U.S. patent application number 12/711447 was filed with the patent office on 2010-08-26 for microporation of tissue for delivery of bioactive agents.
Invention is credited to Jonathan A. Eppstein.
Application Number | 20100217212 12/711447 |
Document ID | / |
Family ID | 23292711 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100217212 |
Kind Code |
A1 |
Eppstein; Jonathan A. |
August 26, 2010 |
Microporation of Tissue for Delivery of Bioactive Agents
Abstract
A method of enhancing the permeability of a biological membrane,
including the skin or mucosa of an animal or the outer layer of a
plant to a permeant is described utilizing microporation of
selected depth and optionally one or more of sonic,
electromagnetic, mechanical and thermal energy and a chemical
enhancer. Microporation is accomplished to form a micropore of
selected depth in the biological membrane and the porated site is
contacted with the permeant. Additional permeation enhancement
measures may be applied to the site to enhance both the flux rate
of the permeant into the organism through the micropores as well as
into targeted tissues within the organism.
Inventors: |
Eppstein; Jonathan A.;
(Atlanta, GA) |
Correspondence
Address: |
KING & SPALDING
1180 PEACHTREE STREET , NE
ATLANTA
GA
30309-3521
US
|
Family ID: |
23292711 |
Appl. No.: |
12/711447 |
Filed: |
February 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772472 |
Feb 6, 2004 |
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12711447 |
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10284408 |
Oct 31, 2002 |
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10772472 |
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09331124 |
Aug 12, 1999 |
6527716 |
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PCT/US97/24127 |
Dec 30, 1997 |
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10284408 |
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Current U.S.
Class: |
604/291 |
Current CPC
Class: |
A61B 1/313 20130101;
A61B 5/15136 20130101; A61B 5/150129 20130101; A61B 5/150091
20130101; A61B 2018/00577 20130101; A61B 5/14514 20130101; A61B
5/150175 20130101; A61B 2018/00642 20130101; A61N 1/0476 20130101;
A61B 5/150022 20130101; A61M 2037/0007 20130101; A61N 1/0412
20130101; A61B 18/10 20130101; A61N 2007/0034 20130101; A61B
5/150099 20130101; A61B 2018/00791 20130101; A61B 18/203 20130101;
A61B 18/04 20130101; A61B 2017/00765 20130101; A61N 1/0428
20130101; A61N 1/327 20130101; A61M 37/0092 20130101; A61B
2017/00026 20130101; A61B 2018/00452 20130101; A61B 2017/00172
20130101; A61N 1/325 20130101; A61B 5/150076 20130101; A61B
5/150083 20130101; A61K 41/0047 20130101 |
Class at
Publication: |
604/291 |
International
Class: |
A61F 7/08 20060101
A61F007/08; A61M 37/00 20060101 A61M037/00 |
Claims
1-73. (canceled)
74. An apparatus for delivering a formulation into an organism
comprising: a supply of a dry powder formulation; and a heat
conducting element, which, when put in substantial physical contact
with a selected area of a biological membrane of the organism, is
capable of porating the biological membrane at the selected area to
form at least one micropore 1-1000 .mu.m in diameter, by delivering
sufficient energy to the selected area such that the temperature of
tissue-bound water and other vaporizable substances in the selected
area is elevated above the vaporization point of the water and
other vaporizable substances, thereby removing the biological
membrane in the selected area, and wherein the apparatus enables
the dry powder formulation, when the dry powder formulation is put
in contact with the selected area, to be taken up through the
micropore into the organism, wherein the apparatus delivers the dry
powder formulation into the organism.
75. The apparatus of claim 74, wherein the dry powder formulation
comprises a peptide(s), protein(s), vaccine antigen, DNA or
RNA.
76. The apparatus of claim 74, wherein the dry powder formulation
comprises adenovirus.
77. The apparatus of claim 74, wherein the dry powder formulation
comprises microparticles.
78. The apparatus of claim 77, wherein said microparticles comprise
a bioactive agent(s).
79. The apparatus of claim 78, wherein said bioactive agent(s) is
selected from the group consisting of peptide(s), protein(s),
vaccine antigen(s), DNA or RNA.
80. The apparatus of claim 79, wherein said DNA or RNA is naked,
fragmented, encapsulated or coupled to another agent.
81. An apparatus for delivering a bioactive agent into an organism
comprising: a heat conducting element, which, when put in
substantial physical contact with a selected area of a biological
membrane of the organism, is capable of porating the biological
membrane at the selected area to form at least one micropore 1-1000
.mu.m in diameter, by delivering sufficient energy to the selected
area such that the temperature of tissue-bound water and other
vaporizable substances in the selected area is elevated above the
vaporization point of the water and other vaporizable substances,
thereby removing the biological membrane in the selected area, and
wherein the apparatus enables the bioactive agent, when the
bioactive agent is put in contact with the selected area, to be
taken up through the micropore into the organism, wherein said
bioactive agent is put in contact with the selected area in a form
selected from the group consisting of a tablet and a bio-erodable
matrix, wherein said bio-erodable matrix is fabricated in a manner
to allow the bioactive agent to be released into the organism via
the micropore, wherein the apparatus delivers the bioactive agent
into the organism.
82. A system for stimulating an immune response in an organism,
comprising: a supply of a permeant; and a heat conducting element,
which, when put in substantial physical contact with a selected
area of a biological membrane of the organism, is capable of
porating the biological membrane at the selected area to form at
least one micropore 1-1000 .mu.m in diameter and at a depth
coincident with increased concentration of langerhans cells, by
delivering sufficient energy to the selected area such that the
temperature of tissue-bound water and other vaporizable substances
in the selected area is elevated above the vaporization point of
the water and other vaporizable substances, thereby removing the
biological membrane in the selected area, and wherein the system
enables the permeant, when the permeant is put in contact with the
selected area, to be taken up through the micropore into the
organism, wherein the system stimulates the immune response in the
organism.
83. The system of claim 82, wherein the depth of the micropore is
180 microns to 250 microns.
84. The system of claim 82, wherein the organism is an animal.
85. The system of claim 84, wherein the animal is a human.
86. The system of claim 82, wherein the permeant is a vaccine.
87. The system of claim 86, wherein the vaccine comprises DNA or
RNA.
88. The system of claim 82, wherein the permeant is introduced into
the epidermis via formed micropores at a depth coincident with
increased concentration of langerhans cells.
89. The system of claim 82, wherein the surface area of the
selected area of the biological membrane is greater than the total
area of the micropores.
90. A system for delivering a formulation into an organism
comprising: a supply of a dry powder formulation; and a heat
conducting element, which, when put in substantial physical contact
with a selected area of a biological membrane of the organism, is
capable of porating the biological membrane at the selected area to
form at least one micropore 1-1000 .mu.m in diameter, by delivering
sufficient energy to the selected area such that the temperature of
tissue-bound water and other vaporizable substances in the selected
area is elevated above the vaporization point of the water and
other vaporizable substances, thereby removing the biological
membrane in the selected area, and wherein the system enables the
dry powder formulation, when the dry powder formulation is put in
contact with the selected area, to be taken up through the
micropore into the organism, wherein the system delivers the dry
powder formulation into the organism.
91. The system of claim 90, wherein the dry powder formulation
comprises a peptide(s), protein(s), vaccine antigen(s), DNA or
RNA.
92. The system of claim 90, wherein the dry powder formulation
comprises microparticles.
93. The system of claim 92, wherein said microparticles comprise a
bioactive agent(s).
94. The system of claim 93, wherein said bioactive agent(s) is
selected from the group consisting of peptide(s), protein(s),
vaccine antigen(s), DNA or RNA.
95. The system of claim 94, wherein said DNA or RNA is naked,
fragmented, encapsulated or coupled to another agent.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates generally to the field of
transmembrane delivery of drugs or bioactive molecules to an
organism. More particularly, this invention relates to a minimally
invasive to non-invasive method of increasing the permeability of
the skin, mucosal membrane or outer layer of a plant through
microporation of this biological membrane, which can be combined
with sonic, electromagnetic, and thermal energy, chemical
permeation enhancers, pressure, and the like for selectively
enhancing flux rate of bioactive molecules into the organism and,
once in the organism, into selected regions of the tissues
therein.
[0002] The stratum corneum is chiefly responsible for the well
known barrier properties of skin. Thus, it is this layer that
presents the greatest barrier to transdermal flux of drugs or other
molecules into the body and of analytes out of the body. The
stratum corneum, the outer horny layer of the skin, is a complex
structure of compact keratinized cell remnants separated by lipid
domains. Compared to the oral or gastric mucosa, the stratum
corneum is much less permeable to molecules either external or
internal to the body. The stratum corneum is formed from
keratinocytes, which comprise the majority of epidermal cells, that
lose their nuclei and become corneocytes. These dead cells comprise
the stratum corneum, which has a thickness of only about 10-30
.mu.m and, as noted above, is a very resistant waterproof membrane
that protects the body from invasion by exterior substances and the
outward migration of fluids and dissolved molecules. The stratum
corneum is continuously renewed by shedding of corneum cells during
desquamination and the formation of new corneum cells by the
keratinization process.
[0003] Underlying the stratum corneum is the viable cell layer of
the epidermis and the dermis, or connective tissue layer. These
layers together make up the skin. Microporation of these underlying
layers (the viable cell layer and dermis) has not previously been
used but may enhance transdermal flux. Deep to the dermis are the
underlying structures of the body, including fat, muscle, bone,
etc.
[0004] Microporation of the mucous membrane has not been used
previously. The mucous membrane generally lacks a stratum corneum.
The most superficial layer is the epithelial layer which consists
of numerous layers of viable cells. Deep to the epithelial layer is
the lamina propria, or connective tissue layer.
[0005] Microporation of plants has been previously limited to
select applications in individual cells in laboratory settings.
Plant organisms generally have tough outer layers to provide
resistance to the elements and disease. Microporation of this tough
outer layer of plants enables the delivery of substances useful for
introduction into the plant such as for conferring the desired
trait to the plant or for production of a desired substance. For
example, a plant may be treated such that each cell of the plant
expresses a particular and useful peptide such as a hormone or
human insulin.
[0006] The flux of a drug or analyte across the biological membrane
can be increased by changing either the resistance (the diffusion
coefficient) or the driving force (the gradient for diffusion).
Flux may be enhanced by the use of so-called penetration or
chemical enhancers. Chemical enhancers are well known in the art
and a more detailed description will follow.
[0007] Another method of increasing the permeability of skin to
drugs is iontophoresis. Iontophoresis involves the application of
an external electric field and topical delivery of an ionized form
of drug or an un-ionized drug carried with the water flux
associated with ion transport (electro-osmosis): While permeation
enhancement with iontophoresis has been effective, control of drug
delivery and irreversible skin damage are problems associated with
the technique.
[0008] Sonic energy has also been used to enhance permeability of
the skin and synthetic membranes to drugs and other molecules.
Ultrasound has been defined as mechanical pressure waves with
frequencies above 20 kHz, H. Lutz et al., Manual of Ultrasound 3-12
(1984). Sonic energy is generated by vibrating a piezoelectric
crystal or other electromechanical element by passing an
alternating current through the material, R. Brucks et al., 6
Pharm. Res. 697 (1989). The use of sonic energy to increase the
permeability of the skin to drug molecules has been termed
sonophoresis or phonophoresis.
[0009] Although it has been acknowledged that enhancing
permeability of the skin should theoretically make it possible to
transport molecules from inside the body through the skin to
outside the body for collection or monitoring, practicable methods
have not been disclosed. U.S. Pat. No. 5,139,023 to Stanley et al.
discloses an apparatus and method for noninvasive blood glucose
monitoring. In this invention, chemical permeation enhancers are
used to increase the permeability of mucosal tissue or skin to
glucose. Glucose then passively diffuses through the mucosal tissue
or skin and is captured in a receiving medium. The amount of
glucose in the receiving medium is measured and correlated to
determine the blood glucose level. However; as taught in Stanley et
al., this method is much more efficient when used on mucosal
tissue, such as buccal tissue, which results in detectable amounts
of glucose being collected in the receiving medium after a lag time
of about 10-20 minutes. However, the method taught by Stanley et
al. results in an extremely long lag time, ranging from 2 to 24
hours depending on the chemical enhancer composition used, before
detectable amounts of glucose can be detected diffusing through
human skin (heat-separated epidermis) in vitro. These long lag
times may be attributed to the length of time required for the
chemical permeation enhancers to passively diffuse through the skin
and to enhance the permeability of the barrier stratum corneum, as
well as the length of time required for the glucose to passively
diffuse out through the skin. Thus, Stanley et al. clearly does not
teach a method for transporting blood glucose or other analytes
non-invasively through the skin in a manner that allows for rapid
monitoring, as is required for blood glucose monitoring of diabetic
patients and for many other body analytes such as blood
electrolytes.
[0010] While the use of sonic energy for drug delivery is known,
results have been largely disappointing in that enhancement of
permeability has been relatively low. There is no consensus on the
efficacy of sonic energy for increasing drug flux across the skin.
While some studies report the success of sonophoresis, J. Davick et
al., 68 Phys. Ther. 1672 (1988); J. Griffin et al., 47 Phys. Ther.
594 (1967); J. Griffin & J. Touchstone, 42 Am. J. Phys. Med, 77
(1963); J. Griffin et al., 44 Am. J. Phys. Med. 20 (1965); D. Levy
et al., 83 J. Clin. Invest. 2074); D. Bommannan et al., 9 Pharm.
Res. 559 (1992), others have obtained negative results, H. Benson
et al., 69 Phys. Ther. 113 (1988); J. McElnay et al., 20 Br. J.
Clin. Pharmacol. 4221 (1985); H. Pratzel et al., 13 J. Rheumatol.
1122 (1986). Systems in which rodent skin were employed showed the
most promising results, whereas systems in which human skin was
employed have generally shown disappointing results. It is well
known to those skilled in the art that rodent skin is much more
permeable than human skin, and consequently the above results do
not teach one skilled in the art how to effectively utilize
sonophoresis as applied to transdermal delivery and/or monitoring
through human skin.
[0011] A significant improvement in the use of ultrasonic energy in
the monitoring of analytes and also in the delivery of drugs to the
body is disclosed and claimed in copending applications Ser. No.
08/152,442 filed Nov. 15, 1993, now U.S. Pat. No. 5,458,140, and
Ser. No. 08/152,174 filed Dec. 8, 1993, now U.S. Pat. No.
5,445,611, both of which are incorporated herein by reference. In
these inventions, the transdermal sampling of an analyte or the
transdermal delivery of drugs, is accomplished through the use of
sonic energy that is modulated in intensity, phase, or frequency or
a combination of these parameters coupled with the use of chemical
permeation enhancers. Also disclosed is the use of sonic energy,
optionally with modulations of frequency, intensity, and/or phase,
to controllably push and/or pump molecules through the stratum
corneum via perforations introduced by needle puncture, hydraulic
jet, laser, electroporation, or other methods.
[0012] The formation of micropores (i.e. microporation) in the
stratum corneum to enhance the delivery of drugs has been the
subject of various studies and has resulted in the issuance of
patents for such techniques.
[0013] Jacques et al., 88 J. Invest. Dermatol. 88-93 (1987),
teaches a method of administering a drug by ablating the stratum
corneum of a region of the skin using pulsed laser light of
wavelength, pulse length, pulse energy, pulse number, and pulse
repetition rate sufficient to ablate the stratum corneum without
significantly damaging the underlying epidermis and then applying
the drug to the region of ablation. This work resulted in the
issuance of U.S. Pat. No. 4,775,361 to Jacques et al. The ablation
of skin through the use of ultraviolet-laser irradiation was
earlier reported by Lane et al., 121 Arch. Dermatol. 609-617
(1985). Jacques et al. is restricted to use of few wavelengths of
light and expensive lasers.
[0014] Tankovich, U.S. Pat. No. 5,165,418 (hereinafter, "Tankovich
'418"), discloses a method of obtaining a blood sample by
irradiating human or animal skin with one or more laser pulses of
sufficient energy to cause the vaporization of skin tissue so as to
produce a hole in the skin extending through the epidermis and to
sever at least one blood vessel, causing a quantity of blood to be
expelled through the hole such that it can be collected. Tankovich
'418 thus is inadequate for noninvasive or minimally invasive
permeabilization of the stratum corneum such that a drug can be
delivered to the body or an analyte from the body can be
analyzed.
[0015] Tankovich et al., U.S. Pat. No. 5,423,803 (hereinafter,
"Tankovich '803") discloses a method of laser removal of
superficial epidermal skin cells in human skin for cosmetic
applications. The method comprises applying a light-absorbing
"contaminant" to the outer layers of the epidermis and forcing some
of this contaminant into or through the intercellular spaces in the
stratum corneum, and illuminating the infiltrated skin with pulses
of laser light of sufficient intensity that the amount of energy
absorbed by the contaminant will cause the contaminant to explode
with sufficient energy to tear off some of the epidermal skin
cells. Tankovich '803 further teaches that there should be high
absorption of energy by the contaminant at the wavelength of the
laser beam, that the laser beam must be a pulsed beam of less than
1 .mu.s duration, that the contaminant must be forced into or
through the upper layers of the epidermis, and that the contaminant
must explode with sufficient energy to tear off epidermal cells
upon absorption of the laser energy. This invention also fails to
disclose or suggest a method of drug delivery or analyte
collection.
[0016] Raven et al., WO 92/00106, describes a method of selectively
removing unhealthy tissue from a body by administering to a
selected tissue a compound that is highly absorbent of infrared
radiation of wavelength 750-860 nm and irradiating the region with
corresponding infrared radiation at a power sufficient to cause
thermal vaporization of the tissue to which the compound was
administered but insufficient to cause vaporization of tissue to
which the compound had not been administered. The absorbent
compound should be soluble in water or serum, such as indocyanine
green, chlorophyll, porphyrins, heme-containing compounds, or
compounds containing a polyene structure, and power levels are in
the range of 50-1000 W/cm.sup.2 or even higher.
[0017] Konig et al., DD 259351, teaches a process for thermal
treatment of tumor tissue that comprises depositing a medium in the
tumor tissue that absorbs radiation in the red and/or near red
infrared spectral region, and irradiating the infiltrated tissue
with an appropriate wavelength of laser light. Absorbing media can
include methylene blue, reduced porphyrin or its aggregates, and
phthalocyanine blue. Methylene blue, which strongly absorbs at
600-700 nm, and a krypton laser emitting at 647 and 676 nm are
exemplified. The power level should be at least 200
mW/cm.sup.2.
[0018] It has been shown that by stripping the stratum corneum from
a small area of the skin with repeated application and removal of
cellophane tape to the same location one can easily collect
arbitrary quantities of interstitial fluid, which can then be
assayed for a number of analytes of interest. Similarly, the
`tape-stripped` skin has also been shown to be permeable to the
transdermal delivery of compounds into the body. Unfortunately,
`tape-stripping` leaves a open sore which takes weeks to heal, and
for this, as well as other reasons, is not considered as an
acceptable practice for enhancing transcutaneous transport in wide
applications.
[0019] As discussed above, it has been shown that pulsed lasers,
such as the excimer laser operating at 193 nm, the erbium laser
operating near 2.9 .mu.m or the CO.sub.2 laser operating at 10.2
.mu.m, can be used to effectively ablate small holes in the human
stratum corneum. These laser ablation techniques offer the
potential for a selective and potentially non-traumatic method for
opening a delivery and/or sampling hole through the stratum
corneum. However, due to the prohibitively high costs associated
with these light sources, there have been no commercial products
developed based on this concept. The presently disclosed invention,
by defining a method for directly conducting thermal energy into or
through the biological membrane with very tightly defined spatial
and temporal resolution, makes it possible to produce the desired
micro-ablation of the biological membrane very low cost energy
sources.
[0020] In view of the foregoing problems and/or deficiencies, the
development of a method for safely enhancing the permeability of
the biological membrane for minimally invasive or noninvasive
monitoring of body analytes in a more rapid time frame would be a
significant advancement in the art. It would be another significant
advancement in the art to provide a method of minimally invasively
or non-invasively enhancing the transmembrane flux rate of a drug
into a selected area of an organism.
[0021] Significant advancements in the delivery of drugs and other
compounds are being made through the use of various techniques that
increase the permeability of a biological membrane, such as the
skin or mucosal membrane. Even more promising advances have been
made through techniques for creating micropores, as disclosed in
the aforementioned applications.
[0022] Nevertheless, it is desirable to improve upon these
technologies by forming micropores at selected depths in the
biological membrane and to deliver both small and large compounds,
in terms of molecular weight and size, through the micropores into
the body.
BRIEF SUMMARY OF THE INVENTION
[0023] This invention provides a method for enhancing the
transmembrane flux rate of a permeant into a selected site of an
organism comprising the steps of enhancing the permeability of said
selected site of the organism to said permeant by means of (a)
porating a biological membrane at said selected site by means that
form a micropore in said biological membrane, thereby reducing the
barrier properties of said biological membrane to the flux of said
permeant and (b) contacting the porated selected site with a
composition comprising an effective amount of said permeant,
whereby the transmembrane flux rate of said permeant into the
organism is enhanced.
[0024] This invention further provides the method of enhancing the
transmembrane flux rate further comprising applying to said site of
said organism an enhancer to increase the flux of said permeant
into said organism. The invention also provides the method wherein
said enhancer comprises sonic energy, and more specifically,
wherein the said sonic energy is applied to said site at a
frequency in the range of about 10 Hz to 1000 MHz, and wherein said
sonic energy is modulated by means of a member selected from the
group consisting of frequency modulation, amplitude modulation,
phase modulation, and combinations thereof. Alternatively, the said
enhancer comprises an electromagnetic field, and, more
specifically, iontophoresis or a magnetic field, or a mechanical
force, chemical enhancer, or thermal enhancer. Additionally, the
invention further provides a method wherein any of the methods of
sonic, electromagnetic, mechanical, thermal, or chemical
enhancement may be applied in any combination thereof to increase
the transmembrane flux rate of said permeant into or through said
micropore.
[0025] This invention also provides a method of further enhancing
the transmembrane flux rate with an enhancer, wherein said
enhancers at said site are applied so as to increase the flux rate
of the permeant into tissues surrounding the micropore. The said
enhancer can comprise sonic energy. Furthermore, the said sonic
energy is applied to said site at a frequency in the range of about
10 Hz to 1000 MHz, wherein said sonic energy is modulated by means
of a member selected from the group consisting of frequency
modulation, amplitude modulation, phase modulation, and
combinations thereof. Alternatively, the said enhancer comprises
sonic or thermal energy, electroporation, iontophoresis, chemical
enhancers, mechanical force, or a magnetic field, or any
combination thereof.
[0026] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant further comprising applying
to said site of said organism an enhancer, wherein any of the
methods of methods of sonic or thermal energy, electroporation,
iontophoresis, chemical enhancers, mechanical force, or a magnetic
field may be applied in any combination thereof further comprising
the method of combining sonic or thermal energy, electroporation,
iontophoresis, chemical enhancers, mechanical force, or a magnetic
field to increase the flux rate of the permeant into tissues
surrounding the micropore.
[0027] The invention also includes the method of further enhancing
the tranmembrane flux rate within and beneath the outer layer
wherein said porating of said biological membrane in said site is
accomplished by means selected from the group consisting of (a)
ablating the biological membrane by contacting said site, up to
about 1000 .mu.m across, of said biological membrane with a heat
source such that a micropore is formed in said biological membrane
at said site; (b) puncturing said biological membrane with a
micro-lancet calibrated to form a micropore of up to about 1000
.mu.m in diameter; (c) ablating the biological membrane by a beam
of sonic energy onto said biological membrane up to about 1000
.mu.m in diameter; (d) hydraulically puncturing said biological
membrane with a high pressure jet of fluid to form a micropore of
up to about 1000 .mu.m in diameter and (e) puncturing said
biological membrane with short pulses of electricity to form a
micropore of up to about 1000 .mu.m in diameter. Further, the
invention includes the method wherein said porating is accomplished
by contacting said site, up to about 1000 .mu.m across, with a heat
source to conductively transfer an effective amount of thermal
energy to said site such that the temperature of some of the water
and other vaporizable substances in said site is elevated above
their vaporization point creating a micropore to a selected depth
in the biological membrane at said site or wherein said porating is
accomplished by contacting said site, up to about 1000 .mu.m
across, with a heat source to conductively transfer an effective
amount of thermal energy to said site such that the temperature of
some of the tissue at said site is elevated to the point where
thermal decomposition occurs creating a micropore to a selected
depth in the biological membrane at said site. Additionally, the
invention includes the method of porating said biological membrane
in said site further comprising treating at least said site with an
effective amount of a substance that exhibits sufficient absorption
over the emission range of a pulsed light source and focusing the
output of a series of pulses from said pulsed light source onto
said substance such that said substance is heated sufficiently to
conductively transfer an effective amount of thermal energy to said
biological membrane to elevate the temperature to thereby create a
micropore. The invention also includes the method wherein said
pulsed light source emits at a wavelength that is not significantly
absorbed by said biological membrane. The invention further
provides the method wherein said pulsed light source is a laser
diode emitting in the range of about 630 to 1550 nm, wherein said
pulsed light source is a laser diode pumped optical parametric
oscillator emitting in the range of about 700 and 3000 nm, wherein
said pulsed light source is a member selected from the group
consisting of arc lamps, incandescent lamps, and light emitting
diodes. The invention also includes the method further comprising
providing a sensing system for determining when the micropore in
the biological membrane has reached the desired dimensions,
including width, length, and depth, and, further, wherein said
sensing system comprises light collection means for receiving light
reflected from said site and focusing said reflected light on a
detector for receiving said light and sending a signal to a
controller wherein said signal indicates a quality of said light,
and a controller coupled to said detector and to said light source
for receiving said signal and for shutting off said light source
when a preselected signal is received, or, alternatively, an
electrical impedance measuring system which can detect the changes
in the impedance of the biological membrane at different depths
into the organism as the micropore is formed.
[0028] The invention also provides the method of enhancing the
tranmembrane flux rate within and beneath the outer layer further
comprising cooling said site and adjacent tissues such that said
site and adjacent tissues are in a cooled condition. The said
cooling means comprises a Peltier device.
[0029] The invention also includes the method of enhancing the
transmembrane flux within and beneath the outer layer further
comprising, prior to porating said site, illuminating at least said
site with light such that said site is sterilized.
[0030] This invention also includes the method of enhancing the
transmembrane flux within and beneath the outer layer further
comprising contacting said site with a solid element, wherein said
solid element functions as a heat source to conductively transfer
an effective amount of thermal energy to said biological membrane
to elevate the temperature to thereby create a micropore. Further,
said heat source is constructed to modulate the temperature of said
site to greater than 100.degree. C. within about 10 nanoseconds to
50 milliseconds and then returning the temperature of said site to
approximately ambient temperature within about 1 millisecond to 50
milliseconds and wherein a cycle of raising the temperature and
returning to ambient temperature is repeated one or more times
effective for porating the biological membrane to the desired
depth. The invention further includes the method of using a heat
source wherein said returning to approximately ambient temperature
of said site is carried out by withdrawing said heat source from
contact with said site and wherein the modulation parameters are
selected to reduce sensation to the animal subject.
[0031] The invention includes the method for enhancing
transmembrane flux rates using a heat source and sensing system
further comprising providing means for monitoring electrical
impedance between said solid element and said organism through said
site and adjacent tissues and means for advancing the position of
said solid element such that as said poration occurs with a
concomitant change in impedance, said advancing means advances the
solid element such that the solid element is in contact with said
site during heating of the solid element, until the selected
impedance is obtained. Further, the invention includes this method
further comprising means for withdrawing said solid element from
contact with said site wherein said monitoring means is capable of
detecting a change in impedance associated with contacting a
selected layer underlying the surface of said site and sending a
signal to said withdrawing means to withdrawn said solid element
from contact with said site.
[0032] The method of enhancing the transmembrane flux rate using a
solid element wherein said solid element is heated by delivering an
electrical current through an ohmic heating element and, further,
wherein said solid element is formed such that it contains an
electrically conductive component and the temperature of said solid
element is modulated by passing a modulated electrical current
through said conductive element. Additionally, the invention
includes the method wherein said solid element is positioned in a
modulatable magnetic field wherein energizing the magnetic field
produces electrical eddy currents sufficient to heat the solid
element.
[0033] The invention also includes the method of enhancing the
transmembrane flux rate wherein said porating is accomplished by
puncturing said site with a micro-lancet calibrated to form a
micropore of up to about 1000 .mu.m in diameter, by a beam of sonic
energy directed onto said site to form a micropore of up to about
1000 .mu.m in diameter, by hydraulically puncturing said biological
membrane with a high pressure jet of fluid to form a micropore of
up to about 1000 .mu.m in diameter, or, alternatively, by
puncturing said biological membrane with short pulses of
electricity to form a micropore of up to about 1000 .mu.m in
diameter.
[0034] The invention further comprises the method of enhancing the
transmembrane flux rate of a permeant wherein said permeant
comprises a nucleic acid. More specifically, the invention includes
the method wherein said nucleic acid comprises DNA or wherein the
nucleic acid comprises RNA.
[0035] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant wherein the micropore in the
biological membrane extends into a portion of the outer layer of
the biological membrane ranging from 1 to 30 microns in depth,
extends through the outer layer of the biological membrane ranging
from 10 to 200 microns in depth, extends into the connective tissue
layer of the biological membrane ranging from 100 to 5000 microns
in depth, or extends through the connective tissue layer of the
biological membrane ranging from 1000 to 10000 microns in
depth.
[0036] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant, wherein the micropore
penetrates the biological membrane to a depth determined to
facilitate desired activity of the selected permeant.
[0037] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant wherein the permeant
comprises a polypeptide, including wherein the polypeptide is a
protein or a peptide, and further including wherein the peptide
comprises insulin or a releasing factor; a carbohydrate, including
wherein the carbohydrate comprises a heparin; an analgesic,
including wherein the analgesic comprises an opiate; a vaccine; or
a steroid.
[0038] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant wherein the permeant is
associated with a carrier. The invention further includes the
method wherein the carrier comprises liposomes; lipid complexes;
microparticles; or polyethylene glycol compounds. More
specifically, the invention further includes the method wherein the
permeant is a vaccine in combination with the method wherein the
permeant is associated with a carrier.
[0039] The invention further includes the method of enhancing the
transmembrane flux rate of a permeant wherein the permeant
comprises a substance which has the ability to change its
detectable response to a stimulus when in the proximity of an
analyte present in the organism.
[0040] An object of the invention is to provide a method for
controlling transmembrane flux rates of drugs or other molecules
into the body and, if desired, into the bloodstream through minute
perforations in the biological membrane, including stratum corneum
or other layers of the skin or in the mucosa or outer layers of a
plant.
[0041] It is still another object of the invention to provide a
method of delivering drugs into the body through micropores in the
biological membrane in combination with sonic energy, permeation
enhancers, pressure gradients, electromagnetic energy, thermal
energy, and the like.
[0042] An object of the invention is to minimize the barrier
properties of the biological membrane using poration to
controllably collect analytes from within the body through
perforations in the biological membrane to enable the monitoring of
these analytes.
[0043] It is also an object of the invention to provide a method of
monitoring selected analytes in the body through micropores in the
biological membrane in combination with sonic energy, permeation
enhancers, pressure gradients, electromagnetic energy, mechanical
energy, thermal energy, and the like.
[0044] These and other objects may be accomplished by providing a
method for monitoring the concentration of an analyte in an
individual's body comprising the steps of enhancing the
permeability of the biological membrane of a selected area of the
individual's body surface to the analyte by means of.
[0045] (a) porating the biological membrane of the selected area by
means that form a micropore in the biological membrane optionally
without causing serious damage to the underlying tissues, thereby
reducing the barrier properties of the biological membrane to, the
withdrawal of the analyte;
[0046] (b) collecting a selected amount of the analyte; and
[0047] (c) quantitating the analyte collected.
[0048] In one preferred embodiment, the method further comprises
applying sonic energy to the porated selected area at a frequency
in the range of about 5 kHz to 100 MHz, wherein the sonic energy is
modulated by means of a member selected from the group consisting
of frequency modulation, amplitude modulation, phase modulation,
and combinations thereof. In another preferred embodiment, the
method comprises contacting the selected area of the individual's
body with a chemical enhancer with the application of
electromagnetic, thermal, mechanical, or sonic energy to further
enhance analyte withdrawal.
[0049] Porating of the biological membrane is accomplished by means
selected from the group consisting of (a) ablating the biological
membrane by contacting a selected area, up to about 1000 .mu.m
across, of the biological membrane with a heat source such that the
temperature of tissue-bound water and other vaporizable substances
in the selected area is elevated above the vaporization point of
the water and other vaporizable substances thereby removing the
biological membrane in the selected area; (b) puncturing the
biological membrane with a micro-lancet calibrated to form a
micropore of up to about 1000 .mu.m in diameter; (c) ablating the
biological membrane by focusing a tightly focused beam of sonic
energy onto the stratum corneum; (d) hydraulically puncturing the
biological membrane with a high pressure jet of fluid to form a
micropore of up to about 1000 .mu.m in diameter and (e) puncturing
the biological membrane with short pulses of electricity to form a
micropore of up to about 1000 .mu.m in diameter.
[0050] One preferred embodiment of thermally ablating the
biological membrane comprises treating at least the selected area
with an effective amount of a dye that exhibits strong absorption
over the emission range of a pulsed light source and focusing the
output of a series of pulses from the pulsed light source onto the
dye such that the dye is heated sufficiently to conductively
transfer heat to the stratum corneum to elevate the temperature of
tissue-bound water and other vaporizable substances in the selected
area above the vaporization point of the water and other
vaporizable substances. Preferably, the pulsed light source emits
at a wavelength that is not significantly absorbed by skin. For
example, the pulsed light source can be a laser diode emitting in
the range of about 630 to 1550 nm, a laser diode pumped optical
parametric oscillator emitting in the range of about 700 and 3000
nm, or a member to selected from the group consisting of arc lamps,
incandescent lamps, and light emitting diodes. A sensing system for
determining when the barrier properties of the stratum corneum have
been surmounted can also be provided. One preferred sensing system
comprises light collection means for receiving light reflected from
the selected area and focusing the reflected light on a photodiode,
a photodiode for receiving the focused light and sending a signal
to a controller wherein the signal indicates a quality of the
reflected light, and a controller coupled to the photodiode and to
the pulsed light source for receiving the signal and for shutting
off the pulsed light source when a preselected signal is
received.
[0051] In another preferred embodiment, the method further
comprises cooling the selected area of biological membrane and
adjacent tissues with cooling means such that said selected area
and adjacent tissues are in a selected cooled, steady state,
condition prior to, during, and/or after poration.
[0052] In still another preferred embodiment, the method comprises
ablating the biological membrane such that interstitial fluid
exudes from the micropores, collecting the interstitial fluid, and
analyzing the analyte in the collected interstitial fluid. After
the interstitial fluid is collected, the micropore can be sealed by
applying an effective amount of energy from the laser diode or
other light source such that interstitial fluid remaining in the
micropore is caused to coagulate. Preferably, vacuum is applied to
the porated selected area to enhance collection of interstitial
fluid.
[0053] In yet another preferred embodiment, the method comprises,
prior to porating the biological membrane, illuminating at least
the selected area with light such that the selected area
illuminated with the light is sterilized.
[0054] Another preferred method of porating the biological membrane
comprises contacting the selected area with a solid element such
that the temperature of the selected area is raised from ambient
temperature to greater than 100.degree. C. within about 10
nanoseconds to 50 ms and then returning the temperature of the
selected area to approximately ambient skin temperature within
about 1 to 50 ms, wherein this cycle of raising the temperature and
returning to approximately ambient temperature is repeated a number
of time effective for reducing the barrier properties of the
biological membrane. Preferably, the step of returning to
approximately ambient temperature is carried out by withdrawing the
solid element from contact with the biological membrane. It is also
preferred to provide means for monitoring electrical impedance
between the solid element and the body through the selected area of
biological membrane and adjacent tissues and means for advancing
the position of the solid element such that as the ablation occurs
with a concomitant reduction in resistance, the advancing means
advances the solid element such that the solid element is in
contact with the biological membrane during heating of the solid
element. Further, it is also preferred to provide means for
withdrawing the solid element from contact with the biological
membrane, wherein the monitoring means is capable of detecting a
change in impedance associated with contacting a layer underlying
the biological membrane or a layer thereof and sending a signal to
the withdrawing means to withdrawn the solid element from contact
with the biological membrane. The solid element can be heated by an
ohmic heating element, can have a current loop having a high
resistance point wherein the temperature of the high resistance
point is modulated by passing a modulated electrical current
through said current loop to effect the heating, or can be
positioned in a modulatable alternating magnetic field of an
excitation coil such that energizing the excitation coil with
alternating current produces eddy currents sufficient to heat the
solid element by internal ohmic losses.
[0055] A method for enhancing the transmembrane flux rate of an
active permeant into a selected area of a body comprising the steps
of enhancing the permeability of the biological membrane layer of
the selected area of the body surface to the active permeant by
means of
[0056] (a) porating the biological membrane of the selected area by
means that form a micropore in the biological membrane optionally
without causing serious damage to the underlying tissues and
thereby reducing the barrier properties of the biological membrane
to the flux of the active permeant; and
[0057] (b) contacting the porated selected area with a composition
comprising an effective amount of the permeant such that the flux
of the permeant into the body is enhanced.
[0058] In a preferred embodiment, the method further comprises
applying energy to the porated selected area for a time and at an
intensity and a frequency effective to create a fluid streaming
effect and thereby enhance the transmembrane flux rate of the
permeant into the body.
[0059] A method is also provided for applying a tattoo to a
selected area of skin on an individual's body surface comprising
the steps of:
[0060] (a) porating the stratum corneum of the selected area by
means that form a micropore in the stratum corneum optionally
without causing serious damage to the underlying tissues and
thereby reduce the barrier properties of the stratum corneum to the
flux of a permeant; and
[0061] (b) contacting the porated selected area with a composition
comprising an effective amount of a tattooing ink as a permeant
such that the flux of said ink into the body is enhanced.
[0062] A method is still further provided for reducing a temporal
delay in diffusion of an analyte from blood of an individual to
said individual's interstitial fluid in a selected area of
biological membrane comprising applying means for cooling to said
selected area of skin.
[0063] A method is yet further provided for reducing evaporation of
interstitial fluid and the vapor pressure thereof, wherein said
interstitial fluid is being collected from a micropore in a
selected area of the biological membrane of an individual,
comprising applying means for cooling to said selected area of
biological membrane.
[0064] In accordance with still further embodiments, the present
invention is directed to a method for delivering bioactive agents
into the body through micropores formed at selected depths in a
biological membrane, such as the skin or mucous membrane or outer
layer of a plant. The method involves porating an outer layer of
the biological membrane through any of the poration techniques
known in the art, but to a sufficient and desired depth into or
through the biological membrane, and contacting the porated site
with an effective quantity of the bioactive agent of low or high
molecular weight and size. This process can be enhanced by applying
further permeation enhancement measures either before, during or
after the bioactive agent is delivered. For example, sonic energy,
iontophoresis, magnetic fields, electroporation, chemical
permeation enhancer; osmotic pressure and atmospheric pressure
measures may be applied to the porated site to enhance the
permeability of layers beneath the outer layer of the biological
membrane.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0065] FIG. 1 shows a schematic representation of a system for
delivering laser diode light and monitoring the progress of
poration.
[0066] FIG. 2 shows a schematic representation of a closed-loop
feedback system for monitoring poration.
[0067] FIG. 3A shows a schematic representation of an optical
poration system comprising a cooling device.
[0068] FIG. 3B shows a top view of a schematic representation of an
illustrative cooling device according to FIG. 3B.
[0069] FIG. 4 shows a schematic representation of an ohmic heating
device with a mechanical actuator.
[0070] FIG. 5 shows a schematic representation of a high resistance
current loop heating device.
[0071] FIG. 6 shows a schematic representation of a device for
modulating heating using inductive heating.
[0072] FIG. 7 shows a schematic representation of a closed loop
impedance monitor using changes in impedance to determine the
extent of poration.
[0073] FIGS. 8A-D show cross sections of human skin treated with
copper phthalocyanine and then subjected, respectively, to 0, 1, 5,
and 50 pulses of 810 nm light with an energy density of 4000
J/cm.sup.2 for a pulse period of 20 ms.
[0074] FIGS. 9-11 show graphic representations of temperature
distribution during simulated thermal poration events using optical
poration.
[0075] FIGS. 12 and 13 show graphic representations of temperature
as a function of time in the stratum corneum and viable epidermis,
respectively, during simulated thermal poration events using
optical poration.
[0076] FIGS. 14-16 show graphic representations of temperature
distribution, temperature as a function of time in the stratum
corneum, and temperature as a function of time in the viable
epidermis, respectively, during simulated thermal poration events
using optical poration wherein the tissue was cooled prior to
poration.
[0077] FIGS. 17-19 show graphic representations of temperature
distribution, temperature as a function of time in the stratum
corneum, and temperature as a function of time in the viable
epidermis, respectively, during simulated thermal poration events
wherein the tissue was heated with a hot wire.
[0078] FIGS. 20-22 show graphic representations of temperature
distribution, temperature as a function of time in the stratum
corneum, and temperature as a function of time in the viable
epidermis, respectively, during simulated thermal poration events
wherein the tissue was heated with a hot wire and the tissue was
cooled prior to poration.
[0079] FIGS. 23 and 24 show graphic representations of temperature
distribution and temperature as a function of time in the stratum
corneum, respectively, during simulated thermal poration events
wherein the tissue is heated optically according to the operating
parameters of Tankovich '803.
[0080] FIG. 25 shows a graphic representation of interstitial fluid
(ISF;) and blood (*) glucose levels as a function of time.
[0081] FIG. 26 shows a scatter plot representation of the
difference term between the ISF glucose and the blood glucose data
of FIG. 25.
[0082] FIG. 27 shows a histogram of the relative deviation of the
ISF to the blood glucose levels from FIG. 25.
[0083] FIG. 28 shows a cross section of an illustrative delivery
apparatus for delivering a drug to a selected area on an
individual's skin.
[0084] FIGS. 29A-C show graphic representations of areas of skin
affected by delivery of lidocaine to selected areas where the
stratum corneum is porated (FIGS. 29A-B) or not porated (FIG.
29C).
[0085] FIG. 30 shows a plot comparing the amount of interstitial
fluid harvested from micropores with suction alone ( ) and with a
combination of suction and ultrasound (*).
[0086] FIGS. 31, 32, and 33 show a perspective view of an
ultrasonic transducer/vacuum apparatus for harvesting interstitial
fluid, a cross section view of the same apparatus, and cross
sectional schematic view of the same apparatus, respectively.
[0087] FIGS. 34A-B show a top view of a handheld ultrasonic
transducer and a side view of the spatulate end thereof,
respectively.
[0088] FIG. 35 is a graphical representation showing the enhancing
effects of microporation in the transdermal delivery of
testosterone.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0089] It is to be understood that this invention is not limited
to"the particular configurations, process steps, and materials
disclosed herein as such configurations, process steps, and
materials may vary somewhat. It is also to be understood that the
terminology employed herein is used for the purpose of describing
particular embodiments only and is not intended to be limiting
since the scope of the present invention will be limited only by
the appended claims and equivalents thereof.
[0090] It must be noted that, as used herein the singular forms
"a," "an," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to a
method for delivery of "a drug" includes reference to delivery of a
mixture of two or more drugs, reference to "an analyte" includes
reference to one or more of such analytes, and reference to "a
permeation enhancer" includes reference to a mixture of two or more
permeation enhancers or techniques such as a combination of
ultrasound and electroporation.
[0091] Thus, as used herein, the singular form may be used
interchangeably with the plural form, and vice versa, i.e. "layer"
could mean layers or "layers" could mean layer.
[0092] As used herein, "including" or "includes" or the like means
including, without limitation.
[0093] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0094] As used herein "organism" means the entire animal or plant
being acted upon by the methods described herein.
[0095] As used herein, "poration," "microporation," or any such
similar term means the formation of a small hole or pore to a
desired depth in or through the biological membrane, such as skin
or mucous membrane, or the outer layer of an organism to lessen the
barrier properties of this biological membrane to the passage of
analytes from below the surface for analysis or the passage of
permeants or drugs into the body for selected purposes, or for
certain medical or surgical procedures. The microporation process
referred to herein is distinguished from the openings formed by
electroporation principally by the minimum dimensions of the
micropores which shall be no smaller than 1 micron across and at
least 1 micron in depth, whereas the openings formed with
electroporation are typically only a few nanometers in any
dimension. Nevertheless, electroporation is useful to facilitate
uptake of selected permeants by the targeted tissues beneath the
outer layers of an organism after the permeant has passed through
the micropores into these deeper layers of tissue. Preferably the
hole or micropore will be no larger than about 1 mm in diameter,
and more preferably no larger than about 300 .mu.m in diameter, and
will extend to a selected depth, as described hereinafter.
[0096] As used herein, "micropore" or "pore" means an opening
formed by the microporation method.
[0097] As used herein "ablation" means the controlled removal of
material which may include cells or other components comprising
some portion of a biological membrane or tissue caused by any of
the following: kinetic energy released when some or all of the
vaporizable components of such material have been heated to the
point that vaporization occurs and the resulting rapid expansion of
volume due to this phase change causes this material, and possibly
some adjacent material, to be removed from the ablation site;
thermal, mechanical, or sonic decomposition of some or all off the
tissue at the poration site.
[0098] As used herein ablation of a tissue or puncture of a tissue
may be achieved utilizing the same energy source.
[0099] As used herein, "tissue" means any component of an organism
including but not limited to, cells, biological membranes, bone,
collagen, fluids and the like comprising some portion of the
organism.
[0100] As used herein, "sonic" or "acoustic" are interchangeable
and cover the frequency space from 0.01 Hz and up.
[0101] As used herein, "ultrasonic" describes a subset of sonic
comprising frequencies greater or equal to 20,000 Hz with no upper
limit.
[0102] As used herein "puncture" or "micro-puncture" means the use
of mechanical, hydraulic, sonic, electromagnetic, or thermal means
to perforate wholly or partially a biological membrane such as the
skin or mucosal layers of an animal or the outer tissue layers of a
plant.
[0103] To the extent that "ablation" and "puncture" accomplish the
same purpose of poration, i.e. the creating a hole or pore in the
biological membrane optionally without significant damage to the
underlying tissues, these terms may be used interchangeably.
[0104] As used herein, "penetration enhancement" or "permeation
enhancement" means an increase in the permeability by utilization
of a permeation enhancer of a biological membrane such as the skin
or mucosal or buccal membrane or a plant's outer layer of tissue to
a bioactive agent, drug, analyte, dye, stain, microparticle,
microsphere, compound, or other chemical formulation (also called
"permeant"), i.e., so as to increase the rate at which a bioactive
agent, drug, analyte, stain, micro-particle, microsphere, compound,
or other chemical formulation permeates the biological membrane and
facilitates the withdrawal of analytes out through the biological
membrane or the delivery of substances through the biological
membrane and into the underlying tissues. The enhanced permeation
effected through the use of such enhancers can be observed, for
example, by observing diffusion of a dye, as a permeant, through
animal or human skin using a diffusion apparatus.
[0105] As used herein, "penetration enhancer," "permeation
enhancer," "enhancer," and the like includes all substances and
techniques that increase the flux of a permeant, analyte, or other
molecule across the skin, and is limited only by functionality. In
other words, all cell envelope disordering compounds and solvents
and physical techniques such as electroporation, iontophoresis,
magnetic fields, sonic energy, thermal energy, or mechanical
pressure or manipulation such as a local massaging of the site and
any chemical enhancement agents are intended to be included.
[0106] As used herein " chemical enhancer" means a substance that
increases the flux of a permeant or analyte or other substance
across a biological membrane and is limited only by function.
[0107] As used herein, "dye," "stain," and the like shall be used
interchangeably and refer to a biologically suitable chromophore
that exhibits suitable absorption over some or all of the emission
range of a pulsed light source used to ablate tissues to form
micropores therein.
[0108] As used herein, "transdermal" or "percutaneous" or
"transmembrane" or "transmucosal" or "transbuccal" means passage of
a permeant into or through the biological membrane or tissue to
achieve effective therapeutic blood levels or tissue levels of a
drug, or the passage of a molecule present in the body ("analyte")
out through the biological membrane or tissue so that the analyte
molecule may be collected on the outside of the body.
[0109] As used herein, the term "bioactive agent," "permeant,"
"drug," or. "pharmacologically active agent" or "deliverable
substance" or any other similar term means any chemical or
biological material or compound suitable for delivery by the
methods previously known in the art and/or by the methods taught in
the present invention, that induces a desired effect, such as a
biological or pharmacological effect, which may include but is not
limited to (1) having a prophylactic effect on the organism and
preventing an undesired biological effect such as preventing an
infection, (2) alleviating a condition caused by a disease, for
example, alleviating pain or inflammation caused as a result of
disease, (3) either alleviating, reducing, or completely
eliminating the disease from the organism, and/or (4) the placement
within the viable tissue layers of the organism of a compound or
formulation which can react, optionally in a reversible manner, to
changes in the concentration of a particular analyte and in so
doing cause a detectable shift in this compound or formulation's
measurable response to the application of energy to this area which
may be electromagnetic, mechanical or acoustic. The effect may be
local, such as providing for a local anesthetic effect, or it may
be systemic. This invention is not drawn to novel permeants or to
new classes of active agents other than by virtue of the
microporation technique, although substances not typically being
used for transdermal, transmucosal, transmembrane or transbuccal
delivery may now be useable. Rather it is directed to the mode of
delivery of bioactive agents or permeants that exist in the art or
that may later be established as active agents and that are
suitable for delivery by the present invention.
[0110] Such substances include broad classes of compounds normally
delivered into the organism, including through body surfaces and
membranes, including skin as well as by injection, including
needle, hydraulic, or hypervelocity methods. In general, this
includes but is not limited to: Polypeptides, including proteins
and peptides (e.g., insulin); releasing factors, including
Luteinizing Hormone Releasing Hormone (LHRH); carbohydrates (e.g.,
heparin); nucleic acids; vaccines; and pharmacologically active
agents such as antiinfectives such as antibiotics and antiviral
agents; analgesics and analgesic combinations; anorexics;
antihelminthics; antiarthritics; antiasthmatic agents;
anticonvulsants; antidepressants; antidiabetic agents;
antidiarrheals; antihistamines; antiinflammatory agents;
antimigraine preparations; antinauseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics; antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations including
potassium and calcium channel blockers, beta-blockers,
alpha-blockers, and antiarrhythmics; antihypertensives; diuretics
and antidiuretics; vasodilators including general coronary,
peripheral and cerebral; central nervous system stimulants;
vasoconstrictors; cough and cold preparations, including
decongestants; hormones such as estradiol, testosterone,
progesterone and other steroids and derivatives and analogs,
including corticosteroids; hypnotics; immunosuppressives; muscle
relaxants; parasympatholytics; psychostimulants; sedatives; and
tranquilizers. By the method of the present invention, both ionized
and nonionized permeants may be delivered, as can permeants of any
molecular weight including substances with molecular weights
ranging from less than 50 Daltons to greater than 1,000,000
Daltons.
[0111] As used herein, an "effective" amount of a permeant means a
sufficient amount of a compound to provide the desired local or
systemic effect and performance at a reasonable benefit/risk ratio
attending any treatment. An "effective" amount of an enhancer as
used herein means an amount selected so as to provide the desired
increase in tissue permeability and the desired depth of
penetration, rate of administration, and amount of permeant
delivered.
[0112] As used herein, "carriers" or "vehicles" refer to carrier
materials without significant pharmacological activity at the
quantities used that are suitable for administration with other
permeants, and include any such materials known in the art, e.g.,
any liquid, gel, solvent, liquid diluent, solubilizer,
microspheres, liposomes, microparticles, lipid complexes, or the
like, that is sufficiently nontoxic at the quantities employed and
does not interact with the drug to be administered in a deleterious
manner. Examples of suitable carriers for use herein include water,
buffers, mineral oil, silicone, inorganic or organic gels, aqueous
emulsions, liquid sugars, lipids, microparticles, waxes, petroleum
jelly, and a variety of other oils and polymeric materials.
[0113] As used herein, a "biological membrane" means a tissue
material present within a living organism that separates one area
of the organism from another and, in many instances, that separates
the organism from its outer environment. Skin and mucous and buccal
membranes are thus included as well as the outer layers of a plant.
Also, the walls of a cell or a blood vessel would be included
within this definition.
[0114] As used herein, "mucous membrane" or "mucosa" refers to the
epithelial linings of the mouth, nasopharynx, throat, respiratory
tract, urogenital tract, anus, eye, gut and all other surfaces
accessible via an endoscopic device such as the bladder, Colon,
lung, blood vessels, heart and the like.
[0115] As used herein, the "buccal membrane" includes the mucous
membrane of the mouth.
[0116] As used herein, "outer layer" and "connective-tissue layer"
are parts of the biological membrane and have the following
meanings. "Outer layer" means all or part of the epidermis of the
skin, or the epithelial lining of the mucous membrane or the outer
layer of a plant. The most superficial portion of the animal
epidermis is the stratum corneum, as is well known in the art. The
deeper portion of the epidermis is called, for simplicity, the
"viable cell layer" hereinafter. Beneath the outer layer is the
"connective tissue layer." The connective tissue layer means the
dermis in the skin or the lamina propria in the mucous membrane or
other underlying tissues in plants or animals.
[0117] As used herein, "organism" or "individual" or "subject" or
"body" refers to any of a human, animal, or plant to which the
present invention may be applied.
[0118] As used herein, "analyte" means any chemical or biological
material or compound suitable for passage through a biological
membrane by the technology taught in this present invention, or by
technology previously known in the art, of which an individual
might want to know the concentration or activity inside the body.
Glucose is a specific example of an analyte because it is a sugar
suitable for passage through the skin, and individuals, for example
those having diabetes, might want to know their blood glucose
levels. Other examples of analytes include, but are not limited to,
such compounds as sodium, potassium, bilirubin, urea, ammonia,
calcium, lead, iron, lithium, salicylates, antibodies, hormones, or
an exogenously delivered substance and the like.
[0119] As used herein, "into" or "in" a biological membrane or
layer thereof includes penetration in or through only one or more
layers (e.g., all or part of the stratum corneum or the entire
outer layer of the skin or portion thereof).
[0120] As used herein, "through" a biological membrane or layer
thereof means through the entire depth of the biological membrane
or layer thereof.
[0121] As used herein, "transdermal flux rate" is the rate of
passage of any analyte out through the skin of a subject or the
rate of passage of any bioactive agent, drug, pharmacologically
active agent, dye, particle or pigment in and through the skin
separating the organism from its outer environment. "Transmucosal
flux rate" and "transbuccal flux rate" refer to such passage
through mucosa and buccal membranes and "transmembrane flux rate"
refers to such passage through any biological membrane.
[0122] As used herein, "transdermal," "transmucosal," "transbuccal"
and "transmembrane" may be used interchangeably as appropriate
within the context of their use.
[0123] As used herein, the terms "intensity amplitude,"
"intensity," and "amplitude" are used synonymously and refer to the
amount of energy being produced by a sonic, thermal, mechanical or
electromagnetic energy system.
[0124] As used herein, "frequency modulation" or "sweep" means a
continuous, graded or stepped variation in the frequency of a
sonic, thermal, mechanical or electromagnetic energy in a given
time period. A frequency modulation is a graded or stepped
variation in frequency in a given time period, for example 5.4-5.76
MHz in 1 sec., or 5-10 MHz in 0.1 sec., or 10-5 MHz in 0.1 sec., or
any other frequency range or time period that is appropriate to a
specific application. A complex modulation can include varying both
the frequency and intensity simultaneously. For example, FIGS. 4A
and 4B of U.S. Pat. No. 5,458,140 could, respectively, represent
amplitude and frequency modulations being applied simultaneously to
a single sonic energy transducer.
[0125] As used herein, "amplitude modulation" means a continuous,
graded or stepped variation in the amplitude or intensity of a
sonic, thermal, mechanical or electromagnetic energy in a given
time period.
[0126] As used herein "phase modulation" means the timing of a
sonic, thermal, mechanical or electromagnetic energy or signal has
been changed relative to its initial state. An example is shown in
FIG. 4C of U.S. Pat. No. 5,458,140. The frequency and amplitude of
the signal can remain the same. A phase modulation can be
implemented with a variable delay such as to selectively retard or
advance the signal temporarily in reference to its previous state,
or to another signal.
[0127] As used herein "signal," or "energy" may be used
synonymously. The sonic, thermal, mechanical or electromagnetic
energy, in its various applications such as with frequency,
intensity or phase modulation, or combinations thereof and the use
of chemical enhancers combined with sonic, thermal, mechanical or
electromagnetic energy, as described herein, can vary over a
frequency range of between about 0.01 Hz to 1000 MHz, with a range
of between about 0.1 Hz and 30 MHz being preferred.
[0128] As used herein, "non-invasive" means not requiring the entry
of a needle, catheter, or other invasive instrument into a part of
the subject including the skin or a mucous membrane.
[0129] As used herein, "minimally invasive and "non-invasive" are
synonymous.
[0130] As used herein, "microparticles" or "microspheres" or
"nanoparticles" or "nanospheres" or " liposomes" or "lipid
complexes" may be used interchangeably.
Means for Poration of the Biological Membrane
[0131] The formation of a micropore in the biological membrane can
be accomplished by various state of the art means as well as
certain means disclosed herein that are improvements thereof. While
the following techniques and examples are made with respect to
porating the biological membrane, it should be understood that the
improvements described herein also apply to porating the mucous or
buccal membrane or the outer layers of a plant.
[0132] The use of laser ablation as described by Jacques et al. in
U.S. Pat. No. 4,775,361 and by Lane et al., supra, certainly
provide one means for ablating the stratum corneum using an excimer
laser. At 193 nm wavelength, and 14 ns pulse width, it was found
that about 0.24 to 2.8 .mu.nm of stratum corneum could be removed
by each laser pulse at radiant exposure of between about 70 and 480
mJ/cm.sup.2. As the pulse energy increases, more tissue is removed
from the stratum corneum and fewer pulses are required for complete
poration of this layer. The lower threshold of radiant exposure
that must be absorbed by the stratum corneum within the limit of
the thermal relaxation time to cause suitable micro-explosions that
result in tissue ablation is about 70 mJ/cm.sup.2 within a 50
millisecond (ms) time. In other words, a total of 70 mJ/cm.sup.2
must be delivered within a 50 ms window. This can be done in a
single pulse of 70 mJ/cm.sup.2 or in 10 pulses of 7 mJ/cm.sup.2, or
with a continuous illumination of 1.4 watts/cm.sup.2 during the 50
ms time. The upper limit of radiant exposure is that which will
ablate the stratum corneum without damage to underlying tissue and
can be empirically determined from the light source, wavelength of
light, and other variables that are within the experience and
knowledge of one skilled in this art.
[0133] By "delivery", in the context of the application of energy,
is meant that the stated amount of energy is absorbed by the tissue
to be ablated. At the excimer laser wavelength of 193 nm,
essentially 100% absorption occurs within the first 1 or 2 .mu.m of
stratum corneum tissue. Assuming the stratum corneum is about 20
.mu.m thick, at longer wavelengths, such as 670 nm, only about 5%
of incident light is absorbed within the 20 .mu.m layer. This means
that about 95% of the high power beam passes into the tissues
underlying the stratum corneum where it will likely cause
significant damage. In the context of delivery of a bioactive
agent, the term means providing the bioactive agent to the desired
location.
[0134] The ideal is to use only as much power as is necessary to
perforate the biological membrane or other selected skin, mucosal,
or tissue layers without causing bleeding, thermal, or other
unacceptable damage to underlying and adjacent tissues from which
analytes are to be extracted or permeants delivered.
[0135] It would be beneficial to use sources of energy more
economical than energy from excimer lasers. Excimer lasers, which
emit light at wavelengths in the far UV region, are much more
expensive to operate and maintain than, for example, diode lasers
that emit light at wavelengths in visible and IR regions (600 to
1800 nm). However, at the longer wavelengths, the biological
membrane becomes increasingly more transparent and absorption
occurs primarily in the underlying tissues.
[0136] The present invention facilitates a rapid and minimally
traumatic method of eliminating the barrier function of the
biological membrane to facilitate the transmembrane transport of
substances into the body when applied topically or to access the
analytes within the body for analysis. The method utilizes a
procedure which begins with the contact application of a small area
heat source to the targeted area of the biological membrane.
[0137] The heat source must have several important properties, as
will now be described. First, the heat source must be sized such
that contact with the biological membrane is confined to a small
area, typically about 1 to 1000 .mu.m in diameter. Second, it must
have the capability to modulate the temperature of the biological
membrane at the contact point from ambient surface temperature to
greater than the vaporization point of a sufficient amount of the
components within the biological membrane and then return to
approximately ambient temperature with cycle times to minimize
collateral damage to viable tissues and trauma to the subject. This
modulation can be created electronically, mechanically, or
chemically.
[0138] Additionally, for selected applications, an inherent depth
limiting feature of the microporation process can be facilitated if
the heat source has both a small enough thermal mass and limited
energy source to elevate its temperature such that when it is
placed in contact with tissues with more than 30% water content,
the thermal dispersion in these tissues is sufficient to limit the
maximum temperature of the heat source to less than 100 C. This
feature effectively stops the thermal vaporization process once the
heat probe had penetrated through the stratum corneum into or
through the lower layers of the epidermis.
[0139] However, if one utilizes a heat probe which can continue to
deliver sufficient energy into or through the hydrated viable
tissue layers beneath the outer layer of the biological membrane,
the poration process can continue into the body to a selected
depth, penetrating through deeper layers including, e.g., in the
case of the skin, through the epidermis, the dermis, and into the
subcutaneous layers below if desired. The concern when a system is
designed to create a micropore extending some distance into or
through the viable tissues beneath the stratum corneum, mucosal or
buccal membranes is principally how to minimize damage to the
adjacent tissue and the sensation to the subject during the
poration process. Experimentally, we have shown that if the heat
probe used is a solid, electrically or optically heated element,
with the active heated probe tip physically defined to be no more
than a few hundred microns across and protruding up to a few
millimeters from the supporting base, that a single pulse, or
multiple pulses of current can deliver enough thermal energy into
or through the tissue to allow the ablation to penetrate as deep as
the physical design allows, for example, until the support base
acts as a component to limit the extent of the penetration into or
through the tissue, essentially restricting the depth to which the
heat probe can penetrate into a micropore to contact fresh,
unporated tissue. If the electrical and thermal properties of said
heat probe, when it is in contact with the tissues, allow the
energy pulse to modulate the temperature of said probe rapidly
enough, this type of deep tissue poration can be accomplished with
essentially no pain to the subject. Experiments have shown that if
the required amount of thermal energy is delivered to the probe
within less than roughly 20 milliseconds, that the procedure is
painless. Conversely, if the energy pulse must be extended beyond
roughly 20 milliseconds, the sensation to the subject increases
rapidly and non-linearly as the pulse width is extended.
[0140] An electrically heated probe design which supports this type
of selected depth poration can be built by bending a 50 to 150
micron diameter tungsten wire into a sharp kink, forming a close to
180 degree bend with a minimal internal radius at this point. This
miniature `V` shaped piece of wire can then be mounted such that
the point of the `V` extends some distance out from a support piece
which has copper electrodes deposited upon it. The distance to
which the wire extends out from the support will define the maximum
penetration distance into or through the tissue when the wire is
heated. Each leg of the tungsten `V` will be attached to one of the
electrodes on the support carrier which in turn can be connected to
the current pulsing circuit. When the current is delivered to the
wire in an appropriately controlled fashion, the wire will rapidly
heat up to the desired temperature to effect the thermal ablation
process in a single pulse or in multiple pulses of current. By
monitoring the dynamic impedance of the probe and knowing the
coefficient of resistance versus temperature of the tungsten
element, closed loop control of the temperature of the contact
point can easily be established. Also; by dynamically monitoring
the impedance through the body from the contact point of the probe
and a second electrode placed some distance away, the depth of the
pore can be estimated based on the different impedance properties
of the tissue as one penetrates deeper into the body.
[0141] An optically heated probe design which supports this type of
selected depth poration can be built by taking an optical fiber and
placing on one end a tip comprised of a solid cap or coating. A
light source such as a laser diode will be coupled into the other
end of the fiber. The side of tip facing the fiber must have a high
enough absorption coefficient over the range of wavelengths emitted
by the light source that when the photons reach the end of the
fiber and strike this face, some of them will be absorbed and
subsequently cause the tip to heat up. The specific design of this
tip, fiber and source assembly may vary widely, however fibers with
gross diameters of 50 to 1000 microns across are common place items
today and sources emitting up to thousands of watts of optical
energy are similarly common place. The tip forming the actual heat
probe can be fabricated from a high melting point material, such as
tungsten and attached to the fiber by machining it to allow the
insertion of the fiber into a cylindrical bore at the fiber end. If
the distal end of the tip has been fabricated to limit the thermal
diffusion away from this tip and back up the supporting cylinder
attaching the tip to the fiber within the time frame of the optical
pulse widths used, the photons incident upon this tip will elevate
the temperature rapidly on both the fiber side and the contact side
which is placed against the tissues surface. The positioning of the
fiber/tip assembly onto the tissue surface, can be accomplished
with a simple mechanism designed to hold the tip against the
surface under some spring tension such that as the tissue beneath
it is ablated, the tip itself will advance into the tissue. This
allows the thermal ablation process to continue into or through the
tissue as far as one desires. An additional feature of this
optically heated probe design is that by monitoring the black body
radiated energy from the heated tip that is collected by the fiber,
a very simple closed loop control of the tip temperature can be
effected. Also, as described earlier, by dynamically monitoring the
impedance through the body from the contact point of the probe and
a second electrode placed some distance away, the depth of the pore
can be determined based on the different impedance properties of
the tissue as one penetrates deeper into the body. The relationship
between pulse width and sensation for this design is essentially
the same as for the electrically heated probe described
earlier.
[0142] Impedance can be used to determine the depth of a pore made
by any means. It can be used as an input to a control system for
making pores of selected depth. The impedance measured may be the
complex impedance measured at a frequency selected to highlight the
impedance properties of the selected tissues in a selected
organism.
[0143] An additional feature of this invention is the large
increase in efficiency which can be gained by combining the
poration of the outer layers of the biological membrane with other
permeation enhancement techniques which can now be optimized to
function on the various barriers to effective delivery of the
desired compound into or through the internal spaces it needs to go
to be bio-effective. In particular, if one is delivering a DNA
compound either naked, fragmented, encapsulated or coupled to
another agent, it is often desired to get the DNA into the living
cells without killing the cell to allow the desired uptake and
subsequent performance of the therapy. It is well know in the art
that electroporation, iontophoresis, and ultrasound can cause
openings to form, temporarily, in the cell membranes and other
internal tissue membranes. By having breached the stratum corneum
or mucosal layer or outer layer of a plant and if desired the
epidermis and dermis or deeper into a plant, electroporation,
iontophoresis, magnetic fields, and sonic energy can now be used
with parameters that can be tailored to act selectively on these
underlying tissue barriers. For example, for any electromagnetic or
sonic energy enhancement means, the specific action of the
enhancement can be designed to focus on any part of the pore, e.g.,
on the bottom of the pore by the design of the focusing means
employed such as the design of the electrodes, sonic and magnetic
field forming devices and the like. Alternatively, the enhancer can
be focused more generally on the entire pore or the area
surrounding the pore. In the case of electroporation, where pulses
exceeding 50 to 150 volts are routinely used to electroporate the
stratum corneum or mucosal layer, in the environment we present,
pulses of only a few volts can be sufficient to electroporate the
cell, capillary or other membranes within the targeted tissue. This
is principally due to the dramatic reduction in the number of
insulating layers present between the electrodes once the outer
surface of the biological membrane has been opened. Similarly,
iontophoresis can be shown to be effective to modulate the flux of
a fluid media containing the DNA through the micropores with very
small amounts of current due to the dramatic reduction in the
physical impedance to fluid flow through these porated layers.
[0144] Whereas ultrasound has previously been used to accelerate
the permeation of the stratum corneum or mucosal layer, by
eliminating this barrier via the micropores, we have created the
opportunity to utilize sonic energy to permeabilize the cell,
capillary or other structures within the targeted tissue. As in the
cases of electroporation and iontophoresis, we have demonstrated
that the sonic energy levels needed to effect a notable improvement
in the trans-membrane flux of a substance are much lower than when
stratum corneum or mucosal layers are left intact. The mode of
operation of all of these active methods, electroporation,
iontophoresis, magnetic fields, mechanical forces or ultrasound,
when applied solely or in combination, after the poration of
biological membrane has been effected is most similar to the
parameters typically used in in vitro applications where single
cell membranes are being opened up for the delivery of a
substance.
[0145] With the heat source placed in contact with the surface of
the biological membrane, it is cycled through a series of one or
more modulations of temperature from an initial point of ambient
temperature to a peak temperature in excess of 123.degree. C. and
back to ambient surface temperature. To minimize or eliminate the
animal's sensory perception of the microporation process, these
pulses are limited in duration, and the interpulse spacing is long
enough to allow cooling of the viable tissue layers in the
biological membrane, and most particularly the innervated tissues,
to achieve a mean temperature within the innervated tissues of less
than about 45 C. These parameters are based on the thermal time
constants of the human skin's viable epidermal tissues (roughly
30-80 ms) located between the heat probe and the innervated tissue
in the underlying dermis. The result of this application of pulsed
thermal energy is that enough energy is conducted into or through
the stratum corneum within the tiny target spot that the local
temperature of this volume of tissue is elevated sufficiently
higher than the vaporization point of the tissue-bound water
content in the stratum corneum. As the temperature increases above
100 C, the water content of the stratum corneum (typically 5% to
15%) within this localized spot, is induced to vaporize and expand
very rapidly, causing a vapor-driven removal of those corneocytes
in the stratum corneum located in proximity to this vaporization
event. U.S. Pat. No. 4,775,361 teaches that a stratum corneum
temperature of 123.degree. C. represents a threshold at which this
type of flash vaporization occurs. As subsequent pulses of thermal
energy are applied, additional layers of the stratum corneum are
removed until a micropore is formed through the stratum corneum
down to the next layer of the epidermis, the stratum lucidum. By
limiting the duration of the heat pulse to less than one thermal
time constant of the epidermis and allowing any heat energy
conducted into or through the epidermis to dissipate for a
sufficiently long enough time, the elevation in temperature of the
viable layers of the epidermis is minimal. This allows the entire
microporation process to take place without any sensation to the
subject and no damage to the underlying and surrounding tissues. If
the heat probe can achieve temperatures greater than 300 degrees C.
some of the poration may be due to the direct thermal decomposition
of the tissue.
[0146] The present invention comprises a method for painlessly, or
with little sensation, creating microscopic holes, i.e. micropores,
from about 1 to 1000 .mu.m across, in a biological membrane of an
organism. The key to successfully implementing this method is the
creation of an appropriate thermal energy source, or heat probe,
which is held in contact with the biological membrane. The
principle technical challenge in fabricating an appropriate heat
probe is designing a device that has the desired contact with the
biological membrane and that can be thermally modulated at a
sufficiently high frequency.
[0147] It is possible to fabricate an appropriate heat probe by
contacting the biological membrane with a suitable light-absorbing
compound, such as a dye or stain, or any thin film or substance
selected because of its ability to absorb light at the wavelength
emitted by a selected light source. In this instance, the selected
light source may be a laser diode emitting at a wavelength which
would not normally be absorbed by the biological membrane. By
focusing the light source to a small spot on the surface of the
topical layer of the dye, stain, thin film or substance the
targeted area can be temperature modulated by varying the intensity
of the light flux focused on it. It is possible to utilize the
energy from laser sources emitting at a longer wavelength than an
excimer laser by first topically applying to the stratum corneum a
suitable light-absorbing compound, such as a dye, stain, thin film
or substance selected because of its ability to absorb light at the
wavelength emitted by the laser source. The same concept can be
applied at any wavelength and one must only choose an appropriate
dye or stain and optical wavelength. One need only look to any
reference manual to find which suitable dyes and wavelength of the
maximum absorbance of that dye. One such reference is Green, The
Sigma-Aldrich Handbook of Stains. Dyes and Indicators, Aldrich
Chemical Company, Inc. Milwaukee, Wis. (1991). For example, copper
phthalocyanine (Pigment Blue 15; CPC) absorbs at about 800 nm;
copper phthalocyanine tetrasulfonic acid (Acid Blue 249) absorbs at
about 610 nm; and Indocyanine Green absorbs at about 775 nm; and
Cryptocyanine absorbs at about 703 nm. CPC is particularly well
suited for this embodiment for the following reasons: it is a very
stable and inert compound, already approved by the FDA for use as a
dye in implantable sutures; it absorbs very strongly at wavelengths
from 750 nm to 950 nm, which coincide well with numerous low cost,
solid state emitters such as laser diodes and LEDs, and in
addition, this area of optical bandwidth is similarly not absorbed
directly by the skin tissues in any significant amount; CPC has a
very high vaporization point (>550 C in a vacuum) and goes
directly from a solid phase to a vapor phase with no liquid phase;
CPC has a relatively low thermal diffusivity constant, allowing the
light energy focused on it to selectively heat only that area
directly in the focal point with very little lateral spreading of
the `hot-spot` into the surrounding CPC thereby assisting in the
spatial definition of the contact heat-probe.
[0148] The purpose of this disclosure is not to make an exhaustive
listing of suitable dyes, stains, films or substances because such
may be easily ascertained by one skilled in the art from data
readily available.
[0149] The same is true for any desired particular pulsed light
source. For example, this method may be implemented with a
mechanically shuttered, focused incandescent lamp as the pulsed
light source. Various catalogs and sales literature show numerous
lasers operating in the near UV, visible and near IR range.
Representative lasers are Hammamatsu Photonic Systems Model PLP-02
which operates at a power, output of 2.times.10.sup.-8 J, at a
wavelength of 415 nm; Hammamatsu Photonic Systems Model PLP-05
which operates at a power output of 15 J, at a wavelength of 685
nm; SDL, Inc., SDL-3250 Series pulsed laser which operates at a
power output of 2.times.10.sup.6 J at a wavelength of about 800-810
nm; SDL, Inc., Model SDL-8630 which operates at a power output of
500 mW at a wavelength of about 670 nm; Uniphase Laser Model
AR-081-15000 which operates at a power output of 15,000 mW at a
wavelength of 790-830 nm; Toshiba America Electronic Model TOLD9150
which operates at a power output of 30 mW at a wavelength of 690
nm; and LiCONIX, Model Diolite 800-50 which operates at a power 50
mW at a wavelength of 780 nm.
[0150] For purposes of the present invention a pulsed laser light
source can emit radiation over a wide range of wavelengths ranging
from between about 100 nm to 12,000 nm. Excimer lasers typically
will emit over a range of between about 100 to 400 nm. Commercial
excimer lasers are currently available with wavelengths in the
range of about 193 nm to 350 nm. Preferably a laser diode will have
an emission range of between about 380 to 1550 nm. A frequency
doubled laser diode will have an emission range of between about
190 and 775 nm. Longer wavelengths ranging from between about 1300
and 3000 nm may be utilized using a laser diode pumped optical
parametric oscillator. It is expected, given the amount of research
taking place on laser technology, that these ranges will expand
with time.
[0151] Delivered or absorbed energy need not be obtained from a
laser as any source of light, whether it is from a laser, a short
arc lamp such as a xenon flashlamp, an incandescent lamp, a
light-emitting diode (LED), the sun, or any other source may be
used. Thus, the particular instrument used for delivering
electromagnetic radiation is less important than the wavelength and
energy associated therewith. Any suitable instrument capable of
delivering the necessary energy at suitable wavelengths, i.e. in
the range of about 100 nm to about 12,000 nm, can be considered
within the scope of the invention. The essential feature is that
the energy must be absorbed by the light-absorbing compound to
cause localized heating thereof, followed by conduction of
sufficient heat to the tissue to be ablated within the time frame
allowed.
[0152] In one illustrative embodiment, the heat probe itself is
formed from a thin layer, preferably about 5 to 1000 .mu.m thick,
of a solid, non-biologically active substance placed in contact
with a selected area of an individual's skin that is large enough
to cover the site where a micropore is to be created. The specific
formulation of the chemical compound is chosen such that it
exhibits high absorption over the spectral range of a light source
selected for providing energy to the light-absorbing compound. The
probe can be, for example, a sheet of a solid compound, a film
treated or coated with or containing a suitable light absorbing
compound, or a direct application of the light-absorbing compound
to the skin as a precipitate or as a suspension in a carrier.
Regardless of the configuration of the light-absorbing heat probe,
it must exhibit a low enough lateral thermal diffusion coefficient
such that any local elevations of temperature will remain
sufficiently spatially defined and the dominant mode of heat loss
will preferably be via direct conduction into biological membrane
through the point of contact between the skin and the probe.
[0153] The required temperature modulation of the probe can be
achieved by focusing a light source onto the probe layer and
modulating the intensity of this light source. If the energy
absorbed within the illuminated area is sufficiently high, it will
cause the probe layer heat up. The amount of energy delivered, and
subsequently both the rate of heating and peak temperature of the
probe layer at the focal point, can be easily modulated by varying
the pulse width and peak power of the light source. In this
embodiment, it is only the small volume of probe layer heated up by
the focused, incident optical energy that forms the heat probe,
additional material of this probe layer which may have been applied
over a larger area then the actual poration site is incidental. By
using a solid phase light-absorbing compound with a relatively high
melting point, such as copper phthalocyanine (CPC), which remains
in its solid phase up to a temperature of greater than 550 C, the
heat probe can be quickly brought up to a temperature of several
hundred degrees C., and still remain in contact with the biological
membrane, allowing this thermal energy, to be conducted into or
through the stratum corneum. In addition, this embodiment comprises
choosing a light source with an emission spectrum where very little
energy would normally be absorbed in the tissues of the biological
membrane.
[0154] Once the targeted area has the light-absorbing probe layer
placed in contact to it, the heat probe is formed when the light
source is activated with the focal waist of the beam positioned to
be coincident with the surface of the treated area The energy
density of light at the focal waist and the amount of absorption
taking place within the light-absorbing compound are set to be
sufficient to bring the temperature of the light-absorbing
compound, within the area of the small spot defined by the focus of
the light source, to greater than 123.degree. C. within a few
milliseconds. As the temperature of the heat probe rises,
conduction into or through the biological membrane delivers energy
into or through these tissues, elevating the local temperature of
the biological membrane. When enough energy has been delivered into
or through this small area of biological membrane to cause the
local temperature to be elevated above the boiling point of some of
the water and other vaporizable components contained in these
tissues, a flash vaporization of this material takes place,
removing some portion of the biological membrane at this location
and forming a micropore.
[0155] By turning the light source on and off, the temperature of
the heat probe can be rapidly modulated and the selective ablation
of these tissues can be achieved, allowing a very precisely
dimensioned hole to be created, which can selectively penetrate
only through the first 10 to 30 microns of the biological membrane,
or can be made deeper.
[0156] An additional feature of this embodiment is that by choosing
a light source that would normally have very little energy absorbed
by the biological membrane or underlying tissues, and by designing
the focusing and delivery optics to have a sufficiently high
numerical aperture, the small amount of delivered light that does
not happen to get absorbed in the heat probe itself, quickly
diverges as it penetrates deep into the body. Since there is very
little absorption at the delivered wavelengths, essentially no
energy is delivered to the biological membrane directly from the
light source. This three dimensional dilution of coupled energy in
the tissues due to beam divergence and the low level of absorption
in the untreated tissue results in a completely benign interaction
between the light beam and the tissues, with no damage being done
thereby.
[0157] In one preferred embodiment of the invention, a laser diode
is used as the light source with an emission wavelength of
800.+-.30 nm. A heat-probe can be formed by topical application of
a transparent adhesive tape that has been treated on the adhesive
side with a 0.5 cm spot formed from a deposit of finely ground
copper phthalocyanine (CPC). The CPC exhibits extremely high
absorption coefficients in the 800 nm spectral range, typically
absorbing more than 95% of the radiant energy from a laser
diode.
[0158] FIG. 1 shows a system 10 for delivering light from such a
laser diode to a selected area of an individual's biological
membrane and for monitoring the progress of the poration process.
The system comprises a laser diode 14 coupled to a controller 18,
which controls the intensity, duration, and spacing of the light
pulses. The laser diode emits a beam 22 that is directed to a
collection lens or lenses 26, which focuses the beam onto a mirror
30. The beam is then reflected by the mirror to an objective lens
or lenses 34, which focuses the beam at a preselected point 38.
This preselected point corresponds with the plane of an xyz stage
42 and the objective hole 46 thereof, such that a selected area of
an individual's biological membrane can be irradiated. The xyz
stage is connected to the controller such that the position of the
xyz stage can be controlled. The system also comprises a monitoring
system comprising a CCD camera 50 coupled to a monitor 54. The CCD
camera is confocally aligned with the objective lens such that the
progress of the poration process can be monitored visually on the
monitor.
[0159] In another illustrative embodiment of the invention, a
system of sensing photodiodes and collection optics that have been
confocally aligned with the ablation light source is provided. FIG.
2 shows a sensor system 60 for use in this embodiment. The system
comprises a light source 64 for emitting a beam of light 68, which
is directed through a delivery optics system 72 that focuses the
beam at a preselected point 76, such as the surface of an
individual's skin 80. A portion of the light contacting the skin is
reflected, and other light is emitted from the irradiated area. A
portion of this reflected and emitted light passes through a filter
84 and then through a collection optics system 88, which focuses
the light on a phototodiode 92. A controller 96 is coupled to both
the laser diode and the photodiode for, respectively, controlling
the output of the laser diode and detecting the light that reaches
the photodiode. Only selected portions of the spectrum emitted from
the skin pass through the filter. By analyzing the shifts in the
reflected and emitted light from the targeted area, the system has
the ability to detect when the stratum corneum has been breached,
and this feedback is then used to control the light source,
deactivating the pulses of light when the microporation of the
stratum corneum is achieved. By employing this type of active
closed loop feedback system, a self regulating, universally
applicable device is obtained that produces uniformly dimensioned
micropores in the stratum corneum, with minimal power requirements,
regardless of variations from one individual to the next.
[0160] In another illustrative embodiment, a cooling device is
incorporated into the system interface to the skin. FIG. 3A shows
an illustrative schematic representation thereof. In this system
100, a light source 104 (coupled to a controller 106) emits a beam
of light 108, which passes through and is focused by a delivery
optics system 112. The beam is focused by the delivery optics
system to a preselected point 116, such as a selected area of an
individual's skin 120. A cooling device 124, such as a Peltier
device or other means of chilling, contacts the skin to cool the
surface thereof. In a preferred embodiment of the cooling device
124 (FIG. 3B), there is a central hole 128 through which the beam
of focused light passes to contact the skin. Referring again to
FIG. 3A, a heat sink 132 is also preferably placed in contact with
the cooling device. By providing a cooling device with a small hole
in its center coincident with the focus of the light, the tissues
in the general area where the poration is to be created may be
cooled to 5.degree. C. to 10.degree. C. This cooling allows a
greater safety margin for the system to operate in that the
potential sensations to the user and the possibility of any
collateral damage to the epidermis directly below the poration site
are reduced significantly from non-cooled embodiment. Moreover, for
monitoring applications, cooling minimizes evaporation of
interstitial fluid and can also provide advantageous physical
properties, such as decreased surface tension of such interstitial
fluid. Still further, cooling the tissue is known to cause a
localized increase in blood flow in such cooled tissue, thus
promoting diffusion of analytes from the blood into the
interstitial fluid and promoting diffusion of delivered permeants
away from the pore site or into the tissue underlying the pore.
[0161] The method can also be applied for other micro-surgery
techniques wherein the light-absorbing compound/heat-probe is
applied to the area to be ablated and then the light source is used
to selectively modulate the temperature of the probe at the
selected target site, affecting the tissues via the
vaporization-ablation process produced.
[0162] A further feature of the invention is to use the light
source to help seal the micropore after its usefulness has passed.
Specifically, in the case of monitoring for an internal analyte, a
micropore is created and some amount of interstitial fluid is
extracted through this opening. After a sufficient amount of
interstitial fluid had been collected, the light source is
reactivated at a reduced power level to facilitate rapid clotting
or coagulation of the interstitial fluid within the micropore. By
forcing the coagulation or clotting of the fluid in the pore, this
opening in the body is effectively sealed, thus reducing the risk
of infection. Also, the use of the light source itself for both the
formation of the micropore and the sealing thereof is an inherently
sterile procedure, with no physical penetration into the body by
any device or apparatus. Further, the thermal shock induced by the
light energy kills any microbes that may happen to be present at
the ablation site.
[0163] This concept of optical sterilization can be extended to
include an additional step in the process wherein the light source
is first applied in an unfocused manner, covering the target area
with an illuminated area that extends 100 .mu.m or more beyond the
actual size of the micropore to be produced. By selecting the area
over which the unfocused beam is to be applied, the flux density
can be correspondingly reduced to a level well below the ablation
threshold but high enough to effectively sterilize the surface of
the skin. After a sufficiently long exposure of the larger area,
either in one continuous step or in a series of pulses, to the
sterilizing beam, the system is then configured into the sharply
focused ablation mode and the optical microporation process
begins.
[0164] Another illustrative embodiment of the invention is to
create the required heat probe from a solid element, such as a
small diameter wire. As in the previously described embodiment, the
contacting surface of the heat probe must be able to have its
temperature modulated from ambient biological membrane temperatures
to temperatures greater than 123.degree. C., within the required
time allowed of, preferably, between about 1 microsecond to 50
milliseconds at the high temperature (on-time) and at least about 1
to 50 ms at the low temperature (off-time). In particular, being
able to modulate the temperature up to greater than 150.degree. C.
for an "on" time of around 5 ms and an off time of 50 ms produces
very effective thermal ablation with little or no sensation to the
individual.
[0165] Several methods for modulating the temperatures of the solid
element heat probe contact area may be successfully implemented.
For example, a short length of wire may be brought up to the
desired high temperature by an external heating element such as an
ohmic heating element used in the tip of a soldering iron. FIG. 4
shows an ohmic heating device 140 with a mechanical actuator. The
ohmic heating device comprises an ohmic heat source 144 coupled to
a solid element heat probe 148. The ohmic heat source is also
coupled through an insulating mount 152 to a mechanical modulation
device 156, such as a solenoid. In this configuration, a steady
state condition can be reached wherein the tip of the solid element
probe will stabilize at some equilibrium temperature defined by the
physical parameters of the structure, i.e., the temperature of the
ohmic heat source, the length and diameter of the solid element,
the temperature of the air surrounding the solid element, and the
material of which the solid element is comprised. Once the desired
temperature is achieved, the modulation of the temperature of the
selected area of an organism's biological membrane 160 is effected
directly via the mechanical modulation device to alternatively
place the hot tip of the wire in contact with the biological
membrane for, preferably, a 5 ms on-time and then withdraw it into
the air for, preferably, a 50 ms off-time.
[0166] Another illustrative example (FIG. 5), shows a device 170
comprising a current source 174 coupled to a controller 178. The
current source is coupled to a current loop 182 comprising a solid
element 186 formed into a structure such that it presents a high
resistance point. Preferably, the solid element is held on a mount
190, and an insulator 194 separates different parts of the current
loop. The desired modulation of temperature is then achieved by
merely modulating the current through the solid element. If the
thermal mass of the solid element is appropriately sized and the
heat sinking provided by the electrodes connecting it to the
current source is sufficient, the warm-up and cool-down times of
the solid element can be achieved in a few milliseconds. Contacting
the solid element with a selected area of biological membrane 198
heats the biological membrane to achieve the selected ablation.
[0167] In FIG. 6 there is shown still another illustrative example
of porating the biological membrane with a solid element heat
probe. In this system 200, the solid element 204 can be positioned
within a modulatable alternating magnetic field formed by a coil of
wire 208, the excitation coil. By energizing the alternating
current in the excitation coil by means of a controller 212 coupled
thereto, eddy currents can be induced in the solid element heat
probe of sufficient intensity that it will be heated up directly
via the internal ohmic losses. This is essentially a miniature
version of an inductive heating system commonly used for heat
treating the tips of tools or inducing out-gassing from the
electrodes in vacuum or flash tubes. The advantage of the inductive
heating method is that the energy delivered into the solid element
heat probe can be closely controlled and modulated easily via the
electronic control of the excitation coil with no direct electrical
connection to the heat probe itself. If the thermal mass of the
solid element heat probe and the thermal mass of the biological
membrane in contact with the tip of the probe are known,
controlling the inductive energy delivered can allow precise
control of the temperature at the contact point 216 with the
biological membrane 220. Because the biological membrane tissue is
essentially non-magnetic at the lower frequencies at which
inductive heating can be achieved, if appropriately selected
frequencies are used in the excitation coil, then this alternating
electromagnetic field will have no effect on the organism's
tissues.
[0168] If a mechanically controlled contact modulation is employed,
an additional feature may be realized by incorporating a simple
closed loop control system wherein the electrical impedance between
the probe tip and the subject's skin is monitored. In this manner,
the probe can be brought into contact with the subject's skin,
indicated by the step-wise reduction in resistance once contact is
made, and then held there for the desired "on-time," after which it
can be withdrawn. Several types of linear actuators are suitable
for this form of closed loop control, such as a voice-coil
mechanism, a simple solenoid, a rotary system with a cam or
bell-crank, and the like. The advantage is that as the thermal
ablation progresses, the position of the thermal probe tip can be
similarly advanced into the biological membrane, always ensuring
good a contact to facilitate the efficient transfer of the required
thermal energy. Also, for poration of skin, the change in the
conductivity properties of the stratum corneum and the epidermis
can be used to provide an elegant closed loop verification that the
poration process is complete, i.e., when the resistance indicates
that the epidermis has been reached, it is time to stop the
poration process. Similar changes in impedance can be used to
control the depth of penetration to other layers as well.
[0169] FIG. 7 shows an illustrative example of such a closed loop
impedance monitor. In this system 230, there is an ohmic heat
source 234 coupled to a wire heat probe 238. The heat source is
mounted through an insulating mount 242 on a mechanical modulator
246. A controller 250 is coupled to the wire and to the skin 254,
wherein the controller detects changes in impedance in the selected
area 258 of skin, and when a predetermined level is obtained the
controller stops the poration process.
[0170] Along the same line as hydraulic poration means are
microlancets adapted to just penetrate the stratum corneum for
purposes of administering a permeant, such as a drug, through the
pore formed or to withdraw an analyte through the pore for
analysis. Such a device is considered to be "minimally invasive" as
compared to devices and/or techniques which are non-invasive. The
use of micro-lancets that penetrate below the stratum corneum for
withdrawing blood are well known. Such devices are commercially
available from manufacturers such as Becton-Dickinson and Lifescan
and can be utilized in the present invention by controlling the
depth of penetration. As an example of a micro-lancet device for
collecting body fluids, reference is made to Erickson et al.,
International Published PCT Application WO 95/10223 (published 20
Apr. 1995). This application shows a device for penetration into or
through the dermal layer of the skin, without penetration into
subcutaneous tissues, to collect body fluids for monitoring, such
as for blood glucose levels.
[0171] Poration of a biological membrane can also be accomplished
using sonic means. Sonic-poration is a variation of the optical
means described above except that, instead of using a light source,
a very tightly focused beam of sonic energy is delivered to the
area of the stratum corneum to be ablated. The same levels of
energy are required, i.e. a threshold of 70 mJ/cm.sup.2/50 ms still
must be absorbed. The same pulsed focused ultrasonic transducers as
described in parent applications Ser. Nos. 08/152,442 (now U.S.
Pat. No. 5,458,140) and Ser. No. 08/152,174 (now U.S. Pat. No.
5,445,611) can be utilized to deliver the required energy densities
for ablation as are used in the delivery of sonic energy which is
modulated in intensity, phase, or frequency or a combination of
these parameters for the transdermal sampling of an analyte or the
transdermal delivery of drugs. This has the advantage of allowing
use of the same transducer to push a drug through the stratum
corneum or pull a body fluid to the surface for analysis to be used
to first create a micropore.
[0172] Additionally, electroporation or short bursts or pulses of
electrical current can be delivered to the stratum corneum with
sufficient energy to form micropores. Electroporation is known in
the art for producing pores in biological membranes and
electroporation instruments are commercially available. Thus, a
person of skill in this art can select an instrument and conditions
for use thereof without undue experimentation according to the
guidelines provided herein.
[0173] The micropores produced in the biological membrane by the
methods of the present invention allow high flux rates of a variety
of molecular weight therapeutic compounds to be delivered
transmembranely. In addition, these non-traumatic microscopic
openings into the body allow access to various analytes within the
body, which can be assayed to determine their internal
concentrations.
Example 1
[0174] In this example, skin samples were prepared as follows.
Epidermal membrane was separated from human cadaver whole skin by
the heat-separation method of Klingman and Christopher, 88 Arch.
Dermatol. 702 (1963), involving the exposure of the full thickness
skin to a temperature of 60.degree. C. for 60 seconds, after which
time the stratum corneum and part of the epidermis (epidermal
membrane) were gently peeled from the dermis.
Example 2
[0175] Heat separated stratum corneum samples prepared according to
the procedure of. Example 1 were cut into 1 cm.sup.2 sections.
These small samples were than attached to a glass cover slide by
placing them on the slide and applying an pressure sensitive
adhesive backed disk with a 6 mm hole in the center over the skin
sample. The samples were then ready for experimental testing. In
some instances the skin samples were hydrated by allowing them to
soak for several hours in a neutral buffered phosphate solution or
pure water.
[0176] As a test of these untreated skin samples, the outputs of
several different infrared laser diodes, emitting at roughly 810,
905, 1480 and 1550 nanometers were applied to the sample. The
delivery optics were designed to produce a focal waist 25 .mu.m
across with a final objective have a numerical aperture of 0.4. The
total power delivered to the focal point was measured to be between
50 and 200 milliwatts for the 810 and 1480 nm laser diodes, which
were capable of operating in a continuous wave (CW) fashion. The
905 and 1550 nm laser diodes were designed to produce high peak
power pulses roughly 10 to 200 nanoseconds, long at repetition
rates up to 5000 Hz. For the pulsed lasers the peak power levels
were measured to be 45 watts at 905 nm and 3.5 watts at 1550
nm.
[0177] Under these operating conditions, there was no apparent
effect on the skin samples from any of the lasers. The targeted
area was illuminated continuously for 60 seconds and then examined
microscopically, revealing no visible effects. In addition, the
sample was placed in a modified Franz cell, typically used to test
transdermal delivery systems based on chemical permeation
enhancers, and the conductivity from one side of the membrane to
the other was measured both before and after the irradiation by the
laser and showed no change. Based on these tests which were run on
skin samples from four different donors, it was concluded that at
these wavelengths the coupling of the optical energy into or
through the skin tissue was so small that no effects are
detectable.
Example 3
[0178] To evaluate the potential sensation to a living subject when
illuminated with optical energy under the conditions of Example 2,
six volunteers were used and the output of each laser source was
applied to their fingertips, forearms, and the backs of their
hands. In the cases of the 810, 905 and 1550 nm lasers, the subject
was unable to sense when the laser was turned on or off. In the
case of the 1480 nm laser, there was a some sensation during the
illumination by the 1480 nm laser operating at 70 mW CW, and a
short while later a tiny blister was formed under the skin due to
the absorption of the 1480 nm radiation by one of the water
absorption bands. Apparently the amount of energy absorbed was
sufficient to induce the formation of the blister, but was not
enough to cause the ablative removal of the stratum corneum. Also,
the absorption of the 1480 nm light occurred predominantly in the
deeper, fully hydrated (85% to 90% water content) tissues of the
epidermis and dermis, not the relatively dry (10% to 15% water
content) tissue of the stratum corneum.
Example 4
[0179] Having demonstrated the lack of effect on the skin in its
natural state (Example 3), a series of chemical compounds was
evaluated for effectiveness in absorbing the light energy and then
transferring this absorbed energy, via conduction, into or through
the targeted tissue of the stratum corneum. Compounds tested
included India ink; "SHARPIE" brand indelible black, blue, and red
marking pens; methylene blue; fuschian red; epolite #67, an
absorbing compound developed for molding into polycarbonate lenses
for protected laser goggles; tincture of iodine;
iodine-polyvinylpyrrolidone complex ("BETADINE"); copper
phthalocyanine; and printers ink.
[0180] Using both of the CW laser diodes described in Example 2,
positive ablation results were observed on the in vitro samples of
heat-separated stratum corneum prepared according to Example 1 when
using all of these products, however some performed better than
others. In particular the copper phthalocyanine (CPC) and the
epolite #67 were some of the most effective. One probable reason
for the superior performance of the CPC is its high boiling point
of greater the 500.degree. C. and the fact that it maintains its
solid phase up to this temperature.
Example 5
[0181] As copper phthalocyanine has already been approved by the
FDA for use in implantable sutures, and is listed in the Merck
index as a rather benign and stabile molecule in regard to human
biocompatability, the next step taken was to combine the topical
application of the CPC and the focused light source to the skin of
healthy human volunteers. A suspension of finely ground CPC in
isopropyl alcohol was prepared. The method of application used was
to shake the solution and then apply a small drop at the target
site. As the alcohol evaporated, a fine and uniform coating of the
solid phase CPC was then left on the surface of the skin.
[0182] The apparatus show in FIG. 1 was then applied to the site,
wherein the CPC had been topically coated onto the skin, by placing
the selected area of the individual's skin against a reference
plate. The reference plate consists of a thin glass window roughly
3 cm.times.3 cm, with a 4 mm hole in the center. The CPC covered
area was then positioned such that it was within the central hole.
A confocal video microscope (FIG. 1) was then used to bring the
surface of the skin into sharp focus. Positioning the skin to
achieve the sharpest focus on the video system also positioned it
such that the focal point of the laser system was coincident with
the surface of the skin. The operator then activated the pulses of
laser light while watching the effects at the target site on the
video monitor. The amount of penetration was estimated visually by
the operator by gauging the amount of defocusing of the laser spot
in the micropore as the depth of the micropore increased, and this
can be dynamically corrected by the operator, essentially following
the ablated surface down into the tissues by moving the position of
the camera/laser source along the "z" axis, into the skin. At the
point when the stratum corneum had been removed down to the
epidermis, the appearance of the base of the hole changed
noticeably, becoming much wetter and shinier. Upon seeing this
change, the operator deactivated the laser. In many instances,
depending on the state of hydration of the subject as well as other
physiological conditions, a dramatic outflow of interstitial fluid
occurred in response to the barrier function of the stratum corneum
being removed over this small area. The video system was used to
record this visual record of the accessibility of interstitial
fluid at the poration site.
Example 6
[0183] The procedure of Example 5 was followed except that the CPC
was applied to a transparent adhesive tape, which was then caused
to adhere to a selected site on the skin of an individual. The
results were substantially similar to those of Example 5.
Example 7
[0184] Histology experiments were performed on cadaver skin
according to methods well known in the art to determine ablation
threshold parameter's for given dye mixtures and collateral damage
information. The top surface of the skin sample was treated with a
solution of copper phthalocyanine (CPC) in alcohol. After the
alcohol evaporated, a topical layer of solid phase CPC was
distributed over the skin surface with a mean thickness of 10 to 20
.mu.m. FIG. 8A shows a cross-section of full thickness skin prior
to the laser application, wherein the CPC layer 270, stratum
corneum 274, and underlying epidermal layers 278 are shown. FIG. 8B
shows the sample after a single pulse of 810 nm light was applied
to an 80 um diameter circle with an energy density of 4000 J/cm2,
for a pulse period of 20 ms. It is noteworthy that there was still
a significant amount of CPC present on the surface of the stratum
corneum even in the middle of the ablated crater 282. It should
also be noted that laboratory measurements indicate that only about
10% of the light energy incident on the CPC is actually absorbed,
with the other 90% being reflected or backscattered. Thus the
effective energy flux being delivered to the dye layer which could
cause the desired heating is only about 400 J/cm2. 8C shows the
sample after 5 pulses of 810 nm light were applied, wherein the
stratum corneum barrier was removed with no damage to the
underlying tissue. These results are a good representation of the
"ideal" optically modulated thermal ablation performance. FIG. 8D
shows the sample after 50 pulses were applied. Damaged tissue 286
was present in the epidermal layers due to carbonization of non
ablated tissue and thermal denaturing of the underlying tissue.
FIGS. 8A-8C show separations between the stratum corneum and the
underlying epidermal layers due to an artifact of dehydration,
freezing, and preparations for imaging.
Example 8
[0185] To examine the details of the thermal ablation mechanism, a
mathematical model of the skin tissues was constructed upon which
various different embodiments of the thermal ablation method could
be tried. This model computes the temperature distribution in a
layered semi-infinite medium with a specified heat flux input
locally on the surface and heat removal from the surface some
distance away, i.e. convection is applied between the two. The
axisymmetric, time-dependent diffusion equation is solved in
cylindrical coordinates using the alternating-direction-implicit
(ADI) method. (Note: Constant Temp. B.C. is applied on lower
boundary to serve as z->inf; and zero radial heat flux is
applied on max radial boundary to serve as r->inf). The layers
are parallel to the surface and are defined as: (1) dye; (2)
stratum corneum; (3) underlying epidermis; and (4) dermis. The
depth into the semi-infinite medium and thermal properties, density
(rho), specific heat (c), and conductivity (k) must be specified
for each layer.
[0186] First, a heat-transfer coefficient, h, on the skin is
computed based on the "steady," "1-D," temperature distribution
determined by the ambient air temperature, skin surface
temperature, and dermis temperature. It is assumed that there is no
dye present and provides "h" on the skin surface. The program then
allows one to use this "h" on the dye layer surface or input
another desired "h" for the dye surface. Next, the "steady"
temperature distribution is computed throughout all layers
(including the dye layer) using the specified "h" at the dye
surface. This temperature distribution is the initial condition for
the time-dependent heating problem. This constitutes the "m-file"
initial.m. The program then solves for the time-dependent
temperature distribution by marching in time, computing and
displaying the temperature field at each step.
[0187] Each embodiment of the method described herein, for which
empirical data have been collected, has been modeled for at least
one set of operational parameters, showing how stratum corneum
ablation can be achieved in a precise and controllable fashion. The
output of the simulations is presented graphically in two different
formats: (1) a cross-sectional view of the skin showing the
different tissue layers with three isotherms plotted on top of this
view which define three critical temperature thresholds, and (2)
two different temperature -vs- time plots, one for the point in the
middle of the stratum corneum directly beneath the target site, and
the second for the point at the boundary of the viable cell layers
of the epidermis and the underside of the stratum corneum. These
plots show how the temperature at each point varies with time as
the heat pulses are applied as if one could implant a microscopic
thermocouple into the tissues. In addition, the application of this
model allows investigation of the parametric limits within which
the method can be employed to set the outer limits for two
important aspects of the methods performance. First, general cases
are presented cases that define the envelope within which the
method can be employed without causing pain or undesired tissue
damage.
[0188] For any given heat source, as described in the several
different embodiments of the invention, there is a point at which
the effect on the subject's skin tissues becomes non-optimal in
that the subject perceives a pain sensation, or that the viable
cells in the underlying epidermis and/or dermis sustain
temperatures, which if maintained for a long enough duration, will
render damage to these tissues. Accordingly, a test simulation was
run using the optically heated topical copper phthalocyanine (CPC)
dye embodiment as a baseline method to establish how the thermal
time constants of the different skin tissue layers essentially
define a, window within which the method can be employed without
pain or damage to adjacent tissue layers.
[0189] FIGS. 9 and 10 show schematic cross-sectional views of the
skin and the topical dye layer. In each, figure, three distinct
isotherms are displayed: (1) 123 C, the point at which vaporization
of the water in the tissue produces an ablation of the tissue; (2)
70 C, the point at which viable cells will be damaged if this
temperature is maintained for several seconds; and (3) 45 C, the
average point at which a sensation of pain will be perceived by the
subject. This pain threshold is described in several basic
physiology texts, but experience shows this threshold to be
somewhat subjective. In fact, in repeated tests on the same
individual, different poration sites within a few millimeters of
each other can show significantly different amounts of sensation,
possibly due to the proximity to a nerve ending in relationship to
the poration site.
[0190] The dimensions on the graphs show the different layers of
the dye and skin, as measured in m, with flat boundaries defining
them. Whereas the actual skin tissues have much more convoluted
boundaries, in a mean sense for the dimensions involved, the model
provides a good approximation of the thermal gradients present in
the actual tissues. The dimensions used in this, and all subsequent
simulations, for the thicknesses of the CPC dye layer and the
various skin layers are as follows: dye, 10 m; stratum corneum, 30
m; underlying epidermis, 70 m; and dermis, 100 m.
[0191] Additional conditions imposed on the model for this
particular simulation are shown in the following tables:
TABLE-US-00001 TABLE 1 Initial Conditions for Finite Difference
Thermal Model Ambient Air Temperature Ta = 20 C. Skin Surface
Temperature Ts = 30 C. Dermis Temperature Td = 37 C. Dye
Vaporization Temperature Tvap = 550 C. S.C. Vaporization
Temperature Tc1 = 123 C. Tissue Damage Temperature Tc2 = 70 C.
"Pain" Temperature Tc3 = 45 C. Radius of Irradiated Area R.sub.hot
= 30 m Energy Density Applied FLUX = 400 Joules/cm.sup.2
TABLE-US-00002 TABLE 2 Parameter Dye S.C. Epidermis Dermis Thermal
0.00046 .00123 0.00421 0.00421 Conductivity Density 0.67 1.28 1.09
1.09 Specific Heat 0.8 1.88 3.35 3.35
[0192] When these simulations are run, the following conservative
assumptions are imposed:
[0193] 1. While some portion of the stratum corneum may be shown as
having a temperature already exceeded the ablation threshold for
thermal vaporization of the water content, this event is not
modeled, and the subsequent loss of heat energy in the tissues due
to this vaporization is not factored into the simulation. This will
cause a slight elevation in the temperatures shown in the
underlying tissues from that point on in the simulation run.
[0194] 2. Similarly, when some portion of the copper phthalocyanine
(CPC) dye layer is shown to have reached its vaporization point of
550.degree. C., this event is not modeled, but the temperature is
merely hard-limited to this level. This will also cause a slight
elevation of the subsequent temperatures in the underlying layers
as the simulation progresses.
[0195] Even with these simplifications used in the model, the
correlation between the predicted performance and the empirically
observed performance based on both clinical studies and
histological studies on donor tissue samples is remarkable. The key
data to note in FIGS. 9 and 10 are the length of time which the
heat pulse is applied, and the location of the three different
threshold temperatures displayed by the isotherms.
[0196] In FIG. 9, with a pulse length of 21 milliseconds, the
70.degree. C. isotherm just crosses the boundary separating the
stratum corneum and the viable cell layers in the epidermis. In in
vitro studies on donor skin samples under these conditions, fifty
pulses of thermal energy delivered 50 milliseconds apart cause
detectable damage to this top layer of living cells (see FIG. 8D).
However, it was also shown in the in vitro studies that five pulses
of heat energy at these same operating parameters, did not produce
any significant damage to these tissues. It seems reasonable that
even though the nominal damage threshold may have been exceeded, at
least in a transient sense, this temperature must be maintained for
some cumulative period of time to actually cause any damage to the
cells. Nevertheless, the basic information presented by the
simulation is that if one keeps the "on-time" of the heat pulse to
less than 20 milliseconds with the flux density of 400
Joules/cm.sup.2, then no damage to the living cells in the
underlying epidermis will be sustained, even though the ablation
threshold isotherm has been moved well into or through the stratum
corneum. In other words, by using a low flux density thermal energy
source, modulated such that the "on time" is suitably short,
ablation of the stratum corneum can be achieved without any damage
to the adjacent cells in the underlying epidermis (see FIG. 8C).
This is possible in large part due to the significantly different
thermal diffusivities of these two tissues layers. That is, the
stratum corneum, containing only about 10% to 20% water content,
has a much lower thermal conductivity constant, 0.00123 J/(S m*K),
than the 0.00421 J/(S*cm*K) of the epidermis. This allows the
temperature to build up in the stratum corneum, while maintaining a
tight spatial definition, to the point at which ablation will
occur.
[0197] In FIG. 10, the same simulation scenario started in the
damage threshold critical point run illustrated in FIG. 9 is
carried out farther in time. By leaving the heat pulse on for 58
milliseconds at the same flux density of 400 Joules/cm.sup.2 within
the 60 .mu.m diameter circle of dye being heated, the pain sensory
isotherm at 45.degree. C. just enters the innervated layer of skin
comprised by the dermis. In addition, the damage threshold isotherm
moves significantly farther into the epidermal layer than where it
was shown to be in FIG. 9. Relating this simulation to the numerous
clinical studies conducted with this method, an excellent
verification of the model's accuracy is obtained in that the model
shows almost exactly the duration of `on-time` that the heat probe
can be applied to the skin before the individual feels it. In
clinical tests, a controllable pulse generator was used to set the
"on-time" and "off-time" of a series of light pulses applied to the
topical layer of copper phthalocyanine (CPC) dye on the skin. While
maintaining a constant "off-time" of 80 milliseconds, the "on-time"
was gradually increased until the subject reported a mild "pain"
sensation. Without exception, all of the subjects involved in these
studies, reported the first "pain" at an "on-time" of between 45
and 60 milliseconds, very close to that predicted by the model. In
addition, the site-to-site variability mentioned previously as
regards the sensation of "pain" was noted in these clinical
studies. Accordingly, what is reported as "pain" is the point at
which the first unambiguous sensation is noticeable. At one site
this may be reported as pain, whereas at an adjacent site the same
subject may report this as merely "noticeable."
[0198] One element of this clinical research is the realization
that even at the same site, a non-uniform pulse-train of heat
pulses may work with the subject's psycho-physiological
neuro-perception to cause a genuine reduction in perceived
sensation. For example, a series of shorter length heat pulses can
be used to saturate the neurons in the area, momentarily depleting
the neuro-transmitters available at this synaptic junction and
therefore limiting the ability to send a "pain" message. This then
allows a longer pulse following these short pulses to be less
noticeable than if it were applied at the beginning of the
sequence. Accordingly, a series of experiments was conducted with
some arbitrarily created pulse trains, and the results were
consistent with this hypothesis. An analogy for this situation
might be found in the perception when one first steps into a very
hot bath that is painful at first, but quickly becomes tolerable as
one acclimates to the heat sensation.
Example 9
[0199] An object of this invention is to achieve a painless,
micro-poration of the stratum corneum without causing any
significant damage to the adjacent viable tissues. As described in
the simulation illustrated in Example 8 and FIGS. 9-10, a boundary
appears to exist for any given flux density of thermal energy
within the ablation target spot within which the micro-poration can
be achieved in just such a painless and non-traumatic manner. Both
the in vivo and in vitro studies have shown that this is the case,
and this has permitted development through empirical methods of
some operational parameters that appear to work very well. The
following set of simulations shows how the method works when these
specific parameters are used.
[0200] In the first case, a pulse train of ten pulses, 10
milliseconds "on-time" separated by 10 milliseconds "off-time" is
applied to the CPC-covered skin. FIG. 11 shows the final
temperature distribution in the skin tissues immediately after this
pulse train has ended. As can be seen, the isotherms representing
the three critical temperature thresholds show that stratum corneum
ablation has been achieved, with no sensation present in the dermal
layer nerves and very little cross-over of the damage threshold
into or through the viable cells of the underlying epidermis. As
mentioned previously, it appears that to actually do permanent cell
damage, the epidermal cells must not only be heated up to a certain
point, but they also must be held at this temperature for some
period of time, generally thought to be about five seconds. FIGS.
12 and 13 show the temperature of the stratum corneum and the
viable epidermis, respectively, as a function of time, showing
heating during the "on-time" and cooling during the "off-time" for
the entire ten cycles. Relating this simulation to the in vivo
studies conducted, note that in better than 90% of the poration
attempts with the system parameters set to match the simulation,
effective poration of the stratum corneum was achieved without pain
to the subject, and in subsequent microscopic examination of the
poration site several days later, no noticeable damage to the
tissues was apparent. The in vitro studies conducted on whole
thickness donor skin samples were also consistent with the model's
prediction of behavior.
Example 10
[0201] In conducting both the empirical in vivo studies, and these
simulations, it appears that prechilling of the skin aids in
optimizing the micro-poration process for reducing the probability
of pain or damage to adjacent tissues. In practice, this can easily
be achieved using a simple cold-plate placed against the skin prior
to the poration process. For example, applying a Peltier cooled
plate to the 1 cm diameter circle surrounding the poration target
site, with the plate held at roughly 5.degree. C. for a few
seconds, significantly reduces the temperature of the tissues. A
schematic illustration of an experimental device used for this
purpose in the laboratory is shown in FIGS. 3A-B. By applying
exactly the same ten-cycle pulse train as used in the run
illustrated in Example 9, one can see, by comparing FIG. 11 to FIG.
14, FIG. 12 to FIG. 15, and FIG. 13 to FIG. 16, how much
improvement can be made in the control of the temperature
penetration into or through the skin tissues. Once again, the
relatively low thermal diffusivity and specific heat of the stratum
corneum as compared to the epidermis and dermis is advantageous.
Once cooled, the highly hydrated tissues of the epidermis and
dermis require a much larger thermal energy input to elevate their
temperatures, whereas the stratum corneum, with its relatively dry
makeup, can quickly be heated up to the ablation threshold.
Example 11
[0202] Once the basic thermal conduction mechanism of delivering
the energy into or through the skin tissues underlying the
effective painless ablation and micro-poration of the stratum
corneum is understood, several different specific methods to
achieve the required rapid temperature modulations of the contact
point can be conceived, such as the hot wire embodiments
illustrated in FIGS. 4-7.
[0203] A basic embodiment, as described herein, uses an Ohmic
heating element (FIG. 4), such as the tip of a small cordless
soldering iron, with a suitably sized, relatively non-reactive,
wire wrapped around it with a short amount of the wire left to
protrude away from the body of the heater. When electricity is
applied with a constant current source, the heater will come up to
some temperature and within a few seconds, achieve a steady state
with the convection losses to the surrounding air. Similarly, the
wire, which is a part of this thermal system, will reach a steady
state such that the very tip of the wire can be raised to almost
any arbitrary temperature, up to roughly 1000.degree. C. with these
types of components. The tip can be sized to give exactly the
dimension micropore desired.
[0204] In the laboratory, tungsten wires with a diameter of 80
.mu.m attached to the replaceable tip of a "WAHL" cordless
soldering iron with approximately 2 mm of wire protruding from the
tip have been utilized. With a thermocouple, the temperature of the
tip has been measured at its steady state, and it has been noted
that by varying the constant current settings, steady state
temperatures of greater than 700.degree. C. can easily be reached.
To achieve the desired modulation, a low mass, fast response
electromechanical actuator was coupled to the tip such that the
position of the wire could be translated linearly more than 2 mm at
up to a 200 Hz rate. Then, by mounting the entire apparatus on a
precision stage, this vibrating tip could very controllably be
brought into contact with the skin surface in a manner where it was
only in contact for less than 10 milliseconds at a time, the
"on-time," while an "off-time" of arbitrarily long periods could be
achieved by setting the pulse generator accordingly. These in vivo
studies showed that the poration could actually be achieved before
the subject being porated even knew that the tip of the wire was
being brought into contact with the skin.
[0205] To compare the performance of this embodiment to the
optically heated topical CPC dye embodiment, the following
simulations were run according to the procedure of Example 8.
Essentially, by only varying the initial conditions, the hot wire
embodiment can be run with the identical simulation code. Because
the contact with the wire occurs essentially instantly, there is no
time dependent build-up of heat in the CPC dye layer and when the
wire is physically removed from contact with the skin, there is a
no residual heat still left on the surface as there is with the
heated CPC dye layer. Also, as the wire itself defines the area
targeted for ablation/micro-poration, there should be no lateral
diffusion of thermal energy prior to its application to the stratum
corneum. The comparative performances of the "hot-wire" embodiment
are shown in FIGS. 17-19.
Example 12
[0206] In this example, the procedure of Example 11 was followed
except that the skin was pre-cooled according to the procedure of
Example 10. Similarly, pre-cooling the target site yields similarly
positive results with the "hot-wire" embodiment. The results of the
pre-cooled simulation of the "hot-wire" approach are shown in FIGS.
20-22.
Example 13
[0207] As discussed in the background introduction of this
disclosure, the Tankovich '803 patent appears at first glance to be
similar to the presently claimed invention. In this example, the
simulation model was set up with the operating parameters specified
in Tankovich '803, i.e. a pulse width of 1 s and a power level of
40,000,000 W/cm.sup.2. FIGS. 23 and 24 show that under these
conditions no portion of the stratum corneum reaches the threshold
for flash vaporization of water, 123 C, and thus no
ablation/microporation of the stratum corneum occurs. In practice,
applying this type of high peak power, short duration pulse to the
topical dye layer merely vaporizes the dye off of the surface of
the skin with no effect on the skin. This example, thus,
demonstrates that the conditions specified by Tankovich '803 are
inoperative in the presently claimed invention.
Example 14
[0208] In this example, interstitial fluid obtained after porating
the skin according to the procedure of Example 6 was collected and
analyzed to determine the glucose concentration thereof. Data were
obtained on four non-diabetic subjects and six type I diabetic
subjects undergoing a glucose load test. Subject's ages ranged from
27 to 43. The goal of the study was to examine the utility of the
method for painlessly harvesting enough interstitial fluid (ISF)
from the subjects to allow the ISF samples to be assayed for
glucose content, and then compare these concentrations to the
glucose level presenting in the subject's whole blood.
[0209] All subjects had both the blood and ISF glucose assays
performed with the "ELITE" system from Miles-Bayer. All ten
subjects underwent identical measurement protocols, with
adjustments being made regarding the glucose load and insulin shot
for those subjects with insulin dependent diabetes.
[0210] The basic design of the study was to recruit a modest number
of volunteers, some with diabetes and some without diabetes, from
which a series of sample pairs of ISF and whole blood were drawn
every 3 to 5 minutes throughout the 3 to 4 hour duration of the
study period. Both the blood and the ISF samples were assayed for
glucose and the statistical relationship between the blood glucose
levels and the interstitial fluid determined. To examine the
hypothesized temporal lag of the ISF glucose levels as compared to
the whole blood glucose levels, the study subjects were induced to
exhibit a significant and dynamic change in their glucose levels.
This was accomplished by having each subject fast for 12 hours
prior to beginning the test and then giving the subject a glucose
load after his or her baseline glucose levels have been established
via a set of three fasting blood and ISF glucose levels. After the
baseline levels had been established, the subjects were given a
glucose load in the form of sweet juice based on the following
guidelines: [0211] i. For the control subjects, the glucose load
was calculated based on a 0.75 gram glucose per pound of body
weight. [0212] ii. For the subjects with insulin dependent diabetes
the glucose load was 50 grams of glucose. In addition, immediately
after taking the glucose load the diabetic subjects will self
inject their normal morning dose of fast acting insulin. In the
case where the diabetic subject presents with fasting glucose
levels above 300 mg/dL, they were asked to give themselves their
insulin injection first, and the glucose load was provided after
their blood glucose levels have dropped to below 120 mg/dL.
[0213] Each subject recruited was first given a complete
description of the study in the "Informed Consent" document which
they were required to understand and sign before they were
officially enrolled into the program. Upon acceptance, they
completed a medical history questionnaire. The detailed clinical
procedure implemented was:
[0214] (a) Subject fasted from 9:00 p.m. the night before the study
visit, consuming only water. No caffeine, cigarettes, fruit juice
were allowed during this period.
[0215] (b) Subject arrived at the testing facility by 9:00 a.m. the
next day.
[0216] (c) Subject was seated in a reclining chair provided for the
subject to relax in throughout the study procedure.
[0217] (d) Both whole blood and ISF samples were taken at three to
five minute intervals beginning upon the subject's arrival and
continuing for the next three to four hours. The duration over
which the data were collected was based on when the subject's blood
glucose levels had returned to the normal range and stabilized
after the glucose load. The ISF samples were harvested using the
optical poration, ISF pumping method, described in more detail
below. Each ISF sample was roughly 5 .mu.L by volume to ensure a
good fill of the ELITE test strip. The blood samples were obtained
via a conventional finger prick lancet. Both the ISF and the blood
samples were immediately assayed for glucose with the ELITE home
glucometer system from Miles-Bayer. To improve the estimate of the
`true` blood glucose levels, two separate ELITE assays were be done
on each finger stick sample.
[0218] (e) To facilitate the continued collection of the ISF from
the same site through-out the entire data collection phase for a
given individual, a 5 by 5 matrix of twenty five micropores was
created on the subject's upper forearm, each micropore being
between 50 and 80 .mu.m across and spaced 300 .mu.m apart. A 30
.mu.m diameter teflon disk with a 6 mm hole in the center was
attached to the subject's forearm with a pressure sensitive
adhesive and positioned such that the 6 mm center hole was located
over the 5 by 5 matrix of micropores. This attachment allowed a
convenient method by which a small suction hose could be connected,
applying a mild vacuum (10 to 12 inches of Hg) to the porated area
to induce the ISF to flow out of the body through the micropores.
The top of the teflon disk was fitted with a clear glass window
allowing the operator to directly view the micro-porated skin
beneath it. When a 5 .mu.L bead of ISF was formed on the surface of
the skin, it could easily be ascertained by visually monitoring the
site through this window. This level of vacuum created a nominal
pressure gradient of around 5 pounds/square inch (PSI). Without the
micropores, no ISF whatsoever could be drawn from the subject's
body using only the mild vacuum.
[0219] (f) After the first three sample pairs have been drawn, the
subject was given a glucose load in the form of highly sweetened
orange juice. The amount of glucose given was 0.75 grams per pound
of body weight for the nondiabetic subjects and 50 grams for the
diabetic subjects. The diabetic subjects also self administered a
shot of fast acting insulin, (regular) with the dosage
appropriately calculated, based on this 50 gram level of glucose
concurrent with the ingestion of the glucose load. With the normal
1.5 to 2.5 hour lag between receiving an insulin shot and the
maximum effect of the shot, the diabetic subjects were expected to
exhibit an upwards excursion of their blood glucose levels ranging
up to 300 mg/dL and then dropping rapidly back into the normal
range as the insulin takes effect. The nondiabetic subjects were
expected to exhibit the standard glucose tolerance test profiles,
typically showing a peak in blood glucose levels between 150 mg/dL
and 220 mg/dL from 45 minutes to 90 minutes after administering the
glucose load, and then a rapid drop back to their normal baseline
levels over the next hour or so.
[0220] (g) Following the administration of the glucose load or
glucose load and insulin shot, the subjects had samples drawn,
simultaneously, of ISF and finger prick whole blood at five minute
intervals for the next three to four hours. The sampling was
terminated when the blood glucose levels in three successive
samples indicate that the subject's glucose had stabilized.
[0221] Upon examination of the data, several features were
apparent. In particular, for any specific batch of ELITE test
strips, there exist a distinct shift in the output shown on the
glucometer in mg/dL glucose as compared to the level indicated on
the blood. An elevated reading would be expected due to the lack of
hematocrit in the ISF and to the normal differences in the
electrolyte concentrations between the ISF and whole blood.
Regardless of the underlying reasons for this shift in output, it
was determined via comparison to a reference assay that the true
ISF glucose levels are linearly related to the values produced by
the ELITE system, with the scaling coefficients constant for any
specific batch of ELITE strips. Consequently, for the comparison of
the ISF glucose levels versus the whole blood measurements, first
order linear correction was applied to the ISF data as follows:
ISF.sub.glucose=0.606ISF.sub.ELITE+19.5
[0222] This scaling of the output of the ELITE glucometer when used
to measure ISF glucose levels, allows one to examine, over the
entire data set, the error terms associated with using ISF to
estimate blood glucose levels. Of course, even with no linear
scaling whatsoever, the correlations between the ISF glucose values
and the blood glucose levels are the same as the scaled
version.
[0223] Based on the majority of the published body of literature on
the subject of ISF glucose as well as preliminary data, it was
originally expected that a 15 to 20 minute lag between the ISF
glucose levels and the those presented in the whole blood from a
finger stick would be observed. This is not what the data showed
when analyzed. Specifically, when each individual's data set is
analyzed to determine the time shift required to achieve the
maximum correlation between the ISF glucose levels and the blood
glucose levels it was discovered that the worst case time lag for
this set of subjects was only 13 minutes and the average time lag
was only 6.2 minutes, with several subjects showing a temporal
tracking that was almost instantaneous (about 1 minute).
[0224] Based on the minimal amount of lag observed in this data
set, the graph shown in FIG. 25 presents all ten of the glucose
load tests, concatenated one after another on an extended time
scale. The data are presented with no time shifting whatsoever,
showing the high level of tracking between the ISF and blood
glucose levels the entire clinical data set being dealt with in
exactly the same manner. If the entire data set is shifted as a
whole to find the best temporal tracking estimate, the correlation
between the ISF and blood glucose levels peaks with a delay of two
(2) minutes at an r value of r=0.97. This is only a trivial
improvement from the unshifted correlation of r=0.964. Therefore,
for the remainder of the analysis the ISF values are treated with
no time shift imposed on them. That is, each set of blood and ISF
glucose levels is dealt with as simultaneously collected data
pairs.
[0225] After the unshifted Elite ISF readings had been scaled to
reflect the proportional glucose present in the ISF, it was
possible to examine the error associated with these data. The
simplest method for this is to assume that the average of the two
ELITE finger-stick blood glucose readings is in fact the absolutely
correct value, and then to merely compare the scaled ISF values to
these mean blood glucose values. These data are as follows:
Standard Deviation Blood-ISF, 13.4 mg/dL; Coefficient of Variance
of ISF, 9.7%; Standard Deviation of the Two Elites, 8.3 mg/dL; and
Coefficient of Variance of Blood (Miles), 6%.
[0226] As these data show, the blood based measurement already
contains an error term. Indeed, the manufacturer's published
performance data indicates that the ELITE system has a nominal
Coefficient of Variance (CV) of between 5% and 7%, depending on the
glucose levels and the amount of hematocrit in the blood.
[0227] An additional look at the difference term between the ISF
glucose and the blood glucose is shown in the form of a scatter
plot in FIG. 26. In this figure, the upper and lower bounds of the
90% confidence interval are also displayed for reference. It is
interesting to note that with only two exceptions, all of the data
in the range of blood glucose levels below 100 mg/dL fall within
these 90% confidence interval error bars. This is important as the
consequences of missing a trend towards hypoglycemia would be very
significant to the diabetic user. That is, it would be much better
to under-predict glucose levels in the 40 to 120 mg/dL than to over
predict them.
[0228] Essentially, if one assumes that the basic assay error when
the ELITE system is used on ISF is comparable to the assay error
associated with the ELITE's use on whole blood, then the Deviation
of the ISF glucose from the blood glucose can be described as:
ISF.sub.deviation=[(ISF.sub.actual).sup.2+(ISF.sub.actual).sup.2].sup.1/-
2.
[0229] Applying this equation to the values shown above, one can
solve for the estimated `true` value of the ISF error term:
ISF.sub.actual=[(ISF.sub.deviation).sup.2-(Blood.sub.actual).sup.2].sup.-
1/2.
[0230] Or, solving the equation,
ISF.sub.actual=[(13.4).sup.2-(8.3).sup.2].sup.1/2=10.5 mg/dl.
[0231] A histogram of the relative deviation of the ISF to the
blood glucose levels is shown in FIG. 27.
Drug Delivery Through Pores in the Biological Membrane
[0232] The present invention also includes a method for the
delivery of drugs, including drugs currently delivered
transmembrane, through micropores in the stratum corneum or other
biological membrane. In one illustrative embodiment, the delivery
is achieved by placing the solution in a reservoir over the
poration site. In another illustrative embodiment, a pressure
gradient is used to further enhance the delivery. In still another
illustrative embodiment, sonic energy is used with or without a
pressure gradient to further enhance the delivery. The sonic energy
can be operated according to traditional transdermal parameters or
by utilizing acoustic streaming effects, which will be described
momentarily, to push the delivery solution through the porated
biological membrane.
Example 15
[0233] This example shows the use of stratum corneum poration for
the delivery of lidocaine, a topical analgesic. The lidocaine
solution also contained a chemical permeation enhancer formulation
designed to enhance its passive diffusion across the stratum
corneum. A drawing of an illustrative delivery apparatus 300 is
shown in FIG. 28, wherein the apparatus comprises a housing 304
enclosing a reservoir 308 for holding a drug-containing solution
312. The top portion of the housing comprises an ultrasonic
transducer 316 for providing sonic energy to aid in transporting
the drug-containing solution through micropores 320 in the stratum
corneum 324. A port 328 in the ultrasonic transducer permits
application of pressure thereto for further aiding in transporting
the drug-containing solution through the micropores in the stratum
corneum. The delivery apparatus is applied to a selected area of an
individual's skin such that it is positioned over at least one, and
preferably a plurality, of micropores. An adhesive layer 332
attached to a lower portion of the housing permits the apparatus to
adhere to the skin such that the drug-containing solution in the
reservoir is in liquid communication with the micropores. Delivery
of the drug through the micropores results in transport into the
underlying epidermis 336 and dermis 340.
[0234] Five subjects were tested for the effectiveness of drug
delivery using poration together with ultrasound. The experiment
used two sites on the subjects left forearm about three inches
apart, equally spaced between the thumb and upper arm. The site
near the thumb will be referred to as site 1 the site furthest from
the thumb will be referred to as site 2. Site 1 was used as a
control where the lidocaine and enhancer solution was applied using
an identical delivery apparatus 300, but without any micro-poration
of the stratum corneum or sonic energy. Site 2 was porated with 24
holes spaced 0.8 millimeters apart in a grid contained within a 1
cm diameter circle. The micropores in Site 2 were generated
according to the procedure of Example 6. Lidocaine and low level
ultrasound were applied. Ultrasound applications were made with a
custom manufactured Zevex ultrasonic transducer assembly set in
burst mode with 0.4 Volts peak to peak input with 1000 count bursts
occurring at 10 Hz with a 65.4 kHz fundamental frequency, i.e., a
pulse modulated signal with the transducer energized for 15
millisecond bursts, and then turned off for the next 85
milliseconds. The measured output of the amplifier to the
transducer was 0.090 watts RMS.
[0235] After application of the lidocaine, sensation measurements
were made by rubbing a 30 gauge wire across the test site.
Experiments were executed on both sites, Site 1 for 10 to 12 minute
duration and Site 2 for two 5 minute duration intervals applied
serially to the same site. Both sites were assessed for numbness
using a scale of 10 to 0, where 10 indicated no numbness and 0
indicated complete numbness as reported by the test subjects. The
following summary of results is for all 5 subjects.
[0236] The control site, site 1, presented little to no numbness
(scale 7 to 10) at 10 to 12 minutes. At approximately 20 minutes
some numbness (scale 3) was observed at site 1 as the solution
completely permeated the stratum corneum. Site 1 was cleaned at the
completion of the lidocaine application. Site 2 presented nearly
complete numbness (scale 0 to 1) in the 1 cm circle containing the
porations. Outside the 1 cm diameter circle the numbness fell off
almost linearly to 1 at a 2.5 cm diameter circle with no numbness
outside the 2.5 cm diameter circle. Assessment of site 2 after the
second application resulted in a slightly larger totally numb
circle of about 1.2 cm diameter with numbness falling off linearly
to 1 in an irregular oval pattern with a diameter of 2 to 2.5 cm
perpendicular to the forearm and a diameter of 2 to 6 cm parallel
to the forearm. Outside the area no numbness was noted. A graphic
representation of illustrative results obtained on a typical
subject is shown in FIGS. 29A-C. FIGS. 29A and 29B show the results
obtained at Site 2 (porated) after 5 and 10 minutes, respectively.
FIG. 29C shows the results obtained at Site 1 (control with no
poration).
Sonic Energy and Enhancers for Enhancing Transdermal Flux
[0237] The physics of sonic energy fields created by sonic
transducers can be utilized in a method by which sonic frequency
can be modulated to improve on flux rates achieved by other
methods. As shown in FIG. 1 of U.S. Pat. No. 5,445,611, hereby
incorporated herein by reference, the energy distribution of an
sonic transducer can be divided into near and far fields. The near
field, characterized by length N, is the zone from the first energy
minimum to the last energy maximum. The zone distal to the last
maximum is the far field. The near (N) field pattern is dominated
by a large number of closely spaced local pressure peaks and nulls.
The length of the near field zone, N, is a function of the
frequency, size, and shape of the transducer face, and the speed of
sound in the medium through which the ultrasound travels. For a
single transducer, intensity variations within its normal operating
range do not affect the nature of the sonic energy distribution
other than in a linear fashion. However, for a system with multiple
transducers, all being modulated in both frequency and amplitude,
the relative intensities of separate transducers do affect the
energy distribution in the sonic medium, regardless of whether it
is skin or another medium.
[0238] By changing the frequency of the sonic energy by a modest
amount, for example in the range of about 1 to 20%, the pattern of
peaks and nulls remains relatively constant, but the length N of
the near field zone changes in direct proportion to the frequency.
Major changes the frequency, say a factor of 2 or more, will most
likely produce a different set of resonances or vibrational modes
in the transducer, causing a significantly and unpredictably
different near field energy pattern. Thus, with a modest change in
the sonic frequency, the complex pattern of peaks and nulls is
compressed or expanded in an accordion-like manner. By selecting
the direction of frequency modulation, the direction of shift of
these local pressure peaks can be controlled. By applying sonic
energy at the surface of the skin, selective modulation of the
sonic frequency controls movement of these local pressure peaks
through the skin either toward the interior of the body or toward
the surface of the body. A frequency modulation from high to low
drives the pressure peaks into the body, whereas a frequency
modulation from low to high pulls the pressure peaks from within
the body toward the surface and through the skin to the outside of
the body.
[0239] Assuming typical parameters for this application of, for
example, a 1.27 cm diameter sonic transducer and a nominal
operating frequency of 10 MHz and an acoustic impedance similar to
that of water, a frequency modulation of 1 MHz produces a movement
of about 2.5 mm of the peaks and nulls of the near field energy
pattern in the vicinity of the stratum corneum. From the
perspective of transdermal and/or transmucosal withdrawal of
analytes, this degree of action provides access to the area well
below the stratum corneum and even the epidermis, dermis, and other
tissues beneath it. For any given transducer, there may be an
optimal range of frequencies within which this frequency modulation
is most effective.
[0240] The flux of a drug or analyte across the skin can also be
increased by changing either the resistance (the diffusion
coefficient) or the driving force (the gradient for diffusion).
Flux can be enhanced by the use of so-called penetration or
chemical enhancers.
[0241] Chemical enhancers are comprised of two primary categories
of components, i.e., cell-envelope disordering compounds and
solvents or binary systems containing both cell-envelope
disordering compounds and solvents.
[0242] Cell envelope disordering compounds are known in the art as
being useful in topical pharmaceutical preparations and function
also in analyte withdrawal through the skin. These compounds are
thought to assist in skin penetration by disordering the lipid
structure of the stratum corneum cell-envelopes. A comprehensive
list of these compounds is described in European Patent Application
43,738, published Jun. 13, 1982, which is incorporated herein by
reference. It is believed that any cell envelope disordering
compound is useful for purposes of this invention.
[0243] Suitable solvents 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 (azones) and the like.
[0244] U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985,
contains an excellent summary of prior art and background
information detailing the use of certain binary systems for
permeant enhancement. Because of the completeness of that
disclosure, the information and terminology utilized therein are
incorporated herein by reference.
[0245] Similarly, European Patent Application 43,738, referred to
above, teaches using selected diols as solvents along with a broad
category of cell-envelope disordering compounds for delivery of
lipophilic pharmacologically-active compounds. Because of the
detail in disclosing the cell-envelope disordering compounds and
the diols, this disclosure of European Patent Application 43,738 is
also incorporated herein by reference.
[0246] A binary system for enhancing metoclopramide penetration is
disclosed in UK Patent Application GB 2,153,223 A, published Aug.
21, 1985, and consists 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,
N-methylpyrrolidone and the like.
[0247] 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 as
enhancing the transdermal delivery of steroids such as progesterons
and estrogens. A dual enhancer consisting of glycerol monolaurate
and ethanol for the transdermal delivery of drugs is shown 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 shows penetration-enhancing compositions for topical
application comprising 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.
[0248] Other chemical enhancers, not necessarily associated with
binary systems include DMSO or aqueous solutions of DMSO such as
taught in Herschler, U.S. Pat. No. 3,551,554; Herschler, U.S. Pat.
No. 3,711,602; and Herschler, U.S. Pat. No. 3,711,606, and the
azones (n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in
Cooper, U.S. Pat. No. 4,557,943.
[0249] Some chemical enhancer systems may possess negative side
effects such as toxicity and skin irritation. 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.
[0250] Because the combination of microporation of the stratum
corneum and the application of sonic energy accompanied by the use
of chemical enhancers can result in an improved rate of analyte
withdrawal or permeant delivery through the stratum corneum, the
specific carrier vehicle and particularly the chemical enhancer
utilized can be selected from a long list of prior art vehicles
some of which are mentioned above and incorporated herein by
reference. To specifically detail or enumerate that which is
readily available in the art is not thought necessary. The
invention is not drawn to the use of chemical enhancers per se and
it is believed that all chemical enhancers, useful in the delivery
of drugs through the skin, will function with dyes in optical
microporation and also with sonic energy in effecting measurable
withdrawal of analytes from beneath and through the skin surface or
the delivery of permeants or drugs through the skin surface.
Example 16
[0251] Modulated sonic energy and chemical enhancers were tested
for their ability to control transdermal flux on human cadaver skin
samples. In these tests, the epidermal membrane had been separated
from the human cadaver whole skin by the heat-separation method of
Example 1. The epidermal membrane was cut and placed between two
halves of the permeation cell with the stratum corneum facing
either the upper (donor) compartment or lower (receiver)
compartment. Modified Franz cells were used to hold the epidermis,
as shown in FIG. 2 of U.S. Pat. No. 5,445,611. Each Franz cell
consists of an upper chamber and a lower chamber held together with
one or more clamps. The lower chamber has a sampling port
through-which materials can be added or removed. A sample of
stratum corneum is held between the upper and lower chambers when
they are clamped together. The upper chamber of each Franz cell is
modified to allow an ultrasound transducer to be positioned within
1 cm of the stratum corneum membrane. Methylene blue solution was
used as an indicator molecule to assess the permeation of the
stratum corneum. A visual record of the process and results of each
experiment was obtained in a time stamped magnetic tape format with
a video camera and video cassette recorder (not shown).
Additionally, samples were withdrawn for measurement with an
absorption spectrometer to quantitate the amount of dye which had
traversed the stratum corneum membrane during an experiment.
Chemical enhancers suitable for use could vary over a wide range of
solvents and/or cell envelope disordering compounds as noted above.
The specific enhancer utilized was: ethanol/glycerol/water/glycerol
monooleate/methyl laurate in 50/30/15/2.5/2.5 volume ratios. The
system for producing and controlling the sonic energy included a
programmable 0-30 MHz arbitrary waveform generator (Stanford
Research Systems Model DS345), a 20 watt 0-30 MHz amplifier, and
two unfocused ultrasound immersion transducers having peak
resonances at 15 and 25 MHz, respectively. Six cells were prepared
simultaneously for testing of stratum corneum samples from the same
donor. Once the stratum corneum samples were installed, they were
allowed to hydrate with distilled water for at least 6 hours before
any tests were done.
Example 17
[0252] Effects of Sonic Energy without Chemical Enhancers
[0253] As stated above in Example 16, the heat-separated epidermis
was placed in the Franz cells with the epidermal side facing up,
and the stratum corneum side facing down, unless noted otherwise.
The lower chambers were filled with distilled water, whereas the
upper chambers were filled with concentrated methylene blue
solution in distilled water.
[0254] Heat Separated Epidermis: Immediately after filling the
upper chambers with methylene blue solution, sonic energy was
applied to one of the cells with the transducer fully immersed.
This orientation would correspond, for example, to having the
transducer on the opposite side of a fold of skin, or causing the
sonic energy to be reflected off a reflector plate similarly
positioned and being used to "push" analyte out of the other side
of the fold into a collection device. The sonic energy setting was
initially set at the nominal operating frequency of 25 MHz with an
intensity equivalent to a 20 volt peak-to-peak (P-P) input wave
form. This corresponds to roughly a 1 watt of average input power
to the transducer and similarly, assuming the manufacturer's
nominal value for conversion. efficiency of 1% for this particular
transducer, a sonic output power of around 0.01 watts over the 0.78
cm.sup.2 surface of the active area or a sonic intensity of 0.13
watts/cm.sup.2. Three other control cells had no sonic energy
applied to them. After 5 minutes the sonic energy was turned off.
No visual indication of dye flux across the stratum corneum was
observed during this interval in any of the cells, indicating
levels less than approximately 0.0015% (v/v) of dye solution in 2
ml of receiver medium.
[0255] Testing of these same 3 control cells and 1 experimental
cell was continued as follows. The intensity of sonic energy was
increased to the maximum possible output available from the driving
equipment of a 70 volt peak-to-peak input 12 watts average power
input or (0.13 watts/cm.sup.2) of sonic output intensity. Also, the
frequency was set to modulate or sweep from 30 MHz to 10 MHz. This
20 MHz sweep was performed ten times per second, i.e., a sweep rate
of 10 Hz. At these input power levels, it was necessary to monitor
the sonic energy transducer to avoid overheating. A contact
thermocouple was applied to the body of the transducer and power
was cycled on and off to maintain maximum temperature of the
transducer under 42 C. After about 30 minutes of cycling maximum
power at about a 50% duty cycle of 1 minute on and 1 minute off,
there was still no visually detectable permeation of the stratum
corneum by the methylene blue dye.
[0256] A cooling water jacket was then attached to the sonic energy
transducer to permit extended excitation at the maximum energy
level. Using the same 3 controls and 1 experimental cell, sonic
energy was applied at maximum power for 12 hours to the
experimental cell. During this time the temperature of the fluid in
the upper chamber rose to only 35 C, only slightly above the
approximately 31.degree. C. normal temperature of the stratum
corneum in vivo. No visual evidence of dye flux through the stratum
corneum was apparent in any of the four cells after 12 hrs. of
sonic energy applied as described above.
Example 18
[0257] Effects of Sonic Energy without Chemical Enhancers
[0258] Perforated Stratum Corneum: Six cells were prepared as
described above in Example 16. The clamps holding the upper and
lower chambers of the Franz cells were tightened greater than the
extent required to normally seal the upper compartment from the
lower compartment, and to the extent to artificially introduce
perforations and "pinholes" into the heat-separated epidermal
samples. When dye solution was added to the upper chamber of each
cell, there were immediate visual indications of leakage of dye
into the lower chambers through the perforations formed in the
stratum corneum. Upon application of sonic energy to cells in which
the stratum corneum was so perforated with small "pinholes," a
rapid increase in the transport of fluid through a pinhole in the
stratum corneum was observed. The rate of transport of the
indicator dye molecules was directly related to whether the sonic
energy was applied or not. That is, application of the sonic energy
caused an immediate (lag time approximately <0.1 second) pulse
of the indicator molecules through the pinholes in the stratum
corneum. This pulse of indicator molecules ceased immediately upon
turning off of the sonic energy (a shutoff lag of approximately
<0.1 second). The pulse could be repeated as described.
Example 19
Effects of Sonic Energy and Chemical Enhancers
[0259] Two different chemical enhancer formulations were used.
Chemical Enhancer One or CE1 was an admixture of
ethanol/glycerol/water/glycerol monooleate/methyl laurate in a
50/30/15/2.5/2.5 volume ratio. These are components generally
regarded as safe, i.e. GRAS, by the FDA for use as pharmaceutical
excipients. Chemical Enhancer Two or CE2 is an experimental
formulation shown to be very effective in enhancing transdermal
drug delivery, but generally considered too irritating for long
term transdermal delivery applications. CE2 contained
ethanol/glycerol/water/lauradone/methyl laurate in the volume
ratios 50/30/15/2.5/2.5. Lauradone is the lauryl(dodecyl)ester of
2-pyrrolidone-5-carboxylic acid ("PCA") and is also referred to as
lauryl PCA.
[0260] Six Franz cells were set up as before (Example 16) except
that the heat separated epidermis was installed with the epidermal
layer down, i.e., stratum corneum side facing up. Hydration was
established by exposing each sample to distilled water overnight.
To begin the experiment, the distilled water in the lower chambers
was replaced with methylene blue dye solution in all six cells. The
upper chambers were filled with distilled water and the cells were
observed for about 30 minutes confirming no passage of dye to
ensure that no pinhole perforations were present in any of the
cells. When none were found, the distilled water in the upper
chambers was removed from four of the cells. The other two cells
served as distilled water controls. The upper chambers of two of
the experimental cells were then filled with CE1 and the other two
experimental cells were filled with CE2.
[0261] Sonic energy was immediately applied to one of the two CE2
cells. A 25 MHz transducer was used with the frequency sweeping
every 0.1 second from 10 MHz to 30 MHz at maximum intensity of 0.13
watts/cm.sup.2. After 10-15 minutes of sonic energy applied at a
50% duty cycle, dye flux was visually detected. No dye flux was
detected in the other five cells.
[0262] Sonic energy was then applied to one of the two cells
containing CE1 at the same settings. Dye began to appear in the
upper chamber within 5 minutes. Thus, sonic energy together with a
chemical enhancer significantly increased the transdermal flux rate
of a marker dye through the stratum corneum, as well as reduced the
lag time.
Example 20
Effects of Sonic Energy and Chemical Enhancers
[0263] Formulations of the two chemical enhancers, CE1 and CE2,
were prepared minus the glycerin and these new formulations,
designated CE1MG and CE2MG, were tested as before. Water was
substituted for glycerin so that the proportions of the other
components remained unchanged. Three cells were prepared in
modified Franz cells with the epidermal side of the heat separated
epidermis samples facing toward the upper side of the chambers.
These samples were then hydrated in distilled water for 8 hours.
After the hydration step, the distilled water in the lower chambers
was replaced with either CE1MG or CE2MG and the upper chamber was
filled with the dye solution. Sonic energy was applied to each of
the three cells sequentially.
[0264] Upon application of pulsed, frequency-modulated sonic energy
for a total duration of less than 10 minutes, a significant
increase in permeability of the stratum corneum samples was
observed. The permeability of the stratum corneum was altered
relatively uniformly across the area exposed to both the chemical
enhancer and sonic energy. No "pinhole" perforations through which
the dye could traverse the stratum corneum were observed. The
transdermal flux rate was instantly controllable by turning the
sonic energy on or off. Turning the sonic energy off appeared to
instantly reduce the transdermal flux rate such that no dye was
visibly being actively transported through the skin sample;
presumably the rate was reduced to that of passive diffusion.
Turning the sonic energy on again instantly resumed the high level
flux rate. The modulated mode appeared to provide a regular
pulsatile increase in the transdermal flux rate at the modulated
rate. When the sonic energy was set to a constant frequency, the
maximum increase in transdermal flux rate for this configuration
seemed to occur at around 27 MHz.
[0265] Having obtained the same results with all three samples, the
cells were then drained of all fluids and flushed with distilled
water on both sides of the stratum corneum. The lower chambers were
then immediately filled with distilled water and the upper chambers
were refilled with dye solution. The cells were observed for 30
minutes. No holes in the stratum corneum samples were observed and
no large amount of dye was detected in the lower chambers. A small
amount of dye became visible in the lower chambers, probably due to
the dye and enhancer trapped in the skin samples from their
previous exposures. After an additional 12 hours, the amount of dye
detected was still very small.
Example 21
Effects of Sonic Energy and Chemical Enhancers
[0266] Perforated Stratum Corneum: Three cells were prepared with
heat-separated epidermis samples with the epidermal side facing
toward the upper side of the chamber from the same donor as in
Example 16. The samples were hydrated for 8 hours and then the
distilled water in the lower chambers was replaced with either
CE1MG or CE2MG. The upper chambers were then filled with dye
solution. Pinhole perforations in the stratum corneum samples
permitted dye to leak through the stratum corneum samples into the
underlying enhancer containing chambers. Sonic energy was applied.
Immediately upon application of the sonic energy, the dye molecules
were rapidly pushed through the pores. As shown above, the rapid
flux of the dye through the pores was directly and immediately
correlated with the application of the sonic energy.
Example 22
Effects of Sonic Energy and Chemical Enhancers
[0267] A low cost sonic energy transducer, TDK #NB-58S-01 (TDK
Corp.), was tested for its capability to enhance transdermal flux
rates. The peak response of this transducer was determined to be
about 5.4 MHz with other local peaks occurring at about 7 MHz, 9
MHz, 12.4 MHz, and 16 MHz.
[0268] This TDK transducer was then tested at 5.4 MHz for its
ability to enhance transdermal flux rate in conjunction with CE1MG.
Three cells were set up with the epidermal side facing the lower
chamber, then the skin samples were hydrated for 8 hrs. The dye
solution was placed in the lower chamber. The transducer was placed
in the upper chamber immersed in CE1MG. Using swept frequencies
from 5.3 to 5.6 MHz as the sonic energy excitation, significant
quantities of dye moved through the stratum corneum and were
detected in the collection well of the cell in 5 minutes. Local
heating occurred, with the transducer reaching a temperature of 48
C. In a control using CE1MG without sonic energy, a 24 hour
exposure yielded less dye in the collection well than the 5 minute
exposure with sonic energy.
[0269] This example demonstrates that a low cost, low frequency
sonic energy transducer can strikingly affect transdermal flux rate
when used in conjunction with an appropriate chemical enhancer.
Although higher frequency sonic energy will theoretically
concentrate more energy in the stratum corneum, when used with a
chemical enhancer, the lower frequency modulated sonic energy can
accelerate the transdermal flux rate to make the technology useful
and practical.
Example 23
[0270] Demonstration of molecule migration across human skin: Tests
with the TDK transducer and CE1MG described above were repeated at
about 12.4 MHz, one of the highest local resonant peaks for the
transducer, with a frequency sweep at a 2 Hz rate from 12.5 to 12.8
MHz and an sonic energy density less than 0.1 W/cm.sup.2. The
epidermal side of the heat-separated epidermis was facing down, the
dye solution was in the lower chamber, and the enhancer solution
and the sonic energy were placed in the upper chamber. Within 5
minutes a significant amount of dye had moved across the stratum
corneum into the collection well. Ohmic heating in the transducer
was significantly less than with the same transducer being driven
at 5.4 MHz, causing an increase in temperature of the chemical
enhancer to only about 33 C.
[0271] Even at these low efficiency levels, the results obtained
with CE1MG and sonic energy from the TDK transducer were remarkable
in the monitoring direction. FIGS. 3A and 3B of U.S. Pat. No.
5,445,611 show plots of data obtained from three separate cells
with the transdermal flux rate measured in the monitoring
direction. Even at the 5 minute time point, readily measurable
quantities of the dye were present in the chemical enhancer on the
outside of the stratum corneum, indicating transport from the
epidermal side through the stratum corneum to the "outside" area of
the skin sample.
[0272] To optimize the use of the sonic energy or the sonic
energy/chemical enhancer approach for collecting and monitoring
analytes from the body, means for assaying the amount of analyte of
interest are required. An assay system that takes multiple readings
while the unit is in the process of withdrawing analytes by sonic
energy with or without chemical enhancers makes it unnecessary to
standardize across a broad population base and normalize for
different skin characteristics and flux rates. By plotting two or
more data points in time as the analyte concentration in the
collection system is increasing, a curve-fitting algorithm can be
applied to determine the parameters describing the curve relating
analyte withdrawal or flux rate to the point at which equilibrium
is reached, thereby establishing the measure of the interval
concentration. The general form of this curve is invariant from one
individual to another; only the parameters change. Once these
parameters are established, solving for the steady state solution
(i.e., time equals infinity) of this function, i.e., when full
equilibrium is established, provides the concentration of the
analyte within the body. Thus, this approach permits measurements
to be made to the desired level of accuracy in the same amount of
time for all members of a population regardless of individual
variations in skin permeability.
[0273] Several existing detection techniques currently exist that
are adaptable for this application. See, D. A. Christensen, in 1648
Proceedings of Fiber Optic, Medical and Fluorescent Sensors and
Applications 223-26 (1992). One method involves the use of a pair
of optical fibers that are positioned close together in an
approximately parallel manner. One of the fibers is a source fiber,
through which light energy is conducted. The other fiber is a
detection fiber connected to a photosensitive diode. When light is
conducted through the source fiber, a portion of the light energy,
the evanescent wave, is present at the surface of the fiber and a
portion of this light energy is collected by the detection fiber.
The detection fiber conducts the captured evanescent wave energy to
the photosensitive diode which measures it. The fibers are treated
with a binder to attract and bind the analyte that is to be
measured. As analyte molecules bind to the surface (such as the
analyte glucose binding to immobilized lectins such as concanavalin
A, or to immobilized anti-glucose antibodies) the amount of
evanescent wave coupling between the two fibers is changed and the
amount of energy captured by the detection fiber and measured by
the diode is changed as well. Several measurements of detected
evanescent wave energy over short periods of time support a rapid
determination of the parameters describing the equilibrium curve,
thus making possible calculation of the concentration of the
analyte within the body. The experimental results showing
measurable flux within 5 minutes (FIGS. 3A and 3B of U.S. Pat. No.
5,445,611) with this system suggest sufficient data for an accurate
final reading are collected within 5 minutes.
[0274] In its most basic embodiment, a device that can be utilized
for the application of sonic energy and collection of analyte
comprises an absorbent pad, either of natural or synthetic
material, which serves as a reservoir for the chemical enhancer, if
used, and for receiving the analyte from the skin surface. The pad
or reservoir is held in place, either passively or aided by
appropriate fastening means, such as a strap or adhesive tape, on
the selected area of skin surface.
[0275] An sonic energy transducer is positioned such that the pad
or reservoir is between the skin surface and the transducer, and
held in place by appropriate means. A power supply is coupled to
the transducer and activated by switch means or any other suitable
mechanism. The transducer is activated to deliver sonic energy
modulated in frequency, phase or intensity, as desired, to deliver
the chemical enhancer, if used, from the reservoir through the skin
surface followed by collection of the analyte from the skin surface
into the reservoir. After the desired fixed or variable time
period, the transducer is deactivated. The pad or reservoir, now
containing the analyte of interest, can be removed to quantitate
the analyte, for example, by a laboratory utilizing any number of
conventional chemical analyses, or by a portable device.
Alternately, the mechanism for quantitating the analyte can be
build into the device used for collection of the analyte, either as
an integral portion of the device or as an attachment. Devices for
monitoring an analyte are described in U.S. Pat. No. 5,458,140,
which is incorporated herein by reference.
Example 24
[0276] An alternate method for detection of an analyte, such as
glucose, following the sample collection through the porated skin
surface as described above, can be achieved through the use of
enzymatic means. Several enzymatic methods exist for the
measurement of glucose in a biological sample. One method involves
oxidizing glucose in the sample with glucose oxidase to generate
gluconolactone and hydrogen peroxide. In the presence of a
colorless chromogen, the hydrogen peroxide is then converted by
peroxidase to water and a colored product.
[0277] Glucose Oxidase
[0278] Glucose Gluconolactone+H.sub.2O.sub.2
[0279] 2H.sub.20.sub.2+chromogen H.sub.2O+colored product
The intensity of the colored product will be proportional to the
amount of glucose in the fluid. This color can be determined
through the use of conventional absorbance or reflectance methods.
By calibration with known concentrations of glucose, the amount of
color can be used to determine the concentration of glucose in the
collected analyte. By testing to determine the relationship, one
can calculate the concentration of glucose in the blood of the
subject. This information can then be used in the same way that the
information obtained from a blood glucose test from a finger
puncture is used. Results can be available within five to ten
minutes.
Example 25
[0280] Any system using a visual display or readout of glucose
concentration will indicate to a diagnostician or patient the need
for administration of insulin or other appropriate medication. In
critical care or other situations where constant monitoring is
desired and corrective action needs to be taken almost
concurrently, the display may be connected with appropriate signal
means which triggers the administration of insulin or other
medication in an appropriate manner. For example, there are insulin
pumps which are implanted into the peritoneum or other body cavity
which can be activated in response to external or internal stimuli.
Alternatively, utilizing the enhanced transdermal flux rates
possible with micro-poration of the stratum corneum and other
techniques described in this invention, an insulin delivery system
could be implemented transdermally, with control of the flux rates
modulated by the signal from the glucose sensing system. In this
manner a complete biomedical control system can be available which
not only monitors and/or diagnoses a medical need but
simultaneously provides corrective action.
[0281] Biomedical control systems of a similar nature could be
provided in other situations such as maintaining correct
electrolyte balances or administering analgesics in response to a
measured analyte parameter such as prostaglandins.
Example 26
[0282] Similar to audible sound, sonic waves can undergo
reflection, refraction, and absorption when they encounter another
medium with dissimilar properties [D. Bommannan et al., 9 Pharm.
Res. 559 (1992)]. Reflectors or lenses may be used to focus or
otherwise control the distribution of sonic energy in a tissue of
interest. For many locations on the human body, a fold of flesh can
be found to support this system. For example, an earlobe is a
convenient location which would allow use of a reflector or lens to
assist in exerting directional control (e.g., "pushing" of analytes
or permeants through the porated stratum corneum) similar to what
is realized by changing sonic frequency and intensity.
Example 27
[0283] Multiple sonic energy transducers may be used to selectively
direct the direction of transdermal flux through porated stratum
corneum either into the body or from the body. A fold of skin such
as an earlobe allow transducers to be located on either side of the
fold. The transducers may be energized selectively or in a phased
fashion to enhance transdermal flux in the desired direction. An
array of transducers or an acoustic circuit may be constructed to
use phased array concepts, similar to those developed for radar and
microwave communications systems, to direct and focus the sonic
energy into the area of interest.
Example 28
[0284] In this example, the procedure of Example 19 is followed
with the exception that the heat-separated epidermis samples are
first treated with an excimer laser (e.g. model EMG/200 of Lambda
Physik; 193 nm wavelength, 14 ns pulse width) to ablate the stratum
corneum according to the procedure described in U.S. Pat. No.
4,775,361, hereby incorporated by reference.
Example 29
[0285] In this example, the procedure of Example 19 is followed
with the exception that the heat-separated epidermis samples are
first treated with 1,1'-diethyl-4,4'-carbocyanine iodide (Aldrich,
.sub.max=703 nm) and then a total of 70 mJ/cm.sup.2/50 ms is
delivered to the dye-treated sample with a Model TOLD9150 diode
laser (Toshiba America Electronic, 30 mW at 690 nm) to ablate the
stratum corneum.
Example 30
[0286] In this example, the procedure of Example 29 is followed
with the exception that the dye is indocyanine green (Sigma cat.
no. I-2633; .sub.max=775 nm) and the laser is a model Diolite
800-50 (LiCONiX, 50 mW at 780 nm).
Example 31
[0287] In this example, the procedure of Example 29 is followed
with the exception that the dye is methylene blue and the laser is
a model SDL-8630 (SDL Inc.; 500 mW at 670 nm).
Example 32
[0288] In this example, the procedure of Example 29 is followed
with the exception that the dye is contained in a solution
comprising a permeation enhancer, e.g. CE1.
Example 33
[0289] In this example, the procedure of Example 29 is followed
with the exception that the dye and enhancer-containing solution
are delivered to the stratum corneum with the aid of exposure to
ultrasound.
Example 34
[0290] In this example, the procedure of Example 31 is followed
with the exception that the pulsed light source is a short arc lamp
emitting over the broad range of 400 to 1100 nm but having a
bandpass filter placed in the system to limit the output to the
wavelength region of about 650 to 700 nm.
Example 35
[0291] In this example, the procedure of Example 19 is followed
with the exception that the heat-separated epidermis samples are
first punctured with a microlancet (Becton Dickinson) calibrated to
produce a micropore in the stratum corneum without reaching the
underlying tissue.
Example 36
[0292] In this example, the procedure of Example 19 is followed
with the exception that the heat-separated epidermis samples are
first treated with focused sonic energy in the range of 70-480
mJ/cm.sup.2/50 ms to ablate the stratum corneum.
Example 37
[0293] In this example, the procedure of Example 19 is followed
with the exception that the stratum corneum is first punctured
hydraulically with a high pressure jet of fluid to form a micropore
of up to about 100 .mu.m diameter.
Example 38
[0294] In this example, the procedure of Example 19 is followed
with the exception that the stratum corneum is first punctured with
short pulses of electricity to form a micropore of up to about 100
.mu.m diameter.
Example 39
Acoustic Streaming
[0295] A new mechanism and application of sonic energy in the
delivering of therapeutic substances into the body and/or
harvesting fluids from within the body into an external reservoir
through micro-porations formed in the biological membrane will now
be described. An additional aspect of this invention is the
utilization of sonic energy to create an acoustic streaming effect
on the fluids flowing around and between the intact cells in the
viable tissues beneath the outer layer of an organism, such as the
epidermis and dermis of the human skin. Acoustic streaming is a
well documented mode by which sonic energy can interact with a
fluid medium. Nyborg, Physical Acoustics Principles and Methods, p.
265-331, Vol II-Part B, Academic Press, 1965. The first theoretical
analysis of acoustic streaming phenomenon was given by Rayleigh
(1884, 1945). In an extensive treatment of the subject,
Longuet-Higgins (1953-1960) has given a result applicable to two
dimensional flow that results in the near vicinity of any vibrating
cylindrical surface. A three dimensional approximation for an
arbitrary surface was developed by Nyborg (1958). As described by
Fairbanks et al., 1975 Ultrasonics Symposium Proceedings, IEEE Cat.
#75, CHO 994-4SU, sonic energy, and the acoustic streaming
phenomenon can be of great utility in accelerating the flux of a
fluid through a porous medium, showing measurable increases in the
flux rates by up to 50 times that possible passively or with only
pressure gradients being applied.
[0296] All previous transdermal delivery or extraction efforts
utilizing ultrasound have focused on methods of interaction between
the sonic energy and the skin tissues designed to permeabilize the
stratum corneum layer. The exact mode of interaction involved has
been hypothesized to be due exclusively to the local elevation of
the temperature in the SC layer, and the resultant melting of the
lipid domains in the intercellular spaces between the corneocytes.
Srinivasan et al. Other researchers have suggested that
micro-cavitations and or shearing of the structures in the stratum
corneum opens up channels through which fluids may flow more
readily. In general, the design of the sonic systems for the
enhancement of transdermal flux rates has been based on the early
realization that the application of an existing therapeutic
ultrasound unit designed to produce a "deep-heating" effect on the
subject, when used in conjunction with a topical application of a
gelled or liquid preparation containing the drug to be delivered
into the body, could produce a quantifiable increase in the flux
rate of the drug into the body. In the context of the method taught
herein to create micropores in this biological membrane, the use of
sonic energy may now be thought of in a totally new and different
sense than the classically defined concepts of sonophoresis.
[0297] Based on the experimental discovery mentioned in U.S. Pat.
Nos. 5,458,440 and 5,445,611 that when a small hole existed or was
created in the stratum corneum (SC) in the Franz cells used in the
in vitro studies, that the application of an appropriately driven
ultrasonic transducer to the fluid reservoir on either side of the
porated SC sample, an "acoustic streaming" event could be generated
wherein large flux rates of fluid where capable of being pumped
through this porated membrane.
[0298] With the method taught herein to create the controlled
micro-porations in the biological membrane in the organism, the
application of the fluid streaming mode of sonic/fluid interaction
to the induction of fluid into or out of the organism may now be
practically explored. For example, clinical studies have shown that
by making a series of four 80 .mu.m diameter micropores in a 400
.mu.m square, and then applying a mild (10 to 12 inches of Hg)
suction to this area, an average of about 1 .mu.l of interstitial
fluid can be induced to leave the body for external collection in
an external chamber. By adding a small, low power sonic transducer
to this system, configured such that it actively generates inwardly
converging concentric circular pressure waves in the 2 to 6 mm of
tissue surrounding the poration site, it has been demonstrated that
this ISF flux rate can be increased by 50%.
[0299] By relieving ourselves of the desire to create some form of
direct absorption of sonic energy in the skin tissues (as required
to generate heating), frequencies of sonic energy can be determined
for which the skin tissues are virtually transparent, that is at
the very low frequency region of 1 kHz to 500 KHz. Even at some of
the lowest frequencies tested, significant acoustic streaming
effects could be observed by using a micro-scope to watch an in
vivo test wherein the subject's skin was micro-porated and ISF was
induced to exit the body an pool on the surface of the skin.
Energizing the sonic transducer showed dramatic visual indications
of the amount of acoustic streaming as small pieces of particulate
matter were carried along with the ISF as it swirled about. Typical
magnitude of motion exhibited can be described as follows: for a 3
mm diameter circular pool of ISF on the surface of the skin, a
single visual particle could be seen to be completing roughly 3
complete orbits per second. This equates to a linear fluid velocity
of more than 2.5 mm/second. All of this action was demonstrated
with sonic power levels into the tissues of less than 100
mW/cm2.
[0300] While one can easily view the top surface of the skin, and
the fluidic activity thereon, assessing what is taking place
dynamically within the skin tissue layers in response to the
coupling into these tissues of sonic energy is much more difficult.
One can assume, that if such large fluid velocities (e.g. >2.5
mm/S) may be so easily induced on the surface, then some noticeable
increase in the fluid flow in the intercellular channels present in
the viable dermal tissues could also be realized in response to
this sonic energy input. Currently, an increase in harvested ISF
through a given set of microporations when a low frequency sonic
energy was applied to the area in a circle surrounding the poration
sites has been quantified. In this experiment, an ISF harvesting
technique based solely on a mild suction (10 to 12 inches of HG)
was alternated with using the exact same apparatus, but with the
sonic transducer engaged. Over a series of 10 two-minute harvesting
periods, five with mere suction and five with both suction and
sonic energy active, it was observed that by activating the sonic
source roughly 50% more ISF was collectable in the same time
period. These data are shown in FIG. 30. This increase in ISF flux
rate was realized with no reported increase in sensation from the
test subject due to the sonic energy. The apparatus used for this
experiment is illustrated in FIGS. 31-33. The transducer assembly
in FIGS. 31-33 is comprised of a thick walled cylinder of
piezo-electric material, with an internal diameter of roughly 8 mm
and a wall thickness of 4 mm. The cylinder has been polarized such
that when an electrical field is applied across the metallized
surfaces of the outer diameter and inner diameter, the thickness of
the wall of the cylinder expands or contracts in response to the
field polarity. In practice, this configuration results in a device
which rapidly squeezes the tissue which has been suctioned into the
central hole, causing an inward radial acoustic streaming effect on
those fluids present in these tissues. This inward acoustic
streaming is responsible for bringing more ISF to the location of
the micro-porations in the center of the hole, where it can leave
the body for external collection.
[0301] A similar device shown in FIG. 34A-B was built and tested
and produced similar initial results. In the FIG. 34A-B version, an
ultrasonic transducer built by Zevex, Inc. Salt Lake City, Utah,
was modified by having a spatulate extension added to the sonic
horn. A 4 mm hole was placed in the 0.5 mm thick spatulate end of
this extension. When activated, the principle motion is
longitudinal along the length of the spatula, resulting in
essentially a rapid back and forth motion. The physical
perturbation of the metallic spatula caused by the placement of the
4 mm hole, results in a very active, but chaotic, large
displacement behavior at this point. In use, the skin of the
subject was suctioned up into this hole, and the sonic energy was
then conducted into the skin in a fashion similar to that
illustrated in FIG. 33.
[0302] The novel aspect of this new application of ultrasound lies
in the following basic areas:
[0303] 1. The function of the sonic energy is no longer needed to
be focused on permeabilizing the SC barrier membrane as taught by
Langer, Kost, Bommannan and others.
[0304] 2. A much lower frequency system can be utilized which has
very little absorption in the skin tissues, yet can still create
the fluidic streaming phenomenon desired within the intercellular
passageways between the epidermal cells which contain the
interstitial fluid.
[0305] 3. The mode of interaction with the tissues and fluids
therein, is the so-called "streaming" mode, recognized in the sonic
literature as a unique and different mode than the classical
vibrational interactions capable of shearing cell membranes and
accelerating the passive diffusion process.
[0306] By optimizing the geometric configuration, frequency, power
and modulations applied to the sonic transducer, it has been shown
that significant increases in the fluid flux through the porated
skin sites can be achieved. The optimization of these parameters is
designed to exploit the non-linearities governing the fluid flow
relationships in this microscopically scaled environment. Using
frequencies under 200 kHz, large fluidic effects can be observed,
without any detectable heating or other negative tissue
interactions. The sonic power levels required to produce these
measurable effects are very low, with average power levels
typically under 100 milliwatts/cm2.
[0307] Therefore, the above examples are but representative of
systems which may be employed in the utilization of sonic energy or
sonic energy and chemical enhancers in the collection and
quantification of analytes for diagnostic purposes and for the
transmembrane delivery of permeants. The invention is directed to
the discovery that the poration of the biological membrane followed
by the proper use of sonic energy, particularly when accompanied
with the use of chemical enhancers, enables the noninvasive or
minimally invasive transmembrane determination of analytes or
delivery of permeants. However, the invention is not limited only
to the specific illustrations. There are numerous poration
techniques and enhancer systems, some of which may function better
than another, for detection and withdrawn of certain analytes or
delivery of permeants through the stratum corneum. However, within
the guidelines presented herein, a certain amount of
experimentation to obtain optimal poration, enhancers, or optimal
time, intensity and frequency of applied sonic energy, as well as
modulation of frequency, amplitude and phase of applied sonic
energy can be readily carried out by those skilled in the art.
Therefore, the invention is limited in scope only by the following
claims and functional equivalents thereof.
Further Advancements and Improvements
[0308] Advancements and improvements to the microporation
techniques have been made, particularly suitable for, though not
limited to, delivery applications. One advancement is to porate,
using any one of the aforementioned microporation techniques, to a
selected depth into or through biological membranes, including the
skin, the mucous membrane, or plant outer layer, particularly for
delivery of a drug or bioactive agent into the body. Another
advancement is to deliver bioactive agents into the organism
through micropores formed in the biological membrane. Still another
advancement is to apply permeation enhancement measures before,
during, or after microporation, so as to increase the permeability
of layers within the microporated skin or mucosa when delivering
substances, such as drugs or bioactive agents, thereinto or
therethrough.
[0309] The micropore formed in the biological membrane may extend
to a selected depth. A micropore extending into the epidermis may
penetrate only the stratum corneum or selected depths into the
viable cell layer or underlying connective tissue layer. Similarly,
if formed in the mucous membrane, the micropore may penetrate only
the superficial part of the epithelial layer or selected depths
into the epithelial lining or underlying lamina propria and into
tissue beneath. The micropore depth in either case can extend
through the entire depth of the biological membrane.
[0310] As an example for microporating to a selected depth, if one
utilizes a heat probe which can continue to deliver sufficient
energy into or through the fully hydrated viable cell layers
beneath the stratum corneum, the poration process can continue into
the body to selected depths, penetrating through the epidermis, the
dermis, and into or through the subcutaneous layers below if
desired. The concern when a system is designed to create a
micropore extending some distance into or through the viable
tissues in the epidermis or dermis, or the epithelial lining or
lamina propria, is how to minimize damage to the adjacent tissue
and the sensation to the subject during the poration process.
[0311] Experimentally, we have shown that if the heat probe used is
a solid, electrically or optically heated element, with the active
heated probe tip physically defined to be no more than a few
hundred microns across and protruding up to a few millimeters from
the supporting base, that a single pulse, or multiple pulses of
current can deliver enough thermal energy into the tissue to allow
the ablation,to penetrate as deep as the physical design allows,
that is, until the support base limits the extent of the
penetration into or through the tissue. If the electrical and
thermal properties of said heat probe, when it is in contact with
the tissues, allow the energy pulse to modulate the temperature of
said probe rapidly enough, this type of deep tissue poration can be
accomplished with essentially no pain to the subject. Experiments
have shown that if the required amount of thermal energy is
delivered to the probe within less than roughly 20 milliseconds
(20-50 msec), that the procedure is painless. Conversely, if the
energy pulse must be extended beyond roughly 20 milliseconds (20-50
msec), the sensation to the subject increases rapidly and
non-linearly as the pulse width is extended.
[0312] Similarly, an electrically heated probe design which
supports this type of selected deep poration can be built by
bending a 50 to 150 micron diameter tungsten wire into a sharp
kink, forming a close to 180 degree bend with a minimal internal
radius at this point. This miniature `V` shaped piece of wire can
then be mounted such that this `V` extends some distance out from a
support piece which has copper electrodes deposited upon it. The
distance to which the wire extends out from the support will define
the maximum penetration distance into the tissue when the wire is
heated. Each end of the tungsten `V` will be attached to one of the
electrodes on the support carrier which in turn can be connected to
the current pulsing circuit. When the current is delivered to the
wire in an appropriately controlled fashion, the wire will rapidly
heat up to the desired temperature to effect the thermal ablation
process in a single pulse or in multiple pulses of current. By
monitoring the dynamic impedance of the probe and knowing the
coefficient of resistance versus temperature of the tungsten
element, closed loop control of the temperature of the contact
point can easily be established. Also, by dynamically, monitoring
the impedance through the body from the contact point of the probe
and a second electrode placed some distance away, the depth of the
pore can be determined based on the different impedance properties
of the tissue as one penetrates deeper into the body. Once the
impedance properties of a selected tissue of a selected organism
have been routinely determined, this parameter can be used to
determine the pore depth and can be used in a control system to
control pore depth.
[0313] Likewise, one embodiment of an optically heated probe design
which supports this type of selected depth poration can be built by
taking an optical fiber and placing on one end a tip comprised of a
solid cap or coating. A light source such as a laser diode will be
coupled into the other end of the fiber. The side of tip facing the
fiber must have a high enough absorption coefficient over the range
of wavelengths emitted by the light source that when the photons
reach the end of the fiber and strike this face, some of them will
be absorbed and subsequently cause the tip to heat up. The specific
design of this tip, fiber and source assembly may vary widely,
however fibers with gross diameters of 50 to 1000 microns across
are common place items today and sources emitting up to thousands
of watts of optical energy are similarly common place. The tip
forming the actual heat probe can be fabricated from a high melting
point material, such as tungsten and attached to the fiber by
machining it to allow the insertion of the fiber into a cylindrical
bore at the fiber end. If the distal end of the tip has been
fabricated to limit the thermal diffusion away from this tip and
back up the supporting cylinder attaching the tip to the fiber
within the time frame of the optical pulse widths used, the photons
incident upon this tip will elevate the temperature rapidly on both
the fiber side and the contact side which is placed against the
tissues surface. The positioning of the fiber/tip assembly onto the
tissue surface, can be accomplished with a simple mechanism
designed to hold the tip against the surface under some spring
tension such that as the tissue beneath it is ablated, the tip
itself will advance into the tissue. This allows the thermal
ablation process to continue into or through the tissue as far as
one desires. An additional feature of this optically heated probe
design is that by monitoring the black body radiated energy from
the heated tip that is collected by the fiber, a very simple closed
loop control of the tip temperature can be effected. Also, as
described earlier, by dynamically monitoring the impedance through
the body from the contact point of the probe and a second electrode
placed some distance away, the depth of the pore can be estimated
based on the different impedance properties of the tissue as one
penetrates deeper into the body. The relationship between pulse
width and sensation for this design is essentially the same as for
the electrically heated probe described earlier.
[0314] For example, some vaccine applications are known to be most
effective if delivered into the dermal layer so as to be in
proximity to the Langerhan's or dendritic cells or other cells
important for this immune response. This would imply a poration
depth designed to pass through the epidermis, which in most cases
would be roughly 180 microns to 250 microns deep.
[0315] As another example, when delivering some proteins and
peptides, it is desirable to minimize the immune response to the
permeant at the site of the administration and at the same time
bypass the protease active zones in the skin tissues. In this case
an even deeper pore may be desired, going as deep as 300 microns
into the skin.
[0316] Alternatively, it may be desirable to leave a minimally
thick layer of intact stratum corneum to minimize rapid initial
uptake of a permeant and to provide some retention of the stratum
corneum's barrier function to provide for a controlled release over
a longer period of time.
[0317] An additional feature of this invention is the large
increase in efficiency which can be gained by combining the
poration of the layers of the biological membrane with other
permeation enhancement techniques which can now be optimized to
function on the various barriers to effect delivery of the desired
compound into the internal spaces as necessary for bio-effectivity.
In particular, if one is delivering a nucleic acid compound either
naked, fragmented, encapsulated or coupled to another agent, it is
often desired to get the nucleic acid into the living cells without
killing the cell to allow the desired uptake and subsequent
performance of the therapy. The application of electroporation,
iontophoresis, magnetic fields and thermal and sonic energy can
cause openings to form, temporarily, in the cell membranes and
other internal tissues. Because we have shown how to breach the
stratum corneum or epithelial layer of the mucosal membrane or the
outer layer of a plant, and if desired the epidermis and dermis or
deeper into a plant, electroporation, iontophoresis, magnetic
fields and thermal and sonic energy can now be used with parameters
that can be tailored to act selectively on these underlying tissue
barriers and permeabilize the cell, capillary or other membranes
within the targeted tissue. Electroporation, iontophoresis,
magnetic fields, and thermal and sonic energy were previously
inapplicable for this use.
[0318] In the case of electroporation, where pulses exceeding 50 to
150 volts are routinely used to electroporate the stratum corneum
or outer layer of the mucosal membrane or outer layer of a plant,
in the environment we present, pulses of only a few volts or less
are sufficient to electroporate the cell, capillary or other
membranes within the targeted tissue. This is principally due to
the dramatic reduction in the number of insulating layers present
between the electrodes once the skin, mucosal layer, or outer layer
of a plant has been opened.
[0319] Similarly, iontophoresis can be shown to be effective to
modulate the flux of a fluid media containing the nucleic acid
through the micropores with very small amounts of current due to
the dramatic reduction in the physical impedance to fluid flow
through these porated layers.
[0320] In the case of sonic energy, whereas classically sonic
energy has been used to accelerate the permeation of the stratum
corneum or mucosal layer, by eliminating this barrier, sonic energy
can now be used to permeabilize the cell, capillary or other
membranes within the targeted tissue. As in the cases of
electroporation and iontophoresis, we have demonstrated that the
sonic energy levels needed to effect a notable improvement in the
transmembrane flux of a substance are much lower than when skin or
mucosal layers are left intact. Other permeation enhancement
measures involve changing the osmotic pressure or physical pressure
at the microporated site, for example applying a mild pneumatic
pressure to the permeant reservoir to force a particular fluid flow
into the organism through the micropores
[0321] The mode of operation of all of these active methods,
electroporation, iontophoresis, magnetic or thermal or sonic
energy, when applied solely or in combination, after the poration
of the skin or mucosal layer or the outer layer of a plant has been
effected, has the advantage of being able to use parameters
typically used in in vitro applications where single cell membranes
are opened up for the delivery of a substance. Examples of these
parameters are well known in the literature. For example, Sambvrook
et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
[0322] The micropores produced in the biological membrane by the
methods of the present invention allow high flux rates of large (as
well as small) molecular weight therapeutic compounds to be
delivered transdermally or transmucosally or transmembrane. In
addition, these non-traumatic microscopic openings into the body
allow access to various analytes within the body, which can be
assayed to determine their internal concentrations.
Delivery of Bioactive Agents
[0323] Still another advancement of the present invention involves
the use of poration of the biological membrane for the delivery of
a bioactive agent, e.g., polypeptides, including proteins and
peptides (e.g., insulin); releasing factors; including LHRH;
carbohydrates (e.g., heparin); nucleic acids; vaccines; and
pharmacologically active agents such as antiinfectives such as
antibiotics and antiviral agents; analgesics and analgesic
combinations; anorexics; antihelminthics; antiarthritics;
antiasthmatic agents; anticonvulsants; antidepressants;
antidiabetic agents; antidiarrheals; antihistamines;
antiinflammatory agents; antimigraine preparations; antinauseants;
antineoplastics; antiparkinsonism drugs; antipruritics;
antipsychotics; antipyretics; antispasmodics; anticholinergics;
sympathomimetics; xanthine derivatives; cardiovascular preparations
including potassium and calcium channel blockers, beta-blockers,
alpha-blockers, and antiarrhythmics; antihypertensives; diuretics
and antidiuretics; vasodilators including general coronary,
peripheral and cerebral; central nervous system stimulants;
vasoconstrictors; cough and cold preparations, including
decongestants; hormones such as estradiol, testosterone,
progesterone and other steroids and derivatives and analogs,
including corticosteroids; hypnotics; immunosuppressives; muscle
relaxants; parasympatholytics; psychostimulants; sedatives; and
tranquilizers. By the method of the present invention, both ionized
and nonionized drugs may be delivered, as can drugs of either high,
medium or low molecular weight.
[0324] Delivery of DNA and/or RNA can be used to achieve expression
of a polypeptide, stimulate an immune response, or to inhibit
expression of a polypeptide through the use of an "antisense"
nucleic acid, especially an antisense RNA. The term "polypeptide"
is used herein without any particular intended size limitation,
unless a particular size is otherwise stated, and includes peptides
of any length including proteins. Typical of polypeptides that can
be expressed are those selected from the group consisting of
oxytocin, vasopressin, adrenocorticotrophic hormone, epidermal
growth factor, prolactin, luteinizing hormone releasing hormone,
growth hormone, growth hormone releasing factor, insulin-like
growth factors, insulin, erythropoietin, obesity protein such as
leptin, somatostatin, glucagon, glucagon-like insulinotropic
factors, parathyroid hormone, interferon, gastrin, interleukin-2
and other interleukins and lymphokines, tetragastrin, pentagastrin,
urogastroine, secretin, calcitonin, enkephalins, endorphins,
angiotensins, renin, bradykinin, bacitracins, polymixins,
colistins, tyrocidin, gramicidines, and synthetic analogues,
modifications and pharmacologically active fragments thereof,
monoclonal antibodies and vaccines. This group is not to be
considered limiting; the only limitation to the peptide or protein
drug that may be expressed is one of functionality. Delivery of DNA
and/or RNA is useful in gene therapy, vaccination, and any
therapeutic situation in which a nucleic acid or a polypeptide
should be administered in vivo. E.g., U.S. Pat. No. 5,580,859,
hereby incorporated by reference.
[0325] One illustrative embodiment of the invention is a method for
obtaining long term administration of a polypeptide comprising
porating the biological membrane and then delivering a DNA encoding
the polypeptide through the pores in the biological membrane,
whereby cells of the tissue take up the DNA and produce the
polypeptide for at least one month, and more preferably at least 6
months. Another illustrative embodiment of the invention is a
method for obtaining transitory expression of a polypeptide
comprising porating the biological membrane and then delivering an
RNA or DNA encoding the polypeptide through the pores of the
biological membrane, whereby cells of the tissue (e.g., the skin,
mucous membrane, capillaries, or underlying tissue) take up the RNA
or DNA and produce the polypeptide for less than about 20 days,
usually less than about 10 days, and often less than about 3-5
days. The cells which take up the RNA or DNA could include the
cells of the biological membrane, the underlying tissue or other
target tissue reached by way of the capillaries.
[0326] The DNA and/or RNA can be naked nucleic acid optionally in a
carrier or vehicle, and/or can be contained within microspheres,
liposomes and/or associated with transfection-facilitating
proteins, microparticles, lipid complexes, viral particles, charged
or neutral lipids, carbohydrates, calcium phosphate or other
precipitating agents, and/or other substances for stabilizing the
nucleic acid. The nucleic acid can be contained in a viral vector
that either integrates into the chromosome or is nonintegrating, in
a plasmid, or as a naked polynucleotide. The nucleic acid can
encode a polypeptide, or alternatively, can code for an antisense
RNA, for example for inhibiting translation of a selected
polypeptide in a cell. When the nucleic acid is DNA, it can be a
DNA sequence that is itself non-replicating, but is inserted into a
plasmid wherein the plasmid further comprises a replicator. The DNA
may also contain a transcriptional promoter, such as the CMV IEP
promoter, which is functional in humans. The DNA can also encode a
polymerase for transcribing the DNA. In one preferred embodiment,
the DNA codes for both a polypeptide and a polymerase for
transcribing the DNA. The DNA can be delivered together with the
polymerase or with mRNA coding therefor, which mRNA is translated
in the cell. In this embodiment, the DNA is preferably a plasmid,
and the polymerase is preferably a phage polymerase, such as the T7
polymerase, wherein the T7 polymerase gene should include a T7
promoter.
[0327] The method can be used to treat a disease associated with a
deficiency or absence or mutation of a specific polypeptide. In
accordance with another aspect of the invention, the method
provides for immunizing an individual, wherein such individual can
be a human or an animal, comprising delivering a DNA and/or RNA to
the individual wherein the DNA and/or RNA codes for an immunogenic
translation product that elicits an immune response against the
immunogen. The method can be used to elicit a humoral immune
response, a cellular immune response, or a mixture thereof.
Example 40
[0328] This illustrative example shows the preparation and delivery
of an mRNA.
[0329] In general, it should be apparent that, in practicing the
invention, a suitable plasmid for in vitro transcription of mRNA
can be readily constructed by those of ordinary skill in the art
with a virtually unlimited number of cDNAs. Such plasmids can
advantageously comprise a promoter for a selected RNA polymerase,
followed by a 5' untranslated region, a 3' untranslated region, and
a template for a polyadenylate tract. There should be a unique
restriction site between these 5' and 3' untranslated regions to
facilitate the insertion of any selected cDNA into the plasmid.
Then, after cloning the plasmid containing the selected gene, the
plasmid is linearized by digestion in the polyadenylation region
and is transcribed in vitro to form mRNA transcripts. These
transcripts are preferably provided with a 5' cap. Alternatively, a
5' untranslated sequence such as EMC can be used, which does not
require a 5' cap.
[0330] The readily available SP6 cloning vector, pSP64T, provides
5' and 3' flanking regions from the Xenopus-globin gene, an
efficiently translated mRNA. Any cDNA containing an initiation
codon can be introduced into this plasmid, and mRNA can be prepared
from the resulting template DNA. This particular plasmid can be
digested with BglII to insert any selected cDNA coding for a
polypeptide of interest. Although good results can be obtained with
pSP64T when linearized and then transcribed with SP6 RNA
polymerase, it is preferable to use the Xenopus-globin flanking
sequences of pSP64T with the phage T7 RNA polymerase. This is
accomplished by purifying an approximately 150 by HindIII/EcoRI
fragment from pSP64T and inserting it into a linearized
approximately 2.9 kb HindIII/EcoRI fragment of pIBI131
(commercially available from International Biotechnologies, Inc.,
New Haven, Conn.) with T4 ligase. The resulting plasmid, pXBG, is
adapted to receive any gene of interest at a unique BglII site
situated between the two Xenopus-globin sequences and for
transcription of the selected gene with T7 polymerase.
[0331] A convenient marker gene for demonstrating in vivo
expression of exogenous polynucleotides is chloramphenicol
acetyltransferase, CAT. The CAT gene from the small BamHI/HindIII
fragment of pSV2-CAT (ATCC No. 37155) and the BglII-digested pXBG
are both incubated with the Klenow fragment of E. coli DNA
polymerase to generate blunt ends, and then are ligated with T4 DNA
ligase to form pSP-CAT. This plasmid is then digested with PstI and
HindIII and the small fragment, comprising the CAT gene between the
5' and 3'-globin flanking sequences of pSP64T. The T7
promoter-containing plasmid pIBI131 is also digested with PstI and
HindIII, and the long fragment is purified. This fragment is then
ligated to the CAT gene containing fragment with T4 DNA ligase to
form the plasmid pT7CAT-An.
[0332] The pT7CAT-An plasmid DNA is purified according to methods
well known in the art, e.g. U.S. Pat. No. 5,580,859. The resulting
purified plasmid DNA is then linearized downstream of the
polyadenylate region with an excess of PstI, and the resulting
linearized DNA is then purified and transcribed in vitro according
to the method of Example 5 of U.S. Pat. No. 5,580,859. The
resulting mRNA is then purified according to the method of Example
5 of U.S. Pat. No. 5,580,859, which is sufficiently pure for
delivery according the present invention.
[0333] The purified mRNA is delivered by porating a selected site
on an individual according to the microporation procedures with
selected pore depth which optimizes bioactivity and delivering an
effective amount of mRNA to such site such that the mRNA passes
through the skin or mucous membrane into the underlying tissue,
where the mRNA is taken up by the cells. This delivery through the
porated stratum corneum or mucous membrane can be aided with sonic
energy and/or use sonic energy according to the procedure of
Example 15 and/or with electroporation to enhance cellular uptake,
and/or with a pressure differential for inducing flux through the
pores in the skin or mucous membrane. Moreover, delivery can be
aided by placing the mRNA is a carrier solution, such as a
positively charged lipid complex or liposome, for enhancing the
diffusion of the mRNA through the pores into the body or for
facilitating uptake of the mRNA into cells.
Example 41
[0334] This example shows immunization of an individual with mRNA
encoding the gp120 protein of HIV. The mRNA is prepared according
to the procedure of Example 40 except the gene for gp120
(pIIIenv3-1 from the AIDS Research and Reagent Program, National
Institute of Allergy and Infectious Disease, Rockville, Md.) is
inserted into the plasmid pXBG of Example 40. The mRNA containing
the gp120 gene is delivered according to the procedure of Example
40.
Example 42
[0335] This example shows immunization of an individual with DNA
encoding the gp120 protein of HIV. The gp120 gene is inserted into
a recombinant adenovirus according to the procedure of P. Muzzin et
al., Correction of Obesity and Diabetes in Genetically Obese Mice
by Leptin Gene Therapy, 93 Proc. Nat'l Acad. Sci. USA 14804-14808
(1996); G. Chen et al., Disappearance of Body Fat in Normal Rats
Induced by Adenovirus-mediated Leptin Gene Therapy, 93 Proc. Nat'l
Acad. Sci. USA 14795-99 (1996), hereby incorporated by reference.
The resulting DNA is delivered according to the procedure of
Example 41.
Example 43
[0336] In this example, the procedure of Example 42 is followed
except that DNA encoding glycoprotein D of HSV-2 is substituted for
the DNA encoding gp120 protein and additionally is combined with an
effective amount of the glycoprotein D.
Example 44
[0337] In this example, a nucleic acid encoding the obesity protein
leptin, such as a human leptin or a rat leptin cDNA, C. Guoxun et
al., Disappearance of Body Fat in Normal Rats Induced by
Adenovirus-mediated Leptin, 93 Proc. Nat'l Acad. Sci. USA 14795-99
(1996), or a mouse leptin cDNA, P. Muzzin et al., Correction of
Obesity and Diabetes in Genetically Obese Mice by Leptin Gene
Therapy, 93 Proc. Nat'l Acad. Sci. USA 14804-14808 (1996), both of
which are hereby incorporated by reference, is delivered in an
appropriate plasmid vector. The mammalian expression vector,
pEUK-C1 (Clontech, Palo Alto, Calif.) is designed for transient
expression of cloned genes. This vector is a 4.9 kb plasmid
comprising a pBR322 origin of replication and an ampicillin
resistance marker for propagation in bacteria, and also comprising
the SV40 origin of replication, SV40 late promoter, and SV40 late
polyadenylation signal for replication and expression of a selected
gene in a mammalian cell. Located between the SV40 late promoter
and SV40 late polyadenylation signal is a multiple cloning site
(MCS) of unique XhoI, XbaI, SmaI, SacI, and BamHI restriction
sites. DNA fragments cloned into the MCS are transcribed into RNA
from the. SV40 late promoter and are translated from the first ATG
codon in the cloned fragments. Transcripts of cloned DNA are
spliced and polyadenylated using the SV40 VPI processing signals.
The leptin gene is cloned into the MCS of pEUK-C1 using techniques
well known in the art, e.g. J. Sambrook et al., Molecular Cloning:
A Laboratory Manual (2d ed., 1989), hereby incorporated by
reference. The resulting plasmid is delivered to a human or animal
individual after poration of the skin or mucosal membrane according
to the procedure described above in the previous examples.
Example 45
[0338] Delivery of Heparin. Heparins are useful therapeutic
substances wherein the maintenance of a basal level equivalent to
an intravenous infusion of roughly 1000 to 5000 IU per hour,
subcutaneous injections twice daily of 5000-1000 IU of heparin, or
1500-6000 IU of low molecular weight heparins is a typical clinical
dosage. Normally, heparin would not be considered a good candidate
for a transdermal delivery system because of its relatively high
resistance to crossing the skin due mainly to the molecular weight,
5000 to 30000 Da, of the substance. With the microporation
techniques disclosed herein, a significant flux rate of heparin was
easily achieved when a sufficient quantity of heparin, such as from
a delivery reservoir attached to the skin surface where the
micropores were placed, was administered. A heparin solution was
applied to skin porated to a depth of approximately 100 .mu.m,
allowing either passive diffusion or coupled with iontophoresis
(about 1 mA/cm.sup.2) that was applied for a sufficient period of
time to transport the heparin through the micropores into the
underlying tissues. Evidence of delivery of heparin was observed by
increased capillary dilation and permeability as evidenced by
microscopic examination of the in vivo site for both the passive
and iontophoretically enhanced delivery. In addition to showing a
significant heparin flux using passive diffusion as the main
driving force, heparin, being a highly charged compound, is a
natural candidate for the coupling of an electrical field with the
micropores to allow for an actively controllable flux rate and
higher flux rates than possible through the same number of
micropores than is possible with the passive diffusion method. An
experiment was conducted wherein a site on the volar forearm of a
healthy male volunteer was prepared by creating a matrix of 36
micropores within a 1 square cm area. A small reservoir containing
a sodium heparin solution and the negative electrode for an
iontophoretic system was attached to the site. The positive
electrode was attached to the subject's skin some distance away
using a hydrogel electrode obtained from Iomed, a commercial
supplier of iontophoretic systems. The system was run for ten
minutes at 0.2 milliamperes per square cm. After this period,
microscopic examination of the site showed direct evidence of the
delivery of heparin from the vasodilation of the capillaries and
when a suction force was applied to extract a sample of
interstitial fluid from the micropores, enough red blood cells
exited the capillaries under this force to tint the collected ISF
pink, indicating increased vaso-permeability in the area.
Furthermore, when placed aside to see if the red cells would clot,
no clotting took place, indicating the anticlotting effect of the
heparin present in the tissues at work.
Example 46
[0339] Delivery of Insulin: Insulin, like many compounds normally
present in the healthy individual, is a polypeptide which must be
maintained in individuals, such as diabetics who need exogenous
insulin, at both a basal level and be given in a pulsatile bolus
fashion in response to meals and the subject's activity levels.
Currently this is achieved via subcutaneous injections of fast
acting and slow acting formulations. Because of the molecular
weight of insulin, typically .about.6000, it is not able to be
delivered at clinically useful levels with traditional transdermal
or transmucosal methods. However, by opening the micropores through
the barrier layers of the skin or mucosa, a clear path is provided
allowing the delivery of the insulin into the viable tissues
wherein the interstitial fluid present in these tissues will allow
diffusion (including osmotically driven) of the insulin to and into
the lymph system and capillary bed, delivering clinically useful
amounts. A concentrated insulin solution containing 3500 IU/ml of
recombinant human insulin purchased from Boehringer-Mannhein Co.,
was applied in a reservoir to a crated area of the subject's skin
on the volar forearm covering 4 square cm. The healthy, 44 year
old, male, non-diabetic, subject fasted for 14 hours prior to the
start of the experiment. Intravenous and finger stick blood samples
were drawn periodically prior to and after the delivery phase began
and assayed for glucose, insulin and C-peptide. The finger stick
blood glucose data showed a significant and rapid depression of the
subject's glucose levels after approximately 4 hours, dropping from
100 mg/dl at the start to 67 mg/dl over a ten minute cycle and then
returning to 100 in an additional ten minutes, hypothesized to be
due to the subject's counter-regulatory system engaging and
compensating for the delivered insulin. A repeat of this procedure
with the addition of ultrasound operating at 44 khz, and 0.2
watts/square cm indicated a more rapid delivery of the insulin as
evidenced by the subject's glucose levels which dropped from 109
mg/dl to 78 mg/dl less than 30 minutes after the delivery began. As
in the case of example 45, for heparin delivery, a low current
iontophoretic system can be coupled with the micropores to
facilitate a greater flux rate and provide the ability to modulate
this flux rate by varying the current, allowing a delivery on
demand type of system to be built. Previous work with insulin has
typically shown that relatively high iontophoretic currents are
required to overcome the strong barrier properties of the intact
stratum corneum. By porating the stratum corneum or mucosa, and
optionally setting the poration parameters to make a deeper pore
into or through the targeted biological membrane, a lower current
density is required to produce the desired insulin flux rates.
[0340] Similarly, for uncharged or lower-charged insulin
formulations, an active flux enhancement through the micropores can
be effected by coupling a sonic field or sonophoresis, which may
include frequencies normally described as ultrasonic, to help push
the insulin into the tissues. An additional feature of the sonic
field is its ability to enhance the permeability of the various
barriers within the viable tissues letting the insulin reach a
larger volume of tissue over which the desired absorption into the
blood stream can take place. Modulating the sonic energy has been
shown to be very effective in modulating the total flux of a
compound through the micropores into or through the deeper tissues,
providing a second means of developing a bolus delivery system.
[0341] The exact pathways of absorption of insulin when given as a
subcutaneous injection are still a subject of some debate. One of
the reasons this is still unclear is the widely varying levels of
bio-availability demonstrated within a population, or even the same
subject, on an injection-by-injection basis. One hypothesized
pathway is the direct absorption through the capillaries and into
the blood stream. A method for enhancing this process is to couple
electroporation with the surface poration, where the
electroporation has been specifically optimized to work in the
region of the capillary endothelial membranes, creating
temporarily, a large number of openings to enhance this direct
absorption. As with the iontophoresis and sonophoresis described
previously, the total voltage amplitude levels of the
electroporation system required to effect this type of
electroporation within these tissue layers beneath the outer
surface are often lower than needed to penetrate through an intact
outer surface due to the reduction of the bulk impedance of the
outer layer of the biological membrane.
Example 47
[0342] Delivery of microparticles: The use of liposomes, lipid
complexes, microspheres including nanospheres, PEGellated compounds
(compounds combined with polyethylene glycol) and other
microparticles as part of a drug delivery system is well developed
for many different specific applications. In particular, when
dealing with a compound which is easily broken down by the
endogenous components in the body's tissues such as protease,
nuclease, or carbohydrase enzymes in the skin, tissues, the
macrophages or other cells present in the blood stream or lymph,
increases in bio-availability and/or sustained release can
frequently be realized by utilizing one of these techniques.
Currently, once one has applied one of these techniques, the
formulation is generally delivered via some type of injection. The
present invention, by creating micropores through a biological
membrane (e.g., the skin or a mucous membrane) and into the body to
a selected depth, allows this type of microparticle to be delivered
through the skin or mucosa. As described in the insulin example
above, microporation, electroporation, iontophoresis, sonic energy,
enhancers, as well as mechanical stimulation of the site such as
pressure or massage may be combined in any combination to enhance
the delivery and/or uptake of a specific formulation. In the case
of some engineered microparticles, the pores may have an optimal to
depth designed to bypass certain biologically active zones or place
the particle within the zone of choice. For some microparticle
delivery systems, the energy incident upon the particles after they
have been delivered into or through the tissues beneath the surface
may be used to trigger the accelerated release of the active
compound, thereby allowing the external control of the flux rate of
the therapeutic substance.
Example 48
[0343] Microparticles for implantable analyte monitoring: Another
application of microparticles is to deliver a particle not as a
therapeutic agent but as a carrier of a probe compound which could
be interrogated non-invasively, for example, via electro-magnetic
radiation from an external reader system to obtain information
regarding the levels of a specific analyte in the body. One example
is to incorporate in a porous microsphere a glucose specific
fluorophore compound which, depending on the levels of glucose
present in the surrounding tissues, would alter its fluorescent
response in either amplitude, wavelength, or fluorescent lifetime.
If the fluorophore was designed to be active with an excitation
wavelength ranging from 700 nm to 1500 nm, a low cost infrared
light source such as an LED or laser diode could be used to
stimulate its fluorescent response, which would similarly be in
this range of from 700 nm to 1500 nm. At these wavelengths, the
skin and mucosal tissues absorb very little and would therefore
allow a simple system to be built along these lines.
[0344] Glucose is one candidate analyte, for which experimental
lifetime fluorescence probes have been developed and incorporated
into subcutaneously inserted polymer implants which have been
successfully interrogated through the skin with optical stimulation
and detection methods. It would merely require the reformulation of
these experimental implants into suitably sized microparticles to
allow the delivery into or through the viable tissue layers via the
micropores. However, any analyte could be targeted, and the method
of interrogating the delivered microparticles could be via magnetic
or electric field rather than optical energy.
Example 49
Delivery of a Vaccine
[0345] A bacterial, viral, toxoid or mixed vaccine is prepared as a
solid, liquid, suspension, or gel as required. This formulation
could include any one or combination of peptides, proteins,
carbohydrates, DNA, RNA, entire microorganisms, adjuvants, carriers
and the like. A selected site of an individual is porated (skin or
mucous membrane) according to the procedures described above in
Example 45 and the vaccine is applied to the porated site. The
depth of the micropores may depend on the type of vaccine
delivered. This delivery can be aided with electroporation,
iontophoresis, magnetic or sonic energy, enhancers, as well as
mechanical stimulation of the site such as pressure or massage
according to the procedures described above and/or use
electroporation, iontophoresis, magnetic or sonic energy,
enhancers, as well as mechanical stimulation of the site such as
pressure or massage to enhance cellular uptake. Additional or
reinforcing doses can be delivered in the same manner to achieve
immunization of the individual.
Example 50
[0346] Delivery of Testosterone: A commercially available
testosterone patch, the Androderm.sup.Rpatch from TheraTech, Inc.,
was used in a set of experiments to evaluate the benefits of
microporation as it applies to the delivery of this permeant. A
hypergonadic male subject went off Androderm therapy for two days,
after which a series of venous blood samples were drawn during the
subsequent 24 hour period to establish this subject's baseline
levels of testosterone. Two 2.5 mg Androderm patches were then
installed as recommended by the manufacturer and a similar set of
venous blood sample were drawn to measure the testosterone levels
when the only transdermal flux enhancement method being used was
the chemical permeation enhancers contained in the patch. After two
more days of a washout period, two 2.5 Androderm patches were then
similarly installed, but prior to the installation, the skin
surface at the target sites was porated with 72 micropores per
site, each pore measuring approximately 80 .mu.m in width and 300
.mu.m in length and extending to a depth of 80 to 120 .mu.m. For
the porated delivery phase a similar set of venous blood sample
were drawn to measure the testosterone. The data from all three of
these twenty four hour periods is shown in the FIG. 35 titled
`Effects of Microporation on Transdermal Testosterone Delivery`. A
noteworthy feature of these data is that when the microporations
are present, the testosterone levels in the subjects blood elevate
much more rapidly, essentially preceding the rising edge of the
un-porated cycle by more than four hours. Looking at the slope of
and area under the curve we can calculate that more than a
three-fold flux rate enhancement took place due to the
microporations during the first four hours.
Example 51
[0347] Delivery of Alprostadil: Alprostadil, or PGE1, is a
prostaglandin used therapeutically to treat male erectile
dysfunction via it's vasodilator behavior. The standard delivery
mode for this drug is a direct injection into the base of the penis
or via a suppository inserted into the urethra. A set of
experiments were conducted with two healthy male volunteers. Each
subject had a site of 1 square cm on the base of the penis shaft
prepared by porating 12 to 36 micropores on this area, with the
thermal poration parameters set to create pores roughly 100 microns
deep as measured from the surface of the skin. A concentrated
solution of alprostadil was placed in a small reservoir patch
placed on the poration site, an ultrasonic transducer was then
placed on the top of the reservoir and activated and the subject's
erectile and other clinical responses were recorded on video tape.
Both subjects developed a significant amount of engorgement of the
penis, estimated as achieving 70% of more of a full erection at the
dose applied. In addition, a malar flush response to the systemic
levels of the drug delivered was observed. Over a 30 to 60 minute
delivery period, both subjects developed a profound malar flush
extending from the face, neck, chest and arms. Both the erectile
response and the malar flush provide evidence of the delivery of a
clinically active amount of the drug, a well know vasodilator.
Example 52
[0348] Delivery of Interferon: Interferons are proteins of
approximately 17-22,000 molecular weight, that are administered
clinically to treat a variety of disease states, such as viral
infections (e.g., hepatitis B and C), immune diseases (such as
multiple sclerosis), and cancers (e.g., hairy cell leukemia). Due
to their protein nature, interferons must currently be administered
by injection, as they cannot be given orally and are too large for
traditional transdermal or transmucosal delivery methods. To
demonstrate delivery of an interferon via the microporation
technique, a 100 microliter aliquot of alpha-interferon solution
containing interferon with a specific activity of 100 million
international units of interferon per mg dissolved in 1 ml of
delivery solution, is applied to a 1 square cm area of porated
skin, porated to a depth of 150-180 .mu.m, thus falling short of
the capillary bed, on the thigh of a healthy human subject. Trials
are run using either purely passive diffusion and with the
application of sonic energy to the region at sufficient amplitude,
frequency, and modulation thereof to accelerate the migration of
the interferon through pores into or through the underlying tissues
without causing deleterious heating of the interferon solution.
Venous blood draws are taken at various time intervals for both
trials, and are assayed for interferon levels using
radio-immunoassay and bioassay. Interferon is detected in the serum
over the 4 hour time period monitored. The interferon levels for
the sonically enhanced delivery experiment are detected sooner than
for the passive experiment. In another experiment, the interferon
is administered in dry powder form directly to the micropores in
the porated area of the skin. Interferon is detected in the serum
using the same techniques as described above. In another test, the
interferon solution is applied in a gel with or without a backing
film to the porated tissue of the buccal mucosa. Venous blood is
drawn and assayed for interferon levels. Interferon is detected in
the serum over the 3 hour time period monitored. In another
experiment, the interferon is incorporated into a tablet containing
a bio-erodable matrix, with a mucoadherent polymer matrix that
provided contact of the tablet over the area of buccal mucosa that
is porated. Interferon is detected in the serum using the same
techniques as described above.
Example 53
[0349] Delivery of morphine: A solution of morphine is applied to a
porated area on the volar forearm of the human subjects. A positive
pressure gradient is used to provide a basal delivery rate of the
morphine into the body, as determined by assay of venous blood
draws at appropriate time intervals for the presence of morphine. A
basal level of morphine of approximately 3-6 ng/ml is achieved.
Upon demand, an additional pressure bolus is applied to result in a
spike in the delivery of the morphine. The additional pressure
bolus is achieved in one test by use of ultrasound; or in another
experiment by the use of a pressure spike. This type of delivery,
in which a basal level of the morphine is continuously applied,
with spikes in morphine delivery periodically upon demand, is
useful in treating chronic and breakthrough pain.
Example 54
[0350] Delivery of a disease resistant DNA into a plant: The seeds
of a selected corn plant are microporated. The seeds are placed in
a solution of a permeant formulation containing DNA that encodes
disease resistance proteins. Sonic energy is used, optionally, to
enhance the delivery of the DNA into the corn seeds. The seeds are
germinated and grown to maturity. The resulting seeds of the mature
corn plants now carry the disease resistant gene.
Example 55
[0351] Delivery of DNA into a plant: The seeds of a sugar beet are
microporated. The seeds are placed in a solution of a permeant
formulation containing DNA that encodes human growth hormone.
Electroporation, iontophoresis, sonic energy, enhancers, as well as
mechanical stimulation of the site such as pressure may be used to
enhance the delivery of the DNA into the seeds. The seeds are
germinated and grown to maturity. The resulting mature beet plant
can now be harvested and the human growth hormone extracted for
subsequent purification and clinical use.
Example 56
[0352] An experiment was conducted wherein fluorescent dextran
particles, MW approximately 10,000 Daltons, were applied in an
aqueous solution by means of a reservoir patch over a one square cm
of skin on the volar forearm of a human subject where 36 micropores
extending approximately 80 .mu.m in depth were formed. The
reservoir patch was left in place for 5 minutes. The porated site
and surrounding area were imaged with a fluorescent video
microscope to evaluate the penetration of the permeant into the
tissue. The fluorescence showed that within 5 minutes significant
permeation of dextran occurred more than 2 mm away from the nearest
micropore. The video assay system used 10 minutes later showed
further diffusion so that the fluorescent flush extended 10 mm from
the pores. This experiment gives clear evidence that this technique
allows delivery of permeants with molecular weights of 10,000.
* * * * *