Patent Application: US-88638804-A

Abstract:
methods and compositions for the detection and monitoring of drug therapy are provided . in particular , efficient and sensitive methods for the detection of drug ingestion are provided for determining whether individuals are complying with prescribed therapeutic regimens , and for providing a mechanism for identifying drug - resistant strains of infectious agents . the claimed methods and compositions involve the application of transdermal devices containing detection mechanisms for receiving and recording signals generated by the ingestion of a labeled drug . such devices are attached to the skin for the duration of drug therapy and compliance is determined either by direct reading , or by remote monitoring whereby signals are transmitted from the device and received at an external site such as a healthcare facility .

Description:
the present invention may be understood more readily by reference to the following detailed description of specific embodiments included herein . although the present invention has been described with reference to specific details of certain embodiments thereof , it is not intended that such details should be regarded as limitations upon the scope of the invention . the entire text of the references mentioned herein are hereby incorporated in their entireties by reference . nonadherence to therapeutic regimens has been recognized as a serious threat against the elimination of infectious diseases such as tuberculosis ( tb ). not only does a patient suffer from failing to comply with his prescribed therapy , nonadherence gives rise to other complications such as relapse , continued infectiousness , and is one of the principal causes for the emergence of drug - resistant strains of infectious agents . although programs and initiatives such as directly observed therapy have proven to be valuable , they are often inefficient because they require the involvement of cumbersome procedures which in many cases make therapy burdensome , resulting in ultimate non - compliance . in addition , the emergence of dangerous drug - resistant strains of infectious agents has further heightened the need for ensuring that infected patients comply with prescribed therapy . many people who suffer from disease caused by infectious agents such as mycobacteria , are poor , or live in areas with inadequate access to health care facilities . such people are not easily monitored for compliance , and inexpensive and noninvasive methods for determining compliance are necessary . the present invention provides for monitoring devices comprising detection mechanisms for determining whether a patient has complied with his prescribed drug therapy . in addition to satisfying a compelling public health need to assure compliance with drug therapy , the remotely monitored therapy device / systems of the present invention also function suprisingly well for other applications . for example , other medical situations where prolonged therapy makes compliance difficult , or where nonadherence drives the emergence of drug - resistant strains include the use of antibiotics , including antifungals , immunosuppressive therapy following organ transplantation , and aids therapy . in addition , in order to best interpret clinical trial results , pharmaceutical companies may measure compliance by the methods of the present invention . the present invention provides for a transdermal sensor that , with minimal effort on the part of the patient , accurately detects that a therapeutic drug dose has been ingested . the device may optionally transmit this information to a remote monitoring facility , providing an accurate , real - time record of when each drug dose was ingested . the system is resistant to tampering thereby ensuring accurate detection of compliance . in addition to the drug sensing component , the methods and compositions of the present invention comprise a total of up to four key elements to constitute a remote monitoring device / system : a fluorescent tracer , detection device , signal transmission system and a signal receiving system . the detection methods and compositions described herein may optionally comprise features that enable filtration of interfering signals , data processing , data storage , timing devices , methods for signaling a patient to take medication , display panels and data recovery system . in addition , the detection methods and compositions described herein may further comprise a programming feature enabling the user to program drug schedules for prescribed medications . importantly , the devices of the present invention may also comprise a security or tamper - proof feature that prevents unauthorized use of the device such as modification of settings , data input , or data analysis . the devices of the present invention detect a fluorescent tracer that is incorporated into , or coated onto , the drug tablet . thus any current or future drug , or drug combination tablet , could be modified to be detectable by the methods and compositions of the present invention . there are presently several fluorescent compounds that are fda approved for human use , including but not limited to , fluorescein , indocyanin green and rhodamine b . the tracer compounds used for the present invention are safe for long term use , orally absorbed , and have favorable pharmacokinetics . a minimal requirement for the pharmacokinetics is that the level of circulating tracer falls to a baseline between scheduled drug doses and can be accurately detected . most critically , the tracer is transdermally detectable . the transdermal detection device of the present invention is preferably a small apparatus that can be worn continuously by the patient . some embodiments that may be suitable comprise a wristwatch , earring or necklace - type configuration . the device is able to detect a signal , such as a fluorescent signal , transdermally . transdermal sensing is described for example in the following references , each of which is incorporated by reference in its entirety : lakowicz j . r . emerging application of fluorescence spectroscopy to cellular imaging : lifetime imaging , metal - ligand probes , multi - photon excitation and light quenching , scanning microscopy supplement 10 : 213 - 224 ( 1996 ); tolosa l . et al . optical assay for glucose based on the luminescence decay time of the long wavelength dye cy5 ™, sensors and actuators b 45 : 93 - 99 ( 1997 ); guo et al . use of long - lifetime re ( i ) complex in fluorescence polarization immunoassays of high - molecular - weight analytes , anal . chem . 70 : 632 - 637 ( 1998 ); youn et al . fluorescence energy transfer immunoassay based on a long - lifetime luminescent metal - ligand complex , anal . biochem . 232 : 24 - 30 ( 1995 ); and lakowicz et al . emerging biomedical and advanced applications of time resolved fluorescence spectroscopy , journ . of fluorescence 4 ( 1 ): 117 - 136 ( 1994 ). the teaching of transdermal monitoring in u . s . pat . no . 5 , 628 , 310 by rao et al . which is incorporated herein in its entirety , may also be utilized in carrying out the methods of the present invention . a preferred embodiment of the present invention comprises a device held in close contact with the skin of a person . a highly preferred embodiment comprises a transdermal detection device that is able to detect ingestion of a drug wherein the drug is labeled or tagged with a tracer compound such as a fluorescent dye . the third element of the remotely monitored therapy system comprises a signal transmission system . the signal transmission system relays the detected signal to a receiving base , where the signal can then be interpreted and responded to . in a preferred embodiment , the system operates automatically , requiring no effort on the part of the patient . for example , one preferred embodiment is a portable telephone , using either cellular or digital , technology , or radio - telephone technology . since each of these technologies are experiencing rapid decreases in costs and increasing miniaturization , use of such instruments is both economically and practically feasible . to minimize the effort required on the part of the patient , signal transmission functions automatically , and a reading is transmitted hourly , or according to any desired periodic reading . this allows the receiving system to plot the level of the tracer continuously , and to determine , within any desired period of time , when the therapeutic drug dose was ingested . the fourth principal component of the present invention is a signal receiving system . the signal receiving system receives signals from the transdermal monitoring device via the signal transmission system , and then interprets the signal and triggers and suitable response . such a system may be centrally located for example , at a public health or medical facility assuming responsibility for the patient , or it might be more centralized as is common for cardiac event monitoring . either way , the supervising physician or public health official receives a periodic read out of all patients who have taken their medication as instructed , and a list of any patients who have been nonadherent . in one preferred embodiment of the present invention , the signal transmitting system and the receiving system have two - way communications . this allows enables / enhancers to be incorporated , such as a reminder call to take the medicine , follow - up calls if the reminder went unheeded , and thank you calls when drug ingestion is confirmed . additional calls to remind patients of scheduled clinic visits can also improve adherence with all aspects of treatment . importantly , like directly observed therapy , remotely monitored therapy documents noncompliance , a necessity for making a legally defensible decision to transfer nonadherent patient to a more restrictive therapy option . in another preferred embodiment , a patient ingests a pill ( capsule , etc .) that contains a tracer dye ( fluorophore ) such as indocyanine green ( icg ). the dye is transported through the circulatory system to blood vessels just under the skin . the change in modulation of intensity of the emitted light is detected by an external sensor . the external sensor is embedded within a transdermal device which is placed upon the patient &# 39 ; s skin above a layer of ruthenium doped plastic film . the increase in modulation of the emitted light is compared to the modulated light ( baseline ) prior to ingestion . the magnitude of the measured modulation is proportional to the concentration of the dye . the system monitors the presence of the dye thereby indicating positive or negative compliance . an especially desirable use of the present invention is monitoring compliance with therapeutic regimens for people with tuberculosis . the ability to detect whether a patient has taken his medicine , and thus determine whether the treatment is effective , is highly desirable with the rise in drug - resistant mycobacteria . a preferred method includes attaching a detection device to an individual to monitor drug compliance . during the course of treatment , drug compliance is recorded by a central signal receiving location and reminders are sent to patients when they fail to take their medication . an important result of this system is the ability to detect drug - resistant strains . for example , if a patient is monitored for compliance , and despite confirmed compliance it is discovered that he is still suffering from tuberculosis at the end of the prescribed drug regimen , the supervising healthcare provider may then be alerted to an infection caused by a drug - resistant strain . at this time , the treatment could be changed , the drug sensitivity of the infecting mycobacteria could be determined , or the same treatment could be continued for a longer amount of time . the ease of administration is a particularly beneficial aspect of the present invention . for example , children are not intimidated by the application of a transdermal device , such as a wristwatch , and are not hesitant to wear such a device for a time sufficient to monitor drug therapy . such transdermal devices are also easily stored and transported to isolated places that may lack materials or technical resources to conduct more involved tests such as those requiring chemical analysis . the present invention can be made from inexpensive materials that can be produced at low cost and used by health care organizations . in a preferred embodiment of the present invention , a compliance monitoring system directed to measuring drug compliance by a non - invasive optical measurement is provided . the concept requires labeling a drug with a signalling component such as a red or near infrared fluorophore . as demonstrated in the example , the emission of such fluorophores , even at micromolar concentrations , can be detected through skin . in our novel approach the sensor which is placed against the skin contains a long lifetime fluorophore in a plastic film . the tissue is illuminated with intensity modulated light with a frequency near 2 mhz . the presence of fluorophore in the tissue can be detected from the modulation of the emission , which represents the intensity of the fluorophore in tissues relative to that of the long lived reference . such measurements can be accomplished with simple hand held devices in the doctor &# 39 ; s office or at the point of care . as described in more detail in the examples below , the effectiveness of fluorescent markers was evaluated using indocyanine g ( icg ) and rhodamine 800 ( rh800 ). more specifically , different concentrations of icg and rh800 in 0 . 5 % intralipid ( a model for chicken or bovine tissue ), and in chicken tissue were measured using modulation measurements . these measurements were conducted using a long lifetime ruthenium complex in a film as a reference fluorophore . modulation values at each dye concentration were equivalent to the fractional intensity of the dye emission present in the system , thereby making modulation values a measure of dye concentration . the results of these measurements demonstrated that these dyes can be detected non - invasively through the human skin using appropriate instrumentation . concentrations of as low as 50 nm for rh - 800 ( table ii , as shown in fig2 ) and 250 nm for icg ( table v , as shown in fig2 ) were detected in this study reflecting the sensitivity of this technique . in one embodiment of the present invention , the modulation measurements are used to monitor compliance by formulating medications with icg . icg is currently used for different purposes in humans , and it is known that icg is rapidly cleared from circulation . since administered drugs will not necessarily be cleared from the circulation at the same rate as icg , the methods of the present invention comprise compositions wherein icg is formulated in a time release fashion to market the known clearance rates of administered drugs . evidence for the injection of drugs can then be determined non - invasively by the measurement of the modulation of the emission seen through the skin . with currently available opto - electronics technology , a hand held battery powered hand device , such as a wristwatch , for the measurement of modulation can be readily built ( fig1 ). the light source may comprise a light emitting diode ( led ) or a laser diode . the output of these light sources can be easily modulated to 50 mhz or higher ( 35 - 37 ) and with present technology , a portable monitor can be routinely used each time a chronically ill patient is seen by a health care professional . the compliance monitoring system of the present invention is useful for monitoring therapeutic compliance in a variety of diseases and disorders , including , but not limited to infectious diseases ( staphyloccal , streptococcal , pneumococcal , niesseria , listeriosis , enterobacteriaceae , salmonella , pseudomonas , cholera , treponematoses , histoplasmosis , coccidioidomycosis , mycobacterial , cryptococcosis , aspergillosis , rickettsial , chlamydial , viral , parasitic and sexually transmitted diseases ), immunodeficiency diseases ( acquired immunodeficiency syndrome ), cardiovascular disorders ( endocarditis , pericardial disease , erythromelalgia ), pulmonary disorders ( bronchitis , pneumonia ), gastrointestinal ( functional dyspepsia , peritonitis ), hepatic and biliary disorders , endocrine disorders ( diabetes mellitus , hypoglycemia , hyperthyroidism , hypothyroidism ), cancer , musculoskeletal and connective tissue disorders ( rheumatoid arthritis , gout , osteoporosis ), neurologic and psychiatric disorders ( insomnia , multiple sclerosis , epilepsy ), genitourinary disorders ( renal disease , kidney infection , nephritic syndrome , bacterial pyelonephritis ) and other physiological disorders and diseases . in addition , numerous therapeutic agents may be used in the present invention , including , but not limited to , antimicrobial agents , antibiotics , antivirals , antidepressants , β - lactam antibiotics , aminoglycosides , macrolides , lincomycin , clindamycin , tetracyclines , quinolones , polypeptides , sulfonamides , trimethoprims , sulfamethoxazoles , growth factors , lipids , and neurotransmitters . furthermore , vitamins , and minerals may also labeled for use in the present invention , especially for patients suffering from nutritional or metabolic disorders . the monitoring system of the present invention is also useful when conducting clinical trials for assuring therapeutic compliance by participants . an important advantage of the present invention is that it can function effectively and accommodate discrepancies such as those resulting from variations in dermal pigmentation , thickness , and vascularity . this invention is further illustrated by the following examples , which are not to be construed in any way as imposing limitations upon the scope thereof . on the contrary , it is to be clearly understood that resort may be had to various other embodiments , modifications , and equivalents thereof , which , after reading the description herein , may suggest themselves to those skilled in the art without departing from the spirit of the present invention . the following experiment was conducted in order to measure the presence and concentration of fluorophores in scattering media , including intralipid suspensions , chicken skin and chicken muscle . the fluorophores were rhodamine 800 ( rh800 ) and indocyanine green ( icg ), both of which would be excited at long wavelengths not absorbed by tissues . it is known to be difficult to quantify the fluorescence intensity from probes in scattering media . this problem was solved using modulated excitation near 2 mhz and a long lifetime reference fluorophores in a polymer film placed immediately on the illuminated surface of the sample . under these conditions , the modulation of the emission is a measure of the intensity of the fluorophore ( rh800 or icg ) relative to the long lifetime reference . using this method we were able to measure the concentration dependent intensities of rh800 and icg in an intralipid suspension . additionally , micromolar concentrations of these probes could be detected in chicken muscles , even when the muscle was covered with a layer of chicken skin . based on the findings of this study , we have demonstrated successful use of transdermal detection of long - wavelength fluorophores as a non - invasive method to monitor patient compliance in taking medicines used for treatment of chronic diseases such as tuberculosis and aids . rhodamine 800 ( rh800 ) was obtained from lambda physik , and indocyanine green ( icg ) from sigma ( st . louis , ivio ), and were used without further purification . for aqueous solution , the probes were dissolved in water . intralipid ( 20 %) was obtained from kabivitrum , inc ( clayton , n . c .). the intralipid was diluted 40 - fold into buffer , to 0 . 5 %, to provide a sample with scattering properties comparable to that of tissues like chicken or bovine muscles . based on available data [ 7 ] the effective scattering coefficient ( 1 - g ) ( for 0 . 5 % intralipid can be estimated as 7 . 25 cm − 1 . concentrations of rh800 and icg were determined from the extinction coefficients of 8 . 95 × 10 4 l mol − 1 cm − 1 at 682 nm and at 780 nm , respectively . all fluorescence measurements were performed using front - face illumination and detection , using the sample holder shown in fig1 . the incident light was redirected from the usual position using two mirrors . the position of the sample could be adjusted with a movable stage . the re - emergent light passed through one or more optical filters prior to reaching the destination . the sample consisted of either a cuvette containing the intralipid , or a quartz slide covering the chicken muscle and skin ( fig1 ). excitation at 600 nm was provided by the fundamental output of a rhodamine 6 g dye laser . this dye laser was synchronously pumped by a mode - locked argon ion laser , and cavity dumped at 1 . 88 mhz . frequency domain intensity decay measurements were performed as described previously [ 8 - 11 ]. phase angles and modulation measurements at frequencies greater than 1 . 88 mhz were accomplished using the harmonic content of the picosecond pulses [ 12 - 14 ]. the excitation was polarized vertically , and the emission detected without an emission polarizer . the re - emergent light was either observed directly , or through two cut - off filters ( 630 and 660 nm ) to remove scattered light and / or attenuate the fluorophore emission relative to that of the long lived reference . a long lifetime reference signal was provided by [ ru ( bpy ) 2 ( dppz )]( pf 6 ) 2 in a polyvinyl alcohol ( pva ) film , where bpy is 2 , 2 ′- bipyridine and dppz is dipyrido [ 3 , 2 - a : 2 ′, 3 ′- cl ] phenazine . such metal - ligand complexes are known to display lifetimes from 100 ns to 13 ( s [ 15 - 16 ]. the intensity decay time of [ ru ( bpy ) 2 ( dppz )]( pf 6 ) 2 in the pva film was near 800 ns ( table 1 as shown in fig2 ). the intensity decay data was analyzed in terms of the multi - exponential model i ⁡ ( t ) = ∑ i ⁢ α i ⁢ exp ⁡ ( - t / τ i ) ( 1 ) in this expression α i represents the pre - exponential factors associated with each lifetime τ i . the fractional contribution of each decay time component to the steady state intensity is given by f i = α i ⁢ τ i ∑ j ⁢ α i ⁢ τ i . ( 2 ) the values of σα i and σf i are typically normalized to unity . the mean lifetime is given by τ - = ∑ i ⁢ f i ⁢ τ i = ∑ i ⁢ α i ⁢ τ i 2 α i ⁢ τ i . ( 3 ) in the frequency - domain measurements the measured quantities are the phase shift of the emission ( φ ω ) and its modulation ( m ω ) at the light modulation frequency ω i in radians / sec . the values of α i and τ i are determined by non - linear least squares fitting and minimization of the goodness - of - fit parameter x 2 r . x r 2 = 1 v ⁢ ∑ ω , k ⁢ ( ϕ ω - ϕ ⁢ c ⁢ ⁢ ω δϕ ) 2 + 1 v ⁢ ∑ ω , k ⁢ ( m ω - m ⁢ c ⁢ , ω δ ⁢ ⁢ m ) 2 ( 4 ) in this expression the subscript c refers to calculated values of φ ω and m ω for assumed values of α i and τ i and v is the number of degrees of freedom . δω and δm represent the uncertainties in the measured values . in some cases we performed a global analysis of data measured at more than one fluorophore concentrations . in this case the sum in equation 3 extends over these multiple concentrations ( k ). the values of φ ωc and m ωc can be calculated for many assumed values of α i and τ i . these values are given by m ωc =( n ω 2 + d ω 2 ) 1 / 2 ( 6 ) n ω = ∫ 0 ∞ ⁢ i ⁡ ( t ) ⁢ sin ⁢ ⁢ ω ⁢ ⁢ t ⁢ ⁢ ⅆ t = ∑ i ⁢ α i ⁢ ωτ i 2 1 + ω 2 ⁢ τ i 2 ( 7 ) d = ∫ 0 ∞ ⁢ t ⁡ ( t ) ⁢ cos ⁢ ⁢ ω ⁢ ⁢ t ⁢ ⁢ ⅆ t = ∑ i ⁢ α i ⁢ τ i 1 + ω 2 ⁢ τ i 2 . ( 8 ) the principle of low frequency modulation sensing can be understood by a rearrangement of equations ( 5 - 8 ). suppose the sample displays two decay times . assume further that one decay time is long ( l ) comparable to the modulation frequency and the other is a typical ns decay time ( s ). for these conditions the sine and cosine transforms are given by n ω = f s m s sin ωτ s + f l m l cos ωτ l ( 9 ) d ω = f s m s cos ωτ s + f l m l sin ωτ l ( 10 ) if the ωτ l product is much greater than unity , then the modulation ( m l ) of this component is near zero . under the condition the observed modulation is given by m obs =[( f s m s sin ωτ s ) 2 ( f l m l cos ωτ l ) 2 ] 2 ] 1 / 2 ( 11 ) the modulation frequency can be selected so that m s is near 1 . 0 . then , the observed modulation is for the present studies we used two fluorophores with ns decay times , rh800 and icg , and a long lifetime metal ligand complex . we refer to this long lifetime reference as the ru complex ( fig1 ). absorption and emission spectra of these probes are shown in fig2 . all three fluorophores can be excited at 600 nm . in water rh800 emits maximally near 706 nm , and icg near 805 run . the ru complex is essentially non - fluorescent in water [ 17 - 18 ] but becomes fluorescent in non - polar environments which prevent contact with water . the emission spectra of rh800 and icg in 0 . 5 % intralipid is similar to those observed in water ( fig2 ). indocyanine green ( icg ) is approved for use in humans , and is widely used in opthamology for studies of liver and kidney functions , to measure blood volume , and to estimate the severity of burns [ 19 - 25 ]. while the near infrared ( nir ) absorption and emission of icg in tissues can be readily detected , it is known that the spectral properties of icg are complex [ 29 - 31 ]. icg appears to aggregate in aqueous solution with the aggregates being less fluorescent than the monomeric species . in biological samples icg associates with proteins and lipids , with the intensity being dependent on the total concentration of icg as well as on the concentration of macromolecules which bind icg . hence we examined the dependence of the intensity on the concentration of icg and rh800 . the concentration - dependent intensities of rh800 and icg are shown in fig4 . for both fluorophores the emission intensity initially increased , and then decreased as the probe concentration increased above 10 to 20 μm . the peak intensity was found to occur at lower concentrations in 0 . 5 % intralipid than in water . though not wishing to be bound by the following theory , we believe the self - quenching of rh800 and icg is due to self - association and on energy transfer between the aggregated fluorophores in 0 . 5 % intralipid . the occurrence of the peak intensity at lower probe concentration in 0 . 5 % intralipid suggests that binding to intralipid results in high local concentrations of the fluorophore due to binding to the intralipid micelles . we examined the frequency - domain intensity decay of rh800 and icg in 0 . 5 % intralipid . as the concentration of rh800 increased the frequency response shifted to higher frequency ( fig5 ). these data were analyzed in terms of the multiexponential model ( table ii , as shown in fig2 ). in water , in the absence of intralipid , rh800 displayed a single exponential decay of 0 . 686 ns . in 0 . 5 % intralipid the intensity decay of rh800 becomes more complex . the data could be fit to a two decay time model . the individual decay times and the mean decay times decreased with increasing rh800 concentrations ( fig6 ). similar results are found for icg , with the decay times and mean decay time decreasing with increasing icg concentration ( table iii , as shown in fig2 ). concentrations of rh800 in intralipids were determined by the modulation method . rh800 was dissolved in 0 . 5 % intralipid . a polyvinyl alcohol film containing the ru complex was placed on the illuminated surface of the cuvette . emission spectra are shown in fig7 . the peak near 710 nm is due by rh800 . the signal near 650 nm is due to emission from the ru complex , and also to light scatter from the intralipid . the scattering component was minimized by observing the solution through a combination of two 630 and 660 nm cut - off filters . under these conditions the signal observed through the filters is mostly due to the ru complex and rh800 . frequency - responses of this combined emission from the ru complex and rh800 are shown in fig8 . the most dramatic feature of these data are the modulation values from 2 to 20 mhz . the modulation is essentially independent of frequency , and increases as the rh800 concentration increases . this result is due to the dramatic difference between the decay times of rh800 in intralipid ( 1 . 62 ns ) and of the ru complex ( 834 ns ). the data were globally analyzed in terms of a short and long decay time ( table iv , as shown in fig2 ). three decay times of 1 . 21 , 1 . 62 and 545 ns were found adequate to fit the data of all rh800 concentrations . as the concentration of rh800 increased so did the fractional intensity of the short component ( table iv ). this result can be understood as due to the combined measurements with the long lifetime standard . at frequencies near 5 mhz the observed modulation is similar to the fractional intensity of the short lifetime rh800 . modulation values are 0 . 185 , 0 . 525 and 0 . 812 ( fig8 ). while calculated f s values are 0 . 184 , 0 . 534 and 0 . 803 ( table iv ) at rh800 concentrations of 0 . 05 , 0 . 25 and 1 . 00 μm , respectively . hence , the modulation values reflect the contribution of rh800 to the total emission . the dependence of the modulation on the rh800 concentration is shown in fig9 . for this concentration range the rh800 intensity is essentially linear with its concentration ( fig4 ). however , the observed dependence is hyperbolic ( fig9 ). this is the result of normalizing f s + f l to unity , so that increasing intensities of rh800 shift f s monotonically towards 1 . 0 . the data can be normalized in a different manner to yield a more linear dependence . this is accomplished by normalizing all the values of f s to the same initial value of f l . let f l1 represent the initial fractional intensity of the long lifetime component , and f ln the fractional intensity at some higher concentration of the short lived fluorophore . for the initial conditions at the next higher concentration of the short lived species one has let r = f l / f ln be the ratio of the long lifetime normalized fractional amplitudes . then hence the unnormalized modulated amplitude of the short lived component ( f sn ′) is f sn ’ = rf sn = f sn ⁢ f l1 f l ⁢ ⁢ n ( 16 ) using these recalculated fractional amplitude one expected f sn ′ or m sn ′ approximately linear with the concentration of the short lifetime fluorophore . these values are shown in the lower panel of fig9 , and show that f s increases in a nearly linear fashion with the rh800 concentration . similar experiments were performed with icg in 0 . 5 % intralipid . the emission of icg in intralipid occurs near 820 nm , and is considerably weaker than that of rh800 . emission for the ru complex is seen near 650 nm , along with some components due to scattered light . the scattered light was removed by the combination of two cut - off filters , resulting in the spectra shown as an insert in fig1 . frequency responses for icg in intralipid are shown in fig1 . this sample also had the long lifetime reference . as seen for rh800 , the modulation increased with increasing concentrations of icg . the modulation at 2 mhz increases hyperbolically with icg concentration ( fig1 ). the intensity normalized modulation ( equation 13 ) also increased almost linearly . this is somewhat surprising given the previous observations of decreasing intensity with icg concentration above 20 μm . at present we believe this difference is due to sample - to - sample variations in the icg - intralipid samples . the important point is that the concentration of icg can be estimated from the relatively simple 2 mhz modulation measurement . a particularly advantageous aspect of the present invention is that it is useful for compliance monitoring over a range of fluorophore concentrations , and also for a range of skin types . one feature that makes this possible is that the sensitivity of the invention can be easily adjusted . this is shown in fig1 , where the rh800 concentration was held constant , and the concentration of 1 . tan , k . k . 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