U.S. patent application number 13/919923 was filed with the patent office on 2014-05-08 for smmr (small molecule metabolite reporters) for use as in vivo glucose biosensors.
This patent application is currently assigned to Cercacor Laboratories, Inc. The applicant listed for this patent is Cercacor Laboratories, Inc. Invention is credited to Emile M. Bellott, Dongsheng Bu, James J. Childs, Christopher Lambert, Hubert A. Nienaber, Shirley J. Shi, Zhaolin Wang, Jerome J. Workman, Alex R. Zelenchuk.
Application Number | 20140127137 13/919923 |
Document ID | / |
Family ID | 34752980 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127137 |
Kind Code |
A1 |
Bellott; Emile M. ; et
al. |
May 8, 2014 |
SMMR (SMALL MOLECULE METABOLITE REPORTERS) FOR USE AS IN VIVO
GLUCOSE BIOSENSORS
Abstract
Small Molecule Metabolite Reporters (SMMRs) for use as in vivo
glucose biosensors, sensor compositions, and methods of use, are
described. The SMMRs include boronic acid-containing xanthene,
coumarin, carbostyril and phenalene-based small molecules which are
used for monitoring glucose in vivo, advantageously on the
skin.
Inventors: |
Bellott; Emile M.; (Beverly,
MA) ; Bu; Dongsheng; (Piscataway, NJ) ;
Childs; James J.; (Bolton, MA) ; Lambert;
Christopher; (Hudson, MA) ; Nienaber; Hubert A.;
(Newburyport, MA) ; Shi; Shirley J.; (Lexington,
MA) ; Wang; Zhaolin; (Wellesley, MA) ;
Workman; Jerome J.; (Madison, WI) ; Zelenchuk; Alex
R.; (Stoughton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cercacor Laboratories, Inc |
Irvine |
CA |
US |
|
|
Assignee: |
Cercacor Laboratories, Inc
Irvine
CA
|
Family ID: |
34752980 |
Appl. No.: |
13/919923 |
Filed: |
June 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13215061 |
Aug 22, 2011 |
8466286 |
|
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13919923 |
|
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|
10584821 |
Mar 13, 2008 |
8029765 |
|
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PCT/US2004/043087 |
Dec 23, 2004 |
|
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|
13215061 |
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60571170 |
May 14, 2004 |
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60532667 |
Dec 24, 2003 |
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Current U.S.
Class: |
424/9.6 ; 546/13;
549/213; 562/7 |
Current CPC
Class: |
A61K 49/0041 20130101;
C07F 5/025 20130101; A61K 49/0021 20130101; A61K 49/0052 20130101;
C07F 7/1804 20130101; Y10T 436/144444 20150115 |
Class at
Publication: |
424/9.6 ;
549/213; 546/13; 562/7 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07F 5/02 20060101 C07F005/02 |
Claims
1. A small molecule metabolite reporter compound of the following
formula: ##STR00106## wherein D is a heteroatom; R.sub.1 and
R.sub.2 are different and are selected from the group consisting of
H, OH, NH.sub.2, NO.sub.2, OCH.sub.3, N(CH.sub.3).sub.2, A, or,
R.sub.1 and R.sub.2, taken together with the ring to which they are
attached, form R.sub.7; R.sub.3 and R.sub.4 are different and are
selected from the group consisting of H, ##STR00107## OH,
B(OH).sub.2, M, or R.sub.3 and R.sub.4, taken together with the
ring to which they are attached, form R.sub.8; R.sub.5 and R.sub.6
are different and are selected from the group consisting of H or
##STR00108## wherein Q is H, COOH, B(OH).sub.2, or M; A is OH,
NH.sub.3, ##STR00109## R.sub.7 is ##STR00110## R.sub.8 is
##STR00111## M is ##STR00112## L, when present, is an
amino-containing linking moiety; R.sub.1 and R.sub.2, and R.sub.3
and R.sub.4, are adjacent to each other on the rings on which they
reside; and at least one boronic acid moiety is present; and salts
thereof.
2. The reporter compound of claim 1, wherein D is N or O.
3. (canceled)
4. (canceled)
5. A small molecule metabolite reporter compound of the following
formula: ##STR00113## M is ##STR00114## and L, when present, is an
amino-containing linking moiety; and salts thereof.
6. (canceled)
7. A topical sensor composition comprising a compound of claim 1
and a carrier or binder.
8. A method of measuring a compound or metabolite thereof,
comprising contacting a reporter compound of the following formula:
##STR00115## wherein D is a heteroatom; R.sub.1 and R.sub.2 are
different and are selected from the group consisting of H, OH,
NH.sub.2, NO.sub.2, OCH.sub.3, N(CH.sub.3).sub.2, A, or, R.sub.1
and R.sub.2, taken together with the ring to which they are
attached, form R.sub.7; R.sub.3 and R.sub.4 are different and are
selected from the group consisting of H, , OH, B(OH).sub.2, M, or
R.sub.3 and R.sub.4, taken together with the ring to which they are
attached, form R.sub.8; R.sub.5 and R.sub.6 are different and are
selected from the group consisting of H or ##STR00116## wherein Q
is H, COOH, B(OH).sub.2, or M; A is OH, NH.sub.3, ##STR00117##
R.sub.7 is ##STR00118## R.sub.8 is ##STR00119## M is ##STR00120##
L, when present, is an amino-containing linking moiety; and R.sub.1
and R.sub.2, and R.sub.3 and R.sub.4, are adjacent to each other on
the rings on which they reside; and at least one boronic acid
moiety is present; and salts thereof; which reporter compound is
sensitive to the presence of said compound or metabolite thereof,
with an area of the body where said metabolites may be found, and
detecting a photometric change in said reporter compound indicative
of said metabolite.
9. (canceled)
10. (canceled)
11. The method of claim 8, wherein said area of the body is
skin.
12. The method of claim 8, wherein said area of the body is the
layer of the skin known as stratum corneum.
13. The method of claim 8, wherein said area of the body is the
layer of the skin known as epidermis.
14. The method of claim 8, wherein said area of the body is the
layer of the skin known as dermis.
15. The method of claim 8, wherein said compound is glucose.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/215,061, filed Aug. 22, 2011, which is a continuation of
U.S. application Ser. No. 10/584,821, filed Mar. 13, 2008 now U.S.
Pat. No. 8,029,765, which is a U.S. national phase application
under 35 U.S.C. .sctn.371 of International Application No.
PCT/US2004/043087, filed Dec. 23, 2004, which claims priority under
35 U.S.C. .sctn.119(e) to Provisional Application Nos. 60/532,667,
filed Dec. 24, 2003 and 60/571,170, filed May 14, 2004.
FIELD OF THE INVENTION
[0002] This invention provides compositions and methods for
designing small molecule metabolite reporters (SMMRs) for optical
reporting of cell metabolism and intracellular or extracellular
metabolite or analyte concentrations. The specific application of
this work is to design molecules that are able to optically report
the concentration of the biologically active molecule D-glucose,
including other small molecule analytes or metabolic processes, in
or near human keratinocytes located within the viable epidermis of
human or mammalian skin. The skin glucose levels are then used to
infer blood glucose levels. In particular, this invention provides
compositions and methods for several noninvasive techniques to
determine in vivo blood glucose levels based upon the direct
measurement of glucose levels present in the skin.
BACKGROUND OF THE INVENTION
[0003] Identifying and understanding the risk factors associated
with diabetes is invaluable for the development and evaluation of
effective intervention strategies. Lacking normal regulatory
mechanisms, diabetics are encouraged to strive for optimal control
through a modulated life style approach that focuses on dietary
control, exercise, and glucose self-testing with the timely
administration of insulin or oral hypoglycemic medications.
Invasive forms of self-testing are painful and fraught with a
multitude of psychosocial hurdles, and are resisted by most
diabetics. Alternatives to the currently available invasive blood
glucose testing are highly desirable.
[0004] Conventional approaches seek to reduce or eliminate the skin
trauma, pain, and blood waste associated with traditional invasive
glucose monitoring technologies. In general, noninvasive optical
blood glucose monitoring requires no samples and involves external
irradiation with electromagnetic radiation and measurement of the
resulting optical flux. Glucose levels are derived from the
spectral information following comparison to reference spectra for
glucose and background interferants, reference calibrants, and/or
application of advanced signal processing mathematical algorithms.
Candidate radiation-based technologies include: 1) mid-infrared
(MIR) spectroscopy, 2) near-infrared (NIR) spectroscopy, 3)
far-infrared (FIR) spectroscopy, 4) radio wave impedance, 5)
infrared photoacoustic spectroscopy and 6) Raman spectroscopy. Each
of these methods uses optical sensors, and relies on the premise
that the absorption pattern of infrared light (700-3000 nm) can be
quantitatively related to the glucose concentration. Other
substances such as water, protein, and hemoglobin are known to
absorb infrared light at these wavelengths and easily obscure the
relatively weak glucose signal.
[0005] Other approaches are based on microvascular changes in the
retina; acoustical impedance, NMR spectroscopy and optical
hydrogels that quantify glucose levels in tear fluid. While
putatively noninvasive, these technologies have yet to be
demonstrated as viable in clinical testing.
[0006] Nearly noninvasive techniques tend to rely on interstitial
fluid extraction from skin. This can be accomplished using
permeability enhancers, sweat inducers, and/or suction devices with
or without the application of electrical current. One device
recently approved by the FDA relies on reverse iontophoresis,
utilizing an electrical current applied to the skin. The current
pulls out salt, which carries water, which in turn carries glucose.
The glucose concentration of this extracted fluid is measured and
is proportional to that of blood. This technology, in keeping with
its nearly noninvasive description, is commonly associated with
some discomfort and requires at least twice daily calibrations
against conventional blood glucose measurements (e.g., invasive
lancing).
[0007] Other nearly noninvasive blood glucose monitoring techniques
similarly involve transcutaneous harvesting for interstitial fluid
measurement. Other technologies for disrupting the skin barrier to
obtain interstitial fluid include: 1) dissolution with chemicals;
2) microporation with a laser, sound, or electrical stimulation; 3)
penetration with a thin needle; and/or 4) suction with a pump.
Minimally invasive blood glucose monitoring can also involve the
insertion of an indwelling glucose monitor under the skin to
measure the interstitial fluid glucose concentration. These
monitors typically rely on optical or enzymatic sensors.
Technologically innovative, these in situ sensors have had limited
success. Implantable glucose oxidase sensors have been limited by
local factors causing unstable signal output, whereas optical
sensors must overcome signal obfuscation by blood constituents as
well as interference by substances with absorption spectra similar
to glucose. Moreover, inflammation associated with subcutaneous
monitoring may contribute to systematic errors requiring
repositioning, recalibration or replacement, and more research is
needed to evaluate the effects of variable local inflammation at
the sensor implantation site on glucose concentration and transit
time.
[0008] Interstitial fluid glucose concentrations have previously
been shown to be similar to simultaneously measured fixed or
fluctuating blood glucose concentrations (Bantle et al., Journal of
Laboratory and Clinical Medicine 130:436-441, 1997; Sternberg et
al., Diabetes Care 18:1266-1269, 1995). Such studies helped
validate noninvasive/minimally invasive technologies for blood
glucose monitoring, insofar as many of these technologies measure
glucose in blood as well as interstitial fluid.
[0009] A noninvasive glucose monitor that is portable, simple and
rapid to use, and that provides accurate clinical information is
highly desirable. In particular, the ability to derive primary and
secondary order information regarding real time, dynamic glucose
metabolism (such as the direction and rate of change of
bioavailable glucose distributed within the blood and interstitial
fluid space) is highly desirable.
SUMMARY OF THE INVENTION
[0010] In vivo fluorescence (autofluorescence) has been used for a
number of years to determine the metabolic state and to monitor
pharmaceutical effects in cells and tissues (Dellinger et al.,
Biotechnol Appl Biochem, 28(Pt. 1): 25-32, 1998). Consideration of
the photophysics involved in autofluorescence rapidly leads one to
the conclusion that the use of autofluorescence alone as the
analytic probe imposes some severe limitations on any measurement
technique.
[0011] Fluorescence techniques are capable of detecting molecular
species at picomole levels or less. This sensitivity arises because
of the simplicity of detecting single photons against a dark
background. This advantage disappears if there are other
fluorescent species in the detection volume. Fluorescence intensity
is also not an absolute technique and must be referenced to some
internal standard using a ratiometric or comparative method.
[0012] It has been shown that fluorophores, or colored dyes
utilizing absorption spectroscopy can be used to measure glucose in
solution or serum by using series of separate reagents. These
generic reagents include glucose oxidase (which oxidizes glucose
forming hydrogen peroxide); peroxidase (generally, horseradish
peroxidase: HRP) used to create an oxidizing reaction in the
presence of hydrogen peroxide with the dye or fluorophore; and a
dye reagent or fluorophore, which changes its color or fluorescence
spectrum when brought in contact with hydrogen peroxide, and
peroxidase. The resultant colored or fluorescent species is
measured with a colorimeter or fluorimeter and the amount of
glucose in solution is calculated. In addition, other analytical
techniques have been shown to be commercially useful for measuring
hydrogen peroxide generated from the reaction of glucose oxidase
and glucose.
[0013] The methods and compositions of the present invention
effectively determine the glucose concentration in blood for a
living organism by noninvasive, in vivo measurement of the glucose
level in skin by means of fluorescence measurements of metabolic
indicators/reporters of glucose metabolism. Disclosed are dyes used
as metabolic indicators that allow for specific in vivo monitoring
of metabolites, which are used as indicators of metabolic activity.
Dyes characterized by this invention are referred to herein as a
small molecule metabolite reporters ("SMMRs") or alternatively,
"small molecule multi-domain reporters ("SMMDRs")." In many cases
the description in the specification will apply to both terms.
[0014] The invention relates in one aspect to SMMRs which comprise
novel xanthene-based boronic acid compounds. The SMMRs may be used
in sensor compositions for, e.g., direct measurement of glucose,
and in other diagnostic or analytical methods as described herein.
In an embodiment these compounds are of the following formula
(I):
##STR00001##
wherein [0015] D is a heteroatom; [0016] R.sub.1 and R.sub.2 are
different and are selected from the group consisting of H, OH,
NH.sub.2, NO.sub.2, OCH.sub.3, N(CH.sub.3).sub.2, A, or, R.sub.1
and R.sub.2, taken together with the ring to which they are
attached, form R.sub.7; [0017] R.sub.3 and R.sub.4 are different
and are selected from the group consisting of H,
[0017] ##STR00002## OH, B(OH).sub.2, M, or R3 and R.sub.4, taken
together with the ring to which they are attached, form R.sub.8;
[0018] R.sub.5 and R.sub.6 are different and are selected from the
group consisting of H or
[0018] ##STR00003## wherein Q is H, COOH, B(OH).sub.2, or M; [0019]
A is OH, NH.sub.3,
[0019] ##STR00004## [0020] R.sub.7 is
[0020] ##STR00005## [0021] R.sub.8 is
[0021] ##STR00006## [0022] M is
[0022] ##STR00007## [0023] L, when present, is an amino-containing
linking moiety; [0024] R.sub.1 and R.sub.2, and R.sub.3 and
R.sub.4, are adjacent to each other on the rings on which they
reside; and [0025] at least one boronic acid moiety is present; and
salts thereof.
[0026] "Amino-containing linking moieties" may include moieties
comprising a substituted or unsubstituted amino group, an amido
group or a sulfonamido group. In an embodiment, xanthene-based
boronic acid compounds of the invention include those of the
formula (II):
##STR00008##
wherein [0027] R.sub.1 and R.sub.2 are different and may be A, or,
R.sub.1 and R.sub.2, taken together with the ring to which they are
attached, form R.sub.7; [0028] R.sub.3 and R.sub.4 are different
and are selected from the group consisting of H,
[0028] ##STR00009## OH, B(OH).sub.2, M, or R.sub.3 and R.sub.4,
taken together with the ring to which they are attached, form
R.sub.8; [0029] R.sub.6 is
[0029] ##STR00010## [0030] A is
[0030] ##STR00011## [0031] R.sub.7 is
[0031] ##STR00012## [0032] R.sub.8 is
[0032] ##STR00013## [0033] M is
[0033] ##STR00014## [0034] L, when present, is an amino-containing
linking moiety; and [0035] R.sub.1 and R.sub.2, and R.sub.3 and
R.sub.4, are adjacent to each other on the rings on which they
reside; and salts thereof.
[0036] In another embodiment, xanthene-based boronic acid compounds
of the invention include those of the formula (III):
##STR00015##
wherein [0037] R.sub.1 and R.sub.2 are different and are selected
from the group consisting of H, OH, NH.sub.2, NO.sub.2, OCH.sub.3,
N(CH.sub.3).sub.2, A, or, R.sub.1 and R.sub.2, taken together with
the ring to which they are attached, form R.sub.7; [0038] R.sub.3
and R.sub.4 are different and are selected from the group
consisting of H,
[0038] ##STR00016## OH, M, or R.sub.3 and R.sub.4, taken together
with the ring to which they are attached, form R.sub.8; [0039]
R.sub.5 and R.sub.6 are different and are selected from the group
consisting of H or
[0039] ##STR00017## wherein Q is H or M; [0040] A is OH,
NH.sub.3
[0040] ##STR00018## [0041] R.sub.7 is
[0041] ##STR00019## [0042] R.sub.8 is
[0042] ##STR00020## [0043] M is
[0043] ##STR00021## [0044] L, when present, is an amino-containing
linking moiety; and [0045] R.sub.1 and R.sub.2, and R.sub.3 and
R.sub.4, are adjacent to each other on the rings on which they
reside; and salts thereof.
[0046] Examples of xanthene-based SMMRs of the invention
include:
##STR00022## ##STR00023## ##STR00024## ##STR00025##
[0047] The invention relates in another aspect to SMMRs which
comprise novel phenalene-based boronic acid compounds. In an
embodiment these compounds are phenalene-1-one compounds of the
following formula (IV):
##STR00026##
wherein [0048] M is
[0048] ##STR00027## and [0049] L, when present, is an
amino-containing linking moiety; and salts thereof.
[0050] Examples of such phenalene-based boronic acid compounds
include:
##STR00028##
[0051] The invention relates in another aspect to SMMRs which
comprise novel boronic acid-containing coumarin or carbostyril
derivative compounds. In an embodiment these compounds include
those of the following formula (V):
##STR00029##
wherein [0052] D is a heteroatom (e.g., O or N); [0053] R.sub.9 is
H, OH, CH.sub.3, CF.sub.3, M, or an amino or substituted amino
group; [0054] R.sub.10 is H, CH.sub.3, or M; [0055] R.sub.11,
R.sub.12, and R.sub.13 are individually H, OH, alkoxy, M, or an
amino or substituted amino group; [0056] R.sub.14, when present, is
H or CH.sub.3; [0057] M is
[0057] ##STR00030## and [0058] at least one boronic acid moiety is
present; and salts thereof.
[0059] A substituted amino group may include where R.sub.11,
R.sub.12, and R.sub.13, taken together with the ring to which they
are attached, form a nitrogen-containing polycycle. Examples of
such boronic acid-containing coumarin or carbostyril SMMRs
include:
##STR00031## ##STR00032## ##STR00033## ##STR00034##
[0060] It will be noted that the structure of some of the compounds
of the invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of the invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis. Alkenes
can include either the E- or Z-geometry, where appropriate.
Tautomeric forms of compounds of the invention are also intended to
be included within the scope of the invention, unless indicated
otherwise.
[0061] In another aspect, a chromophore of the following rational
design structure is disclosed:
##STR00035##
wherein Het represents a heteroatomic group, e.g., containing N, O,
or S; B(OH).sub.2; M or R.sub.8 (as set forth in Formula (I)); or
mono or di-substituted N, NO.sub.2 or N(CH.sub.3).sub.2; which
groups may be identical or different. Heteroatomic groups may
include amino, amido, carbonyl, hydroxyl, thiol, and thio.
[0062] This invention also provides for fluorescence measurements
of extracellular and intracellular reporter molecules placed into
the cytosol, nucleus, or organelles of cells within intact, living,
tissue that track the concentration of blood glucose in an
organism. When any one of a series of metabolites is measured using
this technique, the molar concentration of blood glucose can be
calculated. Direct or indirect fluorescence measurements of glucose
using one or more of the following measurements is described: pH
(as lactate/H.sup.+), membrane reduction-oxidation electric
potential, NAD(P)H (nicotinamide adenine dinucleotide (phosphate),
reduced form) for energy transfer, FAD.sup.+ (flavin adenine
dinucleotide, oxidized form) for energy transfer, ATP/ADP ratio,
Ca.sup.2+-pumping rate, Mg.sup.2+-pumping rate, Na.sup.+-pumping
rate, K.sup.+-pumping rate, and vital mitochondrial membrane
stains/dyes/molecules fluorescence response. These analytes,
measured in skin using the techniques taught herein, are used to
provide a complete picture of epidermal skin glycolytic metabolism
where local epidermal analyte (glucose) quantities are proportional
to the concentration of glucose in systemic blood, specifically the
capillary fields within the papillary layer of the dermis (corium).
Temperature and/or nitric oxide measurement may also be combined
with the above measurements for better calibration and
determination of glucose concentrations.
[0063] The invention further provides sensor compositions that are
applied to at least one surface of living tissue, organs,
interstitial fluid, and whole organisms and transported into the
tissue at an effective concentration. The sensor composition can
include at least one SMMR at an effective concentration such that
when the at least one SMMR is brought in contact with one or more
specific metabolites or analytes, a change in fluorescence or
absorption occurs, thereby allowing quantification of the change in
fluorescence or absorption.
[0064] The primary embodiment of this invention utilizes a series
of molecules (SMMRs) specifically designed for topical delivery
onto tissue, such as the viable epidermis, which when applied to
the tissue will report glucose concentration using any one or more
of several reporting mechanisms. The most significant advantage of
the present invention is increased sensitivity in reporting glucose
concentration, while eliminating the requirement to draw body fluid
from the skin as is required by current conventional
techniques.
[0065] The in vivo information obtained when the SMMR is brought in
contact with the one or more metabolites or analytes can include,
but is not limited to, assessment of metabolic function; diagnosis
of metabolic disease state; monitoring and control of disease
state; stress status of cells, tissues and organs; determination of
vitality and viability of cells based on metabolic function;
critical care monitoring; diagnosis and monitoring of
cardiovascular diseases, autoimmune disorders, neurological
disorders, degenerative diseases; determination of metabolic
concentration; and cancer diagnosis, detection, staging and
prognosis.
[0066] For example, the in vivo information obtained may provide
detailed information on glucose metabolism, fructose metabolism and
galactose metabolism; advanced-glycosylated end products;
monitoring and control of diseases such as diabetes, cancer, stress
and organ transplantation.
[0067] The sensor compositions used in these methods for monitoring
the concentration of one or more metabolite(s) or analyte(s) can be
formulated as, but are not limited to, emulsions, ointments,
disposable gel film patches, reservoir devices, creams, paints,
polar solvents, nonpolar solvents, or any combination thereof.
[0068] Penetration of the sensor composition can be accomplished
using an active transport technique or a passive transport
technique, such as, for example, electroporation, laser poration,
sonic poration, ultrasonic poration, iontophoresis,
mechanical-poration, solvent transport, tattooing, wicking,
microneedle or pressurized delivery. In addition, penetration of
the sensor composition to the desired depth can be accomplished by
combining the composition with various molecular size
attachments.
[0069] Typically, the quantification of the change in fluorescence
or absorption is monitored using fluorescence or absorption
spectroscopy.
[0070] An effective concentration of the sensor composition is, for
example, at least between 0.01 to 500 .mu.g/ml, between 0.1 to 500
.mu.g/ml, between 1.0 to 150 .mu.g/ml, between 1 to 100 .mu.g/ml,
and between 10 to 100 .mu.g/ml. The SMMR can be introduced in a low
concentration in a range from 10 .mu.M to 1000 .mu.M and in a
volume from 200 .mu.L to 0.1 .mu.L, respectively (e.g., introducing
the SMMR at a concentration in the range of 200 .mu.L of a 10 .mu.M
SMMR solution to 0.1 .mu.l of a 1000 .mu.M SMMR solution). One
specific application of the sensor composition is, for example, a 5
.mu.L volume of a 400 .mu.M SMMR solution, or a 10 .mu.L volume at
200 .mu.M concentration.
[0071] Once one or more SMMRs are activated as a result of
placement within the skin, fluorescence measurements monitor the
response of the skin cells to glucose. As described herein, the
fluorescence mechanism used is either a direct or indirect
indication of the glucose concentration in the target cell
environment. Fluorescence is typically measured using an optical
reader. The optical reader calculates the skin response to glucose,
applies first principles mathematical models to the response, and
provides a determination of the blood glucose levels. Choosing the
particular commercially available or custom designed optical reader
that is compatible for use with the methods and compositions of
this invention is within the ability of one skilled in the art.
[0072] One embodiment of this invention utilizes indirect means to
measure skin and blood glucose in vivo by placing one or more SMMRs
into the viable epidermis to form a fluorescent product. This
fluorescent product is provided by one of many specifically
described reporting mechanisms, whereby the SMMR fluorescent signal
changes with respect to the effects of glucose concentration on
cell metabolism. The quantity of fluorescence, or the fluorescent
ratio at two or more emission wavelengths, is indicative of the
total glucose concentration within the skin, either intracellular
or extracellular as described here. The skin glucose thus
determined is used to infer blood glucose levels as calibrated and
described herein.
[0073] In another embodiment, a method for monitoring in vivo blood
glucose levels uses SMMRs that directly bind or respond to glucose
itself. The mechanisms of glucose reporting thus does not use cell
metabolism, as in the first embodiment, but rather the SMMR
responds to glucose by one of several direct mechanisms to produce
a fluorescent product. The measured fluorescence is thus a direct
reporter of the interstitial fluid or extracellular glucose
concentrations. Thus, the skin glucose level directly determined in
vivo is used to infer blood glucose levels as calibrated and
described within this invention text.
[0074] This invention describes the unique physicochemical,
photochemical, photophysical and biological properties of SMMR
molecules, as well as their design, synthesis, and application. The
use of an SMMR enables fluorescence measurements from picomolar
through millimolar in vivo glucose levels in living skin tissue, or
interstitial fluid, either of which are indicative of the blood
glucose levels. The invention described here relates to the
indirect or direct determination of skin glucose levels for use in
the monitoring and control of diabetes mellitus. Embodiments of the
invention use SMMR fluorescence to measure skin glucose levels
without withdrawing bodily fluids. When the SMMR-based skin glucose
measurements are made, the blood glucose levels are directly
inferred.
[0075] The quantity of glucose in the epidermis is supplied by mass
transport from the blood vessels and capillary fields located
within the dermis, immediately beneath the epidermis. The movement
of glucose from the blood stream to the epidermis is concentration
dependent and noninsulin regulated providing the basis for
measurement of blood glucose as a direct inference from skin
glucose measurement. The rate of glucose transport into the
epidermis is indicative of the differential concentration between
skin glucose and blood glucose levels. The rate of transport into
the extracellular spaces between human skin cells allows an
accurate first principles mathematical extrapolation of blood
glucose levels. Once modeled, the kinetics of blood glucose
transport to the skin from the blood enables the determination of
the precise first principles mathematical relationship between the
rate of change of skin glucose and the rate of change of blood
glucose. Thus rapid blood glucose concentration changes up or down
can be accurately tracked by knowing the skin glucose mean
concentration levels and the rate of change of skin glucose levels.
First principles mathematical models can be developed for the
individual case, preferably for small local populations, and most
preferably for a universal patient case.
[0076] The SMMR-derived fluorescence reports glucose levels within
or surrounding human keratinocyte cells as an indication of blood
glucose levels. The movement of glucose from the interstitial fluid
surrounding the keratinocytes into the keratinocytes of the
epidermis is concentration dependent and noninsulin regulated. That
is, the glucose is transported into these cells via noninsulin
regulated glucose transporter GluT1 (GenBank Accession Number:
K03195), not insulin regulated glucose transporter GluT4 (GenBank
Accession Number: M91463). This transport mechanism provides the
basis for measurement of blood glucose as a direct inference from
intracellular keratinocyte glucose measurement.
[0077] Also provided are noninvasive methods for monitoring in vivo
blood glucose levels. According to these methods at least one small
molecule metabolic reporter is applied to at least one surface of
skin for a predetermined period of time causing penetration of the
one or more SMMRs to a depth of about 10 .mu.m, wherein the depth
corresponds with the bottom of the dead stratum corneum layer, to
about 175 .mu.m, wherein the depth corresponds with the top of the
dermal layer, into the epidermis. The one or more SMMRs come in
contact with one or more metabolites or analytes and a change in
the concentration of the one or more metabolites or analytes is
monitored by detecting changes in the SMMRs using an optical
reader. The change in the concentration of the one or more
metabolites or analytes is then correlated with in vivo blood
glucose levels.
[0078] Also included in the invention is a reagent strip for use in
a glucose measuring instrument comprising a polymer strip and a
known concentration of at least one small molecule metabolic
reporter, wherein when a sample of a biological fluid containing an
amount of glucose is interacted with the reagent strip, a change in
fluorescence or absorption of the one or more molecular sensor
proteins occurs, and the change is measured by the glucose
measuring instrument, thereby detecting the glucose concentration
of the biological fluid.
[0079] The change in fluorescence or absorption can be monitored
using fluorescence or absorption spectroscopy. Those of ordinary
skill in the art will recognize that any fluorescence or absorption
spectroscopic techniques can be used in accordance with the
invention.
[0080] The invention also provides sensor systems that include a
device having a component that transmits radiation to a material or
tissue, a component that detects radiation emitted from a material
or tissue, and a component to display the detection results, each
component is operably linked. The sensor systems further include an
applicator that delivers the sensor composition of the invention to
the material or tissue. Typically, there is an air interface
between the device and the material or tissue, wherein the air
interface measures a resulting excitation radiation emitted from
the irradiated sensor composition.
[0081] The device included in the sensor system can emit radiation
at one or more wavelengths that have been chosen to specifically
excite the SMMR mixture that is applied to the material or tissue.
The sensor composition can include a reporter dye and a marker dye,
or alternatively, a dye exhibiting a wavelength shift in absorption
or fluorescence emission in the presence of a metabolite. The
sensor composition can be present at a depth from the surface of
the skin of about 10 .mu.m to about 175 .mu.m in the epidermis in a
concentration that is effective for detection of one or more
metabolites or analytes in a biological sample.
[0082] The sensor system can detect radiation at one or more
wavelengths that have been chosen to specifically identify
fluorescence emission that has been scattered back to the system
from the sensor composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] FIG. 1. Glucose pathway of living cells
[0084] FIG. 2. Summary of photochemical reaction pathways
[0085] FIG. 3. Absorption and emission spectra for 5 uM of an
example ratiometric pH reporting dye in PBS. Fluorescence spectra
were measured using 532 nm to excite the dye.
[0086] FIG. 4. Fluorescence emission spectra of an example
ratiometric pH reporting dye loaded into the A-431 cells. Nigericin
is present and allows intracellular pH to equilibrate extracellular
pH.
[0087] FIG. 5. "Absolute" and normalized Fluorescence,
auto-Fluorescence and Air spectra.
[0088] FIG. 6. Difference and relative spectra of normalized
Fluorescence spectra.
[0089] FIG. 7. Glucose, Lactate and SMMR measured ratio
kinetics.
[0090] FIG. 8. Kinetics of physiological parameters and ratio.
[0091] FIG. 9. Normalized and relative changes of ratio, glucose,
and lactate in time.
[0092] FIG. 10. Fluorescence ratio changes after anesthesia (0.1 cc
of ketamine).
[0093] FIG. 11a. Data from a clamp study, comparison between
experimental (YSI values) and calculated values of AR (from the
equation .DELTA.R=.alpha..DELTA.G+.beta..DELTA.L using the
coefficients .alpha. and .beta. from above.
[0094] FIG. 11b. Data from another clamp study, comparison between
experimental (YSI values) and calculated values of AR (from the
equation
.DELTA.R=.alpha..DELTA.G+.beta..DELTA.L+.DELTA.R.sub.a(t-t.sub.a) A
using the coefficients .alpha., .beta. and
.DELTA.R.sub.a(t-t.sub.a) from above.
[0095] FIG. 11c. Data from yet another clamp study, comparison
between experimental (YSI values) and calculated values of AR (from
the
.DELTA.R=.alpha..DELTA.G+.beta..DELTA.L+.DELTA.R.sub.a(t-t.sub.a) A
using the coefficients .alpha., .beta. and
.DELTA.R.sub.a(t-t.sub.a) from above.
[0096] FIG. 12. NADH peak fluorescence signals versus time as
glucose concentration is changed. Glucose concentrations are
annotated in the Figure. The first annotation with 50% PBS is a
control measurement.
[0097] FIG. 13. Glucose concentration is varied from 0 to 66 mg/dL
and the NADH peak signals exhibit corresponding changes in
intensity.
[0098] FIG. 14. NADH measurements versus sequence number. The
glucose values (in mg/dL) for each data point are (from left to
right): 0, 0.1, 0.5, 1.0, 2.0, 5.0, 2.5, 1.0, 0.5, 0.25, 0.16, and
4.0 mg/dL. Each point represents the mean and standard deviation of
5 data points. NOTE: The x-axis label sequence number.
[0099] FIG. 15. Corrected NADH Signal of FIG. 14 for each data
point (as from left to right), corresponding to a concentration 0,
0.1, 0.5, 1.0, 2.0, 5.0, 2.5, 1.0, 0.5, 0.25, 0.16, and 4.0.
[0100] FIG. 16. Corrected NADH Signal versus Glucose concentration
for upward trend and downward trend data together.
[0101] FIG. 17. A membrane-bound Rh123 peak fluorescence signal
change (decrease in signal as an indication of membrane potential
change) versus time as glucose concentration is changed. Glucose
concentrations were increased at the 15-minute point in the Figure.
The first annotation with 50% PBS is a control measurement.
[0102] FIG. 18. Glucose concentration is varied from 0 to 66 mg/dL
and the Rh123 (bottom plot) peak signals exhibit corresponding
changes in intensity due to membrane potential changes.
[0103] FIG. 19. Rh123 fluorescence quenching (as an indication of
membrane potential changes) versus sequence number and glucose
concentration. The glucose values (in mg/dL) for each data point is
(from left to right): 0, 0.1, 0.5, 1.0, 2.0, 5.0, 2.5, 1.0, 0.5,
0.25, 0.16, and 4.0. Each point represents the mean and standard
deviation of 5 data points. The x-axis label is sequence
number.
[0104] FIG. 20. Schematic representation of the products of the
xanthene library in terms of Markush structures and some specific
sub structures.
[0105] FIG. 21a. Absorption and emission spectra for BeXan type
dyes.
[0106] FIG. 21b. Absorption and emission spectra for
fluorescein.
[0107] FIG. 22. Malachite green absorption spectrum (left), with
molecular structure (right).
[0108] FIG. 23. Rhodamine B absorption spectrum (left), with
molecular structure (right).
[0109] FIG. 24. The Boronic acid-diol equilibrium.
[0110] FIG. 25. Typical titration curves of a phenyl boronic analog
(2-4 bound to diol; 1-3 unbound).
[0111] FIG. 26. Argofluor-327d in equilibrium with a diol.
[0112] FIG. 27. Predicted emission wavelengths of several proposed
fluorophores.
[0113] FIG. 28a. The absorption spectra of p-nitroaniline in
different solvents.
[0114] FIG. 28b. The absorption spectra of p-boronic acid aniline
in different solvents.
[0115] FIG. 28c. p-boronic acid spectra for acid (pH 2) and alkali
(pH 12) conditions.
[0116] FIGS. 29a-c. Response of pyrene boronic acid fluorescence in
the presence of glucose (methanol as solvent). Absorption spectra
(a), fluorescence spectra (b), and relative fluorescence intensity
(c) as a function of glucose concentration.
[0117] FIG. 30. Some Bidentate Glucose reporter molecules.
[0118] FIG. 31. Illustrative examples of SMMDR molecules.
[0119] FIG. 32. SMMDR molecule concepts in which phenylboronic acid
is part of a push-pull fluorophore. Carboxamide groups provide
auxiliary binding.
[0120] FIG. 33. In this image, the glucose interacts with a cyclic
peptide that contains four serine residues, eight glycine
structures, and a tryptophan and a tyrosine residue. The dotted
lines represent hydrogen bonds. The two aromatic residues are above
the plane of the peptide ring and in this conformation would be
expected to undergo efficient energy transfer. The model simply
represents a starting point from which a small glucose binding
peptide might be built.
[0121] FIG. 34. Compounds to test cyclic peptide and crown-ether
recognition of glucose.
[0122] FIG. 35. Conceptual illustration of glucose-binding
reporters that operate by repulsion and size exclusion.
[0123] FIG. 36. SMMDR Development strategy: Library generation and
virtual screening.
[0124] FIG. 37. Combinatorial Libraries based on small cyclic
peptides.
[0125] FIG. 38. Illustration of the action of the
2-phenylquinazolin-4(3H)-one compounds.
[0126] FIG. 39. "Push-Pull" Fluorophores.
[0127] FIG. 40. Predicted wavelength changes due to annellation and
substitution.
[0128] FIG. 41. Predicted Wavelength effect of ring annellation in
a boronic acid probe compound.
[0129] FIG. 42. Comparison of Coumarins and Xanthenes.
[0130] FIG. 43. Comparison of Coumarin, Xanthene, and
Seminaphthorhodafluor analogs.
[0131] FIG. 44. Novel Seminaphthorhodafluor compounds with
predicted long wavelength and ratiometric pH properties.
[0132] FIGS. 45a-c. Spectra of esculetin demonstrating absorbance
(a), fluorescence (b), and absorption ratio at 384 nm/344 nm (c) as
a function of pH.
[0133] FIG. 46. Illustration of the excitation scheme and signal
generated by a phase sensitive flash photolysis apparatus.
[0134] FIG. 47. Schematic overview of a phase sensitive flash
photolysis apparatus.
[0135] FIG. 48. Schematic overview of an in vitro glucose probe not
requiring strip use.
[0136] FIG. 49. Strip technology designs when using direct
fluorescence molecules.
[0137] FIG. 50. Absorption and fluorescence spectra of
Argofluor-327d obtained as a function of glucose concentration.
[0138] FIG. 51. Fluorescence and absorption spectra showing effect
of glucose on the complexation of phenyl boronic acid with
esculetin.
[0139] FIG. 52. Glucose response rate using coumarin-boronic
acid-based reporter.
[0140] FIG. 53. Jablonski diagram illustrating electronic states of
fluorescent molecule.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0141] The features and other details of the invention will now be
more particularly described with reference to the accompanying
drawings and pointed out in the claims. It will be understood that
particular embodiments described herein are shown by way of
illustration and not as limitations of the invention. The principal
features of this invention can be employed in various embodiments
without departing from the scope of the invention. All parts and
percentages are by weight unless otherwise specified.
DEFINITIONS
[0142] The singular forms "a", "and" and "the" include plural
referents unless the context clearly dictates otherwise. For
example, the term small molecule metabolic reporter "SMMR" and/or
"small molecule multi-domain reporter "SMMDR" includes one or more
small molecule metabolic reporters "SMMRs" and/or "small molecule
multi-domain reporters "SMMDRs". Those skilled in the art will
recognize that the terms "SMMR" and "SMMRs", and "SMMDR" and
"SMMDRs" are used interchangeably herein.
[0143] The term "biologically active molecule" includes, but is not
limited to, enzymes, coenzymes, metabolites, analytes, reactive
species, polypeptides, proteins, cofactors, small molecules and
other macromolecules of physiological significance including
mixtures or active fragments or subunits thereof. A "small
molecule" includes a molecule from 100 Da to 250 kDa. Molecules of
this molecular weight range have a demonstrated ability for use as
quantitative reporters of glucose activity.
[0144] The terms "small molecule metabolic reporter(s)", "SMMR
(s)", "analyte enhancing molecules", "reporter" and "reporters"
include, but are not limited to, fluorophores, protein-labeled
fluorophores, proteins with a photooxidizable cofactor (such as
FADH contained in a glucose oxidase), and proteins with another
intercalated fluorophore.
[0145] A "chromophore" includes a molecule exhibiting specific
absorption or fluorescence emission when excited by energy from an
external source. This is a more generic term than fluorophore.
[0146] A "fluorophore" includes a molecule exhibiting specific
fluorescence emission when excited by energy from an external
source.
[0147] An "intercalated fluorophore" includes fluorophores that
will fluoresce when intercalated with a molecule. For example,
Glucose Oxidase-Intercalated Fluorophore (GO-IF) is a molecule with
specific glucose binding sites. The fluorescent properties will
change when glucose binds to the molecule, causing a measurable
change.
[0148] A "dye" includes molecules having large absorptivity or high
fluorescence quantum yield and which demonstrates affinity for
certain materials or organic (cellular) structures.
[0149] A "xanthene dye" includes a molecule having a xanthene-like
skeletal structure, which exhibits large absorptivity and high
fluorescence quantum yield and which demonstrates affinity for
certain materials or organic (cellular) structures.
[0150] The phrase "energy transfer from reducing equivalents (e.g.,
NAD/NADH, NAD(P)/NAD(P)H, FAD/FADH.sub.2) indicating SMMRs" refers
to a use of SMMRs whereby the presence of these reducing
equivalents molecules, is detected by excitation of the reducing
equivalents molecules from an external source, energy transfer from
the reducing equivalents molecule(s) to an SMMR, and detection of
the fluorescence emission at the SMMR emission wavelength.
[0151] The phrase "absorption/diffuse reflection or fluorescence
spectrum" refers to two types of spectra measured independently.
The absorption/diffuse reflection spectrum refers to the energy
reflection spectrum from a material reported in either the
dimensions of reflectance or absorbance versus wavelength. The
fluorescence spectrum is measured independently as the fluorescence
emission intensity or the fluorescence lifetime of a fluorophore
following excitation from an external source.
[0152] The phrase "molecular size attachment" refers to the
molecular size in Angstroms (.ANG.), which is related to molecular
weight in Daltons (Da), of an attachment added as an adjunct to an
SMMR. As used herein, "molecular size attachments" includes adducts
to the fluorescent moieties of SMMRs that include, but are not
limited to, structural modifications of fluorescence SMMRs as the
additions to the fluorescence structure of: acetoxy methyl esters,
chloro-methyl derivatives, alkyl chain adducts, highly charged
moieties, enzyme substrate mimics, enzyme cofactor tethers, and
membrane binding tethers. As used herein, a "reporter" includes an
SMMR having the property of optical or fluorescence signal related
to the quantity of analyte in the immediate vicinity of the SMMR.
Thus, as the analyte quantity increases, the fluorescence signal
changes (up or down) in proportion.
[0153] As used herein, a "marker" includes a molecule having the
property of yielding a fluorescence signal that is constant when
applied to target cells or tissues. Its main purpose is for use as
a reference signal channel. As such, it is applied in a ratiometric
measurement for correction of a reporter signal. The variation in
physiological and optical characteristics of individual subjects
requires a reference channel signal to correct or normalize a
reporter channel signal when the ratio of reporter to marker is
used for quantitative applications.
[0154] As used herein, a "sensor" includes a handheld device
capable of making absorption or fluorescence measurements at one or
more wavelengths, and converting the ratios and sums of these
measurements into analyte concentrations. These analyte
concentrations are used to infer the rate or quantity of a specific
metabolic process.
[0155] As used herein, a "metabolite" includes a substance produced
by a metabolic process, such as glycolysis, which can be
quantitatively measured as an indication of the rate or quantity of
a specific metabolic process.
[0156] As used herein, an "analyte" includes a measurable
parameter, using analytical chemistry, which can be quantitatively
measured as an indication of the rate and quantity of a specific
metabolic process. The term analyte is a generic term describing
such concepts as metabolites, ions, processes, conditions,
physico-chemical parameters, or metabolic results that can be used
to infer the rate or quantity of specific metabolic processes.
[0157] As used herein, a "response range" includes an analyte range
(lower and upper limits) over which a metabolic process, and its
measured absorption or fluorescence signal, follow a linear or
defined mathematical function.
[0158] The phrase "physico-chemical parameter" refers to a subset
of broadly defined analyte parameters specifically related to the
physical chemistry constants of materials. These constants can be
used in combination with the measurement of other analytes to infer
the rate or quantity of specific metabolic processes. Such
constants refer specifically to, e.g., atomic mass, Faraday
constant, Boltzmann constant, molar volume, dielectric properties,
and the like.
[0159] As used herein, "wicking" includes the flow of a liquid into
a solid material via the pull of gravity, Brownian motion,
adhesion, mass transport, or capillary action such that a natural
movement of a liquid occurs into a solid material.
[0160] The phrases "direct metabolic reporters," and "indirect
metabolic reporters" refer to the mechanism of action of SMMR for
reporting glucose concentration. Direct metabolic reporters report
the concentration of glucose directly, whereas indirect metabolic
reporters report the concentration of analytes used to infer the
concentration of glucose.
[0161] As used herein, an "octanol-water coefficient (K.sub.ow)"
includes a measure of the extent to which a solute molecule is
distributed between water and octanol in a mixture. The
octanol-water partition coefficient is the ratio of a chemical's
solubility (concentration) in octanol to that in water using a
two-phase mixture at equilibrium.
[0162] As used herein, "toxicity" includes the degree or quality of
being toxic or hazardous to the health and well being of human and
other mammalian organisms, organs, tissues, and cells.
[0163] The phrase "specialized tattoo" or more precisely the
"active viewing window" refers to an area of tissue treated with an
SMMR. That area is used for viewing the fluorescence ratio
measurements of the SMMR interaction with tissue, in order to
directly measure, calculate, or otherwise infer the concentration
of skin and blood glucose or other metabolites of interest.
[0164] As used herein, "organ" includes a structure that contains
at least two different types of tissue functioning together for a
common purpose. Examples of organs in the body include, but are not
limited to, the brain, heart, liver, kidneys, pancreas, stomach,
intestines, lungs, and skin.
[0165] As used herein, a "keratinocyte" includes a living cell
comprising the majority of the epidermis of mammalian skin. The
keratinocyte is unique in both its proximity to the surface of an
organism as well as in its glycolytic behavior. The keratinocyte
metabolizes glucose in such a way as to produce a number of
analytes whereby the glucose concentration within the cell can be
inferred.
[0166] As used herein, a "mammal" includes both a human and a
nonhuman mammal (e.g., rabbit, mouse, rat, gerbil, cow, horse,
sheep, etc.). Transgenic animals are also encompassed within the
scope of the term.
[0167] The noninvasive devices, compositions, and methods of the
present invention directly yield in vivo information for the
assessment of intracellular and extracellular metabolic state, as
well as the stress status of cells, tissues, and organisms. In a
preferred embodiment, the devices, compositions and methods of the
invention can be used to monitor and determine metabolite
concentration levels, and more specifically, determine blood
glucose concentration levels.
[0168] The invention provides noninvasive sensor compositions that
comprise one or more small molecule metabolic reporters ("SMMRs" or
"reporters"). When applied topically to skin, peripheral tissues,
or organs, these reporters are able to penetrate the upper tissue
layers and interact with a specific biologically active molecule in
such a way as to report metabolic or health status, while not
interfering with metabolic function. The reporters provide a
metabolic signal that can be used for multiple purposes including,
but not limited to, assessment of metabolic function (e.g.,
particularly as related to glucose metabolism); diagnosis of
metabolic disease states (e.g., as related to advanced glycosylated
end-products); monitoring and control of disease state; stress
status of cells, tissues and organs; determination of vitality and
viability of cells based on metabolic function; critical care
monitoring; diagnosis and monitoring of cardiovascular diseases,
autoimmune disorders, neurological disorders, degenerative
diseases; determination of metabolic concentration; and cancer
diagnosis, detection, staging and prognosis. Specifically, applying
the reporters of the invention to living peripheral or epithelial
tissue provides detailed information on the state of multiple
metabolic pathways in living organisms that can be analyzed using
low-cost, hand held instrumentation.
[0169] The invention provides techniques whereby one or more
reporters are applied to solid tissue (i.e., are introduced to the
upper cell layers of tissues and organisms following local and/or
topical administration). The reporters are added in trace
quantities (from about 10 to about 1000 .mu.L of 0.1 to 200 .mu.M,
preferably from about 5 to about 100 .mu.L), using a substance that
is transparent to visible light and that has a pre-specified
temporary residence at the application site (e.g., 2 days-up to 30
days, 24-48 hours, preferably 2-6 hours, more preferably 30 seconds
to 5 minutes, and most preferably 5 seconds to 5 minutes).
Contemplated diffusion times include periods less than 48 hrs, 24
hrs, 10 hrs, 6 hrs, 2 hrs, 1 hr, 30 min, 15 min, 10 min, 5 min, 1
min, 30 sec, 10 sec, or 1 sec. Reporters that are placed on skin
are able to penetrate the skin and be transported to a depth from
the surface of from about 10 .mu.m to about 300 .mu.m into the
tissue and are brought in contact with a specific metabolite,
wherein a change in fluorescence or absorption (e.g., measured
using fluorescence or absorption spectroscopy) of the one or more
reporters occurs, thereby allowing quantification of the change in
fluorescence or absorption that provides detailed in vivo
information regarding picomolar through millimolar cellular
metabolite and precursor levels for living tissue, organs,
interstitial fluid, and whole organisms.
[0170] The reporters can be monitored noninvasively using any
low-cost instrumentation capable of directly analyzing the
metabolic state in tissue (e.g., using optical instrumentation).
The reporters are chosen to specifically enhance the signal of
pre-specified analytes in order to assess metabolic state of a
tissue or organism and to yield detailed, real-time information
regarding the state of intracellular and extracellular
metabolism.
Discussion of Properties of SMMR Compounds: Physicochemical,
Photochemical, Photophysical and Biological
[0171] SMMR compounds consist of elements of molecular
sub-structure which confer the special properties required to
fulfill their specific metabolic reporter function: [0172] 1. A
fluorescent reporter, with specific photophysical properties,
[0173] 2. Chemical functional groups that confer affinity to
metabolites, enzymes, cell organelles, membranes, or glucose
itself, [0174] 3. Structural features that confer specificity
between the target of interest and similar targets, which are
present in the biological medium (e.g., glucose versus
fructose).
[0175] The chemistry of small molecule metabolic reporter (SMMR)
compounds combines a number of parameters that, in general, results
in the following characteristics: SMMR compounds are nontoxic; they
have high molar absorption coefficients, and a high quantum yield
of fluorescence. They have a large Stokes shift; they are readily
taken up by cells, and are retained in the active form at the
target tissue. They undergo a large change in fluorescence in
response to the metabolism monitored, they are photostable, they do
not exhibit excited state chemistry, and they are eventually lost
from the body by shedding of the stratum corneum. The molecular
design behind these characteristics is the subject of this
invention.
Toxicity
[0176] The metabolic impact of the compounds to be used as SMMRs is
low because of a number of properties that are common to these
molecules. When minute quantities may be absorbed into the body
they are readily eliminated from the system via biotransformation
(metabolism) of the aromatic rings through hydroxylation by a
variety of nonspecific enzymes contained within the microsomes of
the liver endoplasmic reticulum. Small, more water soluble
metabolites result, which are then eliminated from the body by
passing through the glomeruli into the proximal tubules of the
kidney and into the urine. SMMRs have strict requirements relative
to toxicity. Four main criteria must be met: (1) they do not bind
to DNA, (2) they do not disrupt cell membranes, (3) they are used
at low concentration (e.g., 50 .mu.L of 250 .mu.M), and (4) the
SMMR is delivered to a limited volume of tissue, typically the
viable epidermis.
[0177] The Activity Index (A.I.) of the SMMR compounds is an
indication of the effective dose required to elicit an appropriate
response to a metabolic signal or glucose. It is indicated by the
ratio of the Toxic Dose (T.D.) to the Effective Dose (E.D.) as in
equation (1). Note that the A.I. for any proposed SMMR must be
greater than 1.0 and ideally should be 10,000 or more.
A . I . = T . D . E . D . ( EQ . 1 ) ##EQU00001##
[0178] A better indication of the safety of the SMMR would be
indicated by the Minimum Activity Index (A.I..sub.min) as the ratio
of the Maximum Tolerated Dose (T.D..sub.max) for 100 percent of the
tested group, indicating a maximum dose at which no adverse effects
occur, to the Maximum Effective Dose used (E.D..sub.max), whereby
the maximum signal occurs as equation (2). The larger the
A.I..sub.min the better toxicity to effective signal
characteristics the SMMR possesses. This number must always exceed
1.0 with values greater than 5.0 considered optimum.
A . I . min = T . D . max E . D . max ( EQ . 2 ) ##EQU00002##
[0179] SMMR molecules with quantum yield (.phi..sub.F) values
substantially less than unity (e.g., less than 0.3) or those with
longer fluorescent lifetimes may show phototoxicity via a
photodynamic effect, a process that is unrelated to the inherent
toxicity of the SMMR. Phototoxicity arises from reactions of an
excited state or from a reactive intermediate generated by the
excited state. Phototoxicity can be minimized by using low light
doses combined with high quantum yields (e.g., less than 5 mW
excitation with a .phi..sub.F greater than 0.6), thereby decreasing
the energy available to form damaging oxygen radicals, and the
number of excited states generated, respectively.
[0180] As a general rule, exogenous materials such as drugs or,
with reference to this embodiment, SMMRs that interact with more
than one metabolic pathway have a reduced likelihood of becoming
clinically significant due to the availability of compensatory
pathways if one is inhibited. Interactions, and hence toxicity, are
likely to be increased if the SMMR is an inhibitor or inducer of a
particular enzyme, if the response of the SMMR is critically
dependent on the concentration and particularly if turnover of the
SMMR occurs via a single specific pathway (see, for example,
Johnson, M. D. et al. Clinically significant drug interactions,
Postgraduate Medicine 1999; 105(2): 193-222).
[0181] To a large extent the rationale for SMMR design has avoided
many of these problems by targeting normal overall changes in the
chemistry of the cell rather than the concentration or activity of
a specific metabolite. For example, the monitoring of reducing
equivalents within a cell by energy transfer does not affect the
overall intracellular concentration of those equivalents nor are
there interactions between the enzymes responsible for metabolism
and the SMMRs. For some molecules, the toxicity or carcinogenicity
is not related to the parent molecule but the metabolite of the
molecule. This is often a result of activity of the liver on the
molecule. For example, metabolites of benzene are formed by the
action of cytochrome P450 in the liver to form, epoxides, phenol,
catechol and muconaldehyde. Many of these compounds are extremely
reactive and toxic and they may be metabolized further to other
toxic materials.
Molar Absorption Coefficient
[0182] The molar absorption coefficient (s) of a typical SMMR is
high (greater than 50,000 dm.sup.3 mol.sup.-1 cm.sup.-1). This
implies that the probability of a transition from
S.sub.0.fwdarw.S.sub.1 is high. The amount of light absorbed is
given by the Beer-Lambert law as equation (3).
log I 0 I 1 = A = cl ( EQ . 3 ) ##EQU00003##
[0183] Where, I.sub.0 is the intensity of the incident light,
I.sub.t is the intensity of the transmitted light, A is the
absorbance, .di-elect cons. is the molar absorption coefficient, c
is the concentration and 1 is the pathlength. This expression can
also be rearranged to give the fraction of incident light that is
absorbed (I.sub.a) as described in equation (4).
I.sub.a=1-10.sup.-A (EQ. 4)
[0184] For an SMMR concentration of 10 .mu.M in a sample thickness
of 100 .mu.m (typical skin thickness for skin epidermis), a molar
absorption coefficient of 50,000 dm.sup.3 mol.sup.-1 cm.sup.-1
results in 11% of the incident light being absorbed. Ultimately,
the more light absorbed the more will be converted into
fluorescence. In designing an SMMR a high molar absorption
coefficient (.di-elect cons.) is important; practically this means
values greater than 50,000 dm.sup.3 mol.sup.-1 cm.sup.-1.
[0185] The probability of the S.sub.0.fwdarw.S.sub.1 transition
occurring can be explained in molecular terms by consideration of
the type of bonding that is present in the SMMR. A high probability
for the transition requires good overlap between the orbitals in
the ground and excited state. Such overlap is found for .pi.-.pi.*
transitions and charge transfer states. Transitions involving
nonbonding electrons, i.e., n-.pi.* are not as probable and hence
the molar absorption coefficients are lower for these type of
transitions. However, the electrons in a nonbonding orbital are
higher in energy than electrons in a bonding orbital and therefore
the n-.pi.* transition occurs at lower energy than the .pi.-.pi.*
transition.
[0186] Because of the low probability of the n-.pi.* transition the
excited state generated by such a transition is expected to be
longer lived. Since the balance between the rate constants for
radiative and nonradiative decay determines fluorescence quantum
yield a long lifetime, in general, allows a greater probability for
radiationless decay to occur. Hence, n-.pi.* transitions tend to be
nonfluorescent.
Fluorescence Quantum Yield
[0187] Following absorption of a photon the SMMR is promoted to an
electronically excited state. The molecule undergoes vibrational
changes and interactions with the solvent that result in relaxation
to the state from which fluorescence occurs. If this state only
undergoes spontaneous emission then the yield of fluorescence is
high (.phi..sub.F=1). If the state undergoes any other process,
such as internal conversion or other photophysical change, then
.phi..sub.F<1. The fluorescence quantum yield (.phi..sub.F) of
an ideal SMMR is close to unity. Compounds with .phi..sub.F less
than unity are likely to be less photostable and more
photoreactive. The factors that are common in the design of an SMMR
that result in a high quantum yield are:
[0188] 1. Rigidity of the molecule. Constraining a molecule limits
the number of vibrational modes by which the excited state can be
deactivated. Binding of ethidium bromide to DNA for example,
increases the quantum yield of the molecule by 30 times.
[0189] 2. Lack of heavy atom effect. Heavy atom substituents, such
as iodine or bromine, cause spin orbit coupling in a molecule and
facilitate intersystem crossing. The excited singlet state for such
a molecule can readily form a triplet state. Not only does this
process decrease the fluorescence quantum yield but it also
generates a potentially long-lived reactive state.
[0190] 3. Bonding character. As mentioned above, n-.pi.*
transitions are, in general, not only weakly absorbing but also
nonfluorescent. .pi.-.pi.* transitions have high molar absorption
coefficients and are fluorescent. For molecules that have both an
electron donating and withdrawing group attached to the .pi.
system, the transitions that can occur are described as charge
transfer. Typical electron donating groups include amine and
hydroxyl groups and withdrawing groups include carbonyl and nitro
groups. The transitions are intense and if the transition is of the
lowest energy, they are also fluorescent.
[0191] From this discussion, it is apparent that the intensity or
brightness of fluorescence is determined by the product of the
molar absorption coefficient and the quantum yield (.di-elect
cons..phi..sub.F). If brightness is sufficiently high, then even at
low concentration, the SMMR absorbs excitation light strongly and
efficiently converts this energy to fluorescence. As a general rule
then the minimum requirement for an SMMR is that .di-elect
cons.>50,000 dm.sup.3 mol.sup.-1 cm.sup.-1 and
.phi..sub.F>0.2; thus brightness or .di-elect
cons..phi..sub.F.gtoreq.25,000 dm.sup.3 mol.sup.-1 cm.sup.-1.
Stokes Shift
[0192] The Stokes shift is the difference in energy between the
lowest energy absorption and the highest energy emission of a
molecule. The advantage in having a large Stokes shift is that it
is much easier, from a practical standpoint, to optically eliminate
the influence of the excitation light on the detected light, i.e.,
the bandpass filter requirements are simplified.
[0193] Consideration of a Jablonski diagram (FIG. 53) would imply
that for an S.sub.0 to S.sub.1 transition, involving only the
lowest vibrational levels, the energy of the fluorescence should be
the same as the absorption. This is almost never precisely the
case. The main reasons are threefold:
[0194] 1. Consideration of the bond order in the transition. In a
.pi.-.pi.* transition the electron distribution in the excited
state involves nonbonding orbitals. As a result the bond length and
the magnitude of the vibration in that bond increases. This process
results in a loss of energy and therefore the fluorescent
transition occurs from an excited state slightly lower in energy
that the state generated by the absorption.
[0195] 2. The lower bond order in the excited state literally
causes the molecule to expand. This change in volume is measurable
using photoacoustic spectroscopy. For a molecule in solution to
expand requires work to be done in pushing back the solvent. This
work results in a loss of energy in the excited state and an
increase in the Stokes shift. The volume change increases as the
size of the .pi. system increases.
[0196] 3. The greater the degree of flexibility in the molecule the
more the number of vibrational modes available to the molecule. For
a complex molecule energy may be lost from parts of the molecule
not associated with the chromophore. Any mechanism that causes the
molecule to lose energy, including solvent and intermolecular
interactions, will lead to a decrease in the energy of the observed
fluorescence.
[0197] It is noted that factors that serve to increase the
magnitude of the Stokes shift also serve to lower the overall
fluorescence quantum yield. A novel method to increase the
separation of the excitation and emission wavelengths for an SMMR
is to covalently link two fluorescent probes together. In practice
the molecule that absorbs the light need not even be fluorescent
provided its excited state lifetime is long enough to transfer
energy to the acceptor molecule that fluoresces. This kind of
system increases the design flexibility of the SMMR. Proposed
examples to monitor pH include a molecule where both the donor and
acceptor are sensitive to pH and because of electrostatic changes
associated with pH changes, the energy transfer process is also
sensitive. Ratiometric measurements of the donor and acceptor
fluorescence show very large changes as a function of pH.
[0198] An ideal SMMR has a Stokes shift of about 50 nm or more.
Xanthene dyes typically have a Stokes shift of 5-15 nm but this
value is also dependent on the pKa of the molecule. As an example,
BeXan type dyes exhibit Stokes shifts of about 40 nm for the acid
form and 60 nm for the basic forms of the dye.
Predicting Spectroscopic Properties from Molecular Structure
[0199] The prediction of spectroscopic properties such as
absorption and emission spectra are very difficult. The absorption
of a photon involves the promotion of an electron from a ground to
excited state. There is currently no molecular calculation
algorithm nor automated software package that can take into account
the chemical microenvironment of a molecule. The position of the
S.sub.1.rarw.S.sub.0 transition is calculated by considering the
energy of the molecule in its ground equilibrium configuration and
the energy of the excited state in the same geometry, which is not
an equilibrium conformation for the excited state. This is a
consequence of the fact that an electronic transition occurs with
no change in the geometry of the molecule during the transition
(the Born-Oppenheimer approximation). The intensity of the
absorption is dependent on the probability of the transition. In
general .pi.*.rarw..pi. transitions are intense and have molar
absorption coefficients of 10.sup.5 dm.sup.3 mol.sup.-1 cm.sup.-1
or greater. High probability is favored by strong overlap between
the ground and excited state orbitals.
Affinity to Target Site
[0200] Designing SMMR compounds to have an affinity for specified
cellular locations, membranes, or structures helps reduce noise in
an SMMR measurement. When the SMMR is targeted to specific cell
locations, both the immediate chemical microenvironment of the SMMR
molecule as well as its location for optical measurement can be
more closely controlled. SMMRs can be designed to have high
affinity for membranes, organelles, charged structures including
specific membrane layers, biopolymers, protein or enzyme binding
sites, and regions of the cell that are particularly hydrophobic or
hydrophilic. Absolute specificity may be conferred on an SMMR by
binding it to a variety of other membrane specific binding
substituents including the use of antibodies.
[0201] Examples of an SMMR structure designed to confer target
affinity are given here:
[0202] Membrane Affinity:
[0203] Amphipathic molecules have a high affinity for membranes.
Hydrophilic molecules become amphipathic when an alkyl chain is
linked to the structure. This linkage is not necessarily covalent
in nature. Electrostatic complexes of cationic detergents and
methyl viologen, for example, are stable and bind strongly to
membranes. In binding to a membrane, the alkyl chain is solubilized
in the hydrophobic core of the membrane and the hydrophilic head
group is located at the surface of the membrane. This type of
molecule is particularly effective at monitoring changes at the
interface between the bulk phase and the membrane that include
membrane potential and pH changes, either of which may be used to
track glucose concentration at the cell.
[0204] Enzyme Binding Site Affinity:
[0205] SMMR affinity for an enzyme-binding site may be conferred by
covalently linking a model enzyme substrate or enzyme cofactor to
the SMMR, e.g., as disclosed in pending U.S. Application No.
60/438,837, entitled "Method for Noninvasive, in vivo monitoring of
Blood Glucose Levels," filed Jan. 9, 2003, that discusses the
binding of an SMMR to the FAD cofactor of glucose oxidase, the
entire disclosure of which is incorporated herein by reference.
Skin Uptake
[0206] One of the most important functions of skin is to protect
the essential tissues of the body from the outside environment. The
layer of skin that forms the physical barrier for the body,
preventing moisture loss, infection and regulating temperature is
the stratum corneum. This cell layer is both hydrophobic as well as
acidic, and thus presents a number of problems to transdermal drug
delivery. The problem is exacerbated in SMMR technology, since the
goal of delivering an SMMR to the epidermis is to have the dye pass
through the stratum corneum but to localize in the living epidermal
layer. The factors that affect skin uptake include molecular size,
hydrophobicity and volatility.
[0207] The ideal characteristics of an SMMR, useful to penetrate
the stratum corneum, include a low molecular weight (less than 600
g mol.sup.-1), a partition coefficient of about 10, yielding good
solubility in lipid and water phases and a low melting point (see,
for example, "Novel mechanisms and devices to enable successful
transdermal drug delivery," B. W. Barry. Eur. J. Pharm. Sci. (2001)
14 101-114). A low melting point correlates with high solubility
since there is little interaction between the molecules.
[0208] For an SMMR that will eventually localize in the cytoplasm
or the interstitial fluid, the molecule must have good water
solubility. However, a hydrophilic molecule will have difficulty in
passing the hydrophobic environment of the stratum corneum. These
opposing properties of the molecule imply that the design of the
molecule has to be altered to accommodate both environments, that
the molecule is amphipathic or that the delivery system can
accommodate both polar and nonpolar molecules. Delivery systems are
customized for individual SMMR properties, and the
preparation/formulation of such delivery systems are within the
skill of one of ordinary skill in the art, taken with the
disclosure herein. It is well known that mixed solvent systems
using mixed organic solvents for the organic phase have a
significantly greater range of properties. Other solvent systems
include the use of surface-active agents to expand the range of
properties of the vehicle.
Cellular Uptake
[0209] To a certain extent the factors that affect uptake in the
skin are the same as those that affect cellular uptake. Overall,
there has to be a balance between movement of the SMMR into the
stratum corneum, diffusion into the epidermis, and then the
competing processes of SMMR uptake by the epidermal cells and loss
of the dye into the dermis. All of these processes may be
considered to be reversible. The most important factors that
facilitate cellular uptake of a small molecule include the
partition coefficient (P), and the molecular size and the diffusion
coefficient in the lipid matrix (D). Diffusion across a biological
membrane is dependent on three factors: the diffusion of the SMMR
from the aqueous phase to the lipid phase, diffusion within the
lipid phase and diffusion from the lipid phase back into an aqueous
phase. For a molecule whose flux across the membrane is
proportional to the concentration gradient the proportionality
constant is called the permeability coefficient (Cp) and it is
directly dependent on the three factors above. The permeability
coefficient is given by the expression in equation (5).
C p = DP x ( EQ . 5 ) ##EQU00004##
[0210] Where x is the distance across the membrane, or more
accurately the distance across the concentration gradient, the
other terms are as defined above. For most SMMRs the permeability
is directly proportional to the partition coefficient although this
relationship does not hold for very small molecules.
[0211] The pK.sub.a is important, since this will determine the
overall charge of the molecule at a particular pH and hence the
partition coefficient. For many pH indicating xanthene compounds
there are two pK.sub.a values of importance. As an example,
fluorescein
##STR00036##
will be considered. The pKa of fluorescein is 6.4. Below this pH,
the hydroxyl group drawn at position 3 is protonated. In practice,
the oxygen atoms at positions 3 and 6 are equivalent, and above pH
6.4 the xanthene ring is negatively charged with electron density
shared between these two positions. The carboxylic acid at the 2'
position is unprotonated until the pH drops to about pH 4.2. Below
a pH of 2.2, the carboxylic acid ring closes at position 9, there
is a hydrogen shift onto the carbonyl at position 6 and the quinoid
structure no longer exists. Under these conditions the compound is
colorless. The relevance of this parameter to drug delivery in the
skin is that this type of compound displays many of the
characteristics that facilitate SMMR delivery.
[0212] SMMRs designed with acidic functionality have greater
potential for skin and cellular uptake. In skin, the stratum
corneum is acidic and the surface of cell membranes is also acidic.
Therefore the use of SMMRs with acidic substitutions result in a
significant fraction of compound molecules being uncharged at pH 5
(close to the values reported in the stratum corneum and at the
cell surface). These molecules are therefore more hydrophobic, may
cross the barrier and once inside the cell, at .about.pH 7 become
deprotonated again. Suitable groups include carboxylic acids (pKa
.about.4-5) and aromatic thiols (pKa .about.8). Both of these
groups are electrostatically neutral when protonated.
[0213] Cellular uptake and retention may be improved by the use of
polyoxyethylene chains or polylysine chains. The purpose of these
substitutions are to give the SMMRs both hydrophobic and
hydrophilic properties. The molecule behaves as a cross-linked
micelle changing its conformation and degree of order depending on
its environment. The chromophore itself is hydrophilic but it can
be solubilized in nonpolar environments by the long alkyl chain. In
a hydrophobic environment, the chain wraps around the molecule and,
if a poly-oxaethylene is used the chain is configured to present
the oxygen atoms of the chain towards the interior of the complex.
In a hydrophilic environment, the chain unwraps and the SMMR is
exposed.
[0214] SMMR species connected by flexible saturated chains behave
as individual chromophores when located in environments where the
monomer is soluble. Under conditions where the monomer is not
soluble, the dimer will fold to present the smallest volume of the
molecule to the environment. This approach has previously been used
to deliver photodynamic therapeutic (PDT) agents to tissues. The
folded dimer, which is often nonfluorescent, may cross the cell
membrane and unfold when it reaches its cytoplasm related target.
The unfolded dimer is again photochemically active.
[0215] An example of a dimeric BeXan type molecule is shown
below:
##STR00037##
[0216] It is readily synthesized from the chloromethyl derivative
of the monomer reacting with a dithioalkane. Thioethers are known
to be biologically stable molecules (see, e.g., Effect of linker
variation on the stability, potency, and efficacy of
carcinoma-reactive BR64-doxorubicin immunoconjugates. P. A. Trail,
D. Willner, J. Knipe, A. J. Henderson, S. J. Lasch, M. E. Zoeckler,
M. D. TrailSmith, T. W. Doyle, H. D. King, A. M. Casazza, G. R.
Braslawsky, J. Brown, S. J. Hofstead, R. S. Greenfield, R. A.
Firestone, K. Mosure, K. F. Kadow, M. B. Yang, K. E. Hellstrom and
I. Hellstrom. Cancer Research, (1997) 57(1) 100-105; and Enhancing
selectivity, stability, and bioavailability of peptidomimetic
estrogen receptor modulators F. Spatola, A. K. Galande, F. M.
Brunel, K. S. Bramlett, and T. P. Burris, Presented at the
18.sup.th American Peptide Symposium, Jul. 19-23, 2003, Boston,
Mass.).
Cellular Retention
[0217] The compounds used as SMMRs must be retained inside the cell
so that repeated applications are not necessary for an SMMR
monitoring device to function over extended periods from 1 hour to
30 days. Many of the same factors that determine how well an SMMR
will cross the membrane also determine whether the compound will
leak out of the cell. The principal factors that influence compound
retention include: charge, size, polarity, pKa, and the presence of
groups that interact with cellular components. Assuming that the
principal mechanism for leakage out of the cell is diffusion across
the membrane, the SMMR compound should be large, hydrophilic and
preferably negatively charged to prevent leakage.
[0218] Molecules with molecular weights of greater than 600 g
mol.sup.-1 will be retained to a much higher degree than smaller
molecules. Negatively charged species are electrostatically
repelled from cell membranes. Cell membranes have a high pH
gradient near the surface. Negatively charged surfaces attract
cationic species, which include the highly mobile hydrogen ion, in
a layer called the Stern layer. This layer, located near the
membrane surface, may be as much as two pH units lower than the
phase of the bulk membrane. Therefore, to maintain charge in the
vicinity of a membrane, it is important that at least some of the
protonatable groups on the molecule have pKa values less than pH
5.
[0219] Quantitatively, leakage from the cell may be measured by
monitoring the fluorescence of the interstitial fluid or medium in
which the cells are bathed. The leakage is dependent on the
concentration gradient and therefore leakage will be higher in a
cell culture type measurement than in a skin type system. For
example, if a 35 mm diameter dish confluent with keratinocytes and
bathed with 1 ml of medium is compared to the same number of cells
stacked as the cells in the epidermis, then the apparent leakage
rate would be at least 30 times higher in the cell culture system.
The leakage rate may ultimately be compared to the target site
affinity (T.S.A.) parameter (equation 3):
T . S . A . = S M M R active S M M R delivered ( EQ . 3 )
##EQU00005##
[0220] The T.S.A. value is related not only to the leakage rate but
also to the rate at which the SMMR is metabolized or photobleached
within the cell. These are the same parameters that would have
prevented the compound entering the cell initially. Therefore,
ideally the SMMR should be converted to this type of molecule after
it has entered the cell. This conversion has been accomplished in a
number of ways.
[0221] Esterification of an SMMR leads to a hydrophobic molecule
that can cross the cell membrane. Once inside the cell, the ester
is cleaved off by esterase enzymes, generating a charged molecule
that cannot readily pass back out of the cell across the membrane.
Reduced compounds such as dihydrorhadamines are hydrophobic and may
be oxidized inside the cell to form the fluorescent, hydrophilic
form of the compound. Other methods that have been used include
substitution of a chloromethyl group that interacts with thiol
groups leading to conjugation with proteins or hydrophilic moieties
preventing leakage from the cell.
[0222] The affinity of the SMMR to the target tissue is also given
as the Target Site Affinity (T.S.A.). The T.S.A. indicates the
percent of SMMR that remains active at the target site after
physical delivery to the site. It is reported as a time dependent
phenomenon relative to 1 hour, 24 hours, and 72 hours. The T.S.A.
for any time period is given in equation (3) as the ratio of moles
of SMMR delivered to moles of SMMR active at the delivery site.
Metabolic Monitoring (Indirect Glucose Measurement)
[0223] SMMR technology is designed to specifically target metabolic
pathways. For SMMRs designed to track glucose these pathways have a
direct relationship with the in vivo glucose concentration. For the
most part biological pathways do not stand in isolation from other
processes that occur in the body. It is therefore possible to
improve the sensitivity of monitoring by targeting more than one
pathway at the same time. For example, it is useful to know the
percentage of metabolism that occurs by oxidative phosphorylation,
and the fraction that occurs by anaerobic metabolism. This
knowledge allows different cell types or cells under different
conditions to be compared. Increasing the number of pathways
monitored increases the specificity of the measurements, the
dynamic range (since measurements can be made under a wider array
of conditions) and decreases the influence of competing processes.
As an example of the use of SMMR technology to monitor glucose
concentration, the pathways that would be targeted are shown in
FIG. 1. The technique to monitor each pathway is given.
[0224] SMMR technology is able to monitor glucose transport through
the use of membrane bound reporters that respond to the activity of
the glucose transporter molecule (GluT). It has previously been
shown that the kinetics of GluT may be monitored from the
autofluorescence of tryptophan residues in the protein. SMMR
technology can monitor the GluT protein either by energy transfer
from tryptophan to the dye, or by monitoring membrane dynamics in
the vicinity of the GluT protein.
[0225] Under conditions where there is excess glucose, cells can
convert glucose to glycogen. Glycogen is stored within the
cytoplasm of cells as small granules. The size of these granules is
fairly uniform and is on the order of tens of nanometers in
diameter. As the amount of glycogen stored increases the number of
granules increases not the size of the granules. Glycogen synthesis
is measured in tissue biopsies using the absorption of the
glycogen:iodine complex at 460 nm. Thus, this aspect of the glucose
metabolism pathway is measurable using optical means.
[0226] In tissue that undergoes primarily anaerobic metabolism, the
products of the glycolysis reaction pathway are lactate and
adenosine triphosphate (ATP). ATP is synthesized from ADP, the
diphosphate analog, and inorganic phosphate. Lactate is generated
as a waste product of the pathway. The lactate concentration within
the cell is dependent on lactate transport out of the cell and the
rate of glycolysis. The extracellular lactate concentration is
dependent on lactate transport and diffusion of lactate into the
blood stream. Published work has correlated the production of
lactate with intracellular pH. Both intra and extracellular pH is
measured using SMMR technology with ratiometric monitoring. To
monitor the pH values simultaneously, dyes with different
spectroscopic properties are used. To use SMMRs with overlapping
spectra requires the SMMRs to be applied to different regions of
the skin and then repetitive measurements to be made at each
site.
Photostability
[0227] The photostability of a fluorophore is a function of the
magnitude of the quantum yield. If the excited state of the SMMR
undergoes any process other than radiative or decay via a
vibrational cascade the possibility for a photochemical reaction to
take place and an attendant loss of photostability. The loss of
fluorescence is the result of a photochemical reaction, often
involving the excited state of the compound, and the generation of
a photoproduct. This process is generally called photobleaching,
which means literally the loss of color. Photobleaching is often an
oxidation process and the degree of photobleaching may be
proportional to the number of excited states generated. Therefore,
photobleaching can be minimized by using low intensity excitation
light, a low oxygen concentration and by increasing antioxidant
concentrations.
Excited State Chemistry
[0228] The processes that can lead to a photochemical reaction
include: energy transfer from the excited singlet state (S.sub.1),
electron transfer from S.sub.1 energy transfer from the excited
triplet state (T.sub.1), electron transfer from T.sub.1 formation
and subsequent reaction of singlet oxygen
(O.sub.2(.sup.1.DELTA..sub.g)). These reactions are summarized in
the following FIG. 2) where: [0229] SMMR+hv represents the
absorption of a photon [0230] .sup.1SMMR* is the first excited
singlet state [0231] .sup.3SMMR* is the first excited triplet state
[0232] S represents some biological substrate [0233] +.cndot. and
.cndot.- represent a semioxidized and semireduced species
respectively O.sub.2(.sup.1.DELTA..sub.g) is singlet oxygen [0234]
i.s.c. is intersystem crossing.
[0235] The k terms in the diagram are the rate constants for each
process. Elt and Ent refer to electron transfer and energy transfer
respectively. For an SMMR to have a high quantum yield the rate
constant for fluorescence has to compete with all of these
processes.
Turnover
[0236] The turnover of the SMMR is related to a number of factors
including: photostability, localization, metabolic activity
involving reaction with the SMMR, leakage out of cells, uptake into
the blood stream, migration into the stratum corneum and loss to
the environment. Some SMMR turnover is an advantage since the
process reduces the potential for a photobleached compound or
compounds to migrate into a nonactive region of the tissue.
Turnover due to photochemical effects has been discussed
earlier.
SMMR Reporting Activity
[0237] The activity of an SMMR is dependent on its response to the
metabolic pathway to which it is targeted as well as its ability to
reach the site of that pathway. The chemical properties of the
compound that determine its potency include pKa, excited state
energy levels, .phi..sub.F, .di-elect cons., octanol:water
partition coefficient, and the selectivity of the SMMR for the
targeted pathway.
[0238] The design of a suitable SMMR involves the correlation of
the chemical properties of the SMMR with the biological reporting
activity of the compound. To be able to do this it is critical that
the reporting activity of the compound be quantified so that
different compounds can be compared and a correlation derived.
[0239] There are several parameters that can be determined to
measure the efficacy of an SMMR. These parameters include: minimum
concentration that can be detected using fluorescence, smallest
change in analyte concentration that results in a measurable
spectroscopic change, and dynamic range in the SMMR response.
[0240] In a series of papers published in the 1960's, Hansch and
co-workers described how certain aspects of a drug structure could
be related to its activity. (Comparison of parameters currently
used in the study of structure-activity relationships. A. Leo, C.
Hansch and C. Church. J. Med. Chem. (1969) 12(5) 766-771; Homolytic
constants in the correlation of chloramphenicol structure with
activity. C. Hansch, E. Kutter and A. Leo. J. Med. Chem. (1969)
12(5) 746-749; Passive permeation of organic compounds through
biological tissue: a nonsteady-state theory. J. T. Penniston, L.
Beckett, D. L. Bentley and C. Hansch. Mol. Pharmacol. (1969) 5(4)
333-341; The linear free-energy relationship between partition
coefficients and the binding and conformational perturbation of
macromolecules by small organic compounds. F. Helmer, K. Kiehs, and
C. Hansch. Biochemistry. 1968 7(8) 2858-2863; Correlation of ratios
of drug metabolism by microsomal subfractions with partition
coefficients. E. J. Lien and C. Hansch. J. Pharm. Sci. (1968) 57(6)
1027-1028.) In particular, the hydrophobicity of a molecule
described how it could partition between tissue and bodily
fluid.
[0241] A similar approach for SMMR design may be used to provide a
semi-empirical approach to SMMR design Two examples are given, one
for an SMMR that monitors a biological pathway, such as glycolysis,
via a change in intracellular pH; and one for an SMMR that is used
to monitor a biological pathway, such as glycolysis, via the
overall reduction potential of the cell through energy
transfer.
WPM Reporting Glycolysis Via a Change in Intracellular pH
[0242] Using an analogous rationale to that described by Hansch, an
empirical equation (4) that would allow the prediction of the
smallest concentration of an SMMR that could be detected from its
fluorescence following application to the skin is provided as
follows.
log ( 1 [ C ] ) = k 1 log A .phi. F + k 2 log P - k 3 ( log P ) 2 +
k 4 pK a + k 5 ( EQ . 4 ) ##EQU00006##
Where:
[0243] C is the smallest concentration that is detectable in the
skin, [0244] A is the absorbance of the solution, [0245]
.phi..sub.F is the quantum yield of the compound, [0246] P is the
octanol water coefficient, [0247] pK.sub.a is the pKa of the
compound, [0248] the constants k.sub.1 through k.sub.5 are
empirically determined constants obtained through linear
regression.
[0249] The determination of the unknown parameters in this equation
requires that at least five times the number of observations be
made, as there are terms in the equation. In the example given here
at least twenty-five observations would have to be made.
[0250] The term k.sub.1 log A.phi..sub.F describes the probability
of the SMMR absorbing a photon and reemitting it as fluorescence.
The higher the absorbance and the quantum yield, the more likely is
the absorption of a photon and the generation of fluorescence. The
term k.sub.2 log P-k.sub.3(log P).sup.2 describes the partition
between hydrophobic and hydrophilic phases within an organism. P is
the octanol water partition coefficient and has been shown to
describe the distribution of a solute between the bulk aqueous
phase and the hydrophobic phase of a lipid bilayer such as a cell
membrane. The optimum value of P is some intermediate value.
Hydrophilic molecules remain in the bulk phase while hydrophobic
molecules are not solubilized and therefore are not carried to the
cell membrane.
[0251] The pK.sub.a is included in this equation because it is
related to the partition coefficient of the SMMR. Protonation of
basic groups and deprotonation of acidic groups lead to an increase
of charge in the molecule and hence increased hydrophilicity.
Equation (5) is a relatively simple equation that merely describes
the factors that control the uptake of a molecule into a cell
membrane. For an SMMR to be effective, the molecule must be
retained in the cell or tissue. To a certain extent, retention is
described by the same factors that describe uptake of the compound.
An equation that describes the response of an SMMR to a change in
pH caused by the activity of a metabolic pathway is given here:
log R = k w log ( A DH .phi. F DH A D .phi. F D ) + k x pK a + k y
log P + k z ( EQ . 5 ) ##EQU00007##
Where:
[0252] R is the difference in the fluorescence under the extreme
conditions of the metabolic pathway, maximum and minimum activity.
[0253] The superscript DH and D refer to the protonated and
deprotonated forms of the compound respectively.
[0254] All other terms are as in equation (1) and the constants
k.sub.w through k.sub.z are determined empirically as before.
Essentially the first two terms in this equation form the
Henderson-Hasselbalch equation (Die Berechnung der Wasserstoffzahl
des Blutes auf der freien und gebundenen Kohlensaure desselben, und
die Sauerstoffbindung des Blutes als Funktion der Wasserstoffzahl.
K. A. Hasselbalch. Biochem. Z. (1916) 78, 112-144). The log P term
appears because the SMMR must localize in a similar region of the
cell as the location of the biochemical pathway.
[0255] Further discussion of the design concept, and compounds and
related constructs are described in the following examples.
Example 1
Using pH to Track D-Glucose Concentration in Living Cells
[0256] For human keratinocytes, the carefully measured
intracellular pH (as a measure of lactate production) is directly
proportional to the concentration of D-glucose entering the cell.
Thus, a decrease of intracellular pH is indicative of an increase
in glucose concentration. The lactate formation within the cell is
in direct proportion to the quantity of glycolysis occurring within
the cell, and this glycolysis is `fueled` by D-glucose and other
simple sugars, such as fructose and galactose. This example
demonstrates the protocol for precise pH measurement within viable
cells, which is directly related to the D-glucose concentration
within viable human keratinocytes.
[0257] A-431 cells obtained from ATCC (#CRL-1555) are seeded at
5.times.10.sup.5 cells in 35 mm culture dishes (Falcon #353801)
containing a #2 25 mm cover glass (VWR#48382-085). Cells are
incubated in 2 mL Dulbecco's Modified Eagle's Medium at 100 mg/dL
D-glucose (Gibco #11966-025) at 90% and Fetal Bovine Serum
(Gibco#26140-087) at 10%. Cells are allowed to reach near
confluence in 6% CO.sub.2 37.degree. C. incubator, over a period of
3 to 5 days.
[0258] The glass cover slip with cells is dipped and rinsed in
Dulbecco's Phosphate-Buffered Saline (D-PBS) (Invitrogen, Catalog
#14040) or pH 7.2 HEPES buffer with composition (mM): NaCl 150, KCl
4.5, MgCl.sub.2 1, CaCl.sub.2 1.8. The cover slip is then mounted
slip on a Sykes-Moore Chamber (#1943-11111, Bellco Glass, Vineland,
N.J.) to form a study chamber. The chamber is always sat on a hot
plate at 36.5.+-.0.5.degree. C. A-431 cells are washed three times
with the buffer. After wash, 1 ml buffer solution with 1 mM (18
mg/dL) D-glucose is added into the chamber. The background spectrum
of A431 cells in buffer solution is then measured by in-house
developed clinical development breadboard (CBB).
[0259] A ratiometric pH reporting dye is used as the in vitro
intracellular pH indicator. 1 mM stock of the ratiometric pH
reporting dye in DMSO is prepared and stored under -20.degree. C.
Cells are loaded in the presence of 0.4.about.2 .mu.M of the dye in
HEPES buffers (pH should be calibrated to 7.22 at 35.degree. C.),
plus 1 mM D-glucose under 36.about.37.degree. C. for 1 hour. During
the time, the cell chamber is shaken gently three times for
homogenous loading. The cell is then washed four times with same
buffer solution. Washing solution should be kept in the cell
chamber for 3 minutes and then decanted. After washing, 1 ml buffer
solution with 1 mM D-glucose is added into the cell chamber.
[0260] Two cell chambers are required for a complete study. One
cell line is for pH change from neutral to acidic and the other is
for the change to basic. About 12 minutes after dye loading, 5
spectra of cell chamber 1 are measured on the CBB as intracellular
pH measurements. The whole solution is next replaced by 1 ml buffer
solution with 15 nigericin (15 .mu.L of 10 mM nigericin), and 5
spectra of pH 7.22 are measured 3 to 5 minutes later. For pH
changes from neutral to pH 6, 10 .mu.L, pH 1.18 HEPES buffer is
added continuously into 1 mL solution contains cells and nigericin.
Each addition provides about 0.2 pH unit decrease. Five spectra at
different pH are measured about 3 to 5 minutes after adding 10
.mu.L pH 1.2 HEPES buffer. The procedure is repeated about 5 times.
For measurements at pH 5, 2.5 uL 1 M HCl in distilled water is
added into the chamber.
[0261] For the pH measurement from neutral to basic, the same
procedure as used for intracellular pH and pH 7.22 are measured on
cell chamber 2. For pH changes from neutral to pH 8.2, 10 .mu.L pH
12.3 HEPES buffer is added continuously into 1 mL solution
containing cells and nigericin. Each addition provides an increase
of approximately 0.2 pH unit. Five spectra at different pH are
measured about 3-5 minutes after adding 10 .mu.L pH 12.3 HEPES
buffer. The procedure is repeated about 4 times. For pH 9
measurements, 5 uL 1 M NaOH in DW is added in the chamber. The
exact pH of 1 ml 7.22 HEPES buffer by adding certain amount of pH
1.2 or pH12.3 buffer should be calibrated by pH meter.
[0262] The basic assumption for the Henderson-Hasselbalch model to
apply is: the fluorescence intensity of each form (protonated and
deprotonated) of the dye is linearly proportional to that form's
concentrations. The application of the modified model, the quantum
yield and photobleaching of two forms should not be affected
differently by environmental effects.
[0263] The governing equation for this model is
pH = pK a - log [ r - r B r A - r .times. F .lamda. 2 B F .lamda. 2
A ] ( EQ . 6 ) ##EQU00008##
where r is the ratio of fluorescence intensity at .lamda..sub.1 to
that at .lamda..sub.2, r.sub.A is the ratio for the fluorescence of
the protonated form, r.sub.B is the ratio for the deprotonated
form, and F.sub..lamda.2.sup.A,B is the fluorescence intensity of
the protonated and deprotonated forms, respectively, at
.lamda..sub.2. The pK.sub.a in Equation 1 is true pK and it can be
expressed as
pK a = pK app + log [ F .lamda. z B F .lamda. z A ] ( EQ . 7 )
##EQU00009##
where pK.sub.app is the apparent pK at the ratio of .lamda..sub.1
over .lamda..sub.2.
[0264] The ratiometric pH reporting dye has a pale color in
dimethyl sulfoxide (DMSO). Aqueous solution of the ratiometric pH
reporting dye will actually exhibit different visible colors at pH
8 to 9. The absorption spectra of the ratiometric pH reporting dye
(5 .mu.M in 0.1 M phosphate buffer) are measured on an HP 8453
UV/Vis spectrometer, an example of which is shown in FIG. 3.
Fluorescence emission spectra excited at 532 nm were measured on a
Fluorolog.RTM. spectrometer (Jobin Yvon Inc., 3880 Park Avenue,
Edison, N.J. 08820-3012 USA) and data are shown in FIG. 4. Samples
were prepared from a series of combinations of 0.1 M monobasic
sodium phosphate and 0.1 M dibasic sodium phosphate. Both sodium
phosphate solutions contain the ratiometric pH reporting dye at 5
.mu.M.
[0265] For the SMMR dynamic study, 12 minutes after SMMR loading,
five spectra of one cell line are measured at 1 spec/min. Preheated
(in water bath) 10 .mu.L 10 g/dL D-glucose or same volume of
control solution is added to the chamber. Twenty five spectra are
measured after solution adding. The first 10 are at 30-second
intervals, and then change to 1 minute. A set of cell lines (4 to
6) is followed above procedure by adding D-glucose and control
solution.
[0266] For the statistical study, 12 minutes after dye loading,
with a 30 second gap, half of total (.about.8) cell lines are added
with 10 .mu.L D-glucose, and another half with 10 .mu.L control
solution. Several rounds of measurements of all cell lines are
carried out at appropriate intervals. The difference between adding
D-glucose and control solution (L-glucose or buffer) will be
studied statistically. The cell viability is assessed and recorded
using fluorescence light microscopy.
Example 2
Using External, In Vivo pH Measurement to Track Blood Glucose or
Blood Lactate
[0267] The rationale for measuring blood glucose levels or blood
lactate levels using external, in vivo optical measurements of SMMR
activity within skin is demonstrated. Fluorescence measurements in
vivo of SMMR placed within the skin during glucose clamp studies
were designed to improve the observation of the correlation between
glucose levels and measured pH changes.
[0268] After improvements of fluorescence measurements, additional
clamp studies with better control over anesthesia were done to
demonstrate the consistency and reliability for the correlation
between glucose levels in blood and the reporter dye fluorescence
ratio.
[0269] This description demonstrates the results of additional
glucose clamp studies during which fluorescence signals were
measured in vivo after dye injections using low-cost components
comprising a fluorescence sensing device.
[0270] An example ratiometric pH reporting dye was prepared as
described by diluting a 1 mM stock aliquot (frozen at -20.degree.
C. in DMSO). The final concentration typically used for placement
into the skin was 20 .mu.M in PBS. The SMMR was delivered by
shallow injection (or topical passive diffusion) into hairless rats
using 100 .mu.L of solution. Multi-injections at the same location
were repeated 4 times at intervals of 1 hour. Clamps were started
on the next day (15-20 hours after last injection) after the
preliminary pre-heating of the rat, i.e., rat had laid on heated
stage about 20 minutes before measurements. During bottom
measurement of the rat the average temperature of the stage was
about 36.5.degree. C., and the standard deviation of the stage
temperature during a single experiment did not exceed 0.5.degree.
C.
[0271] During this study the influence of anesthesia was minimized.
Results of in vivo measurements are shown as in FIGS. 5-8.
[0272] Changes of intensity were not substantial during the
experiments. Anesthesia (0.19 cc of ketamine) was administered by
injection to each rat 75 minutes before measurements. Relative
changes in time of parameter U can be defined as 100
[U(t)-U(0)]/U(0), where t is time (in minutes), and 0 denotes time
at the beginning of measurements. As noted from FIG. 1d, observed
relative changes for glucose and lactate are substantially greater
than the corresponding changes of the fluorescence ratio. To
provide a more meaningful visual comparison between glucose,
lactate and fluorescence ratio changes, a normalization expression
of the independent data are used as:
(U-U.sub.min/U.sub.max-U.sub.min), which refers to the difference
between given value of ratio, lactate, or glucose and it minimum
value divided by the difference between its maximum value and its
minimum value. FIG. 9 shows the normalized and relative changes for
the independent data sets over time:fluorescence ratio, lactate,
and glucose.
[0273] The experimental data allows estimating the sensitivity
coefficients of ratio change to glucose change and lactate change
as given in equation (8).
.DELTA.R=.alpha..DELTA.G+.beta..DELTA.L (EQ. 8)
[0274] Noting that the expression,
.DELTA.R/.DELTA.G=5.times.10.sup.-4 dL/mg and
.DELTA./.DELTA.L=2.times.10.sup.-2 dL/mM, where: L has units mM/dL
and G has units mg/dL.
[0275] Previous clamps with anesthesia (0.1 cc of ketamine) during
measurement provided estimates of the average kinetic changes in
fluorescence ratio, which are shown in FIG. 10.
[0276] Taking into account the influence of three major factors,
namely: anesthesia, lactate and glucose it is possible to compare
experimentally observed changes during clamp studies with
calculated values using an expression such as equation (9):
.DELTA.R=.alpha..DELTA.G+.beta..DELTA.L+.DELTA.R.sub.a(t-t.sub.a)A
(EQ. 9)
[0277] Where t.sub.a is the moment of time when anesthesia was
administered; .DELTA.R.sub.a(t-t.sub.a) is the experimentally
defined ratio changes due to anesthesia with 0.1 cc of ketamine;
and A is amount of ketamine (in 0.1 cc units).
[0278] FIGS. 11a-c show these results. From these results, the
positive correlation between the fluorescence SMMR ratio
measurements and changes in glucose and lactate are
demonstrated.
SMMR Operating Through Energy Transfer
[0279] Assuming that SMMRs can be delivered to the sites at which
energy transfer takes place (as described by equation (4) above,
then a new expression can be derived for the efficiency of an SMMR
monitoring the overall reduction potential of the cell by energy
transfer, i.e.,
.sup.1NAD(P)H.sup..cndot.+SMMR.fwdarw.NAD(P)H+.sup.1SMMR*
[0280] In this process, the excited state of the reduced
nicotinamide interacts with the SMMR to generate the excited state.
The .phi..sub.F of NAD(P)H is less than 0.1. If an SMMR is chosen
with a quantum yield close to unity, then the yield of fluorescence
is increased by at least one order of magnitude, provided the
energy transfer process is efficient, equation (10).
log F = k a log k T ( .tau. D - 1 + k T ) + k b log P + k c ( EQ .
10 ) ##EQU00010##
Where:
[0281] F is the fluorescence response to the change in
intracellular reduction potential, [0282] k.sub.T is the rate of
energy transfer, [0283] .tau..sub.D.sup.-1 is the reciprocal of the
fluorescence lifetime, [0284] P is the partition coefficient,
[0285] and k.sub.a, k.sub.b and k.sub.c are the determined
empirical constants as described previously.
[0286] The rate constant of energy transfer is given by the
expression in equation (11).
k T = ( .phi. F .kappa. 2 .tau. r 6 C n 4 ) J ( .lamda. ) ( EQ . 11
) ##EQU00011##
Where:
[0287] .kappa. is known as the orientation factor, [0288] .tau. is
the fluorescence lifetime of the donor, [0289] .phi..sub.F is the
fluorescence quantum yield of the donor, [0290] C is a collection
of constants, [0291] J(.lamda.) is known as the overlap integral,
[0292] and n is the refractive index of the medium.
[0293] The orientation factor describes how the transition dipoles
of the donor and acceptor molecules align. For two molecules moving
randomly in solution, the value of .kappa..sup.2 is about 0.66, and
for two dipoles perpendicular to each other the value is 0.
[0294] The term k.sub.T/(.tau..sub.D.sup.-1+k.sub.T) in equation
(6) is the efficiency of energy transfer. In words, it is the
number of energy transfer events as a fraction of all decay events.
The energy transfer rate constant must be significantly greater
than the sum of all the rate constants attributed to all other
decay routes for the energy transfer to be efficient.
[0295] The log P (or log P.sub.o/w) value reflects the relative
solubility of any drug in octanol (representing the lipid bilayer
of a cell membrane) and water (the matrix fluid within the cell and
in blood).
Example 3
Using NADH Fluorescence to Track D-Glucose Concentration in Living
Cells
[0296] A set of demonstration experiments for living cells has
shown the expected trend in NADH signals with respect to a change
in glucose concentration, as shown in FIG. 12 (glucose from 0 to
400 mg/dL) and FIG. 13 (glucose from 0 to 66 mg/dL). All glucose
concentrations above .about.5 mg/dL were well above the saturation
limit as can be seen by the lack of any further change in
signals.
[0297] A second set of demonstration experiments was designed to
determine the saturation limit by varying glucose concentration
from 0 to 5 mg/dL and back to zero. The saturation point is
expected to have a value between 0.0 and 2 mg/dL.) As shown in FIG.
14 an approximate linear increase in NADH fluorescence is observed
with glucose concentration from 0 to 5 mg/dL (0.28 mM). FIG. 15 is
a plot of a background subtracted NADH signal where the background
changed linearly as determined from the first zero glucose signal
and the last (near) zero glucose signal (sequence #11). FIG. 16
shows the NADH signal trend versus concentration for living
cells.
Example 4
Using Membrane Potential to Track D-Glucose Concentration in Living
Cells
[0298] A set of demonstration experiments showed the expected trend
in Rh123 fluorescence quenching. The fluorescence quenching is an
indication of membrane potential changes within living cells with
respect to an increase in glucose concentration, as shown in FIG.
17 (glucose from 0 to 400 mg/dL) and FIG. 18 (glucose from 0 to 66
mg/dL).
[0299] A second set of demonstration experiments was designed to
determine the saturation limit for membrane potential-based glucose
analysis by varying glucose 0 to 5 mg/dL and back to zero. The
saturation point value was expected to be between 0.0 and 2 mg/dL.
As shown in FIG. 19 an approximate linear decrease in Rh123
fluorescence with increasing glucose levels was observed from 0 to
5 mg/dL (0.28 mM).
SMMR Fluorophore-Reporter for Glucose and Diol Measurement
[0300] For this application of direct glucose sensing SMMRs, the
objective is to detect and quantify glucose via a small molecule
fluorescent reporter whose photophysical properties are modulated
by binding with D-glucose or other simple sugars or diol molecules.
Such SMMRs report glucose using a reversible binding process and
the molecular structure-activity consists of three mechanistic
parts.
[0301] 1) A fluorophore with suitable photochemical
characteristics;
[0302] 2) A chemical affinity group that binds reversibly with
glucose and similar molecular species (e.g., a boronic
acid-containing component);
[0303] 3) Additional substructural features to favor specificity
for glucose over fructose, galactose, and other biologically active
saccharides, which may be physiologically present near target cells
(i.e., near the intercellular or interstitial spaces of the viable
epidermis).
[0304] Boronic acids are frequently used in saccharide reporter
molecules to provide affinity. Boronic acids undergo reversible
binding with glucose or another sugar or diol-containing molecule
to form a boronate ester with high affinity. (Scheme 3):
##STR00038##
[0305] In the literature, signal transduction of the boronic
acid+sugar-binding phenomenon has been accomplished in two
ways:
[0306] 1) Modulation of Photoinduced Electron Transfer (PET)
quenching, and
[0307] 2) Modulation of Internal Charge Transfer (ICT).
[0308] The PET mechanism of signal transduction is illustrated
schematically in Scheme 4, in the case of an anthracene fluorophore
[See, for example, T. D. James, K. R. A. Samankumara Sandanayake,
R. Iguchi, and S. Shinkai, "Novel Saccharide-Photoinduced Electron
Transfer Sensors Based on the Interaction of Boronic Acid and
Amine", J. Amer. Chem. Soc., 117, 8982 (1995)]. In this example,
the fluorophore-boronic acid is present in a pH dependent
equilibrium, represented by species (1) and (3). For this example,
as pH is increased, the fraction of (3) present increases. In this
form, the electrons of the nitrogen lone pair are available for
fluorescence quenching of the anthracene moiety. At lower pH, the
boron atom participates in a coordinate covalent bond, making the
nitrogen lone pair electrons unavailable for quenching, resulting
in higher fluorescent intensity.
[0309] The presence of a sugar molecule perturbs the (1)-(3)
equilibrium. The boronate ester (2) exhibits a different pKa from
boronic acid (1). Thus, at a given pH the fluorescent-to-quenched
state equilibrium is shifted, resulting in a net increase or
decrease of observed fluorescence intensity, depending on the pH of
the experiment and the relative pKa of the boronic acids (1) and
(2)
##STR00039##
[0310] The ICT mechanism of signal transduction is illustrated
schematically in Scheme 5. In this instance the pH dependent
equilibrium (1)-(3) is perturbed by the presence of a sugar
molecule. At a given value of pH, this results in a different
proportion of the fluorophores (boronic acids and boronate esters)
being present in the hydroxylated form, thus modulating the
electron-withdrawing characteristics of that end of the molecule.
In the case of the boronic chalcone molecule (1), in Scheme 5, this
is manifested as a change in relative fluorescent intensity and
only a slight wavelength shift.
##STR00040##
[0311] Another example of the ICT mechanism from Lakowicz's
laboratory is shown in Scheme 6. In the case of the
N-phenyl-3-nitro-1,8-naphthalimide, the ICT phenomenon manifests
itself in a change in relative fluorescent intensity at two
wavelengths, resulting in a ratiometric probe. The starting point
for these authors' design was the knowledge that suitable
N-phenylnaphthalimides exhibit fluorescent emission at two
wavelengths and that the longer wavelength emission band shows
solvatochromic behavior.
##STR00041##
Designing the Fluorophore-Boronic Acid Reporter
[0312] This compound consists of a fluorophore with long wavelength
excitation and emission and high quantum yield. The phenylboronic
acid moiety is incorporated in the molecular structure as an
essential requirement to add binding affinity for glucose and other
diol-containing compounds. Solvatochromic properties may be
defined. The photochemistry of the fluorophore is sensitive to its
microchemical environment and solvent polarity. Boronic acid can
directly affect photochemical properties such as quantum yield and
wavelength if it is attached to the fluorophore and serves as the
e-withdrawing portion of a "push-pull" system. This can be direct
(when boron is connected directly to the fluorophore), or indirect
(when the phenylboronic acid exerts a "through-bonds"
electron-withdrawing inductive effect).
[0313] Prosthetic group selection is critical for fine-tuning
molecular selectivity. Naturally occurring enzyme active sites and
receptors exhibit exquisite selectivity, even among closely-related
compounds. This is possible due to the receptor's high degree of
spatial and electrostatic complementarity relative to the molecule
in question.
[0314] In the case of glucose-binding proteins, for example, the
selectivity of the enzyme glucose oxidase is achieved by
hydrogen-bonding and close contacts between a glucose molecule and
more than six amino-acid side chains present in the active
site.
[0315] In the realm of small-molecule synthetic glucose reporters,
some reported compounds achieve a relatively good specificity for
glucose over fructose--as much as 10-fold, considering that there
is very little in the way of molecular features (of the synthetic
receptor) to distinguish one from the other. When designing a
synthetic receptor, several strategies can be employed to improve
specificity, namely:
[0316] 1. Multiple Boronic Acid Binding Sites [0317] Spatial
disposition and distance will provide some selectivity among
competing saccharide molecules, due to geometric constraints. (T.
D. James, K. R. A. Samankumara Sandanayake, R. Iguchi, and S.
Shinkai, "Novel Saccharide-Photoinduced Electron Transfer Sensors
Based on the Interaction of Boronic Acid and Amine", J. Amer. Chem.
Soc., 117, 8982 (1995)). Using two or more boronic acid binding
complicates the picture due to the high affinity of the reversible
formation of covalent bonds in the boronate ester. The
equilibrium-binding constant of a single boronic acid based
receptor is in the millimolar (mM) range for glucose. The
corresponding binding constant is micromolar (.mu.M) in the
two-boronic acid case. Thus, at normal physiological
concentrations, the two-boronic probe would be saturated.
[0318] 2. Adjacent H-Bond Donors and Acceptors [0319] Additional
H-bond donors and acceptors contribute only about 6 kcal/Mol.,
each, to ligand-receptor binding. Design of a synthetic probe to
include H-bonding sites provides a means to provide a binding
advantage to molecules, which present H-bondable groups in the
appropriate spatial orientation. Thus the position and orientation
can be designed in by reference to molecular models, to enhance
glucose specificity relative to other saccharides.
[0320] 3. Adjacent Non-Bonded Interactions [0321] Specificity can
be enhanced by model driven choice of other chemical groups, such
as alkyl side chains, which do not add to binding affinity, but
operate by spatial exclusion.
[0322] Several examples of novel boronic acid compounds based on
various fluorophores are given in the following examples.
Example 5
Xanthene Dyes Used to Report Glucose by the ICT Mechanism or
Electronic Perturbation of the Fluorophore
##STR00042## ##STR00043##
[0323] Example 6
Xanthene Dyes Used to Report Glucose by the Pet Mechanism
##STR00044##
[0324] Designing Xanthene-Based Reporters
[0325] A library of xanthene dyes is proposed to form a basis for
Structure-Activity studies with respect to emission wavelength,
ratiometric behavior, and quantum yield. The library of compounds
is made in a combinatorial synthesis paradigm, wherein each
xanthene compound is a result of a cross-product of suitable
building blocks. Substituents are chosen with emphasis on final
products that incorporate electron-donating and
electron-withdrawing groups as well as H-bond donors and acceptors.
Thus the library members will include compounds with push-pull
characteristics and solvatochromic sensitivity.
[0326] The library compounds are generated according to the
reaction sequence outlined in Scheme 7, below. These reactions
employ standard chemical methods. The final reaction yields the
nominal product and byproducts corresponding to disproportionation
and recombination of the building blocks. Final products are
isolated after standard purification techniques such as flash
chromatography and semi-preparative HPLC.
##STR00045##
[0327] The possible products comprising this library are delineated
schematically in FIG. 20 and the accompanying Tables 1, 2, and 3 as
demonstrated.
Tabulated Substituents on Building Blocks Used in the Xanthene
Library.
TABLE-US-00001 [0328] TABLE 1 Xanthene Building Blocks
Incorporating R1, R2, R3, R4, and X Building Block R1 R2 R3 R4 X 1
OH O 2 N(CH.sub.3).sub.2 O 3 OH S 4 SH S 5 SH O 6 OCH.sub.3 O 7
CF.sub.3 O 8 OCH.sub.3 OH O 9 NH.sub.2 OH O 10 NO.sub.2 OH O 11
CF.sub.3 OH O 12 CH.sub.3 OH O 13 CI OH O 14 CN OH O 15 CH.sub.3 OH
CH3 O 16 Br O 17 COOH O 18 NO.sub.2 O 19 OCH.sub.3 O 20 OH CH3 O 21
OAc O 22 N--Me.sub.2 O 23 NH.sub.2 O
TABLE-US-00002 TABLE 2 Xanthene Building Blocks Incorporating R5,
R6, and R7 Building Block R5 R6 R7 1 COOH -- -- 2 COOH -- COOH 3 --
OH -- 4 CH.sub.3 NHAc 5 -- OH --
TABLE-US-00003 TABLE 3 Xanthene Building Blocks Incorporating R8,
R9, R10, 411, and X Building Block R8 R9 R10 R11 X 1 OH -- -- -- O
2 -- OH -- -- O 3 -- -- OH -- O 4 -- -- N--Me.sub.2 -- O 5 -- -- --
Br O 6 -- -- -- COOH O
Appendix 1. List of Building Blocks for the Xanthene Library
Composition
A Components
##STR00046## ##STR00047## ##STR00048## ##STR00049##
[0329] B Components
##STR00050## ##STR00051## ##STR00052## ##STR00053##
[0330] Summary of Design Strategy
[0331] This list comprises 15 A components, 18 B components.
Combination of these materials would result in 270 xanthene dyes.
The list of components is
A Components
Fluoresceins: AF1; AF2
Rhodamines: AR1
[0332] Thio derivatives: AT1; AT2 Rigidified xanthenes: ARg1; ARg2
Quinoid chromophores: AQ1 Push-Pull xanthenes [0333] Push: APus;
APus2 [0334] Pull: APul1; APul2 Naphtho xanthenes: AN1; AN2
Miscellaneous: AM1
[0335] Xanthene analogs: AX1; AX2
B Components
Fluoresceins: BF1
Rhodamines: BR1; BR2; BR3
[0336] Thiol derivatives: BT1; BT2 Heterocyclic analogs: BN1; BN2;
BN3 Naphtho xanthenes: BNap1; BNap2 Push-Pull derivatives [0337]
Push: BPus1; BPus2 [0338] Pull: BPu11; BPu12
Other: BM1; BM2; BM3; BM4
Example 7
Phenalene-1-One Dyes Used as Glucose Reporters
##STR00054##
[0339] Example 8
Coumarin Derivatives Used as Glucose Reporters
##STR00055## ##STR00056##
[0340] Example 9
Use of Coumarin-Boronic Acid SMMRs to Report Glucose in Plasma,
Interstitial Fluid, or Other Body Fluids
[0341] The structure of the glucose sensing deprotected compound
referred to for this invention as (Argofluor-327d) is given here.
The protecting group must be removed before the interaction of the
compound with glucose can be examined.
##STR00057##
[0342] The compound has been prepared by leaving an ethanol
solution of the parent compound to stand overnight at room
temperature in the dark. While not intending to be bound by theory,
it is believed that ethanol replaces the pinacol-protecting group
in an equilibrium driven by the overwhelming concentration of
ethanol. Dilution of this stock solution into aqueous (pH 8) buffer
shows that the largest change in the absorption spectrum within
about 20 minutes, as water displaces the ethanol forming the
deprotected boronic acid. Smaller changes in the spectrum are
observed up to 90 minutes later.
[0343] The absorption and fluorescence spectra of Argofluor-327d
were obtained as a function of glucose concentration. The
absorption spectrum changed by less than 10% at the excitation
wavelength. A plot of the fluorescence intensity from 400 nm to 550
nm as a function of glucose concentration is shown in FIG. 50.
There is no apparent shift in the wavelength maximum. The
excitation wavelength was 375 nm. The long wavelength absorption
maximum was found to be 333 nm. Note for this compound the
fluorescence intensity increases by at least 40% over a glucose
range of 200 mg/dL. A 60% rise in fluorescence intensity was
observed on addition of glucose for a physiological glucose
concentration range of 300 mg/dL. The maximum emission wavelength
position remained constant at all glucose concentrations tested
(i.e., no wavelength EM maximum shift).
In Vitro Experiment (3 mL Total Volume in Buffer Solution)
[0344] While not intending to be bound by theory, it is believed
that the mechanism by which the intensity of fluorescence is
affected by glucose, but not the emission wavelength, involves
modulation of electron density in the coumarin moiety. Calculations
have shown that the electron affinity of bound and free boronic
acid is very different. Free boronic acid is strongly electron
withdrawing while complexed boronic acid is neutral or even
electron donating.
[0345] In light of the magnitude of the effect observed, other
compound analogs are prepared to look for wavelength shift as well
as a strong effect on binding glucose.
[0346] An effect of glucose on the complexation of phenyl boronic
acid with esculetin, i.e.
##STR00058##
has also been observed.
[0347] These two compounds form a complex when the phenyl boronic
acid is present at high concentration. In the presence of glucose,
the complex is disrupted with a change in the fluorescence and
absorption spectra. FIG. 51 shows the fluorescence spectra on top;
and the absorption spectra below. The purpose of an experiment such
as this is that if a complexed pair showed a large spectral change
with glucose concentration then a tethered pair would be
synthesized that would then reasonably be expected to show a large
change in their spectral properties in the presence of glucose.
NMR Observation of Hydrolysis of Protected Boronic Acids
[0348] The timeframe in which a pinacol-protected boronic acid
derivative hydrolyzes and converts into the free boronic acid,
which then could be complexed with glucose can be determined using
NMR.
##STR00059##
[0349] 15 mg protected 4-amino-phenylboronic acid was dissolved in
0.75 ml CD.sub.3OD and the .sup.1H-NMR spectrum was recorded
immediately. A clear spectrum was obtained. The sample was measured
again after 1 h and 15 h. The spectrum was essentially unchanged,
which means, that the pinacol-group is still attached to the boron.
After 15 h two drops of D.sub.2O (ca. 20 mg) were added into the
NMR-sample and the spectrum recorded. This spectrum and a spectrum
after 4 h were also almost unchanged, which means, that the
protecting group is very stable in these media. The same kind of
test was conducted using the protected 3-phenol-boronic acid. It
gave essentially the same results. Understanding the removal of the
protecting group is an important step in the synthesis of boronic
acid analogs.
In-Vivo Glucose Detection Using Coumarin-Boronic Acid-Based
Reporter
[0350] Three separate test series were performed in rats by
injecting 100 microliter of a 1 mM compound solution, followed by
injection of 100 microliter of buffer solution, with and without
glucose at 300 mg/dL concentration. Fluorescence was measured by
excitation at 355 nm and detection of emission at 440 nm. The
typical experiments showed a decreasing baseline of tissue
autofluorescence. Micro-injected dye spots exhibited fluorescence
intensity of more than 10.times. the autofluorescence background,
under the experimental conditions. Injected spots also showed
changing fluorescence intensity (usually declining as the compound
was transported into cells). No effect of glucose could be
discerned in the experimental setup, due to positional sensitivity,
and unknown mechanical factors in acquiring the dye spot and
referencing the intensity.
In-Vitro Detection Using Coumarin-Boronic Acid-Based Reporter
[0351] PHK cells were loaded with AF-327d reporter, at 50
micromolar concentration, in buffer. Fluorescence was measured
using a two-photon fluorescent imaging system at approximately EM
440 nm, and excitation at (2P) 705 nm. Pictures taken 10 minutes
apart show a slight change in fluorescence intensity upon
increasing the glucose concentration to 300 mg/dl in the cell
buffer medium. This is seen clearly, as a 20% decrease in time
course of fluorescence.
Glucose Response Rate Using Coumarin-Boronic Acid-Based
Reporter
[0352] Arg-327 unprotected batches were prepared. One batch was
supplied as a yellow powder and the other as a concentrated
solution in deuterated methanol. A small sample of these materials
was taken and dissolved in 3 ml of pH 8 phosphate buffer. The
fluorescence of these materials was excited at 340 nm and the
emission monitored at 440 nm. The fluorescence was monitored every
15 seconds in a stirred cuvette. After about 3 minutes 100 .mu.L of
300 mg/dL glucose in pH 8 buffer was added. The change in glucose
increased immediately on this timescale and a plot of the
fluorescence intensity as a function of time is shown in FIG. 52.
Similar results were obtained for both samples.
Laboratory Synthesis of Boronic-Acid-Based Glucose Reporters
Preparation of Designation # AF-332: Coupling of a Protected
4-Aminophenylboronic Acid with 6-Methoxy-2-Naphthoic Acid
##STR00060##
[0353] NMR and MS analyses are consistent with the proposed
structure.
Preparation of Designation # AF-333: Coupling of
3-Aminophenylboronic Acid with 6Methoxy-2-Naphthoic Acid
##STR00061##
[0354] NMR spectrum of the isolated product is consistent with the
proposed structure
Preparation of Designation # AF-327 (ZW-17-41)
##STR00062##
[0355] NMR if the isolated compound is consistent with the proposed
structure.
Preparation of Designation # AF-327d (HN-2-58): Synthesis a,
Coupling of 4-Chlorocarbonylphenylboronic Anhydride with Coumarin
151
##STR00063##
[0357] This reaction gives a higher yield of the desired product in
contrast to other experiments using a coupling agent, in which the
isolated yields were less than 5%. A major spot was found in TLC,
which appears to be the desired boronic acid. This compound is
responsive to glucose, again exhibiting a 60% increase in
fluorescence intensity in vitro, when [glucose] is changed from
zero to 300 mg/dL. The NMR is consistent with the proposed
structure.
Preparation of Designation # AF-327d (HN-2-58 and HN-2-64)
Resynthesis: Coupling of 4-Chlorocarbonylphenylboronic Anhydride
with Coumarin 151
##STR00064##
[0358] Preparation of Designation # AF-327d (HN-2-59): Synthesis B,
Coupling of 4-Chlorocarbonylphenylboronic Anhydride with Coumarin
120
##STR00065##
[0359] Resynthesis of AF-327d (HN-2-78): Additional Supply of
AF-327d was Prepared According to the Methods Previously
Reported
##STR00066##
[0361] Approximately 100 mg (13% yield) of pure product and 250 mg
unreacted Coumarin-151 were recovered. Em=440 nm; ex=340 nm.
Preparation of Designation # AF-329 (ZW-17-51)
##STR00067##
[0362] NMR of the purified compound is consistent with the assigned
structure.
Preparation of Designation # AF-329d (HN-2-71) Preparation:
Reaction of 4-Chlorocarbonylphenylboronic Anhydride with
6-Amino-1H-Phenalene-1-One
##STR00068##
[0364] TLC shows some new bright yellow spots. The crude mix shows
a huge Stokes shift. No response to the addition of Glucose could
be observed.
Preparation of Designation # AF-330 (ZW-17-54)
##STR00069##
[0366] A product was isolated by column purification. It is being
analyzed for identity and properties. Paradoxically, it appears
colorless in the bottle.
Preparation of Designation # AF-333-1 (ZW-17-55)
##STR00070##
[0368] A pure product has been obtained. Characterization is in
process.
Preparation of Designation # AF-334 (HN-2-47): Coupling of the Acid
Chloride of Coumarin-343 with 3-Aminophenylboronic Acid
##STR00071##
[0369] Preparation of Designation # AF-334 (HN-2-48): Coupling of
the Acid Chloride of Coumarin 3-Carboxylic Acid with
3-Aminophenylboronic Acid
##STR00072##
[0370] Preparation of Designation # AF-335 (HN-2-47): Coupling of
Coumarin-343 with 3-Aminophenyl-Boronic Acid
##STR00073##
[0372] No useful product was observed, due to limited solubility in
reaction medium.
Preparation of Designation # AF-336 (HN-2-50): Preparation of a
Fluorescein-Like Xanthyl-Boronic Acid
##STR00074##
[0373] Preparation of Designation # AF-337 (HN-2-52): Preparation
of a Rhodamine-Like Xanthyl-Boronic Acid
##STR00075##
[0375] Crude reaction mixture is deep red.
Preparation of Designation # AF-337 (HN-2-77) Preparation of a
Rhodamine-Like Xanthyl-Boronic Acid
##STR00076##
[0377] Reaction carried out at room temperature, or with heating to
reflux, showed no indication of product formation.
Preparation of Designation # AF-338 (HN-2-65): Reaction of
4-Chlorocarbonylphenylboronic Anhydride with Coumarin 500
##STR00077##
[0379] Two batches were combined and worked up to provide 45 mg of
pure compound for additional experiments.
Preparation of Designation # AF-339 (HN-2-70) Synthesis: Coupling
of 4-Chlorocarbonylphenylboronic Anhydride with 4-Nitroaniline
##STR00078##
[0381] This model compound was prepared, to combine the
solvatochromic effects of 4-nitroaniline with glucose binding
capability of phenyl boronic acid. Response to glucose in vitro was
weaker than AF-327.
Preparation of Designation # AF-340 (HN-2-72) Preparation: Coupling
of Chlorocarbonylphenylboronic Anhydride with
8-Hydroxy-1,3,6-Pyrenetrisulfonic Acid, Trisodium Salt. (Pyronin;
D&C Green #8)
##STR00079##
[0383] TLC shows a nonfluorescent spot at Rf=0.3 and a very
fluorescent spot on the starting line. No product was isolated.
Preparation of Designation # AF-341 (HN-2-73) Preparation:
Boronic-Acid Containing (Semi-Rhodafluor) Xanthene Structure
##STR00080##
[0385] No evidence of product formation corresponding to xanthene
structure.
Preparation of Designation # AF-342 (HN-2-69, 75, 76) Preparation:
Boronic-Acid Containing Rhodamine 110 Structure
##STR00081##
[0387] Reaction was attempted in THF; in Acetonitrile; and in DMF.
In all cases, no new product was observed.
Preparation of Designation # AF-343 (EB-16-40): Preparation of a
Coumarin-Boronic Acid Chalcone Compound
##STR00082##
[0389] A rust-colored solid product was formed. No fluorescence or
glucose effect was observed.
Preparation of Designation # AF-344 (EB-16-42): Preparation of a
Coumarin-Boronic Acid Chalcone Compound
##STR00083##
[0391] Besides starting material, a major red fluorescent spot at
Rf=0.25 was observed in TLC. This compound was isolated and
purified. There was no observed effect of glucose in vitro. NMR
suggests that the compound is likely a dimer of Coumarin 334. By
NMR, no spectral features corresponding to the boronic acid
incorporation were observed. A minor orange fluorescent product,
represented by a spot at Rf=0.1 was also inactive in regard to
glucose addition.
Preparation of # AF-345 (EB-16-100): This Compound was Attempted to
Explore Another Fluorophore Family
##STR00084##
[0393] This reaction proceeded in approximately 10 percent yield,
following the procedure previously employed in AF-327d. 11 mg of
pure product was recovered.
[0394] The AF-345 compound is fluorescent em=435 nm; ex=330 nm.
Fluorescence intensity was observed to increase by 30% on addition
of 300 mg/dl glucose concentration.
Comparison Materials
Designations # HN-2-32 and # HN-2-44: Preparation of
3-Phenylboronic Acid 3-Nitro-1,8-Naphthalenedicarboximide (Ref See
Lakowicz, Organic Letters, 9, 1503 (2002))
##STR00085##
[0396] This compound was prepared for comparison to literature
results on the spectroscopic detection of glucose reporting
efficacy.
Designation # HN-2-42: Preparation of Protected 4-Phenylboronic
Acid 3-Nitro-1,8-Naphthalenedicarboximide (Analog of the Lakowicz
Compound Above)
##STR00086##
[0398] TLC of the reaction mixture shows multiple products.
Preparation of Designation # HN-2-44: Preparation of
3-Phenylboronic Acid 3-Nitro-1,8-Naphthalenedicarboximide (See
Lakowicz. Organic Letters, 9, 1503 (2002))
##STR00087##
[0400] The major product isolated has NMR consistent with the
reported structure, but UV which does not fit the literature. A
minor product has the reported UV characteristics, but NMR
inconsistent with the reported structure. It is likely that the
material reported by Lakowicz is a mixture and not a pure
compound.
Example 10
Carbostyril Derivatives Used as Glucose Reporters
##STR00088## ##STR00089##
[0401] Example 11
Use of Tethered Molecules for Glucose Measurement
[0402] Suitable tethers include both ring structures and linear
systems (example given in Scheme 8, below). The advantage of a ring
system is that the structure is semi-rigid which limits the degree
of entropy in the system. If the system is too flexible, the two
chromophores will be less likely to approach each other. In order
of complexity, cyclic tethers include cycloalkanes, crown ethers,
cyclodextrins and cyclic peptides. The number of residues in the
ring system is chosen so that the chromophores come close enough
together to promote complex formation.
##STR00090##
Example 12
Use of a Crown Ether for Glucose Detection
[0403] Substitution of the ring system may at a ring carbon
position for cycloalkanes and crown ethers, at a hydroxyl group in
cyclodextrins or on any of the amino acid residues in a cyclic
peptide (example given in Scheme 9). The cyclic peptide may be
designed to have some specificity for glucose by choosing a peptide
sequence that mimics the binding site found in a protein such as
glucose oxidase or glucose dehydrogenase.
##STR00091##
Example 13
Use of Cyclic Decapeptide Fluorophores for Glucose and Other Diol
Sensing
[0404] This example describes a molecule for use in the detection
and quantification of glucose. This glucose sensing occurs by means
of a fluorescent reporter whose photophysical properties are
modulated by direct binding with glucose or other diol molecules.
Such reporters consist of three mechanistic parts: [0405] 1. A
fluorophore with suitable photochemical characteristics; [0406] 2.
A chemical affinity group that binds reversibly with glucose and
similar molecular species (cyclic peptide); [0407] 3. Additional
substructural features to favor specificity for Glucose over
Fructose and other saccharides.
[0408] A cyclic peptide is proposed as a structural element that
incorporates H-Bond donating and accepting atoms to provide
affinity to glucose (and other biomolecules). Careful choice of
amino acid residues forming the peptide will allow for adjusting
specificity.
[0409] Two proof of concept compounds are demonstrated: Cyclic
peptides "A" and "B" (Scheme 10).
##STR00092##
Cyclic Decapeptide "A"
[0410] Glucose binding to Peptide A is confirmed by: (1)
examination of change in fluorescence polarization of Tryptophan,
or (2) changes in energy transfer from Tyrosine to Tryptophan.
Note: added serines will boost the binding affinity for Glucose by
additional H-bonding sites.
Cyclic Decapeptide "B"
[0411] Glucose binding to Peptide B is confirmed by one or more of
the following: (1) examination of change in fluorescence
polarization of Tryptophan, (2) changes in fluorescent quenching of
tryptophan by the nitrogen of Lysine, (3) change in fluorescence
polarization of tryptophan, or (4) a change in fluorescence
polarization or energy transfer of a fluorophore connected to the
end of the Lysine side chain. Note: added serines will boost the
binding affinity for Glucose by additional H-bonding
Quantitation
[0412] SMMR compounds have a number of quantifiable parameters in
common. They have high molar absorption coefficients (>50,000
dm.sup.3 mol.sup.-1 cm.sup.-1), high fluorescence quantum yield
(>0.2), and they interact only with components of the cell or
biological system that are present in high concentration. This last
requirement helps minimize the toxicity of the SMMR. Competitive
inhibition of enzymatic processes and interference with cellular
processes is minimized if the SMMR does not interact with a
significant fraction of the cells metabolic pathways.
Rational Design for Novel SMMR Compounds
[0413] This section discloses the aspects important to intelligent
design of SMMRs for glucose detection. There are several individual
properties involved during the rational molecular design process
and these are disclosed within the following text.
[0414] The fluorescence quantum yield (.phi..sub.F) of a molecule
is given by equation (12).
.PHI. F = k r k r + k nr ( EQ . 12 ) ##EQU00012##
Where k.sub.r is the radiative rate constant and k.sub.nr is the
nonradiative rate constant. In this discussion, the term k.sub.nr
includes all mechanisms for decay that does not lead to
fluorescence. The .phi..sub.F of BeXan type dyes is typically 0.2
or less. The reasons the value is so low may include the
flexibility which increases the number of vibrational modes
available to the compound, intramolecular reactions or competing
photophysical pathways such as intersystem crossing. All of these
processes increase k.sub.nr for deactivation of the excited state.
For BeXan it is unlikely that flexibility or intramolecular
reactions contribute significantly to k.sub.nr, other molecules of
similar size and substitution have .phi..sub.F values close to
unity. It is more likely that intersystem crossing for the molecule
is significant and is the cause for the low .phi..sub.F. If the
BeXan structure is drawn as shown here then the molecule can be
designed with two linked chromophores (Scheme 11, below):
##STR00093##
a quinoid type structure involving the heteroatom at position 9 and
a substituted naphthalene structure involving the heteroatom at
position 3. Het refers to a heteroatom, most commonly oxygen or
nitrogen. The photochemistry may be interpreted as a combination of
these two chromophores. The validity of this kind of interpretation
is dependent on the fraction time the molecule may be considered in
this configuration as opposed to a configuration involving a
naphthoquinoid and a phenol type structure.
[0415] The configuration of the dye is strongly influenced by the
nature of the heteroatom substitution. The structure of the dye
responsible for the absorption may be estimated by consideration of
the photophysical properties of related compounds and calculations
on them. For example comparison of fluorescein and BeXan type dyes
are shown in FIGS. 21a and 21b, with structures shown in Schemes 11
and 12, respectively.
##STR00094##
##STR00095##
[0416] Table 4 demonstrates the absorption and emission maxima
(i.e., wavelength in nanometers) with corresponding energies for
fluorescein and BeXan visible spectral absorption bands. The table
also demonstrates other spectral properties for a comparison of
these two molecular structures.
TABLE-US-00004 TABLE 4 Absorption and emission position and energy
data for fluorescein and BeXan Absorption Emission Absorption
Emission Wavelength Wavelength Energy Energy (nm) (nm) (kcal/mol)
(kcal/mol) Fluorescein 455, 483 516, 651 62.8, 59.2 55.4, 52.9
Protonated Fluorescein 501 540 57.1 53 Deprotonated BeXan 518, 549
586, 635 55.2, 52.1 48.8, 45 Protonated BeXan 576 640 49.6 44.7
Deprotonated Difference in energy of protonated absorption bands
Fluorescein 3.6 kcal/mol BeXan 3.1 kcal/mol Stokes shift for
deprotonated bands Fluorescein 4.1 kcal/mol BeXan 4.9 kcal/mol
Stokes shift for protonated bands Fluorescein 3.8 kcal/mol BeXan
3.3 kcal/mol Band positions relative to the high-energy protonated
absorption band Absorption Emission Fluorescein (kcal/mol) High
energy protonated band 0 7.4 Low energy protonated band 3.6 9.9
Deprotonated band 5.7 9.8 BeXan (kcal/mol) High energy protonated
band 0 6.4 Low energy protonated band 3.1 10.2 Deprotonated band
5.6 10.5
[0417] These two compounds show very similar visible absorption and
emission spectra. The absorption spectrum of the BeXan type dye is
about 70 nm shifted to the red, probably as a consequence of
extended conjugation compared with fluorescein. The emission
spectrum of BeXan is shifted by as much as 100 nm compared to
fluorescein. Despite this difference in the wavelengths the energy
spacing of the visible bands is also similar. There is a slightly
larger Stokes shift for BeXan, which may have been expected since
the flexibility of the naphthoxanthene structure is likely to be
greater than that for the xanthene ring.
[0418] The similarities in these spectra support the idea that in
the BeXan ring system there is a high degree of delocalization both
in the protonated and the deprotonated form of the dye as is
observed for fluorescein. These similarities also support the idea
that substitution of other heteroatoms onto the xanthene ring
structure should not lead to significant changes to the spectral
properties of the molecule. The parameter that cannot be accounted
for in this kind of analysis is the quantum yield of
fluorescence.
Comparison of the Structures of Two Dyes
[0419] As an example of the principle, Malachite green is an aryl
methine dye that absorbs strongly in the red region of the spectrum
(150,000 dm.sup.3 mol.sup.-1 cm.sup.-1). It is nonfluorescent.
Conjugation in this molecule probably extends over the three phenyl
rings with the positive charge located primarily on the two
substituted rings [FIG. 22 shows the absorption spectrum (left),
and the molecular structures (right)]. Comparison of this structure
with that of Rhodamine B shows the two substituted rings tied
together with an oxygen bridge [FIG. 23 shows the absorption
spectrum (left), and the molecular structures (right)]. The molar
absorption coefficient is 109,000 dm.sup.3 mol.sup.-1 cm.sup.-1.
Molecular modeling shows the phenyl ring twisted out of plane with
respect to the xanthene ring. A consequence of this is that the
molecule has an absorption maximum about 60 nm to the blue,
supporting the view that the phenyl ring is not involved in the
main xanthene chromophore. The molecule is also significantly more
rigid than the aryl methine dye and the quantum yield of
fluorescence is close to unity.
[0420] Consideration of the structures of these two compounds and
their desirable photophysical properties leads to the design of a
new chromophore that may well have a very high molar absorption
coefficient and a high quantum yield of fluorescence, the structure
of which is shown in Scheme 13.
##STR00096##
[0421] Molecular modeling of this compound shows it to be highly
planar and from earlier arguments it would be expected to absorb at
600 nm or higher and to be highly fluorescent. There is little
information concerning this type of structure in the literature but
a synthesis has been published of a molecule
2,6,10-Tris(dialkylamino)trioxatriangulenium
(2,6,10-Tris(dialkylamino)trioxatriangulenium ions. Synthesis,
structure, and properties of exceptionally stable carbenium ions.
B. W. Laursen, F. C. Krebs, M. F. Nielsen, K. Bechgaard, J. B.
Christensen and N. Harrit. J. Am. Chem. Soc. (1998) 120
12255-12263). The rational behind its synthesis however extends to
other molecules with heteroatom substitution including the
carbonyl/hydroxyl derivative, the sulfur analog and mixed
heteroatom analogs such as that proposed in Scheme 14.
##STR00097##
[0422] To the best of our knowledge there is no previous reference
to the oxygen or sulfur analog as proposed in Scheme 15.
##STR00098##
Enhancement of Cellular Retention and Quantum Yield
[0423] The problems that exist for the some dyes that make them
unsuitable as SMMRs are low quantum yield of fluorescence, poor
retention inside cells, and toxicity. These problems may be
addressed to a certain extent by binding the dye to a controlled
size metal particle. The fact that the quantum yield for
fluorescence is determined by the balance between the radiative and
nonradiative rate constants has already been discussed. For dyes
that have low quantum yield of fluorescence the nonradiative rate
constant dominates the radiative rate constant. In the presence of
a strong electric field, the radiative rate constant may be
increased to such an extent that it dominates in determining the
quantum yield.
[0424] This phenomenon is will known near the surface of metals. If
a fluorophore with a low quantum yield is placed within a certain
distance from a metal surface then a dramatic shortening of the
fluorescence lifetime and an enhancement of the fluorescence
quantum yield may be observed. If very small (nm) beads are used to
bind the dye, or the dye resides on the surface of the metal the
dye may stop fluorescing completely. Selecting the optimum bead
size and dye spacing from the surface of the metal has some
important consequences:
[0425] If the bead is too small or if the dye is so close to the
metal that it resides on the surface, the excited state of the dye
is completely quenched. If the particle size is too large, the
particle bound dye will not be taken up by the cell.
[0426] The optimum dye-to-particle spacing and particle size will
lead to a SMMR system that has a quantum yield close to unity.
Because the radiative rate constant is so high the fluorescent
lifetime will be short and as a result there will be less time for
excited state chemistry to take place, reducing photoxicity. Since
the dye is bound to a relatively large bead it cannot migrate away
from the active site and overall toxicity is reduced. With a short
excited state lifetime the photo stability of the molecule will be
improved for the same reason that the phototoxicity is reduced. The
synthesis of these complexes is relatively simple. If the bead is
made from gold then the dye will bind with a free thiol group.
These molecules may be prepared starting with the chloromethyl
BeXan dye. It is reacted at a 1:1 ratio with a dithioalkane. The
free thiol terminus binds to the gold surface.
[0427] Effective concentrations of SMMRs to be applied in
compositions and methods of the invention are in the range of at
least 1 to 500 .mu.g/ml, e.g. 5 to 150 .mu.g/ml or 10 to 100
.mu.g/ml. The concentration of SMMRs used is preferably from 10 to
500 .mu.M, more preferably from 100 to 300 .mu.M, and most
preferably from 150 to 250 .mu.M.
Design Strategy for Boronic Acid Glucose Reporters
Designing Ratiometric Direct Glucose Detection Probes
[0428] A phenyl-boronic acid and a diol, e.g. a molecule of glucose
(or two equivalents of a monohydric alcohol) exist in a 4-way
equilibrium in aqueous solution. This is shown schematically in
FIG. 24 and has been discussed previously.
The pKa of Phenylboronic Acid Compounds and their Interaction with
Diols
[0429] FIG. 25 shows typical schematic curves of the influence of
pH on boronic acid-saccharide binding. In this conceptual example,
the Y-axis represents percent present as the hydroxylated species.
Actual examples are presented in the literature wherein the Y axis
is denominated in terms of an instrument observation like
fluorescence intensity.
[0430] The point illustrated here is that the pH dependent
equilibrium of the "unbound" (1.fwdarw.2) boronic acid has a pKa of
1 to 2 units greater than the "bound" (3.fwdarw.4) (sugar-complex)
form. [pKa of the boronic acid (a Lewis acid) is the pH value at
which the titration curve is at 50% of maximum.]
[0431] A consequence of the difference in the curves is that at a
given pH, (e.g. pH=7) the boronic acid-sugar complex exists mainly
in the hydroxylated form (4), whereas the free boronic acid exists
mainly in the neutral form (1).
[0432] For purposes of creating a biological sensor, the actual pKa
of the bound and unbound forms can be adjusted by engineering the
molecule to include electron-donating or electron-withdrawing
atoms.
[0433] The practical importance of this observation comes into play
when there is some observable property of a boronic acid-containing
molecule that changes depending on the state of hydroxylation. For
example, when a phenyl boronic acid is used in the signal
transduction scheme of a glucose reporter molecule (see below)
maximum dynamic range of the reporter signal may be obtained, when
the pKa's of the boronic acid are adjusted, such that the bound
form has pKa less than 7 and the unbound form has pKa greater than
7. In that case, at pH .about.7 (normal physiological range) the
glucose-bound complex exists primarily in the hydroxylated form,
whereas, in the absence of glucose (or other saccharide) the
neutral form predominates.
[0434] In the case of Argofluor-327d or other
p-carboxamido-phenylboronic acids, the pKa (not measured for
AF-327d) is close to optimal for this phenomenon to take place.
[0435] Thus a large swing in fluorescence intensity has been
observed due to the predominance of species 4, when glucose is
present. The phenylboronic acid species 4 exerts an electron
donating influence, whereas the species 1 is weakly withdrawing. In
the context of an electronic "push-pull" fluorophore such as
Coumarin-151, this translates into increased fluorescence intensity
on hydroxylation. See FIG. 26.
[0436] In practice, AF-327d maintains a constant 440 nm emission
wavelength with and without added glucose at pH=7, in our
experimental measurements. The calculations herein to predict
wavelength would have suggested a red-shift of as much as 79 nm
(536 vs. 457) on hydroxylation--going from species 1 to 4.
[0437] A relatively large wavelength change between forms 1 and 4
would be a necessary precondition to observe resolved fluorescent
emission peaks attributable to the two forms. This would become
useful as a ratiometric probe, which could be accurately calibrated
top glucose concentration. With this in mind, a number of
synthetically accessible probe types were evaluated by calculating
predicted emission wavelengths. Such compounds could form the basis
for a; future chemical synthesis campaign.
[0438] Other model compounds were analyzed to attempt to predict
emission wavelength shift. Examples are presented in FIG. 27.
Compounds such as these, wherein there is a very large predicted
wavelength shift on hydroxylation would be a reasonable place to
start on designing a fluorescent probe with ratiometric
behavior.
Solvatochromism Using Aniline-Boronic Acid-Based Reporters
[0439] Comparison of the electron withdrawing properties of a nitro
group and a boronic acid are similar. The absorption and
fluorescence of p-nitro aniline and p-boronic acid aniline have
been compared in a variety of solvents. These findings are useful
for designing boronic acids that show a ratiometric change in
absorption and or fluorescence in the presence of glucose. The
nitro substituted material is a simpler model for the electron
withdrawing properties of a boronic acid.
[0440] The absorption spectra are shown above (FIG. 28 (a)). The
long wavelength band is a charge transfer band the position of
which is sensitive to the solvent. This behavior is well known. The
material is not fluorescent. The absorption spectra are also
virtually identical in acid and base.
[0441] The corresponding spectra for p-Boronic acid aniline are
shown below (FIG. 28(b)). The p-boronic acid aniline was prepared
from a derivative protected with a pinacol group by dissolving in
ethanol. A small (20 .mu.L) aliquot of this solution was then added
to 3 mL of the solvent.
[0442] The material absorbs at a much shorter wavelength and the
lowest energy band is much less solvent sensitive. The material is
fluorescent. The absorption spectra are also sensitive to pH and
the spectra for acid (pH 2) and alkali (pH 12) conditions are shown
below (FIG. 28(c)).
Glucose Reporting Using Pyrene--Boronic Acid-Based Reporters
[0443] Pyrene boronic acid fluorescence increases in the presence
of glucose, but this effect has only been observed in methanol and
not in aqueous solutions (See FIGS. 29(a)-(c)).
[0444] For both coumarin-based and pyrene-based boronic acids the
quantum yield of fluorescence increases with glucose. The
fluorescence and the absorption spectra remain the same implying
that there is no change in the electronic configuration of the
ground and excited state.
Approaches to Enhance D-Glucose Specificity
[0445] Phenyl Boronic acid analogs bind reversibly to hydroxyl
containing compounds to form a stable boronate ester. This
equilibrium is established in solution, at room temperature with
alcohols, in aqueous solution. The equilibrium is particularly
favorable with diols and other polyhydroxy compounds, when binding
to two vicinal hydroxyl groups allows for the formation of a highly
favorable 5-membered-ring reaction product. Early on Boric Acid (1)
and Phenyl Boronic Acid (2) were studied in relation to their
binding with various sugar molecules.
[0446] Since the 1980's, many investigators have considered phenyl
boronic acids as a suitable reactive moiety to bind to glucose and
other saccharides. Derivatives of Phenylboronic acid have been
invoked in many schemes for the direct measurement of the
concentration of Glucose and other sugars. In particular,
phenylboronic acid has formed a key element in signal transduction
in fluorescence-based glucose assays. (3)
[0447] The majority of the schemes employ boronic acid to bind to
the target saccharide and to modulate photophysical properties of
the fluorophore through a quenching mechanism or direct effect on
the electronic configuration of the fluorophore itself. The
observable effect of saccharide binding manifests itself through
changes in wavelength and/or intensity.
[0448] The boronic acid--sugar detection schemes are specific for
diol-containing compounds. Phenylboronic acid shows a small but
observable specificity in its bonding affinity to various sugars.
The order Fructose>Arabinose, Ribose>Galactose>Glucose has
been observed in the literature, although the relative specificity
spans only 1 to 2 orders of magnitude. (4)
[0449] This is acceptable in the case of laboratory assays in the
absence of interfering compounds. However, in vivo, or with
physiological samples, many additional saccharides, including
glycoproteins are present in unknown large concentrations, which
interfere with a satisfactory assay.
[0450] In nature, glucose and other individual saccharide molecules
are recognized with exquisite specificity. In enzymes (glucose
oxidase, hexokinase) and bacterial periplasmic binding proteins
(PBP) (glucose binding protein; arabinose BP; ribose BP; etc) the
individual sugar molecule is bound in a receptor or active-site by
intermolecular forces, which achieve chemical complementarity with
the molecule in question. This is a delicate balance of nonbonded,
electrostatic, and H-bonding interactions, which exclude undesired
molecules and provide strong affinity to the one native target
molecule. The binding pockets of this family of proteins, favor
H-bonding to the bound sugar by side chains of glutamates
aspartates and asparagines. (5)
[0451] These features can be evaluated by visualization and
inspection of the published crystal structures of the proteins,
with their native saccharide-molecule ligand. All of these proteins
and more particularly, all within a given family, share a common
attribute for specificity-determination. The small saccharide
molecule, i.e. glucose, etc., is bound (indeed almost totally
surrounded) in a concave binding pocket, which effectively excludes
molecules above a size threshold.
[0452] Helling a has reported and patented protein-mediated assay
systems, based on a environmentally-sensitive fluorophore attached
to the appropriate the binding protein (6). For example, a
sensitive and selective glucose reporter is based on
glucose-binding protein, conjugated to NBD, Fluorescein, pyrene,
and other fluorophores. This study also demonstrated that
specificity can be tailored to accommodate man-made small
molecules, by site-directed mutagenesis of the residues that line
the putative monosaccharide binding pocket.
[0453] Thus, the specific reporting of glucose is solved, for small
in vitro assays. The concept faces practical barriers to in vivo
implementation, such as that of delivering a relatively large
protein molecule.
Specificity with Small-Molecule Synthetic Reporters
[0454] In a small-molecule reporter context, strategies may be
enumerated for achieving specificity for glucose (ratio of binding
equilibrium constants) over larger saccharide-containing species:
[0455] 1. Bidentate (or polydentate) reporters, that restrict the
preferred saccharide ligand by size and accessible orientations.
[0456] 2. Reporters with additional auxiliary side chains to
simulate a binding-pocket environment. [0457] 3. Reporters
incorporating a semi-rigid small binding pocket environment to
complement the boronic acid and other "side chains". [0458] 4.
Repulsion of the larger molecules by placement of functionality
similar to silicone polymer.
Bidentate Reporters
[0459] Attempts have been made to improve glucose specificity by
engineering boronic acid based fluorescent sensors that favor one
sugar over another. This has been accomplished by adjusting the
distance of an intramolecular linker, between two boronic acid
groups, thus constraining the geometry into which a sugar molecule
can fit. (7) Several examples from the literature are given in FIG.
30.
Boronic-Acid Reporters with Auxiliary Side Chains
[0460] A similar concept is proposed, using one boronic acid group
and other molecular fragments which provide intermolecular
attraction and constrain the space into which a sugar molecule can
bind. Molecular fragments to be employed in this regard are
selected with a preference for the amino acid side chains that
comprise the binding site of the PBP's (Asp, Asn, Glu, Gln, Ser,
etc). As an initial attempt, neutral fragments (like Asn, Gln, or
Ser) are most likely due to molecular property considerations. Such
a "small molecule" multi-domain reporter (SMMDR) is advantageous in
its ready assembly by general methods of synthetic organic
chemistry and its small size, which facilitates transdermal
delivery, in vivo.
[0461] FIG. 31 shows illustrative examples of SMMDR concepts.
Compounds 1, 2, and 3 modulate fluorescence by sugar displacing the
dihydroxy coumarin fluorophore from the boronic acid group.
Compounds 3, 4, and 5 modulate fluorescence by a photochemically
induced electron transfer mechanism, wherein sugar binds to boron,
displacing the nitrogen proximal to the fluorophore, making its
electron pair available for quenching.
[0462] FIG. 32 shows examples of SMMDR concepts in which compounds
7 thru 11 incorporate phenylboronic acid as an element of a
push-pull fluorophore. Fluorescence intensity and/or wavelength are
modulated by change of the boronic substituent from
electron-withdrawing to electron-donating in the glucose binding
equilibrium. Additional carboxamide H-bonding groups (based on Asn)
are appended in the vicinity of the boronic acid to increase
affinity and geometry-induced selectivity for the sugar of
interest.
[0463] In practice such concept compounds would be refined by
molecular modeling techniques to assure that dimensions and
geometry are optimal for selectivity with the sugar molecule of
interest, and that the auxiliary binding groups provide the maximum
affinity on binding.
Reporters Incorporating a Semi-Rigid Binding Pocket Environment
[0464] An additional refinement of the multi-domain concept is to
add a natural molecular binding pocket, which is an integral part
of the reporter molecule structure. This general concept has been
employed in a number of selective ionophores and used in ion assay
systems. The basic notion is to create a ring or pocket-like
structure, of optimal size, which also possesses some natural
binding affinity toward the analyte of interest. Overall binding
affinity is enhanced over a nonrigid analog, due to a diminished
entropy eanalty on binding. In the case of glucose binding, crown
ethers and cyclic peptides are a logical starting point. These
molecules are advantageous in a) synthetic accessibility, b)
adjustable size, c) general biocompatibility, and d) ability to
attach additional side chains to enhance binding or steric
exclusion.
[0465] The first iteration of such a cyclic peptide might contain
tyrosine and tryptophan residues. The binding of glucose could then
be detected by a change in the efficiency of energy transfer from
tyrosine to tryptophan. Some preliminary modeling of the
interaction of the cyclic peptide with glucose has been done. (FIG.
33). Additional general concepts are illustrated in FIG. 34.
Repulsion and/or Size Exclusion of Larger Molecules
[0466] In the context of the small binding pocket concept proposed
above, compounds could be designed and prepared, in which reporting
of glycoproteins is minimized or eliminated. Two concepts are
proposed to solve this problem: a) incorporating functional groups
that repel the hydrophilic surface of a protein, e.g. the
functionality of a silicone polymer; and b) establishment of sham
binding sites, which when occupied, exclude binding of large
species to the reporter binding site. See FIG. 35.
Combinatorial/Iterative Design Approach
[0467] Due to the subtle distinctions required in molecular
recognition amongst different sugar molecules, the most fruitful
approach to creating and improving ligand-binding specificity is an
evolutionary/combinatorial paradigm. The desired SMMDR structure is
treated as modular and assembled from molecular building blocks.
SMMDR molecules thus conceived are pre-screened and scored by
analysis in silico, prior to beginning synthetic chemistry efforts.
A general flow chart for addressing this problem is illustrated in
FIG. 36.
[0468] This method is applicable to the series of discrete
molecules illustrated above, as well as a series of
combinatorially-generated compounds.
[0469] The library generation/dock and score/virtual screening
paradigm is applicable to any arbitrary set of candidate chemical
compounds. A known chemical synthesis method is not necessary to
conduct this in silico analysis, however a ready means to construct
target structures is advantageous when new compounds are proposed
for testing.
[0470] Over the last decade there has developed a rich literature
on synthesis of diverse combinatorial libraries comprising 3 or
more points of diversity, and considerable stereochemical
variability. Thus, some of the basic library structural types from
the literature may be evaluated to form the basis for SMMDR
libraries. (7) Several concept examples are presented in FIG.
37.
[0471] The ordinary paradigm of drug development relies on
modification of a "small-molecule" to enhance its fit into a
relatively "fixed" receptor, active site, or binding pocket.
Specificity in this interaction (vs. other similar receptors) is
accomplished by adjusting the chemical functionality, size, and
geometry of the small molecule, to enhance nonbonded interactions
(charges, van der Waals interactions, dipoles, etc.) such that the
interaction energy with the target of interest is more favorable
than with other targets.
[0472] One way to consider the specificity problem is that the
small molecule (i.e. glucose) is fixed and a synthetic "receptor"
(SMMR) is adjusted in ways to exclude larger and undesired
saccharides, while enhancing the binding energy between our SMMR
and glucose. By way of nonlimiting example, several approaches are
suggested: [0473] 1. Bidentate and multi-dentate phenylboronic
acid--SMMR's [0474] 2. Artificial binding pockets based on crown
ethers, cyclic peptides, etc. [0475] 3. Multi-dentate SMMR's with
Boronic acid and other interacting side chains based on precedent
from naturally occurring proteins and other literature.
[0476] Nature has solved the specificity problem in enzymes
(hexokinase, glucose oxidase, etc.); Periplasmic binding proteins,
and lectins, by creating a relatively constrained binding pocket
within a large protein molecule.
[0477] There is a trade-off of size and specificity. For example, a
very small SMMR does not embody sufficient molecular features
(information content) for good specificity. A feature-rich large
molecule can be very specific, but may suffer from multiple
practical and economic drawbacks.
Alternate Fluorophores
##STR00099##
[0479] Alternate fluorophores based on carbostyril and quinazoline
have been identified. Characteristics of these alternate
fluorophores include:
[0480] Carbostyrils--Similar structure-activity relationship (SAR)
to coumarins, long-wavelength examples, known SAR to get high
quantum yield.
[0481] Quinazolines--opportunity for enzymatic activation in situ;
and examples with large stokes shift.
[0482] A number of quinazoline derivatives have been reported in
the literature, as fluorescent reporters. They offer the advantage
of facile synthesis of diverse analogs, high quantum yield and
large Stokes shift. The Quinazolin-4(3H)-one analog ELF-97 in
particularly interesting in enzymatic assays. For example, the
weakly-fluorescent, phosphate ester of ELF-97 is hydrolyzed by acid
and alkaline phosphatase enzymes, to yield the insoluble alcohol.
Interestingly, the hydrolyzed form is strongly fluorescent, with a
very large stokes shift and is insoluble in aqueous solution.
[0483] The fluorescence of these compounds arises from the ability
of the molecule to adopt a planar conformation, stabilized by an
intramolecular Hydrogen bond when the bulky phosphate group is
hydrolyzed away (FIG. 38).
[0484] It is likely that an ester or amide of a similar SMMR could
be created, such that the compound would be activated by esterases
or proteases in the interstitial medium of the epidermis. The
resulting insoluble, fluorescent compound may be less likely to
move by diffusion.
[0485] One compound for direct glucose detection, AF-327d, is a
boronic acid analog, based on Coumarin-151. It derives its
fluorescence from a push-pull fluorophore, whose
donating/withdrawing properties are modulated through the acid-base
equilibrium that occurs on the boronic-acid moiety (FIG. 39). This
compound exhibits a stronger response to glucose than other
published compounds. The following characteristics of this compound
can be modified: [0486] 1. Specificity--modifications to
distinguish glucose from other nonglucose saccharides. [0487] 2.
Wavelength--a longer wavelength is desirable, to avoid interference
of autofluorescence in vivo. [0488] 3. Calibration--Ratiometric
self-referencing is will be helpful.
Computational Studies
Wavelength
[0489] Using the correlation of wavelength vs. computed HOMO-LUMO
energy gap, it is possible to predict the fluorescent wavelength of
candidate compounds. Substituent patterns on coumarin, carbostyril,
and xanthene analogs were evaluated. Additional studies assessed
the predicted effects of annellation to the corresponding
naphtho-analogs, and alternate linkages between the fluorophore and
boronic acid units analogous to AF-327d.
[0490] Conclusions from the computational predictions are presented
as relative change in predicted wavelength-- [0491] 1. N,N-dimethyl
substituent increases predicted wavelength about +10 to +12 nm vs.
--NH.sub.2 in coumarin analogs, FIG. 40 (A). [0492] 2. Annellation
to form a naphthocoumarin can increase wavelength dramatically FIG.
40 (B). [0493] 3. Wavelength is sensitive to substitution pattern,
which affects the strength of the push-pull interaction, FIG. 40
(C).
[0494] This wavelength effect is predicted to carry over to a
Naphtho-coumarin analog of AF-327d, as shown in FIG. 41.
[0495] The general influence of ring annellation is also observed
in the case of xanthenes, which can be viewed as a "benzocoumarin"
or a 14-.pi. electron homolog of the coumarin ring system (FIG.
42).
[0496] Starting from a relatively long wavelength analog of
Rhodamine 700, conversion to the semi-naphthorhodafluor provides an
additional dramatic increase in predicted wavelength over the
corresponding coumarin analog (FIG. 43).
[0497] These predictions provide an ideal roadmap for elaboration
of longer-wavelength analogs for our direct glucose probes. The
synthesis of R-700 and a few naphthocoumarin analogs is known in
the literature. Semi-naphthorhodafluors have also been made. Thus
future long wavelength (and near-IR) analogs in these three
families are synthetically accessible through straightforward
reaction schemes.
Ratiometric Behavior
[0498] The ratiometric pH dependent fluorescence of SNARF is well
known. The dual wavelength spectrum around pH=7 is due to the --OH
and --O.sup.- species respectively. In this example, the OH and O--
are the electron donors in a push-pull system, where deprotonation
increases the strength of the donation in a push-pull fluorophore
and allows for additional tautomeric forms.
[0499] Calculations on several analogs with a variety of
substituents at the bottom of the molecule demonstrated a strongly
electron-donating ring that appears to provide a larger wavelength
shift on deprotonation of the hydroxyl group. Examination of
Hammett constants revealed that the carboxylate anions on SNARF
would also be slightly electron donating.
[0500] This knowledge can be used in creating a "ratiometric"
glucose probe, to satisfy the internal referencing situation (FIG.
44).
[0501] Design Strategy for pH-Based Glucose or Lactate
Reporters
Esculetin (6,7 Dihydroxycoumarin) pH Dependency
[0502] The pKa of esculetin was determined to be 7.5. The intensity
of the fluorescence varies with pH but not the emission wavelength.
The absorption spectrum did change with pH, implying the molecule
could be used ratiometrically to measure pH using one monitoring
wavelength and two excitation wavelengths. The relevant spectra are
shown here together with a plot of absorption ratio as a function
of pH (FIGS. 45(a)-(c)).
[0503] Design Strategy for Other Glucose Reporting Structures
Crown Ethers
[0504] The molecular structure shown immediately below is a boron
derivative of a crown ether. Provided the boron still has affinity
for alcohols, the structure might be expected to bind with
monosaccharide. Modeled molecular mechanics of a crown ether with a
fluorescein and rhodamine dye tethered to opposite sides of the
ring. In one embodiment, the crown ether is in a conformation in
which the two dyes are brought into close proximity to one
another.
##STR00100##
[0505] For the molecule drawn above, the electron density is
compared with a conventional boronic acid. Calculations are run
with ZINDO using HyperChem. Calculations with crown ethers and
glucose seem to position the glucose above the plane of the crown
ether.
[0506] It is well known that such compounds have been used to
detect the presence of metal ions. Crown ethers are often used as
phase transfer catalysts in organic chemistry. No reports of
interactions between glucose or monosaccharides with crown ethers
were identified. While not intending to be bound by theory, it is
suggested that modifying the ring with boron as previously
described or by synthesis of a more conventional boronic acid crown
ether derivative would be expected to improve the affinity of the
ether for the saccharide.
##STR00101##
Techniques for Placement of SMMRs into the Epidermis
[0507] For any of the embodiments described herein, a series of
techniques exist that allow the placement of specialized
fluorescent or absorptive molecules (SMMRs) into the epidermis,
epithelial cells, or peripheral cells (for organs or muscle tissue
during invasive surgery). Penetration of the sensor composition can
be accomplished using an active transport technique, such as, for
example, electroporation, laser poration, sonic poration,
ultrasonic poration, iontophoresis, mechanical poration, solvent
transport, direct application by painting, tattooing methods
involving application by needle, an equivalent electrical tattooing
technique; or most preferably by using passive transport using
special solvent and reporter molecule mixtures. Passive transport
may be used to allow small molecules of typically 100 Daltons (Da)
to 1000 Da to enter tissues and cells.
[0508] Exemplary methods for passive transport are pressurized
delivery and wicking. The method is comprised of a direct
measurement of the fluorescence of SMMRs placed within epidermal
cells, i.e., keratinocytes. This fluorescence is measured using
molecules with specific properties for defining glucose metabolism
in epidermis and for inferring the magnitude of the change in
fluorescence signal to blood glucose concentrations.
[0509] Incorporation of a reporter into the tissue without use of
an external device is preferred, due to the reduced cost,
convenience, and ease of use. Such a passive transdermal delivery
solvent system must be accurate and safe. Thus, a more elaborate
solvent regime must be applied than that used for the active
mechanisms such as tattooing, electroporation, and ultrasonic
poration. Suitable solvent systems useful for passive transdermal
delivery include creams, emulsions, and oils. These solvent systems
provide passive transdermal stain delivery into the tissue at a
depth of less than 50 microns. The following additives aid the
process of tissue penetration for SMMR and create a diffusion rate
enhancing solvent system: Soybean Oil, Hazelnut Oil, Jojoba Oil,
Sweet Almond Oil, Olive Oil, Calendula Oil, Apricot Kernel Oil,
Grapeseed Oil, Wheat Germ Oil, refined Light Mineral Oil,
Triundecanoin (Akomed C), Undecanoic acid, Caprylic/Capric
Glycerides (Akoline MCM), Caprylic/Capric Triglycerides, Propylene
glycoldiester of caprylic-/capric acid, Emu oil, all as low
viscosity mixtures, preferably less than 35 cSt at 35.degree. C. In
addition, mixtures of one or more of the above oils in combination
with a nonpolar dilution solvent can also be used. The solvent
system is allowed to passively penetrate the tissue for from about
1 minute, about 5 minutes, about 10 minutes, about 30 minutes to
about 2 hours to allow diffusion of the SMMR into the appropriate
tissue layer(s).
[0510] In addition, penetration of the sensor composition to the
desired depth can be accomplished by combining the composition with
various molecular size attachments.
[0511] After the reporters are injected into, or applied to the
surface of the tissue, they are allowed to penetrate in proximity
to superficial cells of tissues and organs at a depth from the
surface of the cells of from about 10 .mu.m to about 1500 .mu.M.
For measurement of specific metabolites, the preferred placement of
the reporters should be near the surface of the tissue (i.e., about
10 to about 175 .mu.m) yet be representative of the overall
metabolic state of the tissue in which the reporters are placed.
The reporters may also be placed at a greater depth into the
tissue. The precise placement of the reporters is controlled by the
combination of its molecular properties, including: specific
molecular size (i.e., 100 daltons to 100 kilodaltons), polarity,
charge, structure, pKa, solubility, the size and type of molecular
attachments or anchors, the solvent system used, as well as the
specific conditions used for poration (if required). A combination
of these factors provides the ability to control the location,
diffusion rate, and duration or lifetime of the SMMR within the
tissue or organ layers.
[0512] The dyes may be introduced into the skin by passive
diffusion over a period of 24-48 hours, more preferably over a
period of 2-6 hours, and most preferably in 10 seconds to 5
minutes. Contemplated diffusion times include periods less than 48
hrs, 24 hrs, 10 hrs, 6 hrs, 2 hrs, 1 hr, 30 min, 15 min, 10 min, 5
min, 1 min, 30 sec, 10 sec, or 1 sec. With passive absorption, a
molecule is placed on the surface of the skin and allowed to
penetrate in proximity to the epidermal cells (keratinocytes)
directly above the basal layer (stratum basale) at a depth from the
surface of skin from 10 .mu.m to 50 .mu.m and up to 175 .mu.m in
the pits of the stratum basale extending into the dermis between
the dermal papillae. For measurement of glucose, the placement of
the SMMR is below the stratum corneum yet above the dermis, more
specifically in the stratum spinosum or stratum basale immediately
above the upward extensions of the dermal papillae. This SMMR
placement is accomplished by varying the combination of the
polarity and charge on the SMMR, the size of molecular attachments
or anchors, as well as by the polarity and hydrophilicity
characteristics of the solvent system. The specific conditions for
poration or passive diffusion for placement of the SMMR in the skin
are controllable factors. Using any combination of these factors,
it is possible to control the localization of the dye within the
skin layers and target cells.
[0513] Another embodiment of the reporter application involves the
use of a reservoir containing reporter, which is used to
automatically or manually dispense a dose of the reporter mixture
topically prior to poration or passive transport. For measurement
of metabolites and precursors the reporter is placed in the tissue
at a depth of up to 300 .mu.m. A solution of 10-400 .mu.L volume
made from 1-50 .mu.M SMMR in a solvent system penetrates into the
tissue for some period of time to allow activation following
passive diffusion kinetics. Once activated the change in
fluorescence or absorption response of the tissue cells to changes
in extracellular and intracellular metabolite or precursor
concentrations is monitored directly using an optical reader.
Irritant chemicals such as salicylic acid can be used to facilitate
the penetration of reporters into skin or peripheral tissue.
[0514] In another embodiment, a small disposable film patch
composed of polyolefin, polyester, or polyacrylate and having an
SMMR dispersed into a transfer gel applied to the transfer side of
the film patch, is used for SMMR application. The patch is applied
with the gel side toward the skin and the gel contacts the external
surface of the skin. Following the gel application, a poration or
passive transfer technique is used to introduce the mixture into
the appropriate skin layer(s) (as described above). Another
embodiment of the SMMR application involves the use of a reservoir
containing molecular tag or SMMR. This reservoir is used to either
automatically or manually dispense a dose of the SMMR mixture
topically prior to poration or passive transport. A nonlimiting
example of a topical dose is a small dot or spot from 100 .mu.m to
5 mm. A smaller area is preferred in most embodiments, but a larger
area is also contemplated. For measurement of glucose, the SMMR is
placed in the keratinocytes at 30 .mu.m to 50 .mu.m and up to 175
.mu.m so that placement is precisely in the specific layer of the
epidermis (e.g., above the dermal papillae and within or above the
stratum basale), within a comparatively homeostatic keratinocyte
stratum. The molecular tag or SMMR penetrates into the skin for
some period of time (depending upon molecular size and solvent
mixture used) to allow activation following passive diffusion
kinetics (i.e., mass transport). Once activated, the change in
fluorescence response of the skin cells to changes of extracellular
and intracellular glucose is monitored directly using an optical
reader.
[0515] An active mechanism utilizing tissue permeation,
electroporation, laser poration, or ultrasonic poration is another
procedure for introducing SMMRs into the skin. Pulse lengths for
poration technologies are provided below. An example of an
ultrasonic poration device includes those manufactured by Sontra
Medical Corporation, Cambridge Mass. Sontra and other commercial
manufacturers of devices useful for this application have
previously described a method for sensing glucose directly in the
interstitial fluid surrounding the skin cells by removing fluid or
gaining access to removed fluid for analysis. See, e.g., J. A.
Tamada, M. Lesho and M. J. Tierney, "Weekly Feature: Keeping Watch
on Glucose--new monitors help fight the long-term complications of
diabetes." IEEE Spectrum Online, Jun. 10, 2003 at website:
<http://www.spectrum.ieee.org/WEBONLY/publicfeature/apr02/glu.html>
(last visited Jun. 26, 2003). The methods and compositions of the
invention do not remove fluid but, rather, place small quantities
of solution containing low concentrations of SMMRs into the skin
for direct reading of the SMMR fluorescence spectral
characteristics as an indication of both epidermal skin and blood
glucose levels.
[0516] For some reporters above 1,000 daltons in size,
electroporation may be used to introduce reporter into tissue.
Electroporation has been utilized for introducing chemotherapy
treatments, for introduction of DNA into living cells and tissues,
and broadly recommended for introducing materials into tissues for
cosmetic or medical treatment applications. If poration schemes are
used, the optimized settings for an electroporation device are
achieved by commercially available or by a customizable device
having settings that provide conditions as described within this
invention. Commercial systems utilizing a square wave voltage pulse
have been described within the literature, such as those available
from Genetronics Biomedical Corporation, 11199 Sorrento Valley
Road, San Diego, Calif. 92121. Such a small device can be
inexpensively made to have one or more constant settings for the
optimized conditions disclosed for this invention.
[0517] Electroporation uses a short pulse electrical field to alter
cell membrane permeability. Micro-pores form in the membrane of
skin cells allowing the introduction of various molecular size
mixtures into the cells at an appropriate depth of penetration for
this specific inventive application. When the electric field is
discontinued, the cells return to normal and one or more SMMRs
introduced into the cell using the technique remains at the
cellular site specifically within the epidermal cell until either
the dye is chemically degraded and disposed of within the tissue or
is sloughed off in a normal desquamating cycle. The process of
sloughing off (or desquamation) follows a normal ten-day to
twenty-day (typically fourteen-day) cycle as the residence time of
epidermal keratinocytes moving from the basal layer (stratum
basale) to the desquamating layer of the stratum corneum.
[0518] When employed, electroporation is optimized for use in this
invention by selection of voltage range (from about 40 to 90
Volts), gap distance (from about 0 to 2 mm), pulse length (from
about 150 to 250 ms), number or pulses (from about 1-10), pulse
interval (from about 5 to 60 s), specific electrode design, and
desired field strength (from about 40 to 60 V/cm). In addition, the
selection of molecular tag molecules, solvent molecules,
concentration, and lag times relative to measurement onset is
determined as precisely as possible. In certain embodiments,
specific parameters are determined empirically using specific
solvent and SMMR selection. For example, optimization of
electroporation involves the following specifications: [0519] 1.
Output voltage range: 0 to +200 VDC; [0520] 2. Discharge capacitor
(Cdis) values in microfarads are on or about: 200, 500, 700, 1000,
1200, 1500, 1700 .mu.F; [0521] 3. Pulse type: exponential decay;
[0522] 4. Pulse RtCdis decay time constant where Rt (total)=5+Rskin
in parallel with 50 ohms. If Rskin>>50 ohms then Rt=55 ohms
and Rt.times.Cdis=11, 27.5, 38.5, 55, 66, 82.5, 93.5 milliseconds
(ms).
[0523] Electroporation also facilitates the delivery of dyes bound
to large molecules that serve as anchors such as polymer beads,
large polysaccharides, or colloidal particles. These approaches are
contemplated as being within the invention, but are less
advantageous in that the particles are often too massive to pass
through the stratum corneum without active poration or mechanical
injection. Once in the skin, they do not readily dissolve or
organically reabsorb into the body. Such less desirable approaches
would create undesirable particles that would either remain in
place indefinitely or accumulate in lymph nodes, in other
circulatory cavities and/or in other organ sites.
[0524] Reporters of the invention can be made with specific
properties such that they are retained only within skin cells
(keratinocytes) where they report on glycolytic activity and do not
harm or affect cellular metabolism. These reporter compounds are
sloughed off after a few days, even when permanently integrated
into, or attached to, keratinocyte cells. The small quantity of
reporter(s) that diffuse away from the epidermis are rapidly
degraded within the body and are completely eliminated within a few
days. In preferred embodiments, reapplication of the reporter(s) is
relatively easy to perform. The process of sloughing off (or
desquamating) follows a normal ten-day to twenty-day (typically
fourteen-day) cycle as the residence time of epidermal
keratinocytes moves from the basal layer (stratum basale) to the
desquamating layer of the stratum corneum. Thus, reporters are
developed to be applied once every 2 to 3 days, preferably every 3
to 4 days, and more preferably every 5 or more days.
[0525] More advanced solvent systems useful for passive transdermal
delivery include, but are not limited to, e.g., creams, emulsions
(both oil-in-water and water-in-oil), oils (ointments), gel film
patches, a reservoir device, paints, polar solvents and nonpolar
solvents. Nonpolar solvents are preferred, as these are most
miscible with the SMMRs of the invention and the stratum corneum
lipids cementing the keratinocyte lamellae in place. "Lipid solvent
systems" have been reported in the literature for use in
transdermal drug delivery, and are composed to resemble the
chemistry of stratum corneum lipids. Such a mixture may also be
used to place the SMMRs into the appropriate point within the
epidermis. Such a suggested mixture includes: (w/w): ceramide
(50%), cholesterol (28%), palmitic acid (17%) and cholesteryl
sulfate (5%). See, e.g., Downing et al.: Partition of dodecyl
sulfate into stratum corneum lipid liposomes. Arch. Dermatol. Res.
1993, 285:151-157.
[0526] The objective of each of these solvent systems is to provide
passive transdermal SMMR delivery into the skin at a preferred
depth of from about 10 to 175 .mu.m (microns), more preferred from
about 20 to 100 microns, and most preferred from about 20 to 50
microns. For example, the following solvents as additives to the
final SMMR mixtures are added to the skin to initiate passive
transport of the SMMR to the target cellular site. The materials
listed aid the process of skin penetration for SMMRs and create a
diffusion rate enhancing solvent system for transdermal delivery:
dimethyl sulfoxide, ethanol, isopropanol, chloroform, acetic acid,
saturated hydrocarbon solvent (with from 10 to 40 carbons as linear
or branched chained molecules), soybean oil, hazelnut oil, jojoba
oil, sweet almond oil, olive oil, calendula oil, apricot kernel
oil, grapeseed oil, wheat germ oil, refined light mineral oil and
mineral oil spirits, triundecanoin (akomed C), undecanoic acid,
caprylic/capric glycerides (akoline MCM), caprylic/capric
triglycerides, propylene glycoldiester of caprylic-/capric acid,
and emu oil. All are low viscosity mixtures, preferably less than
35 cSt at 35.degree. C. In certain embodiments, mixtures of one or
more of the above oils are used in combination with a nonpolar
dilution solvent.
[0527] Factors that control the depth of penetration of the SMMR
and its compartmentalization into the cells and domains of the
epidermis include the polarity and partition coefficient of the
SMMR as well as the solvent and the molecular size. The SMMR
compound may also be derivatized so that it is readily taken up by
the cell and then acted upon by enzymes that chemically alter the
SMMR to prevent it from leaking out of the cell. One advantage of
this type of approach is that the SMMR is only taken up in its
active form by viable cells. Predictive schemes for determining
appropriate derivatization of SMMR compounds are provided below.
Alternative methods of derivatization well known to those skilled
in the art are also contemplated as part of the invention.
[0528] The physical properties of the solvent system that strongly
influence permeability in the skin include the molecular size, the
vapor pressure, the water solubility, and the octanol water
coefficient. Smaller molecular size increases the diffusion
coefficient. The vapor pressure controls the balance between
diffusion into the skin and evaporation from the surface. The water
solubility and the octanol water partition coefficient determine
the miscibility of the SMMR solution between aqueous interstitial
fluid and hydrophobic core of the cell membrane.
[0529] For a passive solvent delivery system, the depth of
penetration of the SMMR is strongly dependent on the volume of
solvent added. Typically, the volume of SMMR used is from 10 .mu.L
to less than about 100 Preferably, the concentration of SMMR is
from 10 to 500 .mu.M, more preferably from 100 to 300 .mu.M, and
most preferably from 150 to 250 .mu.M. Target cells are exposed to
extracellular concentrations in the range of 1 to 10 .mu.M.
Dilution of the SMMR concentration arises because of the diffusion
properties from the surface of the tissue to the target cell
site.
[0530] The proposed volume range added to the skin or other tissue
is preferably from 1 to more preferably from 5 to 20 and most
preferably from 5 to 15 Alternatively, a gel patch is used
containing an SMMR coated surface of approximately 6 mm in diameter
consisting of a concentration of SMMR preferably from 10 to 500
.mu.M, more preferably from 100 to 300 .mu.M, and most preferably
from 150 to 250 .mu.M.
[0531] Solvent systems used for SMMRs may be adjusted depending
upon their molecular properties and compatibility with the specific
SMMR being delivered. For example, solvent hydrophobicity and
polarity are noted along with the solubility properties of the
SMMR, which will all have an effect on the movement of the SMMR
into the tissue. Each SMMR has a certain affinity for the solvent
and the tissue. The solvent's activity for delivering the SMMR
directly to target tissue is a matter for empirical testing. One
preferred embodiment of the invention uses an SMMR dissolved in
DMSO (dimethyl sulfoxide) and further diluted in a saturated
hydrocarbon solvent (with from 10 to 40 carbons as linear or
branched chained molecules), or an alcohol (with from 2 to 4
carbons) at a volume ratio of 5:95 to 20:80, respectively. The
optimum volume of DMSO in the delivery solvent is less than 20
percent, as the DMSO is used to facilitate dissolution of the SMMR
into the carrier hydrocarbon mixture. The mixture is added to the
tissue in the concentrations and volumes described above.
[0532] A gel patch may be used to apply the SMMR. In one
embodiment, a gel contains the SMMR in a volatile hydrocarbon
solvent in suspension with a polymer such as PVA (polyvinyl
alcohol). When placed against the skin or other living tissue, the
heat of the skin causes the SMMR (dissolved in the PVA-hydrocarbon
solvent) to diffuse into the skin. The final diffusion depth is
controlled by length of application time. Volumes below 100 .mu.L
minimize extraneous transdermal delivery and maximize delivery into
the epidermis target area. Optimum passive solvent delivery is
attained by using a solvent mixture or emulsion that facilitates
the movement of SMMR across the stratum corneum into the epidermis,
but then dissipates rapidly to limit movement of the SMMR away from
the target area. Solvent systems that have the lowest toxicity
include water, saturated hydrocarbon oils, polyethylene glycols and
glycerol. Solvents systems that include alcohols and dimethyl
sulfoxide are less favored in this application since these solvents
are less biologically inert.
[0533] The SMMRs are applied directly to the surface of the skin
and then passively allowed to penetrate the skin for a period of 1
minute to 5 hours, more preferably less than 4 hours, and most
preferably less than 1 hour. Ideally, a solvent delivery system
would be developed to provide SMMR delivery to the target tissue in
less than 1 hour, more preferably less than 30 minutes, and most
preferably in less than 5 minutes. This time period allows the
passive diffusion of the SMMRs into the appropriate epidermal
cells.
[0534] Once the one or more SMMRs are activated as a result of
placement within the skin, measuring fluorescence monitors the
response of the skin cells to glucose. As described herein, the
fluorescence mechanism used is either a direct or indirect
indication of the glucose concentration in the target cell
environment. Fluorescence is typically measured using an optical
reader. The optical reader calculates the skin response to glucose,
applies first principles mathematical models to the response (as
described below and shown in FIG. 7), and provides a determination
of the blood glucose levels (see FIGS. 3, 8-9). The choice of the
particular commercially available or custom designed optical reader
that is compatible for use with the methods and compositions of
this invention is within the ability of one skilled in the art of
the invention.
[0535] Apparatus and Methodology for Glucose Detection Using
SMMRS
Instrumentation Required for Reporter Monitoring
[0536] The instrumentation required to detect changes in reporter
signal may consist of simple light emitting diode sources combined
with low-cost solid-state detectors. The mechanism of signal
extraction relating to a biochemical or physiological process is
derived from the elucidation and measurement of key metabolic
pathways. The reporters are excited, and the remitted energy
detected over the wavelength region of 190 nm to 850 nm (see FIG.
38). The three mechanisms of measurement for metabolites or
precursors using the reporters of the invention include (1) using
reporters to increase the signal-to-noise of native
autofluorescence signals indicative of human reductive metabolism
[FADH.sub.2, NADH, and NAD(P)H], (2) using reporters for selection
and enhancement of specific metabolite and precursor signals in
tissue that are indicative of metabolic state and allow
determination of changes in metabolism [Ca.sup.2+, lactate,
oxygen], and (3) using reporters to directly measure the presence
of intracellular or extracellular molecular metabolites
[protein-FL, and protein-.sup.3FAD*].
[0537] All three mechanisms of signal identification and
enhancement allow utilization of low-cost, hand held
spectrophotometric equipment (e.g., LED excitation and diode
detectors) that is simple in design and does not require advanced
or complicated computational algorithms. Such equipment is not
harmful to subjects and requires just an additional disposable
component (other than a calibration strip) to prepare the subject
for metabolite monitoring. A measurement device approximately the
size of a personal cell phone having quality features, such as
those which allow the user to determine whether a specific
measurement is valid, or whether a repeat measurement is required,
can be used. Such a hand-held, battery powered device is intended
to be used either occasionally, or on a continuous, real-time
monitoring basis for subjects requiring serious health management
regimes. A single calibration allows continuous monitoring for up
to several hours. A calibration technology that utilizes a
calibration strip, which mimics the optical response of the subject
and allows freedom from continuous correction using primary
analysis devices, can be used. Other calibration technologies
contemplated by the invention will be readily discerned by those
skilled in the art.
[0538] As an example, to use the device, the subject or physician
prepares the area to be measured using the enhancement technology,
which is painless and requires a patch (similar in appearance to a
Band-Aid.RTM. bandage), paint, or spray to be applied to the
targeted tissue area. This treatment conditions the tissue area for
from a few minutes up to 30 days, depending upon the SMMR
properties selected and the depth at which it has been deposited in
the subject tissue. The device is then calibrated using a
calibration strip and is ready to make measurements for up to 2
hours or more, without requiring additional calibration. The
subject or physician examines the conditioned area with the sensor
and makes a measurement. Typically, the measurement takes less than
about 5 seconds, and the sensor provides the appropriate metabolite
concentration or reports that a repeat measurement is required.
[0539] In another embodiment, if the photophysics of fluorescent
dyes are considered, the fluorescence changes associated with the
SMMR and the analyte may also be monitored using fluorescence
lifetime technology. One preferred embodiment for such a hand held
device capable of measuring lifetime changes is to use a phase and
modulation spectrometer, which is a device constructed from a radio
frequency modulated light emitting diode and a miniature
photomultiplier or photodiode, whose signal is amplified by a phase
sensitive amplifier. Such devices have been well characterized in
the literature and are commercially available in a variety of
forms. Manufacturers of such devices include: Photon Technology
International, Inc., 1009 Lenox Drive, Lawrenceville, N.J. 08648;
PicoQuant GmbH, Rudower Chaussee 29 (IGZ) 12489 Berlin, Germany;
Tecan Systems Inc., 2450 Zanker Road, San Jose, Calif. 95131;
Thermo Oriel, 150 Long Beach Blvd., Stratford, Conn. 06615. These
devices measure both the degree of modulation of the fluorophore
and the phase shift of the emission relative to the excitation
light, and these two parameters are then related to the lifetime of
the dye. Determination of these parameters at a number of
frequencies increases the accuracy of the device.
Phase Sensitive Flash Photolysis Apparatus
[0540] A suggested sensing apparatus can be miniaturized and
measurement of the phase shift gives an indication of the lifetime
of the transient species. This is an advantage over single pulse
flash photolysis apparatus that typically require mJ pulse energies
to generate transient species with sufficiently high concentration
to be observed. In one embodiment, the apparatus described here is
capable of detecting transients with a change in absorption of
10.sup.-5 (FIG. 46).
[0541] This apparatus is miniaturisable and the phase shift gives
an indication of the lifetime of the transient. This is an
advantage over single pulse flash photolysis apparatus that
typically require mJ pulse energies to generate transient species
with sufficiently high concentration to be observed. In one
embodiment, the apparatus described here is capable of detecting
transients with a .DELTA. absorption of 10.sup.-5.
[0542] The diffuse reflectance light is modulated by the absorption
of the transient therefore the instrument will reject scattered
light. Selectivity is improved over a conventional spectrometer
because the requirements for a transient to be detected are that
the ground state has absorb at the correct wavelength, the
transient species generated has to absorb at the correct wavelength
and the transient generated has to have a defined lifetime. Triplet
states typically have very different absorption spectra than ground
state species and may occur at longer or shorter wavelengths than
the ground state. This is an advantage over fluorescent molecules
with small Stokes shift.
Dual Processor, 1 GHz Computer Set Up with Spartan
[0543] The idea of using light to drive enzymatic reactions is not
new (see, e.g.,
http://chem.ch.huji.ac.i1/.about.eugeniik/photo_enzymes3.htm).
However, taking the cofactor from an enzyme and designing a small
molecule system that can be driven to carry out the same reaction
in the presence of light has not been reported. Enzymatic reactions
are catalyzed essentially by shifting the position of equilibrium
when the reactants are brought into close proximity. Photochemical
reactions are powered by the energy supplied by photons. This is
considerable; to generate the same number of excited states in a
sample with an absorption of 1 and molar absorption coefficient of
10.sup.5 dm.sup.3 mol.sup.-1 cm.sup.-1, with 1 mJ of 400 nm light
would require a temperature of nearly 30,000 degrees Celsius!
[0544] The Brunet paper [Brunet, 2002 #562] refers to some
theoretical studies that show, in the excited state there is an
increase in the dipole moment. This change is brought about by an
increase of electron density on the carbonyl groups. The magnitude
of this change is not sufficient to generate a charge transfer
state but it is obvious if the binding of an analyte interferes
with the charge distribution of the excited state then this would
provide a sensitive transduction mechanism. A change in electron
density with the binging of glucose is caused by the use a boronic
acid. The Brunet behavior can be modeled using Spartan.
[0545] Measured the repetition rate of the nanolase 355 nm tripled
YAG. Using the 500 MHz Tektronix scope and a photodiode the
frequency is 9 kHz. This is consistent for a transient with a
lifetime of about 40 .mu.s.
[0546] Set up the flash photolysis. Calculated using a 5 mJ laser
at 9 kHz, an OD of 1 and a difference absorption coefficient of
45000, then for a 1 cm pathlength an OD should be 0.006. This
should be simple to measure. A change in oxygen concentration with
a porphyrin can also be detected. Subsequent experiments involve
glucose oxidase.
[0547] Apparatus set up. The apparatus has been set up as follows:
The monitoring light wavelength is chosen where there is maximum
difference between the excited state absorption and the ground
state absorption. The excitation wavelength is chosen where there
is maximum absorption of the ground state. The photomultiplier is
coupled to a lock-in amplifier. The trigger diode generated the
signal for the reference channel. The excitation source used in
this experiment was a nanolase tripled YAG that has repetition
frequency of 9 kHz. A telescope was made from a microscope
objective and a cylindrical lens (FIG. 47).
[0548] The experiment was carried out with a solution of
deuteroporphyrin in ethanol. The sample was flushed with nitrogen
first to remove dissolved oxygen. Under these conditions, the
lifetime of the excited triplet state should be about 100 .mu.s.
The difference molar absorption coefficient is about 40,000
dm.sup.3 M.sup.-1 cm.sup.-1. The laser energy was measured at the
sample to be 1.8 mJ. With these figures, it was calculated that the
sample should generate a transient with a difference absorption of
about 0.006. Although the lock-in amplifier detected a signal, the
phase shift of this signal did not change when oxygen was
readmitted into the sample and is probably residual
fluorescence.
[0549] Instead of using the lock-in amplifier, the signal from the
PMT was coupled to a Tektronix 500 MHz digital scope. Averaging 512
pulses, it is possible to observe a change in absorption of about
0.01% that corresponds to an absorption change of about
4.times.10.sup.-5. No transient at all was observed under these
conditions; however, such a small transient can be measured using
this device. It would be expected that even smaller signals could
be observed with the lock-in amplifier. The sensitivity of the
system would be sufficient to observe electron transfer in glucose
oxidase in the presence of glucose.
Chromatography on Saccharide Solutions Containing Glucose
[0550] The purpose of this experiment is to set up a chromatography
system that can be used to separate glucose from other saccharides
and to detect glucose using Arg-327. To set the chromatography
system up we need a rapid reliable test for glucose. HPLC methods
use amperometric methods or refractive index changes to monitor
saccharides. Two well known methods are the Fehling's test for
reducing sugars and to use enzyme activity.
[0551] The Fehling's method uses two solutions commonly known as
Fehling's A and B. Fehling's A consists of 7 g of hydrated
copper(II) sulfate dissolved in 100 mL of distilled water.
Fehling's B is made by dissolving 35 g of potassium sodium tartrate
and 10 g of sodium hydroxide in 100 mL of distilled water. The
Fehling's reagent is made from equal volumes of Fehling's A and
Fehling's B are mixed to form a deep blue solution. The test is
really one for aldehydes and since a small percentage of solvated
glucose exists in an open chain, aldehyde form, it gives a positive
result to the test.
[0552] The cupric ion in the Fehling's A solution acts as a mild
oxidizing agent, the tartrate complexes with the cupric ion and
prevents copper hydroxide from precipitating from solution. The
overall reaction is:
##STR00102##
[0553] Cuprous oxide is insoluble in water and forms a brick red
precipitate. The oxidation of glucose could be carried out
photochemically to generate fluorescent products.
[0554] The method chosen to develop the chromatographic system is
an enzymatic one. The method is quantitative and relatively
specific:
##STR00103##
[0555] An equimolar amount of NADH is generated for the amount of
glucose consumed. The NADH may be monitored by either its
absorption at 340 nm or its emission at 450 nm.
[0556] The proposed experiment is to set up a number of small
packed columns and to determine the optimum conditions for viewing
of the glucose eluting from the column.
In Vitro Glucose Probe not Requiring Strip Use
[0557] This probe is made by attaching Arg-327 to the end of fiber
optic and monitoring the fluorescence of this material using the
following apparatus (FIG. 48).
[0558] The device is calibrated by dipping the end of the fiber in
glucose free medium and a known concentration of glucose. Dipping
the fiber into an unknown glucose solution then gives a
fluorescence response that is correlated with the glucose
concentration. A suitable dye to carry out this experiment is
Arg-327.
[0559] To develop a strip type of device two approaches were used.
First, slurries of materials that may be used as TLC plates were
made and secondly different materials were tried in order to image
spots of glucose placed on the plates.
[0560] Plates were made up by dipping clean microscope slides in
slurries of Silica, TiO.sub.2 and Carbomer 981 in methanol. Of
these, the plates made with TiO.sub.2 came out the most uniform. If
the silica used for these plates is too coarse, commercial plates
can be used. The Carbomer 981 slurry that dries to a clear film is
a commercial material that is used to control viscosity in
cosmetics.
[0561] To view glucose spotted onto commercial silica plates 10%
(w/v) solutions of chloranil and phenyl boronic acid were
co-spotted with the glucose (300-mg/dL) and the result viewed under
UV light. Chloranil was used because it is an oxidant and is known
to undergo a color change when it is reduced. Phenyl boronic acid
was used as a model compound for Arg-327 since it can be viewed
under UV light. There is some indication that the chloranil reacted
with the glucose.
[0562] The experiment was repeated with alkaline glucose.
[0563] Used Arg-327 as one of the visualizing materials. Compared
chloranil and Arg-327 at a concentration of 1% (w/v) with and
without glucose. There is visually little difference between the
spot with and without glucose. A dark spot can be seen with
chloranil and glucose. Also spotted the plate with 1%, 0.1% and
0.01% Arg-327. All three spots are visible but the dynamic range
that can be judged with the eye is poor.
[0564] A sample of Rose Bengal was made up and run on the apparatus
to observe transient absorption.
[0565] Measured fluorescence with fluorolog. Fitted the fluorolog
with the 3 mm fiber bundle. The fluorescence of the Arg-327
decreases by about 30% in the presence of glucose. This is the
opposite of what happens in solution. Plates can be pretreated with
the Arg-327 and/or a gel can be used as the stationary phase.
Strip Technology
[0566] To build a monitor using strip technology different
chemistry to be applied. Molecules bound to gold surfaces can be
used to increase the fluorescence quantum yield. Applying this idea
to strip technology allows one to link fluorophores to a gold
surface. The fluorophore gold linkage is synthesized by
incorporating a thiol group on the chromophore, i.e.
##STR00104##
[0567] The hydrocarbon chain--(CH.sub.2).sub.n-- is of such a
length that the fluorescence of the dye is enhanced by the
proximity of the gold surface [See, for example, "Intrinsic
fluorescence from DNA can be enhanced by metallic particles."
Biochem. Biophys. Res. Commun. Lakowicz, J. R., Shen, B.,
Gryczynski, Z., D'Auria, S, and Gryczynski, I. (2001) 286 875-879
and references therein.] The correct length for the chain is
determined experimentally. If the chain is too short then the
fluorescence of the molecule is quenched completely, if the chain
is too long then there is no surface effect. This phenomenon allows
molecules to be designed that have a sensitive transduction
mechanism for the presence of glucose; their fluorescence
properties are dependent on the presence of the gold surface. Such
technology may be incorporated either into a strip type device or
into a MEMS type device. If a fiber tip was coated with a film of
gold then this type of molecule could be bonded to the fiber by the
interaction of the SH and the gold film.
Compounds Used for Glucose Fluorescent Strip Demonstration
##STR00105##
[0569] AF-327d is a re-synthesis. In parallel, we will make and
test the Benzocoumarin analog, due to the expectation that it may
have a longer emission wavelength, if it's fluorescent.
Strip Design
[0570] The use of strip type technology opens up a number of
possibilities that are not available to noninvasive technologies.
Patents that have been published for strip technology include
features to facilitate blood flow on the strip and to remove
confounding factors such as red blood cells.
[0571] For example, U.S. Pat. No. 5,708,247 issued to Lifescan
includes features such as a silica filter to remove red blood cells
and a mesh to guide the liquid sample to the electrodes on the
strip.
[0572] The construction of a strip for a spectroscopic sensor uses
some of these features to improve selectivity and to ensure rapid
mixing of the blood glucose and the sensor material (FIG. 49).
Prototype substrates have been manufactured from glass, which may
be difficult to engineer but is convenient to use as a reusable
prototyping breadboard in testing.
[0573] The following patterns have been milled in glass. Each of
the patterns described is 0.6 mm deep. Each substrate is 75
mm.times.25 mm.times.1 mm.
[0574] The gray strips are an area where the prototype can be
labeled; the green area is the cutout to a depth of 0.6 mm. Other
shapes were also generated but these were the most accurate and
reproducible to be made. The green portion of each slide may be
filled with aqueous or alcoholic gels, with silica and the third
slide also has a region to apply solvents.
[0575] For the experiments that were carried out with these systems
a silica derivatized with a four carbon chain was used. The silica
was applied in a methanol slurry and the solvent allowed to dry.
The derivatization prevents interaction of the silica with the
boronic acid sensor.
[0576] The technology is using chromatography to separate glucose
in the blood from proteins and cellular material. The development
time for the strip is dependent on the dimensions of the cutout.
The sample is applied at one end of the strip and interaction
between the glucose and the sensor occurs at the other end.
Prototype 2 may be used to look simply at the interaction of the
sensor molecule and glucose.
[0577] Using this type of approach, molecules that have a large
response to the presence of glucose but poor selectivity may be
used as the sensor with interfering substances being removed by the
material on the substrate.
[0578] Silica gels were the only material tried on these plates but
other materials that would be suitable include aqueous and
methanolic gels. The physical dimensions of the strip and the
nature of the gel or silica determine how rapidly the strip
responds to the presence of glucose.
[0579] If the solutions are brought together on plate 2, with no
mixing then the response time may be as much as 100 seconds. By
filling the cavity with small beads or silica, capillary action can
greatly speed up the mixing time.
[0580] This type of technology also lends itself to the use of
fluorescent sensors on gold films. By controlling the distance
between the gold surface and the fluorophore the quantum yield of
the material can be increased to unity. This phenomenon has been
described many times in the literature. The advantage of this kind
of approach to us is that the chemical synthesis of the molecule
can concentrate on the transduction mechanism by which the binding
of the glucose causes a change in the molecule. The quantum yield
of the system is controlled by the gold surface.
REFERENCES
[0581] 1. J. Boeseken, Advan. Carbohydrate Chem., 4, 189 (1949), C.
A. Zittle, Advan. Enzymology, 12, 493 (1951). [0582] 2. H. G.
Kuivila, et al., J. Org. Chem., 19, 780 (1954); J. P. Lorand and J.
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EQUIVALENTS
[0589] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the invention.
Various substitutions, alterations, and modifications may be made
to the invention without departing from the spirit and scope of the
invention. Other aspects, advantages, and modifications are within
the scope of the invention. The contents of all references, issued
patents, and published patent applications cited throughout this
application are hereby incorporated by reference. The appropriate
components, processes, and methods of those patents, applications
and other documents may be selected for the invention and
embodiments thereof.
* * * * *
References