U.S. patent application number 10/869234 was filed with the patent office on 2004-11-25 for transdermal assay with magnetic clamp.
Invention is credited to Chen, Hongming, Cima, Michael J., Gyory, J. Richard, Lemmo, Anthony V..
Application Number | 20040235145 10/869234 |
Document ID | / |
Family ID | 46150218 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235145 |
Kind Code |
A1 |
Cima, Michael J. ; et
al. |
November 25, 2004 |
Transdermal assay with magnetic clamp
Abstract
The transdermal assay apparatus includes first, second, and
third members. The first member has one or more sample surfaces,
each of which is configured to receive a sample thereon. The second
member defines one or more reservoirs, each of which has an opening
on a surface of the second member. Each sample surface is
substantially the same size as each opening. The transdermal assay
apparatus also includes a magnetic clamp configured to clamp a
tissue specimen between the sample surface and the opening. The
magnetic clamp preferably includes a magnet having a strength that
is selected based on the clamping force required between the first
member and the second member. The invention also provides a method
for using a transdermal assay apparatus.
Inventors: |
Cima, Michael J.;
(Winchester, MA) ; Chen, Hongming; (Acton, MA)
; Gyory, J. Richard; (Sudbury, MA) ; Lemmo,
Anthony V.; (Sudbury, MA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
46150218 |
Appl. No.: |
10/869234 |
Filed: |
June 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10869234 |
Jun 15, 2004 |
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10282505 |
Oct 28, 2002 |
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10282505 |
Oct 28, 2002 |
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09904725 |
Jul 13, 2001 |
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6758099 |
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60240891 |
Oct 16, 2000 |
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60220324 |
Jul 24, 2000 |
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60218377 |
Jul 14, 2000 |
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Current U.S.
Class: |
435/287.1 |
Current CPC
Class: |
G01N 33/5088 20130101;
G01N 33/5044 20130101; G01N 33/5008 20130101; B01L 3/50255
20130101; G01N 2500/00 20130101; G01N 13/00 20130101 |
Class at
Publication: |
435/287.1 |
International
Class: |
C12M 001/34; C12M
003/00 |
Claims
We claim:
1. A transdermal assay apparatus, comprising: a) a first lower
member having a sample surface configured to receive an array of
samples and having an array of samples provided on said sample
surface; b) a tissue specimen that overlays said array of samples;
c) a second upper member having an array of reservoirs each having
an opening on the surface of said second member, wherein said
sample surface array and said reservoir array are substantially
aligned with one another when in use; wherein said array of samples
provided on said first member has an active component that
transfers across said tissue specimen into said array of reservoirs
of said second member.
2. The transdermal assay apparatus of claim 1, wherein said sample
surface and said opening are of substantially equal size.
3. The transdermal assay apparatus of claim 1, wherein said first
member includes multiple sample surfaces and said second member
includes multiple reservoirs each having an opening on the surface
of said second member.
4. The transdermal assay apparatus of claim 1, wherein in use said
sample surface and said opening are substantially aligned with one
another along a line substantially perpendicular with said sample
surface.
5. The transdermal assay apparatus of claim 1, wherein said
reservoir is configured to receive at least one fluid medium
therein.
6. The transdermal assay apparatus of claim 1, wherein said first
and second members are made from a material selected from a group
consisting of: stainless steel, plastic, polycarbonate, glass,
aluminum, brass, ceramic, and any combination of the
aforementioned.
7. The transdermal assay apparatus of claim 1, wherein said
apparatus further comprises a liquid source sample.
8. The transdermal assay apparatus of claim 1, wherein said tissue
specimen is skin, lung, tracheal, nasal, placental, vaginal,
rectal, colon, gut, stomach, bladder, corneal tissue, stratum
corneum, living tissue explants, animal tissue, human tissue, or
engineered tissue-equivalents.
9. The transdermal assay apparatus of claim 8, wherein the tissue
specimen is divided into a plurality of segments by mechanical
cutting, scribing, laser cutting, scoring or crimping.
10. The transdermal assay apparatus of claim 8, wherein the tissue
specimen comprises skin tissue.
11. The transdermal assay apparatus of claim 10, wherein the skin
tissue comprises epidermis or stratum corneum.
12. The transdermal assay apparatus of claim 10, wherein the skin
tissue is human skin tissue, animal skin tissue, or engineered skin
tissue.
13. The transdermal assay apparatus of claim 10, wherein the skin
tissue comprises stratum corneum.
14. The transdermal assay apparatus of claim 10, wherein the skin
tissue consists of stratum corneum.
15. The transdermal assay apparatus of claim 1, further comprising
a magnetic clamp configured to clamp said tissue specimen between
said sample surface and said opening.
16. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp comprises: at least one magnet coupled to said first
member; and at least one ferrous insert coupled to said second
member.
17. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp comprises: at least one magnet coupled to said first
member; and at least one magnetic insert coupled to said second
member.
18. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp comprises: at least one magnet coupled to said first
member; and at least a portion of said second member made from a
ferrous material.
19. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp comprises: at least a portion of said first member
made from a magnetic material; and at least a portion of said
second member made from a ferrous material.
20. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp comprises: at least a portion of said first member
made from a magnetic material; and at least a portion of said
second member made from a magnetic material.
21. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp includes a permanent magnet.
22. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp includes an electromagnet.
23. The transdermal assay apparatus of claim 15, wherein said
magnetic clamp includes a magnet having a strength that is selected
based on the clamping force required between said first member and
said second member.
24. The transdermal assay apparatus of claim 15, further comprising
a third member configured to clamp said first member between said
third member and said second member.
25. The transdermal assay apparatus of claim 24, wherein said
magnetic clamp comprises: at least one magnet coupled to said third
member; and at least one ferrous insert coupled to said second
member.
26. The transdermal assay apparatus of claim 25, wherein said third
member comprises a cavity for receiving said at least one magnet
therein.
27. The transdermal assay apparatus of claim 24, wherein said
magnetic clamp comprises: at least one magnet coupled to said third
member; and at least one magnetic insert coupled to said second
member.
28. The transdermal assay apparatus of claim 24, wherein said
magnetic clamp comprises: at least one magnet coupled to said third
member; and at least a portion of said second member made from a
ferrous material.
29. The transdermal assay apparatus of claim 24, wherein said
magnetic clamp comprises: at least a portion of said third member
made from a magnetic material; and at least a portion of said
second member made from a ferrous material.
30. The transdermal assay apparatus of claim 24, wherein said
magnetic clamp comprises: at least a portion of said third member
made from a magnetic material; and at least a portion of said
second member made from a magnetic material.
31. The transdermal assay apparatus of claim 24, wherein: said
first member includes at least one alignment hole; and said third
member comprises at least one alignment post configured to mate
with said at least one alignment hole.
32. The transdermal assay apparatus of claim 24, wherein said third
member is made from a material selected from a group consisting of:
aluminum, steel, brass, plastic, ceramic, and any combination of
the aforementioned.
33. The transdermal assay apparatus of claim 1, wherein said array
of reservoirs contain a reservoir medium.
34. The transdermal assay apparatus of claim 1, wherein said
samples comprise a component-in-common and at least one additional
component, wherein each sample differs from at least one other
sample with respect to at least one of: (i) the identity of the
additional components, (ii) the ratio of the component-in-common to
the additional component, or (iii) the physical state of the
component-in-common.
35. The transdermal assay apparatus of claim 34, wherein the
component-in-common is a pharmaceutical.
36. The transdermal assay apparatus of claim 34, wherein the
component-in-common is a pharmaceutical, a dietary supplement, a
nutraceutical, or an alternative medicine.
37. The transdermal assay apparatus of claim 34, wherein the
additional component is an adhesive, an enhancer, an additive, a
solvent, an excipient, or a combination thereof.
38. The transdermal assay apparatus of claim 37, wherein the
additional component is an adhesive.
39. The transdermal assay apparatus of claim 38, wherein the
adhesive is a polyisobutylene, a silicone, or an acrylic
adhesive.
40. The transdermal assay apparatus of claim 37, wherein the
additional component is an enhancer.
41. The transdermal assay apparatus of claim 40, wherein the
enhancer is a chemical enhancer, a lipid permeation enhancer, a
solubility enhancer, or a combination of enhancers.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/282,505, filed Oct. 28, 2002, which is a
continuation-in-part of U.S. application Ser. No. 09/904,725, filed
Jul. 13, 2001, which claims the benefit of provisional application
No. 60/240,891, filed Oct. 16, 2000; provisional application No.
60/220,324, filed Jul. 24, 2000; provisional application No.
60/218,377, filed Jul. 14, 2000, each of which is hereby
incorporated by reference.
1. FIELD OF THE INVENTION
[0002] The field of the present invention relates to tissue barrier
assays for screening formulations and chemical compositions.
2. BACKGROUND OF THE INVENTION
[0003] In vitro analysis of the movement of compounds (e.g., drugs)
across an epithelial barrier, such as intestinal epithelium or
airway epithelium, is typically performed using an Ussing-type
chamber. To perform a tissue barrier assay using an Ussing-type
chamber, a piece tissue is removed as an intact sheet from the body
and mounted in a device which contains an enclosed, internal hollow
chamber such that it divides the internal chamber into two separate
chambers. Thereafter, biologically compatible solutions are filled
into both chambers, and the drug of interest is added to one
chamber's solution. Samples are then removed from the contralateral
chamber solution at various times to determine the rate at which
the drug moves across the tissue barrier. This type of tissue
barrier assay is cumbersome, inefficient, and only permits a very
limited number of independent samples to be derived from a unit
area of tissue sheet.
[0004] Transdermal delivery of drugs is a type of tissue transfer
that involves transfer of the drug from a transdermal drug delivery
device through the skin and into the patient's blood stream.
Transdermal drug delivery offers many advantages compared to other
methods of drug delivery. One obvious advantage is that needles and
the associated pain are avoided. This is especially desirable for
drugs that are repeatedly administered. Avoiding the unpleasantness
of needles would also lead to improved patient compliance of drug
regimens.
[0005] Another advantage of transdermal drug delivery is its
ability to offer prolonged or sustained delivery, potentially over
several days to weeks. Other delivery methods, such as oral or
pulmonary delivery, typically require that the drug be given
repeatedly to sustain the proper concentration of drug within the
body. With sustained transdermal delivery, dose maintenance is
performed automatically over a long period of time. This is
especially beneficial for drugs with short half-lives in the body,
such as peptides or proteins.
[0006] A final advantage is that drug molecules only have to cross
the skin to reach the bloodstream when given transdermally.
Transdermally administered drugs bypass first-pass metabolism in
the liver, and also avoid other degradation pathways such as the
low pH's and enzymes present in the gastrointestinal tract.
[0007] The skin is the largest organ of the body. It is highly
impermeable to prevent loss of water and electrolytes. It is
subdivided into two main layers: the outer epidermis and the inner
dermis. The epidermis is the outer layer of the skin, 50 to 100
micron thick (Monteiro-Riviere, 1991; Champion, et al., 1992). The
dermis is the inner layer of the skin and varies from 1 to 3 mm in
thickness. The goal of transdermal drug delivery is to get the drug
to this layer of the skin, where the blood capillaries are located,
to allow the drug to be systemically delivered. The epidermis does
not contain nerve endings or blood vessels. The main purpose of the
epidermis is to generate a tough layer of dead cells on the surface
of the skin, thereby protecting the body from the environment. This
outermost layer of epidermis is called the stratum corneum, and the
dead cells that comprise it are called corneocytes or
keratinocytes.
[0008] The stratum corneum is commonly modeled or described as a
brick wall (Elias, 1983; Elite, 1988). The "bricks" are the
flattened, dead corneocytes. Typically, there are about 10 to 15
corneocytes stacked vertically across the stratum corneum
(Monteiro-Riviere, 1991; Champion et al., 1992). The corneocytes
are encased in sheets of lipid bilayers (the "mortar"). The lipid
bilayer sheets are separated by 50 nm. Typically, there are about 4
to 8 lipid bilayers between each pair of corneocytes. The lipid
matrix is primarily composed of ceramides, sphingolipids,
cholesterol, fatty acids, and sterols, with very little water
present (Lampe et al., 1983 [a]; Lampe et al., 1983 [b]; Elias,
1988).
[0009] Although it is the thinnest layer of the skin, the stratum
corneum is the primary barrier to the entry of molecules or
microorganisms across the skin. Most molecules pass through the
stratum corneum only with great difficulty, which is why the
transdermal drug delivery route has not been more widely used to
date. Once the molecules have crossed the stratum corneum,
diffusion across the epidermis and dermis to the blood vessels
occurs rapidly. Thus, most of the attention in transdermal drug
delivery research has been focused on transporting molecules and
drugs across the stratum corneum.
[0010] The most common form of transdermal drug delivery device is
the transdermal drug "patch," where a drug, or pharmaceutical, is
contained within a reservoir placed next to the skin (Schaefer and
Redelmeier, 1996). The drug molecules typically cross the skin by
simple diffusion. Transport is governed by the rate of molecular
diffusion into and out of the skin, and partitioning of the drug
into the skin. Generally speaking, transdermal drug delivery is
limited to small, lipophilic molecules such as scopolamine,
nitroglycerine, and nicotine, which readily permeate the skin. The
delivery is slow, typically taking hours for the drug to cross the
skin, and treatment is only effective when a very small amount of
drug is required to have a biological effect (Guy and Hadgraft,
1989).
[0011] Since transdermal delivery can be slow, many substances have
been used to enhance molecular transport rates. These substances
are known as chemical enhancers or penetration enhancers. Chemical
enhancers increase the flux of a drug through the skin by
increasing the solubility of drug in the stratum corneum or
increasing the permeability of drug in the stratum corneum. There
are many possible enhancers and the selection is further
complicated by the fact that combinations of enhancers are known to
improve drug flux beyond what would be expect due to the presence
of each constituent independently.
[0012] Transdermal drug delivery devices, such as a transdermal
patch, also generally contain an adhesive, which serves to keep the
device in intimate contact with the skin, and may also form the
matrix in which the drug is dissolved or dispersed. There are many
different forms of adhesives that can be used, and it is often a
very difficult problem to select which adhesive to use with any
drug or drug and enhancer.
[0013] Currently, the choice of appropriate adhesive and enhancers
and their relative proportion with respect to the drug is only
determined by general guidelines from what is known to be safe and
what may have been effective with other drugs. The vast majority of
the formulation development is made through trial and error
experimentation.
[0014] Most transdermal transport experiments to date have utilized
a relatively large human skin diffusion cell in which a source side
includes a drug solution with additives and a sink side that
typically includes saline solution or some other solution that is
thought to model the dermis. The skin membrane separates the two
sides of the cell, and is most often stratum corneum cadaver skin
that has been carefully separated from the whole skin sample
supplied by a tissue bank. The volume of the device is typically 5
cc or greater. Samples are periodically taken from the sink side of
the cell to determine the flux of drug through the stratum corneum
film. The entire procedure is very laborious and requires the use
of large quantities of skin, which is extremely difficult to
obtain. Therefore, only a relatively small number of the many
possible combinations of chemical entities can be examined. Also,
only a limited number of formulations can be tested on a single
donor's skin, which makes it more difficult to compare the effect
of those formulations, due to the additional variation in
measurement introduced by the response from different donor's skin
samples.
[0015] Thus, there remains a need in the art for an apparatus and
method for identifying optimal compositions or formulations for
tissue barrier transport, including transdermal transport, of
compounds, pharmaceuticals and other components. Such an apparatus
should be easy to assemble. The apparatus should be sealable,
wherein the required clamping force and sealing pressure between
the drug or pharmaceutical, the skin membrane, and the saline or
other solution used to model the dermis, is even and does not
interfere with the function.
3. SUMMARY OF THE INVENTION
[0016] The present invention relates to high-throughput systems and
methods to prepare a large number of component combinations, at
varying concentrations and identities, at the same time, and
high-throughput methods to test tissue barrier transfer of
components in each combination. The methods of the present
invention allow determination of the effects of additional or
inactive components, such as excipients, carriers, enhancers,
adhesives, and additives, on transfer of active components, such as
pharmaceuticals, across tissue, such as skin, lung tissue, tracheal
tissue, nasal tissue, bladder tissue, placenta, vaginal tissue,
rectal tissue, stomach tissue, gastrointestinal tissue, and eye or
corneal tissue. The invention thus encompasses the high-throughput
testing of pharmaceutical compositions or formulations in order to
determine the overall optimal composition or formulation for
improved tissue transport, including without limitation,
transdermal transport. Specific embodiments of this invention are
described in detail below.
[0017] In one embodiment, the invention concerns an apparatus for
measuring transfer of components across a tissue, comprising a
support plate, an array of samples supported by the support plate,
a membrane or tissue specimen overlaying the array of samples, and
a reservoir plate secured to a side of the membrane or tissue
specimen opposite the array of samples. In one aspect of the
invention, each sample in the array contains a unique composition
or formulation of components, wherein different active components
or different physical states of an active component are present in
one or more of the samples in the sample array.
[0018] In another aspect of the present invention, each sample of
the array includes a component-in-common and at least one
additional component, wherein each sample differs from at least one
other sample with respect to at least one of:
[0019] (i) the identity of the additional components,
[0020] (ii) the ratio of the component-in-common to the additional
components, or
[0021] (iii) the physical state of the component-in-common.
[0022] A "component-in-common" is a component that is present in
every sample in a sample array. In one embodiment, the
component-in-common is an active component, and preferably, the
active component is a pharmaceutical, dietary supplement,
alternative medicine or a nutraceutical. The samples may be in the
form of liquids, solutions, suspensions, emulsions, solids,
semi-solids, gels, foams, pastes, ointments, or triturates.
[0023] In another embodiment, the invention concerns a method of
measuring tissue barrier transport of a sample, comprising:
[0024] (a) preparing an array of samples having an active component
and at least one additional component, wherein each sample differs
from at least one other sample with respect to at least one of:
[0025] (i) the identity of the active component;
[0026] (ii) the identity of the additional components,
[0027] (iii) the ratio of the active component to the additional
components, or
[0028] (iv) the physical state of the active component;
[0029] (b) overlaying the array of samples with a tissue
specimen;
[0030] (c) securing a reservoir plate to a side of the tissue
specimen opposite the array of samples, the plate having an array
of reservoirs corresponding to the array of samples;
[0031] (d) filling the array of reservoirs with a reservoir medium;
and
[0032] (e) measuring concentration of the active component in each
reservoir at one or more time points to determine transport of the
active component from each sample across the tissue specimen.
[0033] In a preferred embodiment, the active component is a
pharmaceutical, a dietary supplement, an alternative medicine, or a
nutraceutical. In another embodiment, the tissue specimen is
skin.
[0034] In another embodiment, the invention concerns a method of
analyzing or measuring flux of a sample across a tissue,
comprising:
[0035] (a) preparing an array of samples having a
component-in-common and at least one additional component, wherein
each sample differs from at least one other sample with respect to
at least one of:
[0036] (i) the identity of an active component;
[0037] (ii) the identity of the additional components,
[0038] (iii) the ratio of the component-in-common to the additional
components, or
[0039] (iv) the physical state of the component-in-common;
[0040] (b) overlaying the array of samples with a tissue
specimen;
[0041] (c) securing a reservoir plate to a side of the tissue
specimen opposite the array of samples, the plate having an array
of reservoirs corresponding to the array of samples;
[0042] (d) filling the array of reservoirs with a reservoir medium;
and
[0043] (e) measuring concentration of the component-in-common in
each reservoir as a function of time to determine flux of the
component-in-common from each sample across the tissue
specimen.
[0044] In an alternative embodiment, the method comprises an
additional step of cutting the tissue specimen to avoid lateral
diffusion between wells. The method preferably comprises analyzing
the tissue specimen for defects, or inhomogeneities, and correcting
for or repairing the defects.
[0045] According to the inventon there is provided a transdermal
assay apparatus. The transdermal assay apparatus includes at least
first and second members. The first member has one or more sample
surfaces, each of which is configured to receive a sample thereon.
The second member defines one or more reservoirs, each of which has
an opening on a surface of the second member. Each sample surface
is substantially the same size as each opening. The transdermal
assay apparatus also includes a magnetic clamp configured to clamp
a tissue specimen between the sample surface and the opening. The
magnetic clamp also seals the various members of the apparatus
together. The magnetic clamp preferably includes a magnet having a
strength that is selected based on the clamping force required
between the first member and the second member.
[0046] The invention also provides a method for using a transdermal
assay apparatus. A sample is placed on a sample surface of a first
member. A reservoir defined by a second member is filled with a
fluid medium, where the reservoir has an opening on a surface of
the second member. A tissue specimen is then placed between the
sample and the opening. The tissue specimen is subsequently clamped
between the sample and the fluid medium at the opening, with a
magnetic clamping force. Periodically, a specimen of the fluid
medium is withdrawn from the reservoir to measure the concenration
of the sample in the fluid medium. This concentration is indicative
of a transfer of the sample across the tissue specimen.
[0047] The above described apparatus and method are preferably used
for high-throughput screening of active component or drug flux
through the stratum corneum recognizing that such flux is
determined, at least in part, by the permeability of the drug
within the tissue in the presence of an enhancer. The permeability
is generally governed by at least two factors: the solubility of
the active component or drug within the stratum corneum and the
diffusivity of the active component or drug within the stratum
corneum. These two factors, solubility and diffusivity, can be
measured independently as a method of indirectly assessing the flux
through the stratum corneum. Thus, an array of wells containing
samples of different compositions of active component and inactive
compounds, including without limitation, compositions comprising
active component/carrier or excipient/, active component/carrier or
excipient/enhancer/, active component/adhesive/enhancer/additive,
are constructed. Known amounts of stratum corneum are added to each
well and the rate at which the active component or drug is taken up
into the tissue sample is measured by extracting the tissue from
similarly prepared wells at different times. Measuring the
concentration after times sufficiently long so that the amount
dissolved is not changing with time can assess the equilibrium
concentration of active component or drug within the tissue. The
product of the rate and solubility is proportional to the
permeability of the active component or drug.
[0048] The high-throughput combinatorial screening systems and
methods of the present invention identify optimal compositions or
formulations to achieve a desired result for such compositions or
formulations, including without limitation, construction of a
transdermal delivery device. In particular, the systems and methods
of the present invention may be used to identify 1) optimal
compositions or formulations comprising one or more active
components and one or more inactive components for achieving
desired characteristics for such compositions or formulations, 2)
optimal adhesive/enhancer/additive compositions for compatibility
with a drug, 3) optimal drug/adhesive/enhancer/additive
compositions for maximum drug flux through stratum corneum, and 4)
optimal drug/adhesive/enhancer/addit- ive composition to minimize
cytotoxicity
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The features and advantages of the present invention will be
better understood by reference to the following detailed
description, which should be read in conjunction with the
accompanying drawings in which:
[0050] FIG. 1 is a schematic diagram of a high-throughput apparatus
for measuring tissue barrier transport, such as transdermal
transport, according to the present invention;
[0051] FIGS. 2A-2D are schematic diagrams of an alternative
embodiment of a high-throughput apparatus for measuring tissue
barrier transport using solid source samples according to the
present invention;
[0052] FIG. 3 is a schematic diagram of an alternative embodiment
of a high-throughput apparatus for measuring tissue barrier
transport according to the present invention;
[0053] FIGS. 4A-4C are schematic diagrams of an alternative
embodiment of a diffusion cell that alone, or as part of a high
throughput apparatus, is used for measuring tissue barrier
transport according to the present invention.
[0054] FIGS. 5A and 5B are schematic diagrams of an apparatus for
filling a sample well in a sample array, such as the sample array
in the high-throughput apparatus shown in FIG. 1;
[0055] FIG. 6 is a schematic diagram of an alternative embodiment
of a high-throughput apparatus for measuring or analyzing tissue
barrier transport using solid source samples according to the
present invention;
[0056] FIG. 7 is a schematic diagram of an alternative embodiment
of a high-throughput apparatus for measuring or analyzing tissue
barrier transport using solid source samples according to the
present invention;
[0057] FIG. 8 is a schematic diagram of an alternative embodiment
of a high-throughput apparatus for measuring or analyzing tissue
barrier transport using solid source samples according to the
present invention;
[0058] FIG. 9 is an exploded view of a another transdermal assay
apparatus according to another embodiment of the invention; and
[0059] FIG. 10 is an exploded view of a yet another transdermal
assay apparatus according to yet another embodiment of the
invention.
5. DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention relates to high throughput
combinatorial systems and methods that improve tissue barrier
transfer of active compounds, such as pharmaceuticals or drugs,
other compounds, or compound combinations. In one embodiment, the
system and methods of the present invention may be used to identify
the optimal components (e.g., solvents, carriers, transport
enhancers, adhesives, additives, and other excipients) for
pharmaceutical compositions or formulations that are delivered to a
patient via tissue transport, including without limitation,
pharmaceutical compositions or formulations administered or
delivered transdermally (e.g., in the form of a transdermal
delivery device), topically (e.g., in the form of ointments,
lotions, gels, and solutions), and ocularly (e.g., in the form of a
solution). As used herein, "high throughput" refers to the number
of samples generated or screened as described herein, typically at
least 10, more typically at least 50 to 100, and preferably more
than 1000 samples.
[0061] The high throughput methods of the present invention can be
performed using various forms of samples. Typically, the methods
are performed either with liquid samples or with solid or
semi-solid samples.
[0062] As used herein, "liquid source" means that the sample
containing the component or components being measured or analyzed
is in the form of a liquid, which includes, without limitation,
liquids, solutions, emulsions, suspensions, and any of the
foregoing having solid particulates dispersed therein.
[0063] As used herein, "solid source" means that the sample
containing the component or components being measured or analyzed
is in the form of a solid or semi-solid, which includes, without
limitation, triturates, gels, films, foams, pastes, ointments,
adhesives, high viscoelastic liquids, high viscoelastic liquids
having solid particulates dispersed therein, and transdermal
patches.
[0064] As used herein, "reservoir medium" or "fluid medium" refers
to a liquid, solution, gel, or sponge that is chemically compatible
with the components in a sample and the tissue being used in an
apparatus or method of the present invention. In one embodiment of
the present invention, the reservoir medium comprises part of the
specimen taken to measure or analyze the transfer, flux, or
diffusion of a component across a tissue barrier. Preferably, the
reservoir medium is a liquid or solution.
[0065] 5.1 Overview of an Apparatus for Measuring Tissue Barrier
Transfer
[0066] FIG. 1 shows a schematic diagram of a preferred embodiment
of a high-throughput apparatus 100 for measuring tissue barrier
transport in a sample array 112 according to the present invention.
Apparatus 100 includes a substrate plate 114 supporting sample
array 112, a tissue specimen 120 and a reservoir plate 130. In this
embodiment, each sample in sample array 112 is placed in a sample
well 116. Attached to the bottom of substrate plate 114 is a base
118 that forms the bottom of each sample well 116. Base 118 is
optionally a membrane made of any suitable material (e.g. a rubber
membrane) in any fashion that permits air to bleed out of sample
well 116 when filling with a sample. Alternatively, base 118 is a
rigid, removable substrate plate such as plate 214 (described infra
with respect to FIGS. 2A-2D) capable of supporting an array of
solid source samples.
[0067] Substrate plate 114 may be any rigid grid or plate capable
of supporting a number of samples. For example, substrate plate 114
may be a 24, 36, 48, 72, 96 or 384 well plate. Preferably,
apparatus 100 comprises one or more sample arrays 112, wherein the
number of sample wells 116 in apparatus 100 is at least 100,
preferably at least 1000, and more preferably at least 10,000.
Preferably, the size of sample well 116 is about 1 mm to about 50
mm, more preferably about 2 mm to about 10 mm, and most preferably
about 3 mm to about 7 mm. For example, a 3 mm well format provides
an array of approximately 30,000 samples for 0.25 m.sup.2 of
skin.
[0068] As used herein, the terms "array" or "sample array" (e.g.
array 112) mean a plurality of samples associated under a common
experiment, wherein each of the samples comprises at least two
components, and at least one of the components being an active
component. In one embodiment of the present invention, one of the
sample components is a "component-in-common", which as used herein,
means a component that is present in every sample of the array,
with the exception of negative controls.
[0069] Sample array 112 is designed to provide a number of
different samples of different compositions, the analysis of which
allows determination of optimal compositions or formulations for
improving transfer of a component across tissue 120. Each sample in
sample array 112 preferably, though not necessarily, differs from
any other sample in the array with respect to at least one of:
[0070] (i) the identity of the active component;
[0071] (ii) the identity of the additional component;
[0072] (iii) the ratio of the active component, or the
component-in-common, to the additional component; or
[0073] (iv) the physical state of the active component, or the
component-in-common.
[0074] An array can comprise 24, 36, 48, 96, or more samples,
preferably at least 1000 samples, more preferably, at least 10,000
samples. An array is typically comprised of one or more sub-arrays.
For example, a sub-array can be a plate having 96 sample wells.
[0075] Overlaying substrate plate 114 and sample array 112 is
tissue specimen 120. Tissue 120 is preferably a sheet of tissue,
such as skin, lung, tracheal, nasal, placental, vaginal, rectal,
colon, gut, stomach, bladder, or corneal tissue. More preferably,
tissue 120 is skin tissue or stratum corneum. If human cadaver skin
is to be used for tissue 120, one known method of preparing the
tissue specimen entails heat stripping by keeping it in water at
60.degree. C. for two minutes followed by the removal of the
epidermis, and storage at 4.degree. C. in a humidified chamber. A
piece of epidermis is taken out from the chamber prior to the
experiments and placed over substrate plate 114. Tissue 120 is
optionally be supported by Nylon mesh (Terko Inc.) to avoid any
damage and to mimic the fact that the skin in vivo is supported by
mechanically strong dermis. Alternatively, other types of tissues
may be used, including living tissue explants, animal tissue (e.g.
rodent, bovine or swine) or engineered tissue-equivalents. Examples
of a suitable engineered tissues include DERMAGRAFT (Advanced
Tissue Sciences, Inc.) and those taught in U.S. Pat. No. 5,266,480,
which is incorporated herein by reference.
[0076] In an alternative embodiment of the present invention,
tissue specimen 120 is divided into a number of segments by cuts
122 between sample wells 116 to prevent lateral diffusion through
tissue specimen 120 between adjacent samples. Cuts 122 may be made
in any number of ways, including mechanical scribing or cutting,
laser cutting, or crimping (e.g., between plates 114 and 130 or by
using a "waffle iron" type embossing tool). Preferably, laser
scribing is used as it avoids mechanical pressure from a cutting
tool which can cause distortion and damage to tissue specimen 120.
Laser cuts 122 are performed with very small kerfs which permit a
relatively high density of samples and a more efficient tissue
specimen utilization. Laser tools are available that produce a
minimal heat affected zone, thereby reducing damage to tissue
specimen 120.
[0077] Reservoir plate 130 (e.g., an open-bottomed titer plate) is
placed on top of tissue 120, on a side of tissue opposite substrate
plate 114. Reservoir plate includes a number of hollow reservoirs
132. When plate 130 is secured in place, each reservoir 132 aligns
over a sample well 116 such that tissue separates each well 116
from reservoir 132. Reservoir plate 130 secures to substrate plate
114 using clamps, screws, fasteners, or any other suitable
attachment means. Plates 130 and 114 preferably secure together
with sufficient pressure so as to create a liquid tight seal around
reservoirs 132. Each reservoir is filled with a reservoir, such as
a saline solution, to receive sample components or compounds that
diffuse across tissue 120 to reservoir 132. In one embodiment, the
reservoir medium is approximately 2% BSA solution in PBS.
[0078] Transfer or flux of components from sample wells 116 across
tissue 120 (i.e., tissue barrier transfer or diffusion) may be
analyzed by measuring component concentration in specimens taken
from reservoirs 132. Comparison of measurements taken from
different samples/reservoirs aids in determining optimal sample
compositions for improving tissue transfer or diffusion of a
desired component (e.g,. a pharmaceutical).
[0079] Preferably the samples are prepared, added to sample wells
and mixed automatically. Similarly, specimen from reservoirs 132
containing transferred or diffused components, and the
concentrations thereof, can be measured and processed
automatically. "Automated" or "automatically" refers to the use of
computer software and robotics to add, mix and analyze the samples,
components, and specimens or diffusion products.
[0080] Samples are added to the sample wells in sample arrays of
the present invention, such as sample array 112 in FIG. 1, using
various deposition or material transfer techniques known to the
skilled artisan, including, without limitation, hand placement,
pipetting, and other manual or automated solid or liquid
distribution systems.
[0081] 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. Additional details regarding
suitable automated dispensing and sampling equipment and methods of
formulating solutions or compositions are disclosed in copending
U.S. patent application Ser. No. 09/540,462 which is herein
incorporated by reference in its entirety.
[0082] Briefly, a number of companies have developed microarray
systems that can be adapted for use in the system described herein,
although all are currently used for the sole purpose of screening
to identify compounds having a particular defined activity, as
opposed to screening of components or compounds having a known
identity in order to identify optimal component combinations to
achieve a desired result. Such systems may require modification,
which is well within ordinary skill in the art. Examples of
companies having microarray systems include Gene Logic of
Gaithersburg, Md. (see U.S. Pat. No. 5,843,767 to Beattie), Luminex
Corp., Austin, Tex., Beckman Instruments, Fullerton, Calif.,
MicroFab Technologies, Plano, Tex., Nanogen, San Diego, Calif., and
Hyseq, Sunnyvale, Calif. These devices test samples based on a
variety of different systems. All include thousands of microscopic
channels that direct components into test wells, where reactions
can occur. These systems are connected to computers for analysis of
the data using appropriate software and data sets. The Beckman
Instruments system can deliver nanoliter samples of 96 or
384-arrays, and is particularly well suited for hybridization
analysis of nucleotide molecule sequences. The MicroFab
Technologies system delivers sample using inkjet printers to
aliquot discrete samples into wells.
[0083] These and other systems can be adapted as required for use
herein. For example, the combinations of active component and
various additional or inactive components at various concentrations
and combinations can be generated using standard formulating
software (e.g., Matlab software, commercially available from
Mathworks, Natick, Mass.). The combinations thus generated can be
downloaded into a spread sheet, such as Microsoft EXCEL. From the
spread sheet, a work list 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 work list can be generated using standard programming
methods according to the automated distribution mechanism that is
being used. The use of so-called work lists simply allows a file to
be used as the process command rather than discrete programmed
steps. The work list combines the formulation output of the
formulating program with the appropriate commands in a file format
directly readable by the automatic distribution mechanism.
[0084] The automated distribution mechanism delivers at least one
active component, such as a pharmaceutical, as well as various
inactive or additional components, such as solvents, carriers,
excipients, and additives, to each sample well. Preferably, the
automated distribution mechanism can deliver multiple amounts of
each component. In one embodiment, the automated distribution
mechanism utilizes one or more micro-solenoid valves.
[0085] Automated liquid and solid distribution systems are well
known and commercially available, such as the Tecan Genesis, from
Tecan-US, RTP, North Carolina. The robotic arm can collect and
dispense active components and inactive components, such as
solutions, solvents, carriers, excipients, additives, and the like,
from a stock plate to a sample well or site. The process is
repeated until an array is completed. The samples are then mixed.
For example, the robotic arm moves up and down in each well plate
for a set number of times to ensure proper mixing.
[0086] In use, apparatus 100 of FIG. 1 is described above as having
reservoir medium above tissue 120 in reservoirs 132 and samples
below tissue 120 in sample wells 116 of array 112. In an
alternative embodiment, the positions are reversed, such that
reservoirs 132 of sample array 112 are below tissue specimen 120
and sample wells 116 are above tissue specimen 120, and a top plate
or top membrane is situated over reservoirs 132 and reservoir plate
130.
[0087] Additional embodiments of the systems and methods of the
present invention are described infra, particularly with respect to
FIGS. 2-8.
[0088] 5.2 Composition of Samples
[0089] Before discussing additional details of the systems and
methods for assessing tissue barrier transfer according to the
present invention, applicants present a discussion of the
composition of samples suitable for use in the present
invention.
[0090] 5.2.1 General Composition Terminology
[0091] As used herein, the term "component" means any substance or
compound. A component can be active or inactive. As used herein,
the term "active component" means a substance or compound that
imparts a primary utility to a composition or formulation when the
composition or formulation is used for its intended purpose.
Examples of active components include pharmaceuticals, dietary
supplements, alternative medicines, and nutraceuticals. Active
components can optionally be sensory compounds, agrochemicals, the
active component of a consumer product formulation, or 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 composition or formulation for
administration of an active component, but does not significantly
share in the active properties of the active component or give rise
to the primary utility for the composition or formulation. Examples
of suitable inactive components include, but are not limited to,
enhancers, excipients, carriers, solvents, diluents, stabilizers,
additives, adhesives, and combinations thereof.
[0092] Preferably, the samples of an array comprise an active
component and inactive components. In one embodiment, the active
components in the samples of an array can be the same or different,
while in another embodiment, the samples in an array comprise an
active component as a 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 selected from the group consisting of
excipients, carriers, solvents, diluents, stabilizers, enhancers,
additives, adhesives, and combinations thereof.
[0093] As used herein, the term "sample" means a mixture of an
active component and one or more additional components or inactive
components. Preferably a sample comprises 2 or more additional
components, more preferably, 3 or more additional components. In
general, a sample will comprise one active component but can
comprise multiple active components. In addition, samples in a
sample array may have one or more 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 .mu.l available from Millipore, Bedford, Mass. The
sample can comprise less than about 100 milligrams of the active
component, preferably, less than about 1 milligram, more
preferably, less than about 100 micrograms, and even more
preferably, less than 100 nanograms. Preferably, the sample has a
total volume of about 1-200 .mu.l, more preferably about 5-150
.mu.l, and most preferably about 10-100 .mu.l. Samples can be
liquid source or solid source samples, which include samples in the
form of solids, semi-solids, films, liquids, solutions, gels,
foams, pastes, ointments, triturates, suspensions, or
emulsions.
[0094] 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 a 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.
[0095] 5.2.2 Active Component and Component-In-Common
[0096] As mentioned above, the component-in-common can be either an
active component, such as a pharmaceutical, dietary supplement,
alternative medicine, or nutraceutical, or an inactive component.
In a preferred embodiment of the present invention, the
component-in-common is an active component, and more preferably a
pharmaceutical. As used herein, the term "pharmaceutical" means any
substance or compound 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.
[0097] 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 sertraline
hydrochloride, venlafaxine, 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 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, ketorolac
tromethamine, morphine, loxoprofen sodium, and ibuprofen;
dermatological products, such as isotretinoin and clindamycin
phosphate; anesthetics, such as propofol, midazolam hydrochloride,
and lidocaine 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.sub.12,
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 compositions or formulations using the systems,
arrays and methods of the present invention.
1TABLE 1 Exemplary Pharmaceuticals Brand Name Chemical Properties
SANDIMMINE 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.
[0098] Still other examples of suitable pharmaceuticals are listed
in 2000 Med Ad News 19:56-60 and The Physicians Desk Reference,
53rd edition, 792-796, Medical Economics Company (1999), both of
which are incorporated herein by reference.
[0099] 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.,
Merck and Co., Inc., Rahway, N.J., 1998; (1997); The Encyclopedia
of Chemical Technology, 24 Kirk-Othomer (4.sup.th ed. at 826); and
Veterinary Drugs in ECT 2nd ed., Vol 21, by A. L. Shore and R. J.
Magee, American Cyanamid Co.
[0100] Other active components suitable for tissue (or
trans-membrane) transfer analysis using the systems and methods of
the present invention include dietary supplements, alternative
medicines, or nutraceuticals.
[0101] 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 Encyclopedia of Chemical
Technology, 11 Kirk-Othomer (4.sup.th ed. at 805-833). Examples of
suitable vitamins are listed in (1998) The Encyclopedia of Chemical
Technology, 25 Kirk-Othomer (4.sup.th ed. at 1) and Goodman &
Gilman 's: The Pharmacological Basis of Therapeutics, 9th Edition,
eds. Joel G. Harman and Lee E. Limbird, McGraw-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.sup.th 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
[0102] 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.
[0103] 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. Linder, ed. Nutritional Biochemistry and
Metabolism with 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.
[0104] Preferably, when the active component is a pharmaceutical, a
dietary supplement, an alternative medicine, or a nutraceutical, at
least one additional component(s) is an excipient. 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 anionic 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 zein; 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 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 that alter the 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.
[0105] Excipients that are typically used in the formation of
transdermal delivery devices, and therefore particularly useful for
formulation of the samples of the present invention, are
penetration enhancers, adhesives and solvents. Each of these is
discussed in more detail below.
[0106] 5.2.3 Penetration Enhancers
[0107] Various types of penetration enhancers may be used to
enhance transdermal transport of drugs. Penetration enhancers can
be divided into chemical enhancers and mechanical enhancers, each
of which is described in more detail below.
[0108] 5.2.3.1 Chemical Enhancers
[0109] Chemical enhancers enhance molecular transport rates across
tissues or membranes by a variety of mechanisms. In the present
invention, chemical enhancers are preferably used to decrease the
barrier properties of the stratum corneum. Drug interactions
include modifying the drug into a more permeable state (a prodrug),
which would then be metabolized inside the body back to its
original form (6-fluorouracil, hydrocortisone) (Hadgraft, 1985); or
increasing drug solubilities (ethanol, propylene glycol). Despite a
great deal of research (well over 200 compounds have been studied)
(Chattaraj and Walker, 1995), there are still no universally
applicable mechanistic theories for the chemical enhancement of
molecular transport. Most of the published work in chemical
enhancers has been done largely based on experience and on a
trial-and-error basis (Johnson, 1996).
[0110] Many different classes of chemical enhancers have been
identified, including cationic, anionic, and nonionic surfactants
(sodium dodecyl sulfate, polyoxamers); fatty acids and alcohols
(ethanol, oleic acid, lauric acid, liposomes); anticholinergic
agents (benzilonium bromide, oxyphenonium bromide); alkanones
(n-heptane); amides (urea, N,N-diethyl-m-toluamide); fatty acid
esters (n-butyrate); organic acids (citric acid); polyols (ethylene
glycol, glycerol); sulfoxides (dimethylsulfoxide); and terpenes
(cyclohexene) (Hadgraft and Guy, 1989; Walters, 1989; Williams and
Barry, 1992; Chattaraj and Walker, 1995). Most of these enhancers
interact either with the skin or with the drug. Those enhancers
interacting with the skin are herein termed "lipid permeation
enhancers", and include interactions with the skin include enhancer
partitioning into the stratum corneum, causing disruption of the
lipid bilayers (azone, ethanol, lauric acid), binding and
disruption of the proteins within the stratum corneum (sodium
dodecyl sulfate, dimethyl sulfoxide), or hydration of the lipid
bilayers (urea, benzilonium bromide). Other chemical enhancers work
to increase the transdermal delivery of a drug by increasing the
drug solubility in its vehicle (hereinafter termed "solubility
enhancers"). Lipid permeation enhancers, solubility enhancers, and
combinations of enhancers (also termed "binary systems") are
discussed in more detail below.
5.2.3.1.1 Lipid Permeation Enhancers
[0111] Chemicals which enhance permeability through lipids are
known and commercially available. For example, ethanol increases
the solubility of drugs up to 10,000-fold and yield a 140-fold flux
increase of estradiol, while unsaturated fatty acids increase the
fluidity of lipid bilayers (Bronaugh and Maibach, editors (Marcel
Dekker 1989) pp. 1-12. Examples of fatty acids which disrupt lipid
bilayer include linoleic acid, capric acid, lauric acid, and
neodecanoic acid, which can be in a solvent such as ethanol or
propylene glycol. Evaluation of published permeation data utilizing
lipid bilayer disrupting agents agrees very well with the
observation of a size dependence of permeation enhancement for
lipophilic compounds. The permeation enhancement of three bilayer
disrupting compounds, capric acid, lauric acid, and neodecanoic
acid, in propylene glycol has been reported by Aungst, et al.
Pharm. Res. 7,712-718 (1990). They examined the permeability of
four lipophilic compounds, benzoic acid (122 Da), testosterone (288
Da), naloxone (328 Da), and indomethacin (359 Da) through human
skin. The permeability enhancement of each enhancer for each drug
was calculated according to E.sub.c pg=P.sub.e pg/P.sub.pg, where
P.sub.e pg is the drug permeability from the enhancer/propylene
glycol formulation and P.sub.pg is the permeability from propylene
glycol alone.
[0112] The primary mechanism by which unsaturated fatty acids, such
as linoleic acid, are thought to enhance skin permeability is by
disordering the intercellular lipid domain. For example, detailed
structural studies of unsaturated fatty acids, such as oleic acid,
have been performed utilizing differential scanning calorimetry
(Barry J. Controlled Release 6,85-97 (1987)) and infrared
spectroscopy (Ongpipattanankul, et al., Pharm. Res. 8, 350-354
(1991); Mark, et al., J. Control. Rd. 12, 67-75 (1990)). Oleic acid
was found to disorder the highly ordered SC lipid bilayers, and to
possibly form a separate, oil-like phase in the intercellular
domain. SC Lipid bilayers disordered by unsaturated fatty acids or
other bilayer disrupters may be similar in nature to fluid phase
lipid bilayers.
[0113] A separated oil phase should have properties similar to a
bulk oil phase. Much is known about transport a fluid bilayers and
bulk oil phases. Specifically, diffusion coefficients in fluid
phase, for example, dimyristoylphosphatidylcholine (DMPC) bilayers
Clegg and Vaz In "Progress in Protein-Lipid Interactions" Watts,
ed. (Elsevier, N.Y. 1985) 173-229; Tocanne, et al., FEB 257, 10-16
(1989) and in bulk oil phase Perry, et al., "Perry's Chemical
Engineering Handbook" (McGraw-Hill, NY 1984) are greater than those
in the SC, and more importantly, they exhibit size dependencies
which are considerably weaker than that of SC transport Kasting, et
al., In: "Prodrugs: Topical and Ocular Delivery" Sloan. ed. (Marcel
Dekker, NY 1992) 117-161; Ports and Guy, Pharm. Res. 9, 663-339
(1992); Willschut, et al. Chemosphere 30, 1275-1296 (1995). As a
result, the diffusion coefficient of a given solute will be greater
in a fluid bilayer, such as DMPC, or a bulk oil phase than in the
SC. Due to the strong size dependence of SC transport, diffusion in
SC lipids is considerably slower for larger compounds, while
transport in fluid DMPC bilayers and bulk oil phases is only
moderately lower for larger compounds. The difference between the
diffusion coefficient in the SC and those in fluid DMPC bilayers or
bulk oil phases will be greater for larger solutes, and less for
smaller compounds. Therefore, the enhancement ability of a bilayer
disordering compound which can transform the SC lipids bilayers
into a fluid bilayer phase or add a separate bulk oil phase should
exhibit a size dependence, with smaller permeability enhancements
for small compounds and larger enhancement for larger
compounds.
[0114] A comprehensive list of lipid bilayer disrupting agents is
described in European Patent Application 43,738 (1982), which is
incorporated herein by reference. Exemplary compounds are
represented by the formula:
[0115] R--X, wherein R is a straight-chain alkyl of about 7 to 16
carbon atoms, a non-terminal alkenyl of about 7 to 22 carbon atoms,
or/& branched-chain alkyl of from about 13 to 22 carbon atoms,
and X is --OH, --COOCH.sub.3, --COOC.sub.2H.sub.5, --OCOCH.sub.3,
--SOCH.sub.3, --P(CH.sub.3).sub.2O,
COOC.sub.2H.sub.4OC.sub.4H.sub.4OH, --COOCH(CHOH).sub.4CH.sub.3OH,
--COOCH.sub.2CHOHCH.sub.3, COOCH.sub.2CH(OR")CH.sub.2OR",
--(OCH.sub.2CH.sub.2).sub.mOH, --COOR', or --CONR.sub.12, where R'
is H, --CR.sub.3, --C.sub.2H.sub.5, --C.sub.2H.sub.7 or
--C.sub.2H.sub.4OH; R" is --H, or a non-terminal alkenyl of about 7
to 22 carbon atoms; and m is 2-6; provided that when R" is an
alkenyl and X is --OH or COOH, at least one double bond is in the
cis-configuration.
5.2.3.1.2 Solubility Enhancers
[0116] Another way to increase the transdermal delivery of a drug
is to use chemical solubility enhancers that increase the drug
solubility in its vehicle. This can be achieved either through
changing drug-vehicle interaction by introducing different
excipients, or through changing drug crystallinity (Flynn and
Weiner, 1993).
[0117] Solubility enhancers include water diols, such as propylene
glycol and glycerol; mono-alcohols, such as ethanol, propanol, and
higher alcohols; DMSO; dimethylformamide; N,N-dimethylacetamide;
2-pyrrolidone; N-(2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone,
1-dodecylazacycloheptan-2-one and other
n-substituted-alkyl-azacycloalkyl- -2-ones.
5.2.3.1.3 Combinations of Enhancers (Binary Systems)
[0118] U.S. Pat. No. 4,537,776 to Cooper contains a summary of
information detailing the use of certain binary systems for
penetration enhancement. European Patent Application 43,738, also
describes the use of selected diols as solvents along with a broad
category of cell-envelope disordering compounds for delivery of
lipophilic pharmacologically-active compounds. A binary system for
enhancing metaclopramide penetration is disclosed in UK Patent
Application GB 2,153,223 A, consisting of a monovalent alcohol
ester of a C8-32 aliphatic monocarboxylic acid (unsaturated and/or
branched if C18-32) or a C6-24 aliphatic monoalcohol (unsaturated
and/or branched if C14-24) and an N-cyclic compound such as
2-pyrrolidone or N-methylpyrrolidone.
[0119] Combinations of enhancers consisting of diethylene glycol
monoethyl or monomethyl ether with propylene glycol monolaurate and
methyl laurate are disclosed in U.S. Pat. No. 4,973,468 for
enhancing the transdermal delivery of steroids such as progestogens
and estrogens. A dual enhancer consisting of glycerol monolaurate
and ethanol for the transdermal delivery of drugs is described in
U.S. Pat. No. 4,820,720. U.S. Pat. No. 5,006,342 lists numerous
enhancers for transdermal drug administration consisting of fatty
acid esters or fatty alcohol ethers of C.sub.2 to C.sub.4
alkanediols, where each fatty acid/alcohol portion of the
ester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No.
4,863,970 discloses penetration-enhancing compositions for topical
application including an active permeant contained in a
penetration-enhancing vehicle containing specified amounts of one
or more cell-envelope disordering compounds such as oleic acid,
oleyl alcohol, and glycerol esters of oleic acid; a C.sub.2 or
C.sub.3 alkanol and an inert diluent such as water.
[0120] Other chemical enhancers, not necessarily associated with
binary systems, include dimethylsulfoxide (DMSO) or aqueous
solutions of DMSO such as those described in U.S. Pat. No.
3,551,554 to Herschler; U.S. Pat. No. 3,711,602 to Herschler and
U.S. Pat. No. 3,711,606 to Herschler, and the azones
(n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in U.S.
Pat. No, 4,557,943 to Cooper.
[0121] In PCT/US96/12244 by Massachusetts Institute of Technology,
passive experiments with polyethylene glycol 200 dilaurate (PEG),
isopropyl myristate (IM), and glycerol trioleate (GT) result in
corticosterone flux enhancement values of only 2, 5, and 0.8
relative to the passive flux from PBS alone. However, 50% ethanol
and LA/ethanol significantly increase corticosterone passive fluxes
by factors of 46 and 900.
[0122] Some chemical enhancer systems may possess negative side
effects such as toxicity and skin irritations. U.S. Pat. No.
4,855,298 discloses compositions for reducing skin irritation
caused by chemical enhancer-containing compositions having skin
irritation properties with an amount of glycerin sufficient to
provide an anti-irritating effect. The present invention enables
testing of the effects of a large number of enhancers on tissue
barrier transport, such as transdermal transport, of a compound,
pharmaceutical, or other component.
5.2.3.2 Mechanical Enhancers For Transdermal Delivery
[0123] For convenience, mechanical enhancers are defined as
including almost any extraneous enhancer, such as ultrasound,
mechanical or osmotic pressure, electric fields (electroporation or
iontophoresis) or magnetic fields.
[0124] There have been numerous reports on the use of ultrasound
(typically in the range of 20 kHz to 10 MHz in frequency) to
enhance transdermal delivery. Ultrasound has been applied alone and
in combination with other chemical and/or mechanical enhancers. For
example, as reported in PCT/US96/12244 by Massachusetts Institute
of Technology, therapeutic ultrasound (1 MHz, 1.4 W/cm.sup.2) and
the chemical enhancers utilized together produce corticosterone
fluxes from PBS, PEG, IM, and GT that are greater than the passive
fluxes from the same enhancers by factors of between 1.3 and 5.0.
Ultrasound combined with 50% ethanol produces a 2-fold increase in
corticosterone transport above the passive case, but increase by
14-fold the transport from LA/Ethanol, yielding a flux of 0.16
mg/cm.sup.2/hr, 13,000-fold greater than that from PBS alone.
[0125] Pressure gradients can also be used to enhance movement of
fluids across the skin. Pressure can be applied by a vacuum or a
positive pressure device. Alternatively, osmotic pressure may be
used to drive transdermal transport.
[0126] Similarly, application an of electric current has been shown
to enhance transdermal drug transport and blood analyte extraction.
Such electric current enhances transport by different mechanisms.
For example, application of an electric field provides a driving
force for the transport of charged molecules across the skin and
second, ionic motion due to application of electric fields may
induce convective flows across the skin, referred to as
electro-osmosis. This mechanism is believed to play a dominant role
in transdermal transport of neutral molecules during iontophoresis.
Iontophoresis involves the application of an electrical current,
preferably DC, or AC, at a current density of greater than zero up
to about 1 mA/cm.sup.2. Enhancement of skin permeability using
electric current to achieve transdermal extraction of glucose, was
reported by Tamada, et al., Proceed. Intern. Symp. Control. Rel.
Bioact. Mater. 22, 129-130 (1995).
[0127] Application of magnetic fields to the skin pretreated or in
combination with other permeation enhancers can be used to
transport magnetically active species across the skin. For example,
polymer microspheres loaded with magnetic particles could be
transported across the skin.
[0128] 5.2.4 Adhesives
[0129] Some devices for delivery of an active component or drug
across a tissue barrier, and in particular transdermal delivery
devices such as transdermal patches, typically include an adhesive.
The adhesive often forms the matrix in which the active component
or drug is dissolved or dispersed and, of course, is meant to keep
the device in intimate contact with the tissue, such as skin.
Compatibility of the active component or drug with an adhesive is
influenced by its solubility in that adhesive. Any supersaturated
conditions produced in storage or in use are generally very stable
against precipitation of the active component or drug within the
adhesive matrix. A high solubility is desired in the adhesive to
increase the driving force for permeation through the tissue and to
improve the stability of the device.
[0130] Several classes of adhesive are used, each of which contain
many possible forms of adhesives. These classes include
polyisobutylene, silicone, and acrylic adhesives. Acrylic adhesives
are available in many derivatized forms. Thus, it is often a very
difficult problem to select which adhesive might be best to use
with any particular drug and enhancer. Typically, all ingredients
to be in the device are dissolved in a solvent and cast or coated
onto a plastic backing material. Evaporation of the solvent leaves
a drug-containing adhesive film. The present invention enables
rapid and efficient testing of the effects of various types and
amounts of adhesives in a sample composition or formulation.
[0131] 5.2.5 Solvents
[0132] Solvents for the active component, carrier, or adhesive are
selected based on biocompatibility as well as the solubility of the
material to be dissolved, and where appropriate, interaction with
the active component or agent to be delivered. For example, the
ease with which the active component or agent is dissolved in the
solvent and the lack of detrimental effects of the solvent on the
active component or agent to be delivered are factors to consider
in selecting the solvent. Aqueous solvents can be used to make
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 acceptable for administration to humans in trace amounts,
such as methylene chloride. Other solvents, such as ethyl acetate,
ethanol, methanol, dimethyl formamide (DMF), acetone, acetonitrile,
tetrahydrofuran (THF), acetic acid, dimethyl sulfoxide (DMSO) and
chloroform, and combinations thereof, also may be utilized.
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 will typically be distilled water, buffered saline,
Lactated Ringer's or some other pharmaceutically acceptable
carrier.
[0133] 5.3 Sample Preparation and Screening Methods
[0134] The high throughput screening methods of the present
invention identify, for example, 1) optimal compositions or
formulations comprising one or more active components and one or
more inactive components for achieving desired characteristics for
such compositions or formulations, 2) optimal
adhesive/enhancer/excipient compositions for compatibility with an
active component or drug, 2) optimal active component or
drug/adhesive/enhancer/additive compositions for maximum drug flux
through stratum corneum, and 3) optimal active component or
drug/adhesive/enhancer/additive compositions to minimize
cytotoxicity.
[0135] The basic requirements for sample preparation, processing,
and screening are a distribution mechanism and a testing, or
screening, mechanism. The distribution mechanism adds 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 component(s) and/or the component
concentration, more preferably, two or more variables. For
instance, filling or addition of a sample, such as a pharmaceutical
component and excipients (e.g., enhancers and adhesives) 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. A
testing mechanism is preferably used to test each sample for one or
more properties, such as drug concentration as a function of time.
Preferably, the testing mechanism is automated and driven by a
computer.
[0136] In one embodiment, the system further comprises a processing
mechanism to process the samples after component addition. For
example, after component addition to the sample well but prior to
assembly of the apparatus and in particular placement of the tissue
specimen over the sample well, 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.
[0137] As mentioned supra, a preferred method of using the tissue
barrier transfer device of FIG. 1 entails determining, directly or
indirectly, the presence, absence or concentration of components
(e.g. pharmaceuticals) that diffuse through tissue 120 into
reservoir 132 of the array. Such measurements may be performed by a
variety of means known to those skilled in the art. For example,
any know spectroscopic technique can be used to determine presence,
absence or concentration of a component-in-common. Suitable
measurement techniques include, but are not limited to include
spectroscopy, infrared spectroscopy, near infrared spectroscopy,
Raman spectroscopy, NMR, X-ray diffraction, neutron diffraction,
powder X-ray diffraction, radiolabeling, and radioactivity.
[0138] In one exemplary embodiment, and not by way of limitation,
the passive permeabilities of active components (e.g. a drug)
through human skin can be measured using trace quantities of
radiolabelled active component or drug. According to known methods,
radiolabelled compounds or drugs are rotary evaporated in order to
remove any solvent in which they are shipped and any tritium which
had reverse exchanged into it. The radiolabelled compounds or drugs
are then redissolved in various composition formulations, including
enhancers, carriers, additives, adhesives, and/or other excipients
as described infra, to a typical concentration of 1 .mu.Chi/ml, and
added to the sample wells, such as sample wells 116 of array 112 in
FIG. 1. Passive permeation experiments are then performed. The
reservoir compartments, such as reservoirs 132 of FIG. 1.,
preferably contain, for example, pH 7.4 phosphate buffer saline
(PBS, phosphate concentration=0.01 M, NaCl concentration=0.137 M)
(Sigma Chemical Co.). Other receiver solutions may be used and are
known to those skilled in the art. The concentrations of
radiolabelled component or drug in the sample and reservoir
compartments are measured using a scintillation counter (e.g.,
model 2000 CA, Packard Instruments). Duplicate formulations may be
used in some of the samples and/or repeated experiments may be
performed to optimize reliability of measurements.
[0139] The permeability values can be calculated under steady-state
conditions from the relationship P=(dN.sub.r/dt)/(AC.sub.d) where A
is the surface area of the tissue accessible to a sample, C.sub.d
is the component or drug concentration in the sample, and N.sub.r
is the cumulative amount of component or drug which has permeated
into the receptor reservoir. Inter-subject variability of the human
skin permeability of 40%, is reported by Williams, et al., Int. J.
Pharm. 86, 69-77 (1992). The passive permeability enhancements,
E.sub.p, is calculated relative to the passive permeability from
PBS according to Eq. (1): 1 E p = P ( enhancer ) P ( PBS ) ( 1
)
[0140] where P(enhancer) is the drug permeability from a given
enhancer, and P(PBS) is the drug permeability from PBS. The fluxes
from saturated solutions, J.sup.sat, are calculated from
J.sup.sat=PC.sup.sat, where C.sup.sat is the drug solubility in the
formulation. Flux enhancements, E.sub.J, are calculated using Eq.
(2), 2 E J = it ( enhancer ) J sat ( PBS ) ( 2 )
[0141] where J.sup.sat (enhancer) and J.sup.sat (PBS) are the drug
fluxes from saturated solutions of enhancer and PBS,
respectively.
[0142] 5.4 Correction or Repair of Microdefects in Skin Tissue
Samples
[0143] The present invention includes a methods for repairing
and/or correcting for microscopic defects on tissue specimens, such
as skin. For example, apparatus or a diffusion cell used for study
of transdermal delivery of active components (e.g., pharmaceuticals
or drugs) require skin samples that are free of defect that might
act as diffusional fast transport paths. Such defects can be of
several types with sizes ranging from millimeters to tens of
microns. Physical tears and hair follicles are just two types of
defects that may compromise the interpretation of transport or
diffusion data. Inhomogeneous tissue segments, i.e. segments with
an abnormal amount of defects, will lead to inaccurate and
misleading diffusion measurements, particularly when using
relatively small tissue samples as in the present invention. Rapid
identification of defect locations on the surface of a given tissue
sample may be achieved by image analysis, preferably by high-speed
micro inspection of each tissue segment using video microscopy or
photomicrography.
[0144] According to a preferred embodiment of the invention,
diffusion data related to inhomogeneous tissue segments may be
discarded to avoid inaccurate measurements. Alternatively, if the
effect of defects in a tissue segment can be characterized and/or
quantified, associated diffusion measurements can be mathematically
adjusted to account for the defects.
[0145] In another embodiment of the invention, defects in a tissue
specimen are repaired by feeding the defect locations to an ink jet
printer that is instructed to print wax to cover these locations.
The print pattern is devised so as to cover the entire area of the
defect with some possible overlap on to regions that are free of
defects. Wax print heads print molten wax that solidifies on impact
with the tissue. The solid wax is water-resistant and acts like a
seal to ensure that the repaired region does not contribute to the
diffusional flux during subsequent testing. Droplet placement
preferably is such that overlap is sufficient to make a seal.
[0146] 5.5 Alternative Embodiment for Solid Source Samples (FIGS.
2A-2D)
[0147] FIGS. 2A-2D are schematic diagrams of an alternative
high-throughput apparatus 200 and method for measuring tissue
barrier transfer using a solid source sample. Apparatus 200 is
similar to apparatus 100 of FIG. 1, except that apparatus 200 is
designed for testing solid source samples, such as compositions
containing a semi-solid, such as an adhesive, a relatively flat
transdermal patch, or a film-like sample. Substrate plate 214 is a
dense plate, such as a plastic or glass plate, that supports an
array 212 of samples 216. Each sample includes a combination of
components, including an active component (e.g., a pharmaceutical)
and at least one inactive component. Examples of suitable
components are discussed above with respect to FIG. 1.
[0148] A first step of the method involves creating an array 212 of
different composition regions (i.e., samples 216) on dense
substrate 214. The array may be produced in any number of ways, but
one simple method is to use combinatorial dispensing equipment to
make solutions of all the constituents in a convenient solvent.
Suitable dispensing equipment and methods of formulating solutions
or compositions are discussed above and disclosed in U.S. patent
application Ser. No. 09/540,462, which is herein incorporated by
reference in its entirety.
[0149] In a preferred embodiment, the formulated solutions are
contained in the wells of a microtiter plate similar to substrate
plate 114 (of FIG. 1) that includes a sample array 112 of sample
wells 116 and separable dense bottom plate 214 rather than base
118. The solvent is then evaporated and each of the samples in the
wells is allowed to dry to leave a film at the bottom. This
evaporation process mimics the manufacturing process used to make
various tissue transfer devices, such as transdermal patches. The
upper plate may then be removed to yield the array shown in FIG.
2A. The samples 216 can be any shape, and preferably are generally
round in shape as shown in FIG. 2A.
[0150] It should be noted that the plates of this format can be
used to assess the stability of the compositions or formulations,
such as drug/adhesive/enhancer solutions, toward precipitation of
the active component, such as a pharmaceutical or drug. Optical
examination of each of the films will reveal if precipitation has
occurred, since the precipitates may cause increased light
scattering when the sample is illuminated. Crystallization can also
be optically detected by observing birefringence of crystals (for
compounds that are birefringent). Alternative means may be used
when the film is already sufficiently opaque to preclude the
scattering method. One such method is second harmonic generation
(SHG) which easily detects the presence of crystals in the film. It
is also possible to use microfocus X-ray diffraction to detect the
presence of crystals.
[0151] Referring to FIG. 2B, the next step of the present method is
to prepare a tissue specimen 220 that is to be used in the study. A
specimen 220, such as a specimen of stratum corneum, may be
conventionally prepared or obtained as described above. It is most
convenient, however, that the sample specimen 220 should be
sufficiently large to cover whatever plate format is used for the
study. For example, it should be sufficiently large to cover a 96
well microtiter plate. Thus, a separate tissue sample is prepared
for each plate 214 of the study. The tissue is then placed on plate
214 so as to cover each of the sample regions, as shown in the FIG.
2B. Care is taken to insure that no air pockets are present under
tissue 220. One approach is to lay tissue 220 down on plate 214
starting at one edge and gently proceeding across the surface of
the plate. The air is expelled ahead of the tissue/plate contact
line.
[0152] Referring to FIG. 2C, in one embodiment of the present
invention, the region of tissue 220 above each sample region may
now be physically sectioned or isolated into segments 224 from
neighboring regions to ensure that lateral diffusion does not occur
between adjacent samples. As described above, this can be done in
any number of ways, such as mechanical scribing or cutting, laser
cutting or crimping along cuts 222.
[0153] Each of the tissue segments 224 on each plate 214 may now be
imaged and characterized by video microscopy. Automated image
recognition can be used to identify and record those tissue
segments that are damaged or otherwise inhomogeneous. As described
above, damaged or inhomogeneous tissue segments 224 may be
replaced, repaired or ignored. Alternatively, data associated with
damaged or inhomogeneous segments 224 may be adjusted to account
for the defects. Optionally, tissue 220 may be imaged and replaced
or repaired prior to sectioning. In yet another alternative method,
the tissue 220 is sectioned and/or imaged before placing tissue
segments 224 over samples 216.
[0154] Referring to FIG. 2D, a next step in the present method is
to place a reservoir plate 230, similar to reservoir plate 130 of
FIG. 1 or an open-bottomed titer plate, over the tissue segments
224 as shown. Reservoir plate 230 includes a number of hollow
reservoirs 232. When plate 230 is secured in place, each reservoir
232 aligns over a sample and tissue such that a tissue segment 224
separates each sample from reservoir 232. Reservoir plate 230
secures to substrate plate 214 using clamps, screws, fasteners, or
any other suitable attachment means. Plates 230 and 214 preferably
secure together with sufficient pressure so as to create a liquid
tight seal around reservoirs 232. Each reservoir is filled with a
reservoir medium, preferably a liquid or solution, such as a saline
solution, to receive sample compounds that diffuse across tissue
segments 224 to reservoir 232. In one embodiment, the reservoir
medium is approximately 2% BSA solution in PBS.
[0155] Incubation of the apparatus 200 with automated periodic
sampling and makeup of the reservoir 232 solution is used to assess
the permeability of the active component for all the samples of the
combinatorial study.
[0156] Although the embodiments of the invention described herein
are directed to movement of compounds across a tissue, the systems
and methods of the present invention are suitable for studying
movement of compounds across any membrane or other barrier.
[0157] 5.6 Alternative Embodiment Using Indirect Measurement (FIG.
3)
[0158] In FIG. 3, another embodiment of the invention, apparatus
300 relates to a method of high-throughput screening of active
component flux through a tissue specimen, such as the stratum
corneum, recognizing that such flux is determined, at least in
part, by the permeability of the active component (such as a
pharmaceutical or drug) within the tissue in the presence of an
enhancer. The permeability is generally governed by at least two
factors: the solubility of the active component within the tissue
(such as the stratum corneum) and the diffusivity of the active
component within the tissue specimen. These two factors, solubility
and diffusivity, are measured independently as a method of
indirectly assessing the flux through the tissue specimen.
[0159] Referring to FIG. 3, an array 312 of wells 316 containing
samples (e.g. solutions 338) of different compositions of active
components and inactive components (e.g.,
pharmaceutical/adhesive/enhancer/additive) is constructed. Known
amounts of tissue segments 340, e.g. stratum corneum, are added to
each well. Alternatively, a tissue segment is placed on or over
each well 316 (similar to the arrangement shown in FIGS. 1, 2C and
2D) such that each segment is in contact with a sample solution
338. The rate at which a component (e.g., a drug, or
pharmaceutical) is taken up into the tissue sample may be measured
by extracting the tissue 340 from similarly prepared wells 316 at
different times and measuring the presence, absence, or
concentration of the component. Measuring the concentration after
times sufficiently long so that the amount dissolved is not
changing with time can assess solubility, or the equilibrium
concentration of the component within the tissue 340. The product
of the rate and solubility is proportional to the permeability of
the component.
[0160] 5.7 Alternative Tissue Barrier Transfer Apparatus (FIGS.
4A-4C)
[0161] Referring to FIG. 4A, an alternative embodiment of the
apparatus of FIG. 1 is diffusion cell 400. Diffusion cell 400
includes a sink plate 410, a source plate 430, and a tissue
specimen 420 disposed between sink plate 410 and source plate 430.
Sink plate 410 includes a sink well 412 for holding reservoir
medium as described above with respect to FIG. 1. Sink well 410 is
shown as having a cylindrical shape with an open end, however it
may be rectangular, hexagonal, spherical, elliptical, or any other
shape. Sink plate 410 includes at least one access port 416 along
an edge of sink well 412 that fluidly communicates with sink well
412. Sink plate 410 also preferably includes a surface feature 414
configured to mate with source plate 430 and form a tight seal with
tissue specimen 420.
[0162] In one preferred embodiment, tissue specimen 420 is skin
tissue, but may be any tissue or membrane as described above with
respect to tissue specimen 120 of FIG. 1. Tissue specimen 420 is
cut, formed or otherwise dimensioned to cover sink well 412 and
surface feature 414. Tissue specimen 420 is placed such that it
preferably does not completely cover access port 416.
[0163] Referring to FIG. 4C, source plate 430 includes a source
reservoir, or well 432 that has open ends and aligns with sink well
412 when source plate 430 is placed on tissue specimen 420. A
passage 436 also passes through source plate 430 and is
approximately adjacent to, but not in communication with, source
well 432. Passage 436 is configured to align with access port 416
to provide access to the reservoir medium in sink well 412 without
removing source plate 430.
[0164] Referring again to FIG. 4A, source plate 430 also preferably
includes a surface feature 434 that is configured and dimensioned
to mate with surface feature 414 of sink plate 410 and form a seal
with tissue specimen 420 around the perimeter of sink well 412 and
source well 432. For example, in one embodiment surface feature 414
is a convex ring extending from the surface sink plate 420 around
the open perimeter of sink well 412; and surface feature 434 is a
concave ring formed in source plate 430 configured to mate with
surface feature 414.
[0165] In another embodiment of the present invention, a number
diffusion cells 400 are attached or formed together to create an
array of diffusion cells similar to array 112 of FIG. 1.
[0166] Exemplary uses of the apparatus of FIGS. 4A-4C are the same
as those described above with respect to FIG. 1, except that access
port 416 and passage 436 allow addition or removal of reservoir
medium from sink well 412 without removing source plate 430 or
tissue 420. Preferably, the reservoir medium used in diffusion cell
400 is a liquid or solution. In an alternative method of using
diffusion cell 400, the placement of reservoir medium and sample
could be reversed as in FIG. 1; for example, reservoir medium could
be placed above tissue specimen 420 in source well 432 and sample
could be held in sink well 412. In such an embodiment, sample may
be added or removed through passage 436 and access port 416.
[0167] 5.8 Method For Filling or Adding Samples (FIGS. 5A &
5B)
[0168] FIGS. 5A and 5B show a schematic drawing of an apparatus 500
for use in adding or filling a sample 530 into a sample well 522 in
a sample array, such as sample array 112 shown in FIG. 1, wherein
the occurrence of air pockets or bubbles between the sample 530 and
a tissue 524 is avoided. In the sample array, the tissue 524 is
located between a sample well 522, which is located in a substrate
plate, such as substrate plate 114 shown in FIG. 1, and a reservoir
526, which is located in a reservoir plate, such as reservoir plate
130 shown in FIG. 1. In the filling method of the present
invention, a feed canula 510, having a sample feed source 514 and
an air evacuation space 512, punctures a base membrane 520 which
covers one side a the sample well 522 to be filled with sample
530.
[0169] Then, sample feed source 514 is extended into sample well
522 until it is in contact with tissue 524. Sample 530 is then fed
through sample feed source 512, and as sample 530 begins to fill
sample well 522, air is forced out of sample well 522 through air
evacuation space 512 in feed canula 510. When the desired amount of
sample 530 is filled into sample well 522, sample feed source 512
and feed canula 510 are completely withdrawn from base membrane 520
and sample well 522.
[0170] In a preferred embodiment of the filling method of the
present invention, while sample 530 is being fed into sample well
522, sample feed source 514 retracts at a rate that is synchronized
with the fill rate for sample 530 into sample well 522 such that at
all times during the filling process, the outlet of sample feed
source 514 is inside extruded sample 530 in sample well 522. When
the desired amount of sample 530 is filled into sample well 522,
both sample feed source 512 and feed canula 510 are completely
withdrawn from base membrane 520 and sample well 522.
[0171] In a preferred embodiment, base membrane 520 is a rubber
membrane.
[0172] The filling method of the present invention can be performed
by hand or using automated dispensing means, wherein sample wells
in a sample array are filled using automated dispensing equipment
that is capable of dispensing the same or different samples to
multiple sample wells in one or more sample arrays in a fast,
accurate, and controlled approach.
[0173] Sample 530 dispensed in accordance with the filling method
of the present invention is preferably a liquid source sample.
[0174] 5.9 Alternative Embodiments For Solid Source Samples (FIGS.
6-9)
[0175] FIG. 6 shows an exploded view, schematic diagram of a
preferred embodiment of a high-throughput apparatus 600 for
measuring tissue barrier transport in an array of solid source
samples 630 according to the present invention. Apparatus 600
comprises a base plate 610 supporting a spacer plate 620, an array
of solid source samples 630, a tissue specimen 640, a reservoir
plate 650 having an array of donor reservoirs 654, and a clamping
means, such as shoulder screws 660 with threads 662.
[0176] Preferably, base plate 610 is made aluminum, and spacer
plate 620 and reservoir plate 650 are made of clear plastic or
polycarbonate.
[0177] Base plate 610 has screw holes 612 which are drilled to mate
with threads 662 on shoulder screws 660, such that when screws 660
are fed threw the apparatus into screw holes 612 and tightened, the
apparatus is clamped together. When the apparatus is clamped
together, a seal is formed between reservoir plate 650 and tissue
specimen 640. There can be any number of screw holes 612 located
around the edges of base plate 610, but preferably, the number of
screw holes 612 is at least 4, and more preferably between 4 and 8.
In a preferred embodiment, base plate 610 further comprises an
array of guide marks 614, which can be any array formation, such as
2.times.2, 4.times.4, 6.times.6, and 8.times.12, which are used to
help align various components of apparatus 600 during assembly.
[0178] Screw holes 622 and screw holes 652 in spacer plate 620 and
reservoir plate 650, respectively, are drilled to allow the neck
and threads 662 of shoulder screws 660 to smoothly pass through,
but not the head of shoulder screw 660 (as shown for shoulder
screws 760 and 860 in FIGS. 7 and 8, respectively). There can be
any number of screw holes 622 and screw holes 652 located around
the edges of spacer plate 620 and reservoir plate 650,
respectively, but preferably, the number of screw holes 622 and
screw holes 652 is at least 4, and more preferably between 4 and 8.
In a preferred embodiment, there is at least a screw hole at each
corner of both spacer plate 620 and reservoir plate 650.
[0179] In an alternative embodiment, apparatus 600 further
comprises a top plate located above reservoir plate 650, which is
made out of the same material as base plate 610 (e.g., aluminum)
and is either an open frame having screw holes matching screw holes
652 in reservoir plate 650 or is a "solid" plate having the same
screw holes and array of reservoirs as screw holes 652 and donor
reservoirs 654 on reservoir plate 650.
[0180] Apparatus 600 is assembled by first placing spacer plate 620
on top of base plate 610 and aligning screw holes 622 in spacer
plate 620 with screw holes 612 in base plate 610. An array of solid
source samples 630 is created on spacer plate 620 in a pattern
corresponding to the pattern of donor reservoirs 654 in reservoir
plate 650, and guide marks 614 on base plate 610 are used to ensure
that each sample 630 is placed such that it aligns with a donor
reservoir 654 in top plate 650. The size of samples 630 are
commensurate with the size of donor reservoirs 654.
[0181] Each sample 630 includes a combination of components,
including an active component (e.g., a pharmaceutical) and at least
one inactive component. Examples of suitable components are
discussed above with respect to FIG. 1.
[0182] A sheet of tissue specimen 640 is placed over the array of
samples 630 in a manner which avoids formation of air pockets
between tissue specimen 640 and samples 630. Then, reservoir plate
650 having an array of donor reservoirs 654 is placed over the skin
such that screw holes 652 on top plate 650 align with the
corresponding screw holes 622 of spacer plate 620.
[0183] The resulting assembled apparatus 600 is then clamped
together by sliding shoulder screws 660 with threads 622 through
aligned screw holes 652 of assembled apparatus 600, and each
shoulder screw 660 is tightened so as to form a seal between
reservoir plate 650 and tissue specimen 640. Preferably, a shoulder
screw 660 should be used in at least each of the four corners of
the assembled apparatus 600.
[0184] A reservoir medium is added to donor reservoirs 654 of
assembled apparatus 600, and at an appropriate time or various time
intervals, specimens are withdrawn from donor reservoirs 654 and
used to measure the transfer or flux of components, such as active
components and components-in-common, in samples 630 across tissue
specimen 640. If multiple specimens are taken, after a volume of
specimen is removed from a donor reservoir 654, an equal volume of
reservoir medium is added to the same donor reservoir 654.
[0185] The size of donor reservoirs 654 is about 1 mm to about 50
mm, more preferably about 2 mm to about 10 mm, and most preferably
about 3 mm to about 7 mm.
[0186] FIG. 7 shows a compressed view, schematic diagram of a
high-throughput apparatus 700 for measuring tissue barrier
transport in an array of solid source samples according to the
present invention. Apparatus 700 is the similar to apparatus 600,
except the array of donor reservoirs 754 is an 8.times.12 array for
a total of 96, wherein each reservoir is no more than 6 mm in
diameter. Apparatus 700 comprises a base plate 710 supporting a
spacer plate 720, an array of solid source samples (such as samples
630 shown in FIG. 6), a tissue specimen (such as tissue specimen
640 shown in FIG. 6), a reservoir plate 750 having an array of
donor reservoirs 754, and a clamping means, such as shoulder screws
760.
[0187] FIG. 8 shows a compressed view, schematic diagram of a
high-throughput apparatus 800 for measuring tissue barrier
transport in an array of solid source samples according to the
present invention. Apparatus 800 is the similar to apparatus 600
and apparatus 700, except the array of donor reservoirs 854 is a
16.times.24 array for a total of 384 donor reservoirs 854 wherein
each reservoir is no more than 3 mm in diameter. Apparatus 800
comprises a base plate 810 supporting a spacer plate 820, an array
of solid source samples (such as samples 630 shown in FIG. 6), a
tissue specimen (such as tissue specimen 640 shown in FIG. 6), a
reservoir plate 850 having an array of donor reservoirs 854, and a
clamping means, such as shoulder screws 860.
[0188] Variations to the apparatuses of FIGS. 7 & 8 are the
same as those described for FIG. 6, and other variations to and
exemplary uses of the apparatuses of FIGS. 6-8 are the same as
those described above with respect to FIG. 1, where applicable.
[0189] FIG. 9 is an exploded view of a another transdermal assay
apparatus according to another embodiment of the invention. The
transdermal assay apparatus 900 is similar to the apparatuses 600,
700, and 800 described above, as the apparatus 900 is also used for
measuring the transport of a sample through a tissue barrier or
specimen. The sample is preferably a solid source sample, but
alternatively may be any other suitable sample.
[0190] Transdermal assay apparatus 900 comprises first and second
members 902 and 904, respectively. The first and second members are
preferably made from stainless steel, coated appropriately to
obtain the desired chemical resistance. These members could also be
made from a clear plastic, such as polycarbonate, other plastics,
or glass. The first member 902 has opposing sides 905 and 922.
These opposing sides are preferably substantially planar surfaces
that are substantially parallel to one another. One side 905
includes one or more sample surfaces 906 configured to receive one
or more samples 908 thereon. The sample surfaces 906 are preferably
circular and arranged in an array, as shown.
[0191] The second member 904 also has opposing sides 910 and 914
that are also preferably substantially planar surfaces that are
substantially parallel to one another. The second member 904
defines one or more reservoirs 912 therein. The size of each
reservoir 912 is similar to that described above in relation to
FIGS. 7-8. Each reservoir has an opening 934 on the side 910 of the
second member 904. In a preferred embodiment, each opening 934 is
substantially the same size as each sample surface 906.
[0192] In use, each sample surface 906 is configured to
substantially align with a corresponding opening 934 in a direction
substantially perpendicular to the sample surface 906. Accordingly,
in use, each sample 908 of an array of samples is configured to
substantially align with a corresponding opening 934 of an array of
openings. In one embodiment, the reservoirs 912 extend entirely
through the second member 904 from the surface 910 to the opposing
side 914, in a similar manner to that described above.
[0193] The transdermal assay apparatus 900 also preferably
comprises a third member 916. The third member 916 is preferably
made from aluminum, but could also be made from a number of other
materials, such as steels, brass, plastics, ceramics or the like,
that can be manufactured into a part that provides the desired
geometry and dimensional stability. The third member 916 has
opposing sides 918 and 920 that are also preferably substantially
planar surfaces that are substantially parallel to one another. The
third member 916 preferably includes a cavity 924 therein for
receiving a magnet 926. In an alternative embodiment, the magnet
926 is permanently formed in the third member 916, bonded to the
third member, or the third member 916 itself is the magnet 926,
i.e., the third member is made from a magnetic material. The magnet
926 may be a permanent magnet, electromagnet, or the like.
[0194] The magnet 926 forms one portion of a magnetic clamp used to
clamp first 902, second 904, and third members 916 together. The
other portion of the magnetic clamp preferably comprises one or
more inserts 928 embedded in the second member 904. In a preferred
embodiment, these inserts 928 are made from a ferrous material that
is attracted to the magnet 926. In an alternative embodiment, the
inserts 928 are magnetic inserts that are attracted to the magnet
926, i.e., the magnetic inserts and the magnet 926 are arranged to
have opposite polarities facing one another. These magnetic inserts
are preferably permanent magnets, but alternatively may be
electromagnets. In a preferred embodiment, the first and third
members are made from a ferrous or magnetic material (such as 430
stainless steel). These magnetic members are then attracted to hold
by the magnet in the third member to provide the desired clamping
force.
[0195] The third member also preferably includes at least one, but
preferably two or more, alignment posts 930 configured to mate with
complementary alignment holes 932 in the first member 902. The
alignment posts are preferably made from a ferrous material to aid
the attraction between the magnet 926 and the inserts 928, and to
hold the magnet 926 in place in the cavity 924.
[0196] It should be appreciated that in alternative embodiments the
magnetic clamp may take on various other forms. For example, the
magnetic clamp may comprise one or more magnets and/or ferrous
inserts disposed in, on, or near the first member 902, second
member 904, and/or third member 916. In addition, these members may
be fully or partially made from ferrous and/or magnetic materials.
In addition, the strength of the magnet 926, or the gap between the
magnet(s) and insert(s) or magnetic material(s) is preferably
chosen to provide the required clamping force between the members.
In other words, the magnet 926 can be replaced by a stronger or
weaker magnet to alter the resulting clamping force and to optimize
the sealing among the members of the apparatus 900.
[0197] In a preferred method of use, the magnet 926 is bonded to
the third member 916. A sample 908 is arranged on each sample
surface 906 of the first member 902, such that each sample surface
906 will align with a corresponding opening 934. The size of the
samples 908 are commensurate with the size of each opening 934.
Each sample 908 includes a combination of components, including an
active component (e.g., a pharmaceutical) and at least one inactive
component. Examples of suitable components are discussed above with
respect to FIG. 1.
[0198] The second member 902 is then placed onto the third member
916 such that the alignment posts 930 mate with the alignment holes
932. This aligns the second member 902 in a predetermined position
on the third member 916. A sheet of tissue specimen 936 is placed
over the samples 908 in a manner which avoids formation of air
pockets between the tissue specimen 936 and the samples 908.
[0199] The second member 904 is then placed over the tissue
specimen 936, such that the tissue specimen 936 is exposed to each
reservoir's opening 934. A magnetic attraction between the magnet
926 and inserts 928, preferably along the alignment posts 930,
aligns the second member 904 with the first member 902, such that
each sample 908 is aligned under a corresponding opening 934. This
magnetic attraction between the magnet 926 and inserts 928 also
creates a clamping force that clamps the first, second, and third
members together, thereby clamping the tissue specimen between each
sample 908 and each opening 934.
[0200] A fluid medium is added to the reservoirs 912 of assembled
apparatus 900, and at an appropriate time or various time
intervals, specimens of the fluid medium are withdrawn from
reservoirs 912 and used to measure the transfer or flux of
components, such as active components and components-in-common, in
the samples 908 across the tissue specimen 936. If multiple
specimens are taken after a volume of specimen is removed from a
reservoir 912, an equal volume of fluid medium is added to the same
reservoir 912.
[0201] FIG. 10 is an exploded view of a yet another transdermal
assay apparatus 1000 according to yet another embodiment of the
invention. The transdermal assay apparatus 1000 is similar to the
transdermal assay apparatus 900 (FIG. 9) described above. Unlike
the transdermal assay apparatus 900 (FIG. 9), the transdermal assay
apparatus 1000 includes a sample plate and flexible magnet, as
described below. In addition, the sample is preferably a solid
source sample, but alternatively may be any other suitable
sample.
[0202] Transdermal assay apparatus 1000 comprises a first member
1010 and a second member 1002. These members are preferably made
from stainless steel, coated appropriately to obtain the desired
chemical resistance. These members could also be made from a clear
plastic, such as polycarbonate, other plastics, or glass. These
members also preferably have opposing planar surfaces and are
substantially parallel to one another. The second member 1002
defines one or more reservoirs 1014 therein, each having an
opening, as described above in relation to FIG. 9.
[0203] In use, a flexible magnet 1008 is disposed between the first
and second plates. A sample array, preferably disposed on a sample
plate 1006 is disposed between the flexible magnet 1008 and the
second plate. Similarly, a tissue sample 1004 is disposed between
the sample plate 1006 and the second plate 1002.
[0204] The sample plate 1006 includes one or more sample surfaces
1012 configured to receive one or more samples thereon. The sample
surfaces 1012 are preferably circular and arranged in an array, as
shown. In use, each sample surface 1012 is configured to
substantially align with a corresponding opening of each reservoir
1014 in a direction substantially perpendicular to the sample
surface 1012. Accordingly, in use, each sample of an array of
samples is configured to substantially align with a corresponding
opening of an array of openings. In one embodiment, the reservoirs
1014 extend entirely through the second member 1002, in a similar
manner to that described above.
[0205] The flexible magnet 1008 forms one portion of a magnetic
clamp used to clamp the first 1010 and second 1002 members
together. The second plate 1002 is preferably made from a ferrous
material that is attracted to the flexible magnet 1008. In an
alternative embodiment, ferrous inserts are placed in the second
member 1002.
[0206] The transdermal assay apparatus 1000 is used as described
above.
6. EXAMPLE
[0207] The following Example further illustrates the method and
arrays of the present invention. It is to be understood that the
present invention is not limited to the specific details of the
Example provided below.
Example 1
Nicotine Permeation Across Human Cadaver Skin
[0208] Human cadaver skin epidermis was prepared by first
separating skin from the underlying fat and then separating the
epidermis by heat treatment at 60.degree. C. for 90 seconds using
standard techniques.
[0209] A NICODERM CQ.RTM. brand nicotine Step 1 (21 mg/24 hours)
transdermal patch (sold by GlaxoSmithKline, Research Triangle Park,
N.C. USA) was punched into {fraction (5/16)}" diameter circles,
keeping the backing and release liners on the resulting punched
samples until such were deposited in the test apparatus.
[0210] An apparatus as described in FIG. 6 was assembled, wherein
each plate in the apparatus was a rectangular shape having
dimensions of 5.030" (127.76 mm) by 3.365" (85.48 mm). The
apparatus was assembled by first placing a {fraction (1/8)}" (3.175
mm) thick clear polycarbonate spacer plate 620 on top of an
aluminum base plate 610 and aligning screw holes 622 in the spacer
plate with screw holes 612 in the base plate. Thereafter, a
4.times.4 sample array was created on spacer plate 620, as
described in Table 2 below:
2TABLE 2 4 .times. 4 Test Array 1 2 3 4 A sample sample sample
sample* B sample sample empty (control) sample C empty (control)
sample sample sample D sample* sample empty (control) sample
[0211] Punched samples were placed on spacer plate 620 in the
4.times.4 array of Table 2 one at a time, and the location of each
array sample was selected using guide marks 614 on base plate 610
to ensure that each array sample was placed such that it aligned
with a donor reservoir 654 in reservoir plate 650. There were 96,
0.020" (0.508 mm) deep, guide marks 614 on base plate 610, arranged
in a 12.times.8 array which was located 11.24 mm in from the edges
of the long sides of base plate 610, and 14.38 mm in from the edges
of its short sides. At the time of placement, the release liner on
each sample was removed to expose the drug reservoir/adhesive of
the sample.
[0212] In array samples A4 and D1, the drug reservoir of the sample
patch came off of the backing with the release liner. Array
locations B3, C1, and D3 were controls without any samples in order
to determine the potential impact of lateral diffusion on
transdermal transport measurements with this apparatus.
[0213] Once all the samples were placed in the array, the piece of
heat-stripped human cadaver skin, the size of which was larger than
the array of samples, was gently and slowly placed over the samples
so as to avoid any air pockets between the skin and the samples.
The skin was oriented with the stratum corneum next to the samples.
Then, a {fraction (1/4)}" (6.35 mm) thick clear polycarbonate
reservoir plate 650 having an 4.times.4 array of {fraction (1/4)}"
(6.35 mm) diameter donor reservoirs 654 was placed over the skin
such that all of screw holes 652 on reservoir plate 650 were
aligned with the corresponding screw holes 622 of spacer plate
620.
[0214] The resulting assembled apparatus 600 was clamped together
by sliding a shoulder screw 660 with threads 622 through aligned
screw holes 652 at each of the four corners of the assembled
apparatus, and tightening each shoulder screw 660 so as to form a
seal between reservoir plate 650 and the skin. The screw holes 612
on base plate 610 had a 10-24 tap, ranging between 0.250" (6.35 mm)
and 0.188 (4.775 mm), which gripped threads 662 of screws 660 as
the screws were tightened, thereby clamping the apparatus
together.
[0215] 75 ml of Dulbecco's Phosphate Buffered Saline (PBS) was
added to each donor reservoir in the array as the reservoir
medium.
[0216] After 2 hours, a 50 ml test aliquot of reservoir medium was
removed from each donor reservoir 654, and at that time, an
additional 50 ml of PBS was added into each donor reservoir 654.
Each of the 2-hour test aliquots was placed in an HPLC vial and
diluted to 500 ml by addition of 450 ml of 50:50 (v/v) 50 mM
potassium phosphate (adjusted to pH 3.0 with phosphoric acid) and
acetonitrile.
[0217] The foregoing process was repeated at 3 hours, 4 hours, and
5 hours. At the end of the sampling phase of the experiment, each
donor reservoir 654 in the array resulted in four (4) 50 ml test
aliquots that were diluted as set forth above, except that the test
aliquot taken at 3 hours for the B3 donor reservoir was diluted to
950 ml rather than 500 ml.
[0218] The nicotine content in each test aliquot was then
determined by HPLC analysis.
[0219] The components of the HPLC system used to analyze the test
aliquots were a Waters 2790 Separations Module, a Waters Photodiode
Array Detector Model 996, and Waters Millennium 32 v3.2
Chromatography Software (Waters Corp., Milford Mass.).
[0220] The HPLC analysis was performed using a Platinum EPS C18
column (Alltech Associates, Muskegan, Mich.) with dimensions of 250
mm.times.4.6 mm and a 5 mm particle size. The mobile phase was
50:50 (v/v) 50 mM potassium phosphate (adjusted to pH 3.0 with
phosphoric acid): acetonitrile, with a flow rate of 1.0 ml/minute.
Detection was performed by measuring UV absorbance at a wavelength
of 260 nm. The run time was 4 minutes. Injection volume was 10 ml.
Column temperature was ambient.
[0221] Quantification of nicotine content in each test aliquot was
performed by comparison to a calibration curve generated using a
set of nicotine standards (Sigma). Nicotine quantitation was shown
to be linear over a range of 1-100 mg/ml. Potential chromatographic
interference with this method of other components (e.g., fat,
protein) in the skin was ruled out by direct analysis.
[0222] The results of the HPLC analysis are set forth in Tables 3
and 4 below:
3TABLE 3 Nicotine Concentration Of Diluted Test Aliquots
Concentration (mg/ml) 2 hour 3 hour 4 hour 5 hour A1 39.7 37.7 33.0
33.5 A2 51.2 26.7 42.5 42.4 A3 49.3 40.7 30.5 33.2 A4 1.1 1.4 1.6
1.8 B1 11.3 16.8 17.1 16.7 B2 25.4 33.0 36.6 30.1 B3 2.2 2.5 4.8
6.8 B4 28.1 36.1 33.3 32.7 C1 1.5 3.2 2.7 2.6 C2 37.5 37.0 33.4
29.1 C3 10.1 12.9 13.1 15.2 C4 27.2 32.7 38.0 34.2 D1 1.0 1.2 1.4
1.6 D2 15.4 25.2 28.8 29.5 D3 2.6 4.0 4.2 4.7 D4 37.4 36.1 39.3
31.5
[0223]
4TABLE 4 Nicotine Concentration Of Original Test Aliquots
Concentration (mg/ml) 2 hour 3 hour 4 hour 5 hour A1 397.0 377.0
330.0 335.0 A2 512.0 267.0 425.0 424.0 A3 493.0 407.0 305.0 332.0
A4 11.0 14.0 16.0 18.0 B1 113.0 168.0 171.0 167.0 B2 254.0 330.0
366.0 301.0 B3 22.0 25.0 48.0 68.0 B4 281.0 361.0 333.0 327.0 C1
15.0 32.0 27.0 26.0 C2 375.0 370.0 334.0 291.0 C3 101.0 129.0 131.0
152.0 C4 272.0 327.0 380.0 342.0 D1 10.0 12.0 14.0 16.0 D2 154.0
252.0 288.0 295.0 D3 26.0 40.0 42.0 47.0 D4 374.0 361.0 393.0
315.0
[0224] The accumulation of nicotine in each donor reservoir was
calculate according to the following equations, Eq. (3)-(6):
Ac.sub.2hr(mg)=[C.sub.2].times.0.075 ml (3)
AC.sub.3hr(mg)=[C.sub.3].times.0.075 ml+([C.sub.2].times.0.05 ml)
(4)
AC.sub.4hr(mg)=[C.sub.4].times.0.075
ml+(([C.sub.2]+[C.sub.3]).times.0.05 ml) (5)
AC.sub.5hr(mg)=[C.sub.5].times.0.075
ml+(([C.sub.2]+[C.sub.3]+[C.sub.4]).t- imes.0.05 ml) (6)
[0225] The results of the nicotine accumulation calculation for
each donor reservoir in the array are set forth in Tables 6A and 6B
below:
5TABLE 6A Nicotine Accumulation For Reservoirs A1 to B4 Nicotine
Accumulation (mg) A1 A2 A3 A4 B1 B2 B3 B4 0 hr 0 0 0 0 0 0 0 0 2 hr
29.775 38.400 36.975 0.825 8.475 19.050 1.650 21.075 3 hr 48.125
45.625 55.175 1.600 18.250 37.450 4.663 41.125 4 hr 63.450 70.825
67.875 2.450 26.875 56.650 7.075 57.075 5 hr 80.325 92.000 85.150
3.400 35.125 70.075 10.975 73.275
[0226]
6TABLE 6B Nicotine Accumulation For Reservoirs C1 to D4 Nicotine
Accumulation (mg) C1 C2 C3 C4 D1 D2 D3 D4 0 hr 0 0 0 0 0 0 0 0 2 hr
1.125 28.125 7.575 20.400 0.750 11.550 1.950 28.05 3 hr 3.150
46.500 14.725 38.125 1.400 26.600 4.300 45.775 4 hr 4.375 62.300
21.325 58.450 2.150 41.900 6.450 66.225 5 hr 5.650 75.775 29.450
74.600 3.000 56.825 8.925 80.025
[0227] As shown in the foregoing results, the active component,
nicotine, was detected in donor reservoirs, and thus nicotine
crossed the skin barrier. These results also indicate that the
detection or measuring method used was sufficiently sensitive to
detect the transported nicotine. There clearly was transdermal
movement of the active component, nicotine, and most of the samples
demonstrated similar rates of transport.
[0228] In addition, substantially lower amounts of nicotine were
detected in donor reservoirs that were not located over samples,
demonstrating that lateral diffusion of nicotine to adjacent
"wells" was sufficiently slower than direct transdermal movement.
Thus, this experiment clearly demonstrates the ability of this
apparatus to measure transport of a component across a tissue
barrier.
[0229] The foregoing descriptions of specific embodiments of the
present invention are presented for purposes of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise forms disclosed, obviously many
modifications and variations are possible in view of the above
teachings. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
applications, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated.
Furthermore, the order of steps in the method are not necessarily
intended to occur in the sequence laid out. It is intended that the
scope of the invention be defined by the following claims and their
equivalents. In addition, a number of references have been cited
herein, all of which are hereby incorporated herein by
reference.
* * * * *