ELSEVIER
`
`Journal
`
`of Photochemistry
`
`and Photobiology
`
`B: Biology
`
`40
`
`( 1997)
`
`3 13-3 19
`
`Pharmacokinetics of Saminolevulinic acid-induced protoporphyrin IX in
`skin and blood
`
`K. Rick a.1, R. Sroka a, H. Stepp a, M. Kriegmair b, R.M. Huber ‘, K. Jacob d, R. Baumgartner a,*
`’ Laser-Research
`Laboratory
`at
`the Department
`of Urology,
`University
`of Munich. Munich.
`Germany
`’ Department
`of Urology,
`lJniver.si&
`of Munich, Munich,
`Germany
`’ Department
`of Medicine,
`University
`of Munich, Munich,
`Germany
`d Institute
`of Clinical
`Chemistry,
`University
`of Munich, Munich,
`Germany
`
`Received
`
`10 March
`
`1997; accepted
`
`11 July 1997
`
`Abstract
`
`used for the diagnosis and
`is increasingly
`IX (PPIX)
`synthesized protoporphyrin
`of endogenously
`The fluorescence and photosensitivity
`treatment of malignant and certain non-malignant
`diseases. A selective accumulation of PPIX can be induced by application of 5-aminolevulinic
`acid
`(5ALA),
`which
`is a precursor
`of PPIX
`in
`the cellular
`biosynthetic
`pathway
`of heme.
`locations of the skin after oral ingestion and to
`The purpose of this study was to monitor
`the in vivo accumulation
`of PPIX
`in different
`determine
`the pharmacokinetics
`of 5-ALA
`and PPIX
`in human
`blood
`plasma
`for
`various
`routes
`of application.
`At
`the same
`time we wanted
`to achieve an optimal
`treatment scheme but also study possible side-effects of 5-ALA
`administration.
`in the skin showed
`intensities of PPIX
`After oral application
`of 5-ALA
`in a concentration of 40 mg kg
`’ body weight,
`the fluorescence
`maxima between 6.5 and 9.8 h depending on the location and decreased to values lower
`than 5% related
`to the maximum after a mean time
`of about
`40 h. The measured
`absolute
`intensities
`of PPIX
`fluorescence
`varied
`strongly
`between
`different
`patients
`and different
`locations
`on
`one patient.
`In the plasma of blood samples, PPIX could be detected via its fluorescence
`for all studied routes of application with the exception
`of the ointment, where PPIX
`levels were below
`the detection
`limit of I pg 1-l. The highest mean concentration
`of 742 p,g I- ’ PPIX
`in the
`plasma was measured 6.7 h after oral application. For inhalation of 5-ALA,
`a mean maximum concentration
`of I2 ug 1~ ’ could be detected
`4.1 h after application,
`for intravesical
`instillation,
`the mean maximum concentration was found
`to be I pg I- ’ 2.9 h after
`application.
`The
`kinetics of 5-ALA
`in the plasma peaked much earlier with a maximum concentration
`of 32 mg I
`’ about 30 min. after oral administration.
`The 5-ALA
`levels
`did not exceed
`normal
`reference
`values
`after
`topical
`application.
`of our experiments suggest that for a systemic application
`of 5-ALA
`The
`results
`0 1997 Elsevier Science S.A.
`
`in sensitive patients cannot be excluded.
`
`side-effects
`
`Keywords:
`
`Porphyrins;
`
`Fluorescence:
`
`Photodynamic
`
`diagnosis;
`
`Photodynamic
`
`therapy:
`
`Side-effects
`
`1.
`
`Introduction
`
`Usually, photodynamic diagnosis and therapy require the
`administration of exogenous photosensitizers. Besides syn-
`thetic photosensitizers
`like Photofrin@, there are several
`potential endogenous sensitizers synthesized during the bio-
`synthesis of heme. Under physiological conditions, a feed-
`back loop controls the metabolic steps of heme biosynthesis
`and excludes an accumulation of any photosensitizing con-
`
`acid
`5-aminolevulinic
`IX; 5.ALA,
`PPIX, protoporphyrin
`Abbreviations:
`Klinik
`Urologische
`author.
`Laser-Forschungslabor,
`* Corresponding
`der
`23, D-
`Marchioninistrasse
`Ludwig-Maximilians-Universitat
`Munchen,
`Tel.:
`81377 Munich, Germany.
`+49
`89 70906162;
`Fax:
`+49 89 70906142;
`e-mail:
`baumgartner@life.med.uni-muenchen.de
`’ Part of thesis
`
`101 l-1344/97/$17.00
`PIISlOll-1344(97)00076-6
`
`0 1997 Elsevier
`
`Science S.A. All
`
`rights
`
`reserved
`
`[ I]. The intracel-
`centrations of the involved intermediates
`lular concentration of free heme in the liver inhibits
`the
`synthesis of 5aminolevulinic
`acid (5ALA)
`and thus all
`following metabolites as a rate-limiting
`factor
`[ 21. Enzy-
`matic defects associated with the metabolic steps result in
`abnormal quantities of the corresponding porphcyrins in the
`biosynthetic pathway. The clinical manifestation of the
`resulting cutaneous porphyrias include acute or chronic pho-
`tosensitization of the skin [ 11. Investigations of Berlin et al.
`in 1956 [ 31 showed that the administration of an excess of
`exogenous 5-ALA bypasses the cellular feedback control
`mechanism even in normal organisms. The resulting accu-
`mulation of protoporphyrin
`IX (PPIX), an immediate pre-
`cursor of heme in the biosynthesis, leads to comparable
`
`

`

`314
`
`K. Rick
`
`et al. /Journal
`
`of Photochemistry
`
`trnd Photobiology
`
`B: Biology
`
`40 (1997)
`
`313-319
`
`symptomatic photosensitization of the skin as seen in the
`cutaneous porphyrias [ 1,4].
`The quantitative expression and regulation of heme pro-
`duction differs from tissue to tissue. Changes in heme bio-
`synthesis of certain malignant cells, in particular, seem to
`correlate with a preferential accumulation of 5-ALA induced
`PPIX [ 41. At present, the tumorselective potential of PPIX
`and its clinical value for photodynamic diagnosis and treat-
`ment of neoplastic lesions are part of extensive investigations
`[ 5-121. Clinical experience has been reported for systemic
`and different topical routes of 5-ALA application
`[ 13-l 81.
`Considering both the many different application schemes
`as well as the possible systemic side-effects of the 5-ALA
`administration, a thorough knowledge of the time course and
`the distribution of 5-ALA and the induced PPIX are of sig-
`nificant importance. Moreover,
`the phamracokinetics of 5-
`ALA and its metabolites in vitro and in vivo provide the basis
`for a better understanding of the involved mechanisms. The
`systemic load of patients with 5-ALA and PPIX at different
`times after application can be evaluated analytically by means
`of a biochemical assay of the blood samples from the patients.
`In addition, the skin photosensitization due to PPIX after oral
`ingestion of 5-ALA can be estimated by measuring the fluo-
`rescence intensity of PPIX in the skin. The purpose of this
`study was to monitor PPIX on different locations of the skin
`after oral ingestion and to determine kinetics of 5-ALA and
`PPIX
`in human blood plasma
`for various
`routes of
`application.
`
`2. Materials and methods
`
`2.1. PPIXJuorescence kinetics in the skin
`
`11 patients (mean age 70 a, SD 6.2 a, range 62 to 79 a)
`proposed
`for photodynamic diagnosis or treatment were
`
`included in this investigation. After informed consent had
`been obtained, all patients received 40 mg kg- ’ body weight
`5-ALA dissolved in mineral water per o’s in a single bolus.
`All patients were kept in a darkened room for 24 h after 5-
`ALA ingestion.
`For a reproducible recording of PPIX fluorescence kinetics
`at a constant distance to the skin, a handheld detection device
`was designed to allow for a homogenous illumination of the
`probing location by the light of a modified Xenon short arc
`lamp (D-Light, Storz, Tuttlingen, Germany) (Fig. 1).
`To ensure an efficient excitation of PPIX within its absorp-
`tion band centered at A ~408 nm, the light source was
`equipped with a bandpass filter system transmitting blue-
`violet light in the spectral range of h = 375 to 440 nm. The
`total irradiation of the tissue was limited to 4 mJ cm-’
`for
`each measurement and location in order to prevent photo-
`bleaching effects [ 191.
`As shown in Fig. 1, the remitted fluorescence light was
`collected by a fused silica fiber (HCN6001, core diameter 600
`pm) and analyzed spectroscopically by means of an inten-
`sified optical multichannel analyzer (0-SMA 3, SI Spectros-
`copy Instruments, Gilching, Germany). A sufficientblocking
`of the excitation light was obtained by a combination of two
`longpass filters (KV 450, GG 455, Schott, Mainz, Germany)
`mounted behind
`the entrance slit of the monochromator.
`Emission spectra were recorded in the spectral range between
`A = 450 and 750 nm and corrected according to the back-
`ground and the wavelength sensitivity of the system.
`To separate the specific fluorescence of PPIX from the
`autofluorescence of the skin, the ratio of the integral autoflu-
`orescence intensities at h = 635 + 5 nm and A = 590 f 5 nm
`was calculated for all spectra recorded from the skin prior to
`the application of 5-ALA and free of any porphyrin peaks. If
`this ratio is applied, the contribution of the autofluorescence
`to the integral fluorescence intensity at it = 635 Z!Z 5 nm can
`
`

`

`K. Rick
`
`et 01. /Journal
`
`of Phutochrmistr?;
`
`and Photobiology
`
`B: Biology
`
`40 (1997)
`
`313-319
`
`315
`
`Table
`Doses
`
`I
`and number
`
`of patients
`
`for different
`
`routes of application
`
`Delivery
`
`Oral
`Inhalation
`Intravesical
`Ointment
`
`instillation
`
`Dose
`
`40 mg/kg
`500 rn&
`lg(2Q1
`200 mg
`
`body weight
`i 10%)
`
`( 10%)
`
`n
`
`12
`5
`5
`1
`
`be determined and then subtracted for each set of spectra.
`Subsequently, the resulting specific fluorescence intensities
`for PPIX at A = 635 i 5 nm were plotted as a function of time.
`
`2.2. Pharmacokinetics of 5-ALA and PPIX in blood plasma
`
`Pharmacokinetic measurements in human blood plasma
`were performed
`for different routes of 5-ALA application.
`The applied doses for clinical 5-ALA assisted photodynamic
`diagnosis or therapy are listed in Table I.
`is
`The application procedure for oral ingestion of 5-ALA
`similar to the one described in the context of the skin meas-
`urements. For inhalation of S-ALA, a concentration of 10%
`(500 mg 5-ALA
`in 5 ml isotonic saline) was applied 30 to
`40 min. by means of a medical nebulizer (Inhalierboy, Pari,
`Stamberg, Germany).
`Intravesical instillation of the 5-ALA
`solution ( 1 g 5-ALA in 50 ml 1 M NaHCO,) was performed
`via a catheter and incubated for a period of 2 to 4 h. For
`topical application as an ointment, a water-in-oil based cream
`containing 10% of 5-ALA
`(200 mg 5-ALA
`in 2 g cream)
`was applied and covered for 6 h with an occlusion pad.
`Venous blood samples of all patients were collected imme-
`diately before and at various times between one and 48 hours
`after 5-ALA application. Blood was collected into EDTA
`tubes and protected from light during the whole preparation
`procedure. The separated plasma was stored at a temperature
`below 20 “C until analyzed.
`The amount of 5-ALA
`in the plasma of two patients was
`determined by an HPLC method using fluorescence detection
`as described by Tomokuni et al. [ 201.
`PPIX was extracted from the plasma using a mixture of
`acetonitrile and ethanol ( 1:8) as a solvent. Plasma and sol-
`vent in the ratio of 1:9 were mixed for one minute by a sonifier
`(Sonifier 250, Branson, Danbury, CT, USA). After one hour
`the mixture was centrifuged in an ultra centrifuge (Cryofuge
`800, Heraeus, Hanau, Germany)
`for ten minutes at 57OOg.
`Fluorescence spectra of the supernatant were measured with
`a fluorescence spectrometer (Luminescence SpectrometerLS
`50, Perkin Elmer, iiberlingen, Germany) at a spectra1 reso-
`lution of d A = + 2 nm. According to the Soret band of PPIX
`dissolved in ethanol, emission spectra of the supematant were
`measured at the excitation wavelength of 405 nm. Subse-
`quently, excitation spectra were recorded for all peak wave-
`lengths appearing
`in the emission spectra. All emission
`spectra were corrected as to the background of the solvents
`used and the recovery rate. PPIX concentrations were cal-
`culated on the basis of the PPIX fluorescence measurements
`
`after the calibration of fluorescence intensities obtained from
`standard solutions of PPIX (Porphyrin Products, Logan, UT,
`USA). The PPIX concentrations of these standard solutions
`were determined via the absorbance measured at 405 nm
`(Diode Array Spectrophotometer 8452A, Hewlett Packard)
`using Beer’s law, the extinction coefficient E= 1.71 X IO’ 1
`mol-‘cm-’
`for PPIX in ethanol
`[2l]
`and the molecular
`weight of PPIX=562.7
`g mol-‘. Fluorescence intensities
`were measured in the spectra1 range of A = 635 & 5 nm for a
`series of 10 diluted solutions prepared in the range between
`10 kg I ~ ’ and 10 mg l- ‘, thus showing a linear relationship
`between absorption and fluorescence. By extrapolation
`to
`lower values, PPIX-concentrations of plasma samples could
`be determined down to 1 ~g 1~ ’
`Kinetics based on the achieved values are fitted by using
`least squares fitting procedures
`[22] and rate constants
`according to a one-compartment model for 5-ALA and a
`three-compartment model for PPIX [ 5,6].
`fluomphores was
`Spectral identification of the contributing
`achieved by comparing
`the resulting spectra wilth emission
`and excitation spectra recorded from standard solutions of
`uroporphyrin
`I/III, coproporphyrin
`I/III and protoporphyrin
`IX (Porphyrin Products, Logan, UT, USA) using the above
`mentioned combination of acetonitrile and ethanol ( I :8) as
`a solvent. Mixtures containing uro-, copro- and protoporphy-
`rin IX in varying ratios of concentration showed the contri-
`bution of the different porphyrins to the peak positions of the
`measured emission and excitation spectra.
`By dissolving and extracting a known amount of PPIX in
`fresh and untreated human plasma the recovery rate of the
`procedure was found to be 72%.
`
`3. Results
`
`3.1. PPIXJiuorescence kinetics in the skin
`
`Fig. 2 shows fluorescence spectra (before, 7 h and 50 h
`after application)
`taken from a series of spectra recorded on
`
`-- --
`
`----- -----
`
`Oh Oh
`
`, ,
`
`,, ,,
`
`50 h 50 h
`
`“’
`
`*f’
`
`^-
`
`VT
`
`,\
`\ i \
`‘1
`‘\
`
`--
`
`720
`720
`
`‘.-_
`
`680 680
`
`
`[nm] [nm]
`forearm
`
`of one patient before,
`
`I I
`
`600 600
`
`0
`
`II..
`
`~I~~.II,.I..I....,...I~
`
`640 640
`
`wavelength wavelength
`recorded
`on the
`spectra
`Fig. 2. Fluorescence
`7 h and 50 h after oral application
`of SALA.
`
`

`

`316
`
`K. Rick
`
`et al. /Journal
`
`of Photochemistq
`
`and Photobiology
`
`B: Biology
`
`40 (1997)
`
`313-319
`
`PPIX-fluorescence intensities decreased to values lower than
`5% of the maximum for all locations.
`
`3.2. Pharmacokinetics of S-ALA and PPIX in blood plasma
`
`the plasma
`Both emission and excitation spectra of
`recorded after application of 5-ALA showed the typical shape
`and peak positions of PPIX. There was no spectral indication
`for the presence of other porphyrins. Fig. 4 compares some
`characteristic examples of the kinetics of PPIX in blood
`plasma for various routes of application.
`Table 3 summarizes the results of the determination of
`PPIX in the plasma according to the fitted kinetics and aver-
`aged for all patients: For oral application, we found an aver-
`age peak concentration of 742 kg I-’ PPIX in the blood
`plasma 6.7 h after application. For inhalation, an average peak
`concentration of only 12 kg I- ’ was reached already 4.1 h
`after application. The average peak concentration
`for intra-
`vesical instillation was 1 p,g l- ’ and close to the detection
`limit. For topical application of 5-ALA using an ointment,
`no PPIX could be detected in the plasma. The average time
`to,os at which the PPIX concentration dlecreased to values
`lower than 5% of the maximum was less than 35 hours for
`all routes of delivery.
`In summary, the ratio of the maximum concentrations was
`found to be 742 (oral) : 12 (inhalation)
`: 1 (instillation).
`
`--
`--
`-.--...-
`
`-
`
`oral
`inhalation
`instillation
`
`x 100
`x 100
`
`0
`
`10
`
`30
`20
`[h]
`application
`time after 5-ALA
`of the PPIX
`concentrations
`in the plasma
`Fig. 4. Comparison
`and
`topical
`application.
`
`40
`
`50
`
`after systemic
`
`Table 3
`of the fitted PPIX plasma
`results
`Averaged
`in brackets).
`A mean maximum
`deviations
`time rrnau and decreased
`at the mean peak
`at 4,0s (xd.
`maximum
`= not detected)
`
`standard
`( corresponding
`kinetics
`reached
`concentration
`c,,, was
`to values
`lower
`than 5% of
`the
`
`Application
`
`Oral
`Inhalation
`Instillation
`Ointment
`
`cn,, Ihl (SD)
`
`has [hl (SD!
`
`cm, [&II
`
`(SD)
`
`(0.8)
`6.7
`(0.8)
`4.1
`2.9 (0.5)
`n.d.
`
`35 (4.6)
`21 (7.8)
`n.d.
`n.d.
`
`(87)
`742
`12 (2.9)
`1 (0.8)
`n.d.
`
`0
`
`4,.
`0
`
`I..
`
`.
`
`I,,
`10
`
`Fig. 3. PPIX
`Peak
`values
`
`fluorescence
`at A =635
`
`.
`
`7;;r..
`
`I,,
`
`I,.
`
`I
`
`I..
`
`:.
`50
`
`I,
`
`60
`
`,
`/ I,,
`40
`30
`20
`[h]
`application
`time after 5-ALA
`kinetics
`of forearm
`skin
`(same patient as in Fig. 2).
`f 5 nm are corrected
`for
`autofluorescence
`and
`
`2
`Table
`kinetics
`fluorescence
`fits of the PPIX
`the
`from
`calculated
`parameters
`Mean
`left column
`shows
`the absolute mean
`The
`after oral application
`of 5-ALA.
`as averaged
`for all patients. For
`the second
`peak
`times
`for different
`locations
`the
`ratios
`of
`the peak
`times and
`intensities
`and
`third
`columns
`(relative),
`“back
`of the hand”
`are determined
`individually
`related
`to the
`first
`location
`and averaged
`for
`the patients
`subsequently.
`Since
`the standard
`deviation
`(SD)
`was comparable
`for
`the different
`locations,
`the mean SD
`is calculated
`independently
`of the
`locations
`
`the hand
`
`Location
`
`Back of
`Lip
`Cheek
`Forearm
`SD
`[%I
`
`t,,,
`[hl
`
`6.5
`1.9
`8.7
`9.8
`15
`
`(absolute)
`
`t,,,
`
`(relative)
`
`I,,,
`
`(relative)
`
`1.0
`1.3
`1.5
`1.7
`10
`
`1.0
`9.4
`7.3
`1.4
`25
`
`a patient’s forearm before and at various times between 30
`min and 50 h after oral 5-ALA application.
`In none of the spectra recorded from human skin, any
`indication
`for the presence of porphyrins other than PPIX
`could be found. The fluorescence kinetics for PPIX measured
`on the forearm is shown in Fig. 3 showing a peak after 7 h
`and a fast decrease to baseline levels within 40 h. The highest
`fluorescence intensities were measured for the areas of head
`and neck, especially the lips. It was observed that PPIX flu-
`orescence intensities decreased towards the extremities. The
`lowest signals, however, were detected on the trunk. Table 2
`summarizes the results of the PPIX fluorescence kinetics
`measured on some characteristic locations of the skin.
`As seen in the right column of Table 2, the lowest mean
`intensity was observed at the back of the hand with the max-
`imum intensity already 6.5 h after application. The kinetics
`of the lip with the highest intensities measured peaked after
`7.9 h, followed by the cheek with the maximum
`intensity
`after 8.7 h and the forearm after 9.8 h. After a mean time of
`about 40 h after application (SD 9.3, range 24 to 60 h) the
`
`

`

`K. Rick
`
`et al. /Journal
`
`of Photochemistry
`
`and Photobiology
`
`B: Biology
`
`40 (1997)
`
`313-319
`
`317
`
`5 25
`‘Z
`!!
`E al 20
`2
`8
`m 15
`E
`1
`=
`
`10
`
`2
`ui
`
`5
`
`0
`
`0
`
`10
`
`Fig. 5. Comparison
`
`of the PPIX skin
`
`kinetics
`
`30
`20
`time after 5-ALA
`(a.~.),
`PPIX
`(right
`
`40
`[h]
`application
`axis)
`and SALA
`
`50
`
`0.0
`
`60
`
`(left
`
`axis)
`
`plasma
`
`kinetics
`
`after oral application
`
`The time course of the 5-ALA kinetics (Fig. 5) was much
`faster than all PPIX kinetics. A SALA peak concentration of
`32 mg l- ’ was reached already 30 min. after systemic appli-
`cation with a return to baseline levels in less than 8 h. In
`contrast to these observations, the measured maximum con-
`centrations for topical application did not exceed the detec-
`tion limit of 0.1 mg l- ’ set by the HPLC technique used in
`the experiments [ 201.
`
`4. Discussion
`
`4.1. PPIXJuorescence
`
`kinetics
`
`in the skin
`
`The present study describes the in vivo kinetics of SALA
`induced PPIX for various routes of application. For systemic
`administration
`(oral), the PPIX kinetics in normal skin was
`assessed by in vivo fluorometric measurements. Apart from
`that, we investigated time course and peak levels of both 5-
`ALA and PPIX in human blood plasma for systemic and
`various topical routes of delivery (inhalation,
`instillation,
`ointment).
`locations of the skin
`All kinetics recorded from different
`showed similar time courses, although peak times and inten-
`sities seemed to depend on both location and individual var-
`iations between the patients. After oral administration of
`5-ALA,
`the fluorescence kinetics of PPIX in normal skin
`reached maximum values after about 8 h followed by a
`decline to baseline levels after 40 h.
`The patient-to-patient variability can be studied in Table 2.
`The lower standard deviation for relative t,,,
`(second col-
`umn) versus absolute t,,,
`(first column)
`indicates that, once
`normalized
`individually,
`the variance
`in peak
`times
`decreases. The clearance of PPIX from the sensitized tissue
`appears to be even more sensitive to individual differences
`between the patients, resulting in a higher relative standard
`deviation of the clearance times (22%) compared to the abso-
`lute values of t,,,
`( 15%).
`
`In order to be able to interpret the relative peak intensities
`I,,,,, listed in the last column of Table 2, additional variations
`in the optical properties of the tissue have to bse taken into
`account. Individual variations in the optical tissu’e parameters
`of each location can explain the high standard deviation of
`up to 25% of even the relative values.
`For the routine clinical use of orally administered 5-ALA,
`a detailed knowledge of the induced transient photosensiti-
`zation of the skin is quite important. Considering the good
`correlation between PPIX fluorescence and pbotosensitiza-
`tion of human skin [ 131, we observed
`that the highest risk
`for phototoxic reactions was about 8 h after 5-ALA applica-
`tion. In fact, the exposure to artificial light witlhin this time
`range led to mild erythema in two of the patients. On the
`other hand, our investigations also indicate that with a slight
`individual uncertainty a significant photosensitization of nor-
`mal skin does not persist longer than 40 h after administration.
`In contrast to reports of Edwards [ 231 and Mustajoki [ 241,
`our own yet unpublished observations support the results of
`recently published studies [ 18,251 indicating
`that, besides
`the transient skin photosensitivity, an exogenous systemic 5-
`ALA
`loading might provoke other porphyric or even non-
`porphyric side-effects in morbid patients. Most of our patients
`tolerated an oral dosage of 40 mg kg-’ body weight quite
`well. Only a small group of partly high-risk patients showed
`mild complications from nausea to occasional vomiting and
`neurological symptoms, but also severe side-effects like
`hypotension
`in combination with a remarkable vasodilata-
`tion. The last complication
`in particular, which occurred
`between 2 and 8 h after application, seems to be inconsistent
`with
`the known hypertension
`in the case of acute hepatic
`porphyrias. Thus, a systemic administration of 5-ALA
`in
`excess might result in a clinical manifestation partly different
`to that observed with various kinds of porphyrias.
`
`4.2. Pharmacokinetics
`
`of j-ALA
`
`and PPIX
`
`in blood plasma
`
`To provide a deeper insight into the distribution mecha-
`nisms of 5-ALA and PPIX in the organism and their possible
`
`

`

`318
`
`K. Rick
`
`et al. /Journul
`
`of Photochemistry
`
`md Photobi&gy
`
`B: Biology
`
`40 (1~97)
`
`313-31~
`
`to the observed side-effects, the second part of
`contribution
`our study focused on the pharmacokinetics of both substances
`in blood plasma.
`The examination of porphyrins in the circulation revealed
`a rapid rise of plasma porphyrin levels after oral application
`as well as after inhalation and intravesical instillation of .5-
`ALA. Only in the case of the least invasive application form
`using an ointment no porphyrins could be detected. However,
`only one patient had been evaluated. It can be stated that
`detectable amounts of PPIX might reach the plasma if large
`areas with reduced skin barrier are treated with ointment
`containing 5-ALA.
`On comparing spectra from the plasma with those recorded
`from the series of standard solutions from uro- to protopor-
`phyrin, we found no evidence for any porphyrin other than
`PPIX. In a solution containing predominantly PPIX, even
`small amounts of uro- or coproporphyrin resulted in aremark-
`able blue shift of the maximum, not observed in any of the
`investigated samples. Since the extraction procedure used
`was optimized
`for the highly
`lipophilic PPIX, we cannot
`strictly exclude the presence of a certain amount of the water-
`soluble uro- and coproporphyrins
`in the plasma. Further
`HPLC measurements will have to confirm
`the absence of
`significant concentrations of porphyrins other than PPIX.
`Nevertheless, we suppose that even in the case of an increased
`synthesis of uro- and coproporphyrin,
`their hydrophilic char-
`acter should be responsible for the rapid clearance from the
`body. Therefore an accumulation comparable to that of PPIX
`should occur neither in the skin nor in the circulation.
`The concentrations and peak times of PPIX determined in
`the plasma showed a strong dependence on the mode of
`application. Although all investigated routes of application
`led to a comparable fluorescence ratio between tumor and
`adjacent normal tissue, the mode of application played an
`important role for the systemic load of the patients with PPIX
`and 5-ALA.
`Henderson et al. [ 71 studied the pharmacokinetics of PPIX
`in the circulation of tumor-bearing mice finding a peak con-
`centration of more than 1200 pg l- ’ PPIX 1 h after intraper-
`itoneal injection of 1000 mg kg-’ body weight 5-ALA. After
`a single oral dose of 40 mg kg- ’ body weight, we measured
`an average peak concentration of 742 kg 1~ ’ PPIX in the
`plasma of patients, which is consistent with plasma levels of
`2 to 4 mg I- ’ detected by Webber et al. [26]
`in 4 patients 8
`to 12 h after oral administration of 60 mg kg- ’ body weight
`5-ALA. Normal concentrations of PPIX in plasma measured
`in healthy volunteers have been reported to lie in the range
`ofOtoXpgl-’
`[27].
`In contrast to the at least 1 OO-fold elevated plasma levels
`of PPIX after oral application, the mean maximum concen-
`trations of 12 kg I- ’ obtained for inhalation and even less
`for instillation and ointment are comparable to reference val-
`ues [ 271. Thus, side-effects due to PPIX for any of the topical
`routes of administration can be excluded.
`A similar relationship has to be supposed for the plasma
`levels of 5-ALA after systemic ( L 32 mg 1-l) versus topical
`
`( < 1 mg l- ‘) application. A fast increa’se in circulating 5-
`ALA was noted for oral ingestion. This suggests that there is
`no efficient buffer for 5-ALA
`in the gastrointestinal tract. 5-
`ALA accumulation in the plasma reached a peak level of 32
`mgl-’
`already 30 min. after application. Since this value
`represents the first blood sample we took from the patients,
`we can not rule out an even higher concentration at earlier
`times.
`In contrast to maximum concentrations of less than 0.1
`w 1 ’ of 5-ALA measured for topical aplplication, the values
`for systemic application are utmost unphysiologic
`[ 281 and
`thus may contribute to the mentioned side-effects. The path-
`ogenesis of the neurological dysfunctions
`in acute hepatic
`porphyrias and their correlation to circulating 5-ALA has not
`yet been clearly defined in literature. Nevertheless there is
`the hypothesis that the resemblance of 5-ALA
`to the neuro-
`transmitter 4-aminobutyric acid is responsible for a neuro-
`toxic potential of 5-ALA
`[4]. However, 5-ALA may form
`reactive oxygen species (ROS) by auto-oxidation
`leading to
`the neuropathological manifestations of acute hepatic por-
`phyrias [ 291. Assuming the fact that 5-ALA provokes the
`observed side-effects, a splitting of the administered dose
`over a longer period should result in reduced side-effects in
`combination with a comparable accumulation of PPIX. In
`patients with GI-tract cancer, Regula et al. were able to pro-
`duce a plateau plasma concentration of about 8 mg I-’ 5-
`ALA by administration of a fractionated dosage of 30
`mg kg- ’ body weight, assuming that even higher doses of 5-
`ALA in the circulation might not induce porphyric symptoms
`[ 181. When we compare the times at which side-effects
`occurred in our patients with the kinetics of 5-ALA and PPIX
`plasma levels (Fig. 5)) a connection between side-effects and
`accumulating PPIX in the circulation seems to be more likely
`than a connection with circulating 5-AL.A. Further investi-
`gations will have to give a more detailed insight into the
`correlation between systemic 5-ALA application andpossible
`side-effects.
`the biochemical steps of heme biosynthesis are
`Although
`largely understood [ 1,4], the distribution patterns of 5-ALA
`and PPIX in the organism afterexogenous 5ALAapplication
`are still discussed controversially
`[ 7,131. On the one hand,
`the similar kinetics of PPIX in skin and plasma might support
`the theory of a centralized conversion of 5-ALA in erythro-
`blasts or liver and a subsequent delivery 1.0 other organs via
`the circulation. However,
`the PPIX-fluo,rescence intensity
`contrast of tumor versus adjacent normal tissue is comparable
`after topical and systemic administration
`in contrast to the
`completely different plasma levels for both routes of appli-
`cation. This indicates that PPIX is more likely accumulated
`in epithelial cells by selective in situ synthesis from 5-ALA.
`In contrast to the usually protein-bound PPIX, hydrophilic 5-
`ALA may enter the interstitium easily from the blood vessels.
`Since every nucleated cell in the body must have at least a
`minimum capacity to synthesize PPIX from 5-ALA, an exclu-
`sive PPIX production
`in erythroblasts or liver is highly
`improbable.
`
`

`

`K. Rick
`
`et ul. / Jooumal of Photochemistp
`
`and Photobiology
`
`B: Biolog!
`
`40 (1997)
`
`3/3-3lY
`
`319
`
`[13]
`
`[20]
`
`[23
`
`[24]
`
`We can not rule out that the bulk of circulating PPIX origins
`from the liver and may be responsible for a certain vascular
`photosensitivity
`[7]. Since injected PPIX and 5-ALA
`induced PPIX show a completely different
`tissue specificity
`[ 131, we nevertheless assume that the main part of PPIX in
`the tissue is synthesized in situ.
`
`Acknowledgements
`
`This study was supported by the Federal Ministry of Edu-
`cation and Research (BMBF)
`, grant number 13N63 11. The
`authors like to thank B. Hennel for technical assistance and
`J. Wessels for helpful discussions.
`
`References
`
`[I]
`
`[IO]
`
`Pathophysiology
`U. Mtiller-Eberhard,
`S.S. Bottomley,
`synthesis,
`Semin. Hematol.,
`25 ( 1988) 282-302.
`Porphyrins
`121 M.R. Moore,
`K.E.L. McCall,
`C. Rimington,
`A. Goldberg,
`and enzymes
`of
`the heme
`biosynthetic
`pathway,
`in: Disorders
`Porphyrin
`Metabolism,
`Plenum, New York,
`pp. 21-1 16.
`[3] N.I.
`Berlin,
`A. Neuberger,
`J.J.
`Scott,
`The metabolism
`aminolaevulinic
`acid, Biochem.
`J., 64
`( 1956)
`80-100.
`[4] A.M. Bathe, Porphyrins.porphyrias,cancerandphotodynamic
`- a model
`for carcinogenesis.
`J. Photochem.
`Photobiol.
`(1993)
`5-22.
`fluorescence
`In viva
`R. Sroka. R. Baumgartner,
`[5] P. Heil, S. Stocker,
`instillation
`of
`kinetics
`of
`porphyrins
`following
`intravesical
`aminolevulinic
`acid
`in normal
`and
`tumour
`bearing
`rat bladders,
`Photochem.
`Photobiol.
`B: Biol..
`in press.
`[ 61 R. Sroka, W. Beyer,
`L. Gossner,
`T. Sassy, S. Stocker,
`Pharmacokinetics
`of 5-aminolevulinic-acid-induced
`tumour-bearing
`mice,
`J. Photochem.
`Photobiol.
`13-19.
`D.A. Bellnier,
`L. Vaughan,
`[7] B.W. Henderson,
`Photosensitization
`Oseroff,
`Johnson,
`A.R.
`vasculature
`and skin
`by 5aminoIevulinic
`Photochem.
`Photobiol.,
`62 ( 1995)
`780-789.
`of delta-
`Effectiveness
`[8] Z. Hua, S.L. Gibson.
`T.H.
`Foster,
`R. Hilf,
`as a photosensitizer
`for
`aminolevulinic
`acid-induced
`protoporphyrin
`photodynamic
`therapy
`in viva, Cancer Res., 55 ( 1995)
`1723-1731.
`and
`191 S.
`Iinuma,
`R. Bachor,
`T. Flotte,
`T. Hasan,
`Biodistribution
`phototoxicity
`of 5-aminolevulinic
`acid-induced
`PpIX
`in an orthotopic
`rat bladder
`tumor model,
`J. Urol.,
`153
`( 1995)
`802-806.
`Z. Malik,
`G. Kostenich,
`L. Roitman,
`B. Ehrenberg,
`Topical
`application
`of 5-aminolevulinic
`acid, DMSO
`protoporphyrin
`IX
`accumulation
`in skin
`and
`tumours
`Photochem.
`Photobiol.
`B: Biol., 28 (1995)
`213-218.
`[ 1 l] C.S. Loh, A.J. MacRobert,
`J. Bedwell,
`J. Regula, N. Krasner,
`Bown,
`Oral
`versus
`intravenous
`administration
`of 5aminolaevuIinic
`acid
`for photodynamic
`therapy,
`Br. J. Cancer,
`68 ( 1993)
`41-5 1.
`[ 121 D.L. Campbell,
`E.F. Gudgin-Dickson,
`P.G. Forkett,
`R.H. Pottier,
`Kennedy,
`Detection
`of early
`stages of carcinogenesis
`in adenomas
`murine
`lung
`by 5-aminolevulinic
`acid-induced
`protoporphyrin
`fluorescence,
`Photochem.
`Photobiol..
`64
`( 1996)
`676682.
`
`of
`
`heme
`
`of
`
`of
`
`d-
`
`therapy
`B: Biol.,
`20
`
`5.