxin,
oxycarboxin,
flutolanil, fenfuram, mepronil, and methfuroxam; microtubulin polymerization
inhibitors,
such as thiabendazole, fuberidazole, carbendazim, and benomyl; inhibitors of
sterol
biosynthesis, such as triforine, fenarimol, nuarimol, imazalil, triadimefon,
propiconazole,
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WO 01/09391 PCT/US00/20717
flusilazole, dodemorph, tridemorph, and fenpropidin; and RNA biosynthesis
inhibitors, such
as ethirimol and dimethirimol; phopholipic biosynthesis inhibitors, such as
ediphenphos and
iprobenphos. Other examples of suitable fungicides are listed in Torgeson,
ed., Fungicides:
An Advanced Treatise, Vols. 1 and 2, Academic Press, Inc., New York, 1967 and
(1994)
The Encyclopedia of Chemical Technology, 12 Kirk-Othomer (4'" ed. at 73-227),
all of
which are incorporated herein by reference.
Components to be combined with agrochemicals to form samples include those
substances known in the art of agrochemical compounding, for example, inert
ingredients,
such as solvents, emulsifiers, surfactants, dispersants, stabilizers,
preservatives,
sequestrates, colors, flavors, and fragrances. Examples of suitable components
for samples
comprising agrochemicals are listed in Stevens et al., 1993 Pesticide Science
38:103-122
and Adjuvants for Agrochemicals, ed. C. L. Foy, CRC Press, Boca Raton,
Florida, 1992.
In one aspect of the embodiment concerning arrays of samples, wherein the
component-in-common is an agrochemical, other agrochemicals are additional
components.
That is, each sample can comprise multiple agrochemicals, wherein a particular
agrochemical is present in all the samples (agrochemical-in-common). An array
of such
samples can be used to detect a favorable or synergistic interaction between
agrochemicals,
for example, identification of a pesticide combination that is particularly
advantageous due
to their interaction with each other. Such advantageous combinations can be
unexpected
based on the properties of the agrochemicals in isolation.
In another embodiment, the invention relates to a method to measure or detect
an
interaction between components, comprising:
(a) preparing an array of samples, each sample comprising an agrochemical-in-
common and at least one additional component, wherein each sample differs
from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the agrochemical-in-common to the additional
component; or
(iii) the physical state of the agrochemical-in-common;
(b) testing each sample for a property to generate a data set; and
(c) analyzing the data set to measure or detect the interaction.
Preferably, samples comprising agrochemicals are tested for properties related
to the
use and administration of agrochemicals. Preferred properties for
agrochemicals include
biodegradability (e.g., rate of degradation due to chemical instability or
microbial
metabolism), rate of soil absorption, potency, toxicity, duration of effect,
rate of
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evaporation. Such properties are directly related to solubility (especially
aqueous
solubility), partitioning, and physical and chemical stability, which can be
measured by the
methods discussed above for measuring pharmaceutical formulation properties.
One of skill
in the art can readily develop assays to measure such properties. For
references aiding
S development of assays related to measuring agrochemical properties see
Somasundaram et
al., Pesticide Transformation Products: Fate and Significance in the
Environment, ACS
Symposium Series No. 459, American Chemical Society 1991. Tweedy et al.,
Pesticide
Residues and Food Safety: A Harvest of Viewpoints, ACS Symposium Series No.
446,
American Chemical Society 1991; Van Emon et al., Immunochemical Methods For
Environmental Analysis, ACS Symposium Series No. 442, American Chemical
Society
1990; and Cairns et al., Emerging Strategies for Pesticide Analysis, CRC
Press, Boca
Raton, FL 1992; From the series Modern Methods of Pesticide Analysis, all of
which
references are incorporated herein by reference.
Consumer and Industrial Product Formulations
In another embodiment, the invention relates to an array of samples, each
sample
comprising a component-in-common, wherein the component-in-common is an active
component of a consumer product formulation or an active component of an
industrial
product formulation and at least one additional component, wherein each sample
differs
from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common.
As used herein, a "consumer product formulation" means a formulation for
consumer use, not intended to be absorbed or ingested into the body of a human
or animal,
comprising an active component. Consumer product formulations include, but are
not
limited to, cosmetics, such as lotions, facial makeup; antiperspirants and
deodorants,
shaving products, and nail care products; hair products, such as and shampoos,
colorants,
conditioners; hand and body soaps; paints; lubricants; adhesives; and
detergents and
cleaners.
As used herein an "industrial product formulation" means a formulation for
industrial use, not intended to be absorbed or ingested into the body of a
human or animal.
Industrial product formulations include, but are not limited to, polymers;
rubbers; plastics;
industrial chemicals, such as solvents, bleaching agents, inks, dyes, fire
retardants,
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antifreezes and formulations for deicing roads, cars, trucks, jets, and
airplanes; industrial
lubricants; industrial adhesives; construction materials, such as cements.
One of skill in the art will readily be able to choose active components and
inactive
components used in consumer and industrial product formulations and set up
arrays
S according to the invention for detecting or measuring an interaction between
the active
components and additional components. Data concerning such interactions can
aid in the
optimization of consumer and industrial product formulations. Such active
components and
inactive components are well known in the literature and the following
references are
provided merely by way of example. Active components and inactive components
for use
in cosmetic formulations are listed in (1993) The Encyclopedia of Chemical
Technology, 7
Kirk-Othomer (4'" ed. at 572-619); M.G. de Navarre, The Chemistry and
Manufacture of
Cosmetics, D. Van Nostrand Company, Inc., New York, 1941; CTFA International
Cosmetic Ingredient Dictionary and Handbook, 8th Ed., CTFA, Washington, D.C.,
2000;
and A. Nowak, Cosmetic Preparations, Micelle Press, London, 1991. All of which
are
incorporated by reference herein. Active components and inactive components
for use in
hair care products are listed in (1994) The Encyclopedia of Chemical
Technology, 12 Kirk-
Othomer (4'" ed. at 881-890) and Shampoos and Hair Preparations in ECT 1st
ed., Vol. 12,
pp. 221-243, by F. E. Wall, both of which are incorporated by reference
herein. Active
components and inactive components for use in hand and body soaps are listed
in ( 1997)
The Encyclopedia of Chemical Technology, 22 Kirk-Othomer (4'" ed. at 297-396),
incorporated by reference herein. Active components and inactive components
for use in
paints are listed in (1996) The Encyclopedia of Chemical Technology, 17 Kirk-
Othomer (4'"
ed. at 1049-1069) and "Paint" in ECT 1st ed., Vol. 9, pp. 770-803, by H.E.
Hillman, Eagle
Paint and Varnish Corp, both of which are incorporated by reference herein.
Active
components and inactive components for use in consumer and industrial
lubricants are
listed in ( 1995) The Encyclopedia of Chemical Technology, 15 Kirk-Othomer
(4'" ed. at
463-517); D.D. Fuller, Theory and practice of Lubrication for Engineers, 2nd
ed., John
Wiley & Sons, Inc., 1984; and A. Raimondi and A.Z. Szeri, in E.R. Booser,
eds., Handbook
of Lubrication, Vol. 2, CRC Press Inc., Boca Raton, FL, 1983, all of which are
incorporated
by reference herein. Active components and inactive components for use in
consumer and
industrial adhesives are listed in (1991) The Encyclopedia of Chemical
Technology, 1 Kirk-
Othomer (4'" ed. at 445-465) and LM. Skeist, ed. Handbook ofAdhesives, 3rd ed.
Van
Nostrand-Reinhold, New York, 1990, both of which are incorporated herein by
reference.
Active components and inactive components for use in polymers are listed in
(1996) The
Encyclopedia of Chemical Technology, 19 Kirk-Othomer (4'" ed. at 881-904),
incorporated
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herein by reference. Active components and inactive components for use in
rubbers are
listed in (1997) The Encyclopedia ofChemical Technology, 21 Kirk-Othomer (4'"
ed. at
460-591 ), incorporated herein by reference. Active components and inactive
components
for use in plastics are listed in (1996) The Encyclopedia of Chemical
Technology, 19 Kirk-
Othomer (4'" ed. at 290-316), incorporated herein by reference. Active
components and
inactive components for use with industrial chemicals are listed in Ash et
al., Handbook of
Industrial Chemical Additives, VCH Publishers, New York 1991, incorporated
herein by
reference. Active components and inactive components for use in bleaching
components
are listed in (1992) The Encyclopedia of Chemical Technology, 4 Kirk-Othomer
(4'" ed. at
271-311), incorporated herein by reference. Active components and inactive
components
for use inks are listed in (1995) The Encyclopedia of Chemical Technology, 14
Kirk-
Othomer (4'" ed. at 482-503), incorporated herein by reference. Active
components and
inactive components for use in dyes are listed in (1993) The Encyclopedia of
Chemical
Technology, 8 Kirk-Othomer (4'" ed. at 533-860), incorporated herein by
reference. Active
components and inactive components for use in fire retardants are listed in
(1993) The
Encyclopedia of Chemical Technology, 10 Kirk-Othomer (4'" ed. at 930-1022),
incorporated
herein by reference. Active components and inactive components for use in
antifreezes and
deicers are listed in (1992) The Encyclopedia of Chemical Technology, 3 Kirk-
Othomer (4'"
ed. at 347-367), incorporated herein by reference. Active components and
inactive
components for use in cement are listed in (1993) The Encyclopedia of Chemical
Technology, 5 Kirk-Othomer (4'" ed. at 564), incorporated herein by reference.
In another embodiment, the invention relates to a method to measure or detect
an
interaction between components, comprising:
(a) preparing an array of samples, each sample comprising a component-in-
common, wherein the component-in-common is an active component of a
consumer product formulation or an active component of an industrial
product formulation and at least one additional component, wherein each
sample differs from any other sample with respect to at least one of:
(i) the identity of the additional component,
(ii) the ratio of the component-in-common to the additional component;
or
(iii) the physical state of the component-in-common;
(b) testing each sample for a property to generate a data set; and
(c) analyzing the data set to measure or detect the interaction.
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Preferably, samples comprising the active component of a consumer or
industrial
product formulation and an additional component are tested for properties
related to the use
and administration of the consumer or industrial product formulation.
Preferred properties
for testing samples comprising the active component of an industrial or
consumer product
formulations include solubility (especially aqueous solubility),
biodegradability,
partitioning, and physical and chemical stability. Such properties can be
measured by
adapting the methods discussed above for measuring pharmaceutical formulation
properties.
According to the invention described herein, once an ideal formulation is
identified,
it can be prepared on a bulk scale for use as formulation for delivery of
active components,
using standard scale-up procedures well known in the art. For example, a
promising sample
can be scaled up as a pharmaceutical formulation for delivery of
pharmaceuticals.
Sample Preparation and Testing Systems
The basic requirements for sample preparation, processing, and testing are: (
1 ) a
distribution mechanism to add components to separate sites on an array plate,
such as into
sample wells. Preferably, the distribution mechanism is automated and
controlled by
computer software and can vary at least one addition variable, e.g., the
identity of the
components) and/or the component concentration, more preferably, two or more
variables.
For instance, addition of a pharmaceutical-in-common and excipients to a
sample well
involves material handling technologies and robotics well known to those
skilled in the art
of pharmaceutical process manufacturing. Of course, if desired, individual
components can
be placed into the appropriate well in the array manually. This pick and place
technique is
also known to those skilled in the art. And (2) a testing mechanism to test
each sample for
one or more properties. Preferably, the testing mechanism is automated and
driven by a
computer. Preferably, the system further comprises a processing mechanism to
process the
samples after component addition. For example, after component addition, the
samples can
be processed by stirring, milling, filtering, centrifuging, emulsifying, or
solvent removal
(e.g., lyophilizing) and reconstituting, etc. by methods and devices well
known in the art.
Preferably the samples are processed automatically and concurrently.
Figure 1 is a flow chart depicting preparing an array of samples, processing
the
samples, analyzing the samples for one or more properties, collecting and
storing the data,
and analyzing the data, and detecting or measuring an interaction or detecting
a lack of an
interaction. The first step comprises selecting the component sources 2, i.e.,
a component-
in-common and one or more additional components at one or more concentrations.
Next,
adding the component-in-common and additional components to a plurality of
sample sites,
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WO 01/09391 PCT/US00/20717
such as sample wells on a sample plate to give samples then processing the
samples by, for
example, stirring, milling, filtering, centrifuging, emulsifying, or
concentrating and
reconstituting to form an array of samples 4. Each sample in array 4 can be
tested for one or
more properties and the data collected and stored 6 for subsequent data
analysis 8 to
S measure or identify an interactions) or detect a lack of interactions) 9
between
components. For example, a component-in-common, such as a pharmaceutical, and
additional components, such as excipients or other pharmaceuticals in
different amounts,
different pHs, different physical states, can be distributed in liquid or
solid form, or
combination thereof, into individual sample sites, such as sample wells,
thereby forming a
s~ple array. The different samples comprising different combinations can be
processed
and tested for properties. The data from testing is stored and analyzed,
preferably by a
computer, to measure or identify interactions between the pharmaceutical-in-
common and
excipients or other pharmaceuticals. Such interactions can be used to develop
optimum
formulations to administer the pharmaceutical.
The automated distribution mechanism can distribute or add components in the
form
of liquids, gels, foams, pastes, ointments, triturates, suspensions, or
emulsions or solids,
such as powders, tablets, or pellets. Preferably, solids are in the form of
micropellets or
microtablets, prepared by micropelleting or microtableting. Micropellets can
be prepared
using standard pharmaceutical tableting machines, modified as appropriate.
Such machines
~e well known in the art, for example, see Remington's Pharmaceutical
Sciences, 18th
Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, PA, 1995, Chapter
92,
incorporated herein by reference. Preferably, the tableting machine comprises
a small die,
of from about 1/16" to about 3/16" in diameter. Any required modifications are
easily made
by those skilled in the art. With the appropriate modification, the
microtableting machine
C~ make microtablets of almost any solid component. When the component is an
active
component, such as a pharmaceutical, preferably, it is dispersed within a
matrix of a
compressible, inert carrier material, such as potassium chloride. Preferably,
the ratio of
active component to carrier is about 0.5 to about 10 parts active component to
about 90 to
about 99.5 parts of carrier, more preferably, about 1 part active component to
about 99 parts
of carrier. Preferably, the finished microtablets weigh from about 0.1 to
about 50
milligrams, more preferably, from about 1 to about 10 milligrams, most
preferably, about 5
milligrams. It is also preferable that the finished microtablet contain from
about 1 to about
100 micrograms of the active component, more preferably, from about 10 to
about 75
micrograms, and most preferably, they contain about 50 micrograms. When the
component
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is an inactive component, such as an excipient, it can be dispersed within an
inert matrix as
just described or pelleted in the absence of an inert Garner.
Another non-limiting method of forming micropellets involves forcing a paste
comprising a component and an inert carrier into a mold, drying the paste, and
then ejecting
S the pellet. In this method, the component to be pelleted is first
homogenized with an inert
carrier and a solvent. Preferably, the inert carrier is lactose or mannitol.
Any solvent is
suitable and readily selected by one skilled in the art depending on the
component.
Preferably, the solvent is relatively volatile , more preferably having a
boiling point of about
100 °C or less, for example, alcohols such as methanol or ethanol.
Preferably, the ratio of
solvent to active component/inert carrier mixture is of from about 10:1 to
about 1:10, more
preferably about 6:1 to about 1:1, even more preferably, from about 5:1 to
about 3:1. The
component, solvent, and inert carrier are homogenized to a paste and the
solvent is then
removed at reduced pressure to yield a dry powder. The powder is then mixed
with another
solvent, preferably water, to form a homogeneous paste, which paste is forced
into
individual tube shaped molds. Preferably, the dimensions of the molds is from
about 1/16"
to about 3/16" in depth, preferably, about 1/8" in depth and from about 1/32"
to about 1/8"
in inner diameter, preferably, about 5/64" inner diameter. The pastes are
allowed to dry for
about 1 minute to about S hours, preferably, for about 5 minutes to about 1
hour, more
preferably, for about 10 minutes; at a temperature of from about 15 °C
to about 100 °C,
more preferably, from about 20 °C to about 30 °C; at a pressure
of from about 10 mm/Hg to
about 1000 mm/Hg, preferably, at about 20 mm/Hg. Preferably, the paste-in-mold
is
allowed to dry for about 10 minutes, at about room temperature, and at about
atomospheric
pressure. The dried paste in the form of a micropellet is then ejected from
the molds,
preferably, by inserting a flat head pin through the mold, the pin being of
about the same
diameter, preferably of just a slightly smaller diameter than the inner
diameter of the mold.
The molded micropellets can then be dried under reduced pressure, preferably,
for from
about 6 to about 24 hours, more preferably, about 12 hours; at a temperature
of from about
15 °C to about 100 °C, preferably at about room temperature; and
at a pressure of from
about I Omm/Hg to about 1000 mm/Hg, preferably, about 20 mm/Hg.
A preferred automated mechanism for adding solid components, preferably
micropellets, to sample wells, comprises reservoirs or bins, for each
component. The outlet
of these bins is controlled so that an individual microtablet is "singulated"
and able to be
dispensed to a specified sample well in an array. Once the components and the
component-
in-common are placed in the sample wells of the array, the assay process
continues as
outlined below.
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Figures 2A and 2B are detailed schematics exemplifying a system of the
invention
for preparing an array of samples by depositing components into sample wells,
processing
the samples, testing the samples for one or more properties to generate data,
and collecting,
storing, and analyzing the data to measure or detect interactions or detect a
lack of
interactions. Figures 2A and 2B are directed to low-water-solubility
pharmaceuticals as the
component-in-common and to excipients as the additional components, however,
the
systems and methods depicted in Figures 2A and 2B apply equally to all the
active and
inactive components discussed herein.
Figure 2A depicts a system where an solid component-in-common source 10, such
as a solid-pharmaceutical source and an solid-component source 12, such as a
solid-
excipient source are automatically distributed, preferably in micropellet
form, to sample
sites, such as sample wells in a 96 well filter plate (commercially available,
for example,
from Millipore, Bedford, MA) to give a plurality of dry samples 14. The
combinations of
the component-in-common and various components at various combinations are
generated
using standard software (e.g., Matlab software, commercially available from
Mathworks,
Natick, Massachusetts). The combinations thus generated can be downloaded into
a spread
sheet, such as Microsoft EXCEL. From the spread sheet, a worklist can be
generated for
instructing the automated distribution mechanism to prepare an array of
samples according
to the various combinations generated by the formulating software. The
worklist can be
generated using standard programming methods according to the automated
distribution
mechanism employed. The use of so-called worklists simply allows a file to be
used as the
process command rather than discrete programmed steps. The worklist combines
the
formulation output of the formulating program with the appropriate commands in
a file
format that directly readable by the automatic distribution mechanism.. The
automated
distribution mechanism delivers at least one component-in-common, such as a
pharmaceutical, as well as various additional components, such as excipients,
to each
sample well. Preferably, the automated distribution mechanism can deliver
multiple
amounts of each component. Automated liquid distribution mechanisms are well
known
and commercially available, such as the Tecan Genesis, from Tecan-US, RTP,
North
C~olina. Automated solid distribution mechanisms are readily obtained by
modifying
commercially available robotics systems. The plurality of dry samples 14 thus
formed are
mixed with one or more solvents using the Tecan automated liquid pipetting
system thereby
providing an array of samples 20, wherein each sample comprises a solution or
a
suspension. During or after solvent addition, the samples may be mixed or
agitated,
preferably, however, the force of the liquid addition provides adequate
mixing. If desired,
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WO 01/09391 PCT/US00/20717
suspensions can be filtered to separate the solid and liquid phases thus
forming an array of
filtrates 22. To accomplish filtration, the filter plate comprising the
suspension is placed on
top of a receiver plate containing the same number of sample wells, each of
which
corresponds to a sample well on the filter plate. By applying either
centrifugal or vacuum
force to the filter plate over receiver plate combination, the liquid phase of
the filter plate is
forced through the filter on the bottom of each sample well, into the
corresponding sample
well of the receiver plate. The appropriated centrifuge is available
commercially, for
example, from DuPont, Wilmington, DE. The receiver plate is designed for
analysis of the
individual filtrate samples.
Analysis of the filtrates can provide data concerning the component solubility
using
devices 24, such as UV-Vis spectroscopy (using plate-based readers known to
those skilled
in the art, an example of which is the SpectraMax Plus from Molecular Devices,
Sunnyvale,
CA), GC, HPLC, and LC-MS. In the case of GC, HPLC, and LC-MS, an automated
pipetting station is used for sample introduction, for example, the Genesis
from Tecan or
~y of several devices sold by Gilson, Middleton, WI). Analytial devices used
to measure
stability 26 include, but are not limited to, UV-Vis spectroscopy, MS, GC,
HPLC, or LC
MS. These devices are also modified for use with the array as previously
discussed.
Analytical devices for measuring absorption 28 include the Caco-2 cell line or
Ussing
Chambers. The Caco-2 cell line is known to those skilled in the art to be a
surrogate for
intestinal permeability measurements. There are analysis devices sold
specifically for
absorption assay, namely those sold by Tecan-US (Cell-growth and Cell-feeding
platforms).
Ussing chambers are widely used by those skilled in the art to measure
intestinal
permeability of compounds. There are commercially available devices, such as
those sold
by World Precision Instruments, Sarasota, Florida, for measuring absorption.
Systems for
measuring metabolism 30 include P-450, microsomes and lysosomes, obtainable
from
companies such as In Vitro Technologies, 1450 South Rolling Road, Baltimore,
MD 21227;
see also; Trouet A et al., Methods in Enzymology, Vol. 31, Academic Press,
Inc., New
York, 1987, pp. 287-313 and 323-329) and in vivo assays. Other analytical
devices that can
be used with the methods and arrays of the invention include pH sensors, ionic
strength
sensors, optical spectrometers, devices for measuring turbidity, calorimeters,
infrared
spectrometers, polarimeters, radioactivity counters, conductivity measurers,
and heat of
dissolution measurers. The property-test data is collected and stored 32 then
analyzed 34 to
detect or measure interactions 36.
Data collection and storage 32 preferably, are performed by computers using
the
appropriate software. Such computers and software for data collection and
storage are
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WO 01/09391 CA 02379160 2002-O1-14 pCT/~jS00/20717
readily chosen by one of skill in the art. The samples are first analyzed
using the
appropriate equipment, as just discussed, for the property under study to
generate a data set.
For example, a UV spectrophotometric analysis of each sample can be obtained
using a
Spectramax PIusTM spectrophotometer available from Molecular Devices,
Sunnyvale, CA.
The data is typically collected and stored using software provided by the
instrumentation
manufacturer. For example, the data collected by the Spectramax is collected
and stored
using SoftproTM from Molecular Devices. The data set can then be downloaded to
a
database for analysis.
Data analysis 34 can be performed using visualization software, such as
SPOTFIRE
(commercially available from Spotfire, Inc., Cambridge, MA). The visualized
data can be
analyzed directly to arrive at optimized formulations and interactions 36. Or
the data can be
processed through data mining algorithms so as to optimize the ability of
scientific
personnel to detect complex mufti-dimensional interactions or lack of
interactions between
components or to conduct future experiments to optimize the formulations.
Examples of
suitable data-mining software include, but not limited to, SPOTFIRE; MATLAB
(Mathworks, Natick, Massachusetts); STATISTICA (Statsoft, Tulsa, Oklahoma).
All
resulting analysis files are stored on a central file server, i.e., a data
base, where the files can
be accessed by traditional means known to those skilled in the art.
Figure 2B shows essentially the same process but for liquid components-in-
common
3g~ such as liquid pharmaceuticals and liquid components) 40, such as liquid
excipients.
Such liquid components can be dispensed using an automated liquid distribution
mechanism to make the sample array 42. The solvent is then removed at reduced
pressure
or by evaporation to give an array of dried samples 44. Lyophilization or
other solvent
removal procedures, are readily adapted to arrays 42 by those skilled in the
art.
Commercially available lyophilization devices that can be used without
modification. The
array of dried samples 44 can be reconstituted to an array of reconstituted
samples 46 by
adding one or more component solvents using an automated liquid pipetting
device, as
previously described. The array of reconstituted samples 46 can then be
analyzed as
discussed above for Figure 2A, by.
Although the present invention has been described in considerable detail with
reference to certain preferred embodiments, other embodiments are possible.
Therefore, the
spirit and scope of the appended claims should not be limited to the
description of the
preferred embodiments contained herein.
3 S EXAMPLES
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The present invention will be further understood by reference to the following
non-
limiting example of the arrays and methods disclosed herein. The following
examples are
provided for illustrative purposes only and are not to be construed as
limiting the
invention's scope in any manner.
Griseofulvin, a commercial antibiotic having a complex derivatized benzofuran-
cyclohexane structure, was selected as the component-in-common for use in an
array to
identify excipient combinations that solubilize the pharmaceutical in aqueous
medium more
effectively than the present commercial griseofulvin formulation. The compound
is
marketed for oral administration. A dosage of 3.3 milligrams/lb body weight is
administered per day for children under 50 lbs and 330 milligrams/day is the
typical dosage
for adults. The selected compound is practically insoluble in water, slightly
soluble in polar
organic solvents such as ethanol, methanol, acetone and acetic acid, and
soluble in
dimethylformamide and dioxane. The low-water solubility limits the
bioavailability. The
commercially available preparation, used as a standard, consists of
ultramicrosized crystals
of the compound partially dissolved in a carrier including polyethylene glycol
8000 and
partially dispersed in other inert excipients (corn starch, lactose, magnesium
stearate, and
sodium lauryl sulfate).
Using the methods of the invention, an array of samples, each containing
griseofulvin and various excipient mixtures were rapidly prepared and
systematically
analyzed to identify excipient combinations that interact with each other and
with the
compound to provide aqueous solubility enhanced over the commercial
formulation.
The following GRAS ("generally regarded as safe") excipients (all obtainable
from
Sigma-Aldrich Fine Chemicals or BASF) listed in Table 2 below, were used as
excipients in
the following examples.
30
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WO 01/09391 CA 02379160 2002-O1-14 pCT/US00/20717
Table 2: Excipients Used in the Examples
( 1 gum arabic from acacia tree (a branched polymer
) of galactose, rhamnose,
arabinose, and glucuronic acid, mw approximately
25,000)
(2) (3-cyclodextrin (cycloheptaamylose),
(3) sodium dodecyl sulfate (SDS)
(4) sodium docusate (sulfobutanedioic acid bis[2-ethyl-hexl
ester] or dioctyl
sulfosuccinate)
(5) sodium benzethonium chloride
(6) benzalkonium chloride (alkyldimethylbenzylammonium
chloride)
(7) cetrimide (dodecyltrimethylainmonium bromide)
(8) oleic acid (cis-9-octadecenoic acid)
(9) sodium tartrate dihydrate
(10) polyethylene glycol 1000
( 11 polyethylene glycol 10,000
)
(12) polyvinyl alcohol
(13) POLOXAMER~ 237 (polyoxyalkylene oxide block copolymer)
( 14) polyoxyethylene 40 stearate
(15) polyoxyethylene 100 stearate
(16) TWEEN 80~ (polyoxyethylenesorbitan)
(17) BRIJ 35~ (23 lauryl ether)
(1g) BRIJ 97~ (10 Oleyl ether)
The samples were prepared, processed, and analyzed as demonstrated by the
following
examples.
35
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WO 01/09391 PCT/US00/20717
EXAMPLE 1: Preparation and Identification of Griseofulvin Formulations with
Enhanced
Solubility.
Preparation of formulations with improved solubilities
Each of the 18 excipients were prepared as three aqueous stock solutions at
different
concentrations, i.e., 0.015 milligrams/milliliter, 0.15 milligrams/milliliter,
and 1.5
milligrams/milliliter, to give a total of 54 stock solutions. To each sample
well was added
three different excipients, each excipient chosen from one of its respective
three stock
solutions. Thus, three of the 18 excipients listed above, 20 micro liters
each, were added to
each sample well in the array (Millipore 96 well filter plates, with a volume
of 250 p1,
including one micron pore size polytetrafluoroethylene membranes in the bottom
of each
well using the TECAN~ liquid handling device using the Genesis liquid handling
device
(Tecan-US, RTP, NC). The number of possible unique combinations of 3 different
excipients, each excipient chosen from three different-concentration stock
solutions is
calculated as:
18! x 33
3! x (18-3)!
Giving a grand total of 22,032 unique samples (a total of 66,096 samples for
n=3) were
generated, with 32 unique samples per assay plate and 689 assay plates in
total. All
permutations of excipients, concentrations, and griseofulvin according to the
above equation
were generated using the MatLab program formulating software. The permutations
so
generated were down loaded into a Microsoft EXCEL spread sheet and from this
spread
sheet, a worklist was constructed according to standard programming methods
well known
to those skilled in the art. The work-list is then used to direct the
automated distribution
mechanism to prepare the various permutations of excipients and griseofulvin
generated by
Matlab. The Genesis liquid handling device was used as the automated
distribution
mechanism. The worklist combines the formulation output of the Matlab program
with
Genesis-appropriate commands (as found in the Genesis operating manual) in a
file format
this is directly readable by the Genesis device. Thus, the Genesis liquid
handling device
delivered 20 micro liters of a griseofulvin/dioxane solution (0.15 milligrams
griseofulvin/milliliter dioxane) and the various combinations and
concentrations of
excipients generated by the Matlab program to each sample well. The force
provided by
adding the excipient was enough to adequately mix the components.
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WO 01/09391 PCT/US00/20717
Testing of Samples
All the solvents were removed by lyophilization then water (200 p1) was added
to
each dried well of the filter plates, again using the Genesis liquid handling
device. The
plates were the incubated at 37 °C for 1 hour in a Innova 4200
incubator (New Brunswick
S Scientific, Edison, NJ). The plates were then centrifuged to separate any
undissolved solids,
as previously described. The centrifuge was a Sorvall RT6000B (DuPont,
Wilmington,
DE). The filtrate from each well was collected into UV transparent 96 well
receiver plates
(Corning, Corning, NY) for measurement on a UV plate reader at 290 nm
(SpectraMax
Plus, Molecular Devices, Sunnyvale, CA).
Griseofulvin alone without any excipient was tested to establish the baseline
solubility.
Results
The solubility, measured as absorbance at 290 nm of the filtrates, for 3,500
unique
samples is shown in Figure 3A. The commercial griseofulvin preparation
FULVICIN,
Schering-Plough) alone gave a baseline absorbance (solubility) of 0.3
absorbance units.
Most samples showed improved solubility compared to the commercial
griseofulvin
preparation. Approximately 1,200 samples shown in Figure 3A showed
significantly higher
solubilities than the rest of the samples.
Samples demonstrating 100% increase in solubility compared to griseofulvin
alone
(as measured by absorbance) were identified as lead formulations. Five samples
were
identified and boxed in Figure 3A. The compositions of the 5 lead formulations
(TP 1-TPS)
are listed in Table 3 below.
TABLE 3: 5 Lead Formulations for Solubilizin~ Griseofulvin
TPl TP2 TP3 TP4 TPS
Excipient wt % Excipient wt Excipient wt % Excipient wt % Excipient wt
(10) 83.3 (10) 90% (10) 90% (11) 83.3 (12) 90%
(2) 8.3 % (3) 1% (14) 9% (1) 8.3% (S) 9%
(14) 8.3% (14) 9% (1) I% (7) 8.3% (10) 1%
Figure 3B gives the standard deviation for each of the 3,500 unique samples
(each
tested at n = 3). The majority of the samples tested were reproducible, with
standard
deviations of less than 10%.
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These samples can be further optimized using the same testing technique
described
above, by making additional changes to the concentration of each component in
the
samples.
EXAMPLE 2: Validation of Lead Formulations on a Larger Scale.
The five lead formulations identified from the Example above were validated on
a
lab scale 10,000 times that of the microarray in 96 well plates by weighing
each component
and mixing them in the solid state in scintillation vials. Griseofulvin (30
milligrams) was
weighed into each formulation. Each formulation was formulated three times.
Formulations
TPI-l: 300 milligrams PEG 1000, 30 milligrams beta-cyclodextrin, 30 milligrams
polyoxyethylene 40 stearate
TPI-2: 300 milligrams PEG 1000, 30 milligrams SDS, 3 milligrams
polyoxyethylene 40
stearate
TPI-3: 300 milligrams PEG 1000, 30 milligrams polyoxyethylene 40 stearate, 3
milligrams
acacia
TPI-4: 300 milligrams PEG 10000, 30 milligrams acacia, 30 milligrams cetrimide
TPI-5: 300 milligrams polyvinylalcohol, 30 milligrams benzethonium chloride, 3
milligrams PEG 1000
15 milliliters water was added to each vial and the formulations incubated at
37°C
for 1 hour before they were filtered through 0.2 micron filters to remove any
undissolved
solids. The filtrates were measured using a UV spectrometer at 290 urn in a 1
cm path-
length quartz cuvette.
The commercially available FULVICIN (having 165 milligrams griseofulvin) in
tablet form was ground into powder and an amount containing 30 milligrams
griseofulvin
was tested in the same way as the lead formulations for comparison.
Results
The results from the lab scale dissolution assay are plotted in Figure 4 as
absorbance
at 290 nm, showing the means and standard deviations from the three
measurements. An
increase of up to 300% in solubilities (as measured by UV) was achieved
compared to the
commercial formulation. All five lead formulations identified in the
microarray test were
validated in the solid form in the lab scale ( 10,000x) dissolution assay to
have increased
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WO 01/09391 CA 02379160 2002-O1-14 PCT/US00/20717
solubility compared to the commercially available pharmaceutical, proving the
results from
microarray assay format can now be translated into normal lab scale assays.
EXAMPLE 3: Evaluation of Individual Effect of Each Excipient.
To examine the effect of each excipient on griseofulvin's solubility, the
first three
lead formulations (TPI-1 to TPI-3) identified above in the micro arrays
described above were
"de-convolved" on the lab scale into griseofulvin formulations that contain
(1) one of the
three excipients only, or (2) two of the three excipients in different
combinations (example,
components one and two, two and three, one and three). Solubilities of each
sample were
then measured using the same lab procedures described above for lead
validation, using
absorbance at 290 nm to determine solubility.
The solubilities for the "de-convolved" formulations are shown in Figure 5, 6
and 7
as ratios to their respective lead formulation (It is important to note that
some
reformulations have greater or less solubilities than the lead formulations
identified in
Example 1, but that the results are relative to the starting lead formulation;
not absolute
absorbance).
In Figure S, excipient 14 (polyoxyethylene 40 stearate) (which is present as a
small
percentage in TPI-3, as indicated by the area in the pie chart) yields a
substantial increase in
solubility, which was slightly enhanced by excipient 10 (PEG 1000). Thus, a
positive
solubility interaction was identified between excipient 14 and griseofulvin.
As shown by Figure 5, 14 (polyoxyethylene 40 stearate) was the only important
excipient in TPI-3 and addition of 10 (PEG 1000 and 1 (acacia) had no effect
on overall
solubility. Addition of excipient 2 ((3-cyclodextrin) actually decreased
overall solubility of
the griseofulvin in TPI-l, as shown in Figure 6, demonstrating an antagonistic
solubility
interaction among excipients.
In contrast, as shown by Figure 7, excipient 3 (SDS), 10 (PEG 1000), and 14
(polyoxyethylene 40 stearate) show a synergistic solubility interaction in
enhancing
solubility of griseofulvin.
EXAMPLE 4: Dissolution Rate Comparison under Simulated USP Conditions
The rates of dissolution of TPI-2 and the commercial griseofulvin formulation
( 165
milligrams) were compared at the lab scale using 1000 milliliters deionized
water in 1000
milliliters Erlenmeyer flasks at 37°C stirred at 300 RPM with a 1.5
inch magnetic stir bar.
The rate of dissolution for each formulation was determined separately. Each
formulation
was added to the stirring deionized water and 1 milliliter aliquots were
removed at 0
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WO 01/09391 PCT/US00/20717
seconds, 30 seconds, 1 minute, 3 minutes, 6 minutes, 10 minutes, 1 S minutes,
25 minutes,
40 minutes, and 50 minutes. Each aliquot was added to a 1.5 milliliters
Eppendorf vial,
centrifuged at room temperature at 14,000 RPM for 10 seconds to remove
undissolved
solids and the ultraviolet absorbance determined at 290 nm in a 1 cm path-
length quartz
cuvette.
Results
The rates of dissolution are shown in Figure 8. TPI-2 showed a faster rate of
dissolution as well as a higher equilibrium solubility compared to the
commercial
preparation FULVICIN, further confirming the validity of the lead formulations
selected
from the micro arrays.
These results demonstrate the efficacy of the high throughput formulation and
testing methods, and how it is possible to scale up the results with a high
degree of
reproducibility.
The foregoing has outlined rather broadly the more pertinent and important
features
of the present invention. While it is apparent that the invention disclosed
herein is well
calculated to fulfill the objects stated above, it will be appreciated that
numerous
modifications and embodiments may be devised by those skilled in the art.
Therefore, it is
intended that the appended claims cover all such modifications
25
35
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