ON THE SOURCE OF THE OSCILLATIONS OBSERVED DURING in vivo ZINC PHTHALOCYANINE FLUORESCENCE PHARMACOKINETIC MEASUREMENTS IN MICE

Surface-detected fluorescence spectroscopy can be used to monitor the pharmacokinetics of uptake and clearance of red-absorbing fluorophores such as zinc(II) phthalocyanine (ZnPc) in vivo. When this technique is applied to mice that have been fed on a normal chlorophyll-based diet, and particularly when measurements are performed in the abdominal region, oscillations are sometimes observed superimposed on the pharmacokinetic curve of the ZnPc. An oscillatory signal has also been observed arising from the abdominal region of control mice fed a normal diet but not injected with the ZnPc photosensitizer; this oscillatory component to the signal is reduced when mice are fed a chlorophyll-free diet. The oscillatory signal component has been attributed to fluorescence arising from chlorophyll derivatives (pheophorbide/pheophytin) contained in the rodent food, whose concentration in the measured abdominal region changes substantially with time, presumably due to digestive processes. Thus it is important to be aware of the possibility of such artifactual contributions to in vivo fluorescence pharmacokinetic measurements.     

Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa

We have investigated the pharmacokinetics (PK) of Lutetium Texaphyrin (Lu-Tex), a second-generation photosensitizer, in the Syrian hamster cheek pouch early cancer model. Ten male hamsters, five with chemically induced early squamous cell cancer of the left cheek pouch, received an intracardiac injection of a 10 mg/ml Lu-Tex solution, resulting in a dose of 12 mg Lu-Tex per kg of bodyweight. The PK of the dye have been measured during the 24 h following the injection with an optical-fiber-based spectrofluorometer on the ventral skin, the healthy and the tumoral cheek-pouch mucosa. The Lu-Tex fluorescence is excited at 460 nm and detected around 740 nm. All the measurements yield very similar pharmacokinetic curves. The fluorescence intensity reaches a maximum between two and three hours after the injection and, at its maximum, it is consistently higher (up to 1.5 times) on the tumor than on the healthy mucosa. It remains smaller on the skin than on cheek-pouch mucosa. After 24 h, the Lu-Tex fluorescence is no longer detectable either on the skin, on the lesion or on the healthy mucosa. Moreover, Lu-Tex clearly displays a significant fluorescence selectivity between early carcinoma and healthy mucosa in this model. Furthermore, the inter-animal fluctuations of the fluorescence signal are small (+/-16% on the tumor-bearing mucosa). Eight-minute-long skin-irradiation tests have been performed 24 h after the injection of the Lu-Tex on the ventral skin of 16 additional animals with a solar simulator. No reaction is observed, either macroscopically or microscopically, which further demonstrates, as suggested by the fluorescence measurements, that this photosensitizer is significantly cleared from the skin after 24 h.     

In vitro and in vivo investigations on the photodynamic activity of core-modified expanded porphyrin--ammonium salt of 5,10,15,20-tetrakis-(meso-p-sulfonato phenyl)-25,27,29-trithia sapphyrin

The core modification of expanded porphyrins has been proved to have better photochemical properties, which are favorable for photodynamic therapy (PDT) applications. In this context, this study was aimed to investigate the in vitro and in vivo photodynamic activity of one such core-modified expanded porphyrin, namely, ammonium salt of 5,10,15,20-tetrakis-(meso-p-sulfonato phenyl)-25,27,29-trithia sapphyrin. For the in vitro studies, human erythrocytes were used as a membrane semimodel system to investigate the partitioning ability and drug-uptake characteristics. The partition studies on the membrane semimodel system revealed that maximum partitioning occurs at 12 microgm/mL concentration, and from the drug-uptake studies it is observed that maximum amount of the sensitizer is bound to the erythrocyte membranes during a 45 min incubation period. Photohemolysis studies at different concentrations of the sensitizer and exposure time showed maximum damage at 5 microgm/mL and 30 min exposure time. In vivo studies were performed on 7,12-dimethylbenz(a)nthracene-induced superficial squamous cell carcinoma on mouse skin. The sensitizer at a concentration of 2.5% in 2.0% dimethyl sulfoxide was applied topically on the tumor spot. After 1 h incubation the tumor spot was exposed to laser irradiation from Nd-YAG laser at its second harmonic wavelength of 532 nm. The photodynamic efficacy was estimated by tumor volume measurements at regular intervals after the treatment. One month after PDT exposure a 3.9-fold decrease in the tumor volume was observed with respect to the tumor volume before treatment. The treatment efficacy was further confirmed by histological and fluorescence spectroscopic evaluations of the tissue biopsy sample from the treated area. The results of our study suggest that the ammonium salt of 5,10,15,20-tetrakis-(meso-p-sulfonato phenyl)-25,27,29-trithia sapphyrin may find possible applications in the new modality of cancer treatment.     

In vivo FLUORESCENCE KINETICS OF PHTHALOCYANINES IN A SKIN-FOLD OBSERVATION CHAMBER MODEL: ROLE OF CENTRAL METAL ION AND DEGREE OF SULFONATION

Abstract— The fluorescence pharmacokinetics of a series of metallosulfophthalocyanines, chelated with either aluminum or zinc and sulfonated to different degrees, was studied by fluorescence measurements in vivo . Dyes were administered systemically to female WAG/RIJ rats with an isogeneic mammary carcinoma transplanted into the subcutis in a transparent observation chamber located on their backs. Following an intravenous injection of 2.5 μmol/ kg of the dye, fluorescence dynamics was observed up to 7 h postinjection. The phthalocyanines were excited at 610 nm with a power density of 0.1 mW/cm² without causing photodynamic damage to the vasculature. Fluorescence was detected above 665 nm using a fluorescence imaging system based on an image intensifier. Dye retention in the blood vessels and tumor tissue was expressed as ratios relative to the fluorescence signal of the surrounding subcutaneous tissue. Phthalocyanincs chelated with aluminum gave the highest fluorescence signal with tumor-over-subcutis ratios of up to a value of 4. The zinc complexes exhibited the highest vascular-over-subcutis ratios with maximum values exceeding a value of 6. They also displayed the longest retention times in the vascular system of well over 7 h. Overall, decreasing the degree of sulfonation of the metallophthalocyanines results in lower tumor-over-normal tissue fluorescence ratios, and furthermore aluminum-based dyes seem superior tumor localizers over zinc-based dyes. The advantages of phthalocyanines over porphyrins with respect to tumor localization and photodynamic therapy are discussed.     

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.     

Simulations on the selectivity of 5-aminolaevulinic acid-induced fluorescence in vivo

The knowledge of the exact time course of a photosensitizer in tumour and surrounding host tissue is fundamental for effective photodynamic therapy (PDT) and fluorescence-based diagnosis. In this study the time course of porphyrin fluorescence following topical application of 5-aminolaevulinic acid (ALA) using different formulations, concentrations and incubation times has been measured in amelanotic melanomas (A-Mel-3) (n = 54) grown in transparent dorsal skinfold chambers of Syrian golden hamsters and in human basal cell carcinomas (BCCs) (n = 40) in vivo. To simulate the accumulation of ALA-induced protoporphyrin IX (Pp IX), a three-compartment model has been developed and rate constants have been determined. The kinetics of both the A-Mel-3 tumours and the BCCs show a significantly higher fluorescence intensity in tumour as compared to normal surrounding host tissue. Maximal fluorescence intensity in A-Mel-3 tumours as a percentage of the reference standard used occurs 150 min post incubation (p.i.) using a 1, 3 or 10% (vol.) ALA solution buffered to pH 7.4 and 1 h incubation time. After a 4 h incubation time maximal fluorescence intensity in tumour is measured shortly p.i. A concentration of 10% ALA does not increase the fluorescence intensity as compared to 3% ALA following 4 h incubation, but either 3 or 10% ALA yields a significantly higher fluorescence after 4 h incubation time as compared to 1 h. The fluorescence intensity following an 8 h incubation reaches its maximum directly p.i. for all concentrations and then decreases exponentially. The fluorescence intensity in the surrounding host tissue shows no statistically significant difference regarding concentration or incubation time. At least during the first hour p.i., the fluorescence intensity measured in the surrounding tissue is lower as compared to that in the tumour in all groups. 24 h after topical application hardly any fluorescence is detectable in tumour or surrounding host tissue in all experimental groups. Incubating human BCCs with a 20% ALA cream (water-in-oil emulsion) or a 20% ALA gel (containing 40% dimethyl sulfoxide) for approximately 2 h yields a similar fluorescence intensity directly after incubation for either cream or gel. However, while yielding a maximum 120 min p.i. with cream, the fluorescence intensity increases for a longer time (about 2-3 h p.i.) and up to higher values using the gel formulation. In surrounding normal skin, cream as well as gel formulation yields a similar fluorescence intensity directly after incubation. Afterwards the fluorescence intensity decreases slowly using the cream whereas a further increase of the fluorescence intensity is measured in the normal skin with a maximum 240 min p.i. using the gel formulation. The results of the proposed three-compartment model indicate that the observed selectivity of accumulated porphyrins following topical application of ALA is mainly governed by an increased ALA penetration of the stratum corneum of the skin, an accelerated ALA uptake into the cell and a higher porphyrin formation in tumour as compared to normal skin tissue, but not by a reduced ferrocheletase activity.     

PHOTOBLEACHING OF PORPHYRINS USED IN PHOTODYNAMIC THERAPY AND IMPLICATIONS FOR THERAPY

Abstract— The development of an extraction procedure to quantitate dihematoporphyrin ether (DHE) concentration in tissues correlated to fluorescence measurements from instrumentation developed for in vivo fluorimetry was examined. In vivo fluorometric results from mouse mammary carcinoma (SMT-F) were calibrated against results of the chemical extraction assay quantitated spectrophotometrically. Fluorescence and drug extractable levels increase in a linear fashion with injected dose. Loss of porphyrin fluorescence (photobleaching) and intra-tumoral porphyrin level has been demonstrated both in vitro (NHIK cells) and in vivo (SMT-F tumor) during illumination with light following exposure to Hpd or DHE. This process is essentially independent of porphyrin tumor level in vivo and could lead to tumor protection at very low porphyrin levels. On the other hand, this photobleaching process which occurs concurrent with cellular inactivation and tissue damage due to the photodynamic process can be exploited to protect normal tissue during photodynamic therapy (PDT) and thus greatly enhance the therapeutic ratio. This has been demonstrated in patients undergoing PDT.     

Noninvasive and nondestructive optical spectroscopic measurement of motexafin gadolinium in mouse tissues: comparison to high-performance liquid chromatography

Efficient design of anti-cancer treatments involving radiation- and photo-sensitizing therapeutics requires knowledge of tissue-specific drug concentrations. This study investigates the use of the optical pharmacokinetic system (OPS) to measure concentrations of the anti-cancer agent motexafin gadolinium (MGd) in mouse tissues noninvasively and nondestructively using elastic-scattering spectroscopy. The magnitude of MGd absorbance was quantitated by integration of the MGd peak absorbance area, and MGd concentrations were estimated by comparison with standard curves that were validated by high performance liquid chromatography (HPLC). In tissue-simulating phantoms in vitro, MGd peak absorbance area correlated with MGd concentration. Female C.B-17 SCID mice, bearing subcutaneous MDA-MB-231 human breast cancer xenografts, were dosed with 23 mg/kg MGd i.v. At specific times between 5 min and 24h after dosing, noninvasive OPS measurements were made on skin overlaying the subcutaneous tumor and skin on the opposite flank in vivo, and following exsanguination, nondestructive measurements were made on tumor, skin, and internal tissues in situ. OPS measurements on tissues in vivo detected MGd present in both tissue and blood perfusing the tissue. Both the OPS and the HPLC detected selective localization of MGd in malignant tissues compared with surrounding non-malignant tissues, and neither technique detected MGd in brain tissue. Comparison of MGd concentrations measured by HPLC and OPS is complicated by mismatch between measured tissue volumes, heterogeneous spatial distribution of MGd in tissues, and blood-localized MGd at early time points. Tumor-specific MGd concentrations measured by HPLC correlated with those measured by OPS in vivo and in situ. Best fit lines to the concentration estimates (forced through zero) had slopes of 0.900 and 1.185, respectively; however, the variability was significant (r(2)=0.477 and 0.269). The clinical utility of the OPS to quantitate MGd concentrations remains to be validated.     

Comparative pharmacokinetic and photodynamic studies with zinc(II) phthalocyanine in hamsters bearing an induced or transplanted rhabdomyosarcoma.

Comparative pharmacokinetic studies in hamsters bearing an induced or first-generation transplanted rhabdomyosarcoma that were injected with liposome-incorporated zinc(II) phthalocyanine (ZnPc) show a higher drug concentration in the induced tumour. The selectivity of tumour targeting is underlined by the fact that, 24 h after injection, larger amounts of ZnPc are found in the tumour than in the liver. Photodynamic therapy investigations were carried out using 673 nm light from an argon-dye laser. On the basis of different assessment criteria (changes in mean tumour diameter with time, tumour mass regression, survival time of the treated groups of animals, and histological determination of the necrotic tissue) the photosensitizing effect of ZnPc appears to be comparable for both kinds of tumour in spite of the higher uptake of photosensitizer by the induced tumour.     

In vitro and in vivo fluorescence monitoring of photosensitizers

The use of steady state fluorescence spectroscopy in the detection and monitoring of potential photochemotherapeutic agents is examined. Problems associated with both in vitro and in vivo fluorescence measurements are investigated, and typical data are presented. Recent results on the use of fluorescence in pharmacokinetic studies are discussed, and the relative merits of in vitro vs. in vivo methods are outlined.     

OCTA-ALKYL ZINC PHTHALOCYANINES: POTENTIAL PHOTOSENSITIZERS FOR USE IN THE PHOTODYNAMIC THERAPY OF CANCER

The synthesis, characterization and electronic spectra of a series of nine 1,4,8,11,15,18,22,25-octa-alkyl zinc phthalocyanines (ZnPc), potential photosensitizers for the photodynamic therapy of cancer, are described. The substituents on the phthalocyanine (Pc) macrocycle “red-shift” the absorbance maximum, in cyclohexane, of all nine members of this series to a value of 703 ± 2 nm, with a corrected fluorescence emission maximum for the octadecyl derivative of 715 nm. The solubilities and degree of aggregation of six examples in cyclohexane have been measured. The highest homologue, the octadecyl derivative, remains essentially unaggregated to a concentration of 1.5 ± 10^?4 mol dm^?3. The photostability of this Pc has been examined and the compound shown to be sensitive to photooxidation processes, which lead to its decomposition to 3,6-fcw-decylphthalimide. Known singlet oxygen quenchers inhibit the photodecomposition. In a comparative study, the octadecyl ZnPc underwent a more rapid photodecomposition than the corresponding metal-free derivative.     

Meso-Tetraphenylporphyrin Dimer Derivative as a Potential Photosensitizer in Photodynamic Therapy

Abstract— Studies on the synthesis, preliminary in vivo biological activity, singlet oxygen and fluorescence yields of a di-meric porphyrin (Dl) are described. The pharmacokinetic behavior and photodynamic properties of the dimer Dl were examined in Balb/c mice bearing an MS-2 fibrosarcoma. Compound Dl shows a high selectivity for tumor localization (tumor/peritumoral tissue ratios of dye concentration ranging between ca 100 and 140 at 24 h after drug administration of 5.0-1.0 mg kg_-1 into DL-AL-dipalmitoylphosphatidylcholine liposomes). The photo-therapeutic efficiency of dimer Dl was evaluated by following the growth curves of fibrosarcoma irradiated with red light (600-700 nm) with a total dose of 400 J cm_-2, at 24 h after intravenous injection. Photodynamic therapy-treated tumors showed a significant delay in growth as compared to untreated control mice. The results obtained suggest that the porphyrin dimer Dl may be a promising candidate for further use in PDT experiments.     

TIME-GATED FLUORESCENCE IMAGING FOR THE DIAGNOSIS OF TUMORS IN A MURINE MODEL

A system for time-gated fluorescence imaging was used to perform measurements on tumor-bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns-delayed images with respect to synchronous ones.     

Pharmacokinetics and Tissue Distribution of DVDMS-2 in Tumor-bearing Mice

DVDMS-2 is a novel candidate for photodynamic therapy of tumors. The purpose of the present study was to assess the distribution and elimination of DVDMS-2 in mice bearing hepatoma 22 tumors. DVDMS-2 (1, 2 and 4 mg kg⁻¹) was injected intravenously into the mice, extracted from biological tissues and quantified using a fluorescence assay. The data obtained were processed with WinNonlin pharmacokinetic software. The fluorescence assay established for DVDMS-2 quantification was a rapid, reproducible, sensitive and specific method with good linearity. The pharmacokinetics of DVDMS-2 in tumor-bearing mice conformed to a two-compartment model. DVDMS-2 accumulated in tumor tissue to a greater extent than adjacent tissues (skin, muscle) and sustained a relatively high-level concentration 12 to 24 h following administration, which may be the optimal treatment time point. In conclusion, DVDMS-2 selectively accumulated in tumor tissue and was eliminated at a rapid rate in tumor-bearing mice, suggesting that DVDMS-2 may have few side effects, including skin phototoxicity. The present study established the pharmacokinetic characteristics of DVDMS-2, which may be beneficial in future clinical study.     

PHARMACOKINETICS OF FLUORESCENT POLYPORPHYRIN PHOTOFRIN II IN NORMAL RAT TISSUE AND RAT BLADDER TUMOR

The transient behavior of the molecular components responsible for fluorescence emission of the photosensitizing polyporphyrin Photofrin II has been studied quantitatively in the liver, small intestines, bladder and muscles of rats. Relative concentrations of the substance were determined fluorometrically in vivo using a Kr(+)-laser (wavelength = 406.7 nm) and a mercury arc lamp (wavelength = 405 or 550 nm) for fluorescence excitation of Photofrin II. Fluorescence was detected at the maxima of the emission bands, at 630 or 690 nm. The results of the experiments show that Photofrin II can be clearly detected by its fluorescence in all the organs investigated from 3 h up to at least 28 days after systemic application of the substance. Within this investigational period the fluorescing components of Photofrin II are released continuously from the organs. In all the tissues examined, an initial decrease with time constants between 2 and 42 h followed by a slow decay with time constants between about 300 and 600 h can be observed. In addition the pharmacokinetics of the fluorescent components of Photofrin II in chemically induced rat bladder tumors with different stages of malignancy were compared to healthy rat bladder tissue. In a time range of 2-10 days after intravenous injection Photofrin II shows a fluorescence 2-5 times brighter in rat bladder tumors than in healthy bladder tissue.     

Formation and Photoinduced Electron Transfer in Porphyrin- and Phthalocyanine-Bearing N-Doped Graphene Hybrids Synthesized by Click Chemistry

Graphene doped with heteroatoms such as nitrogen, boron, and phosphorous by replacing some of the skeletal carbon atoms is emerging as an important class of two-dimensional materials as it offers the much-needed bandgap for optoelectronic applications and provides better access for chemical functionalization at the heteroatom sites. Covalent grafting of photosensitizers onto such doped graphenes makes them extremely useful for light-induced applications. Herein, we report the covalent functionalization of N-doped graphene (NG) with two well-known electron donor photosensitizers, namely, zinc porphyrin (ZnP) and zinc phthalocyanine (ZnPc), using the simple click chemistry approach. Covalent attachment of ZnP and ZnPc at the N-sites of NG in NG−ZnP and NG−ZnPc hybrids was confirmed by using a range of spectroscopic, thermogravimetric and imaging techniques. Ground- and excited-state interactions in NG−ZnP and NG−ZnPc were monitored by using spectral and electrochemical techniques. Efficient quenching of photosensitizer fluorescence in these hybrids was observed, and the relatively easier oxidations of ZnP and ZnPc supported excited-state charge-separation events. Photoinduced charge separation in NG−ZnP and NG−ZnPc hybrids was confirmed by using the ultrafast pump-probe technique. The measured rate constants were of the order of 10¹⁰ s,⁻¹ thus indicating ultrafast electron transfer phenomena.     

A pharmacokinetic study to correlate the hypoglycemic effect of phlorizin in rats: Identification of metabolites as inhibitors of sodium/glucose cotransporters

Phlorizin (PRZ) is a natural product that belongs to a class of dihydrochalcones. The unique pharmacological property of PRZ is to block glucose absorption or reabsorption through specific and competitive inhibitors of the sodium/glucose cotransporters (SGLTs) in the intestine (SGLT1) and kidney (SGLT2). This results in glycosuria by inhibiting renal reabsorption of glucose and can be used as an adjuvant treatment for type 2 diabetes. The pharmacokinetic profile, metabolites of the PRZ, and efficacy of metabolites towards SGLTs are unknown. Therefore, the present study on the characterization of hitherto unknown in vivo metabolites of PRZ and pharmacokinetic profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) and accurate mass measurements is undertaken. Plasma, urine, and feces samples were collected after oral administration of PRZ to Sprague–Dawley rats to identify in vivo metabolites. Furthermore, in silico efficacy of the identified metabolites was evaluated by docking study. PRZ at an intraperitoneal dose of 400 mg/kg showed maximum concentration in the blood to 439.32 ± 8.84 ng/mL at 1 h, while phloretin showed 14.38 ± 0.33 ng/mL at 6 h. The pharmacokinetic profile of PRZ showed that the maximum concentration lies between 1 and 2 h after dosing. Decreased blood glucose levels and maximum excretion of glucose in the urine were observed when the PRZ and metabolites were observed in plasma. The identification and characterization of PRZ metabolites by LC/ESI/MS/MS further revealed that the phase I metabolites of PRZ are hydroxy (mono-, di-, and tri-) and reduction. Phase II metabolites are O-methylated, O-acetylated, O-sulfated, and glucuronide metabolites of PRZ. Further docking study revealed that the metabolites diglucuronide metabolite of mono-hydroxylated PRZ and mono-glucuronidation of PRZ could be considered novel inhibitors of SGLT1 and SGLT2, respectively, which show better binding affinities than their parent compound PRZ and the known inhibitors.     

PHOTOBINDING OF CHLORPROMAZINE AND ITS SULFOXIDE IN VITRO AND IN VIVO

Abstract— Photosensitivity as observed after chlorpromazine (CPZ) treatment is enhanced in the UVA- rather than the UVB region, whereas CPZ has its absorption maximum at 305 nm. This long wavelength sensitivity has sometimes been ascribed to CPZ-sulfoxide (CPZSO) which has an absorption maximum at 340 nm. We compared the photobinding properties of CPZSO and CPZ under both in vitro and in vivo conditions.
With 310 and 370 nm lamps CPZSO absorbs twice as much light as CPZ but still binds less efficiently to HSA in vitro. At wavelengths longer than 380 nm CPZSO does not absorb nor photobind to HSA (420 nm lamps) in contrast to CPZ.
In vivo the bioavailability of CPZ and CPZSO in ears, eyes and skin of the back of Wistar rats is comparable, yet irradiation with 370 nm light caused more CPZ-photobinding in these tissues. Chlorpromazine binds relatively more compared to CPZSO, to constituents of deeper lying tissues (dermis). This corresponds with the observation that both the ratio of in vitro CPZ photobinding to CPZSO photobinding, and the penetrating ability of light in the skin increase with wavelength.
In the eyes, where the lens efficiently filters out light with wavelengths shorter than 370 nm, no CPZSO photobinding was observed, in contrast to CPZ; this also corresponds with the in vitro experiments. Therefore it seems more likely that the observed wavelength maximum in the photosensitivity action spectrum after CPZ treatment should be attributed to the non-sulfoxidated drug rather than to the sulfoxidated compound.     

Pharmacokinetics of ICG and HPPH-car for the detection of normal and tumor tissue using fluorescence, near-infrared reflectance imaging: a case study

We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg-1 ICG or 0.3 mg kg-1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5-1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.     

Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin IX in skin and blood

The fluorescence and photosensitivity of endogenously synthesized protoporphyrin IX (PPIX) is increasing used for the diagnosis and treatment of malignant and certain non-malignant diseases. A selective accumulation of PPIX can be induced by application of 5-aminolevulinic acid (5-ALA), which is a precursor of PPIX in the cellular biosynthetic pathway of heme.

The purpose of this study was to monitor the in vivo accumulation of PPIX in different locations of the skin after oral ingestion and to 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.

After oral application of 5-ALA in a concentration of 40 mg kg⁻¹ body weight, the fluorescence intensities of PPIX in the skin showed 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 1 μg l⁻¹. The highest mean concentration of 742 μg l⁻¹ PPIX in the plasma was measured 6.7 h after oral application. For inhalation of 5-ALA, a mean maximum concentration of 12 μg l⁻¹ could be detected 4.1 h after application, for intravesical instillation, the mean maximum concentration was found to be 1 μg l⁻¹ 2.9 h after application. The kinetics of 5-ALA in the plasma peaked much earlier with a maximum concentration of 32 mg l⁻¹ about 30 min. after oral administration. The 5-ALA levels did not exceed normal reference values after topical application.

The results of our experiments suggest that for a systemic application of 5-ALA side-effects in sensitive patients cannot be excluded.