The high photoactivity of m-THPC in photodynamic therapy. Unusually strong retention of m-THPC by RIF-1 cells in culture

5,10,15,20-Tetra(m-hydroxyphenyl)chlorin (m-THPC, Foscan) is an extremely powerful photosensitizer showing up to 200 times the photodynamic activity of Photofrin in patients, in terms of drug/light dose. The influence of treatment conditions on the photodynamic efficacy of m-THPC has been compared to polyhematoporphyrin (PHP), a Photofrin equivalent, and a cationic pyridinium zinc (II) phthalocyanine (PPC), using the RIF-1 cell line. As predicted, the presence of serum during sensitizer incubation reduced the photodynamic efficacy of all three sensitizers. However, the presence of serum during the illumination period only had an inhibitory effect with PHP and PPC but not m-THPC. Quantification of the intracellular levels of sensitizer revealed that this was due to the efflux of PPC and PHP but not m-THPC into the medium, suggesting that m-THPC is tightly sequestered on entering the cell. This may partially explain the high efficacy of m-THPC in clinical photodynamic therapy and also highlights the importance not only of incubation conditions but also illumination conditions when in vitro comparisons are performed.     

Indirect detection of photosensitizer ex vivo

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.     

DETERMINANTS OF PHOTOSENSITIZATION BY PURPURINS

The human colon adenocarcinoma cell line, WiDr, was exposed to Photofrin II, hematoporphyrin derivative (HPD), hematoporphyrin (HP) or tetrasodium-meso-tetra(4-sulfonatophyenyl)porphine (TPPS4) followed by irradiation with light. Clonogenicity was determined and the resultant survival curves compared and shown to be qualitatively similar in shape. However, for equal amounts of drug in the medium, there were large differences in photosensitizing efficiency with Photofrin II approximately 5, 25 and 50 fold more effective than HPD, HP and TTPS4, respectively. For the same power used, all drugs were less efficient photosensitizers under red light (600-1100 nm) than under white light (300-110 nm). For all drugs this could be explained in terms of changes in light absorption over the two wavelength ranges. Differences in clonogenic cell survival could not be explained in terms of differences in singlet oxygen production (from published values). A reduction in drug uptake into the cells was sufficient to explain the differences between Photofrin II, HPD and HP, while TPPS4 was 5-fold less effective compared to other drugs than would be expected from drug uptake measurements. Two methods for measuring drug uptake were compared and shown to give different results for Photofrin II. Measurements of drug fluorescence in 0.1 N NaOH yielded 5-fold lower values than when measurements were in 1 N HCl following heat treatment to monomerise aggregated drug. Clearly the reliability of the method used in determining drug uptake must be carefully ascertained.     

Decreased efficiency of trypsinization of cells following photodynamic therapy: evaluation of a role for tissue transglutaminase

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm-2 (3 J cm-2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm-2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorine (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.     

A comparative study of the cellular uptake and photodynamic efficacy of three novel zinc phthalocyanines of differing charge

Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zinc [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.     

INTRAVENOUS vs INTRAPERITONEAL SENSITIZER: IMPLICATIONS FOR INTRAPERITONEAL PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) is a potential treatment for peritoneal carcinomatosis. However, little data is available regarding the relative distribution of sensitizer to tumor and intra-abdominal organs, optimal route of sensitizer administration, and maximal tolerated light dose. Tumor and normal tissue sensitizer levels were measured by tissue extraction 3, 24, 48 and 72 h after 10 mg/kg of Photofrin II was given intraperitoneally (IP) or intravenously (IV) in a mouse peritoneal tumor model, and the maximal tolerated PDT light dose determined. Equivalent tumor sensitizer levels were obtained regardless of the route of sensitizer administration. Route of administration, however, did affect the kinetics of tumor sensitizer elimination, with the half-time for elimination (T1/2) 113.6 h for IP drug and 60.6 h for IV drug. Route of administration also affected sensitizer levels in several intra-abdominal organs, resulting in somewhat higher tumor to liver and kidney levels at 24 and 72 h after IP sensitizer administration. Despite these tissue distribution differences, route of sensitizer administration did not significantly affect PDT toxicity or mortality when mice were treated with 630 nm light. The maximum tolerated light dose was 1.04 J/cm2. These parameters will prove helpful in designing large scale animal trials assessing the efficacy and safety of intra-abdominal PDT.     

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.     

Kinetic evaluation for measurement of in vivo receptor occupancy by psychotropic drug in brain: implication for human studies.

In vivo receptor occupancy of psychotropic drugs in brain can be estimated by measuring the tissue radioactivity of the tracer, which binds specifically to the receptor unoccupied by the drugs (back titration method). In this study, the validity of this method was evaluated by computer simulation, using various values for the plasma elimination rate, rate of transport across the blood-brain barrier, rate of receptor association and dissociation for both drug and tracer, and the sampling time. The differential equations based on a nonlinear three-compartment model including a plasma pool, precursor pool, and specific binding pool were solved numerically by the Runge-Kutta-Gill method. The receptor occupancy calculated by this method was close to the true value when the plasma concentration and specific binding fraction of the drug did not change greatly during circulation of the tracer. Although the error in calculated occupancy at 5 min after the tracer administration was smaller than that at 20 min, tracer may not greatly accumulate in brain tissue during the initial 5 min in some situations. Our analysis shows that it is necessary to adequately control the elimination rate of drug from plasma and to allow sufficient time for radioactivity to accumulate in the tissue. Therefore, this method requires previous knowledge of the pharmacokinetic behavior of both the drug and the tracer in plasma and tissue. The operation scheme that we suggest for the accurate measurement of the receptor occupancy in vivo can be used in human studies with positron emission tomography and may be useful for therapeutic drug monitoring.     

QUANTITATIVE ANALYSIS OF INTRACELLULAR BEHAVIOUR OF PORPHYRINS

Abstract Fluorometric analysis performed on L 1210 cells after treatment with Photofrin indicated that the interactions with cellular structures induce a significant modification of the equilibria among the different porphyrin species. This modification turned out to be dependent on the uptake and release processes. Thus, a comparative analysis of the dynamic aspects of the drug accumulation process was performed on cells treated with hematoporphyrin, Photofrin and Photofrin II. The results obtained were interpreted taking into account the different chemical composition of the drugs employed. The porphyrin species mainly released seem to be the monomeric ones and 'unfolded oligomers'. The release process results in further modifications of the aggregation and/or configu-rational state of intracellular porphyrins due to altered internal equilibrium.     

BIODISTRIBUTION AND PDT EFFICACY OF A KETOCHLORIN PHOTOSENSITIZER AS A FUNCTION OF THE DELIVERY VEHICLE

Abstract C8KC is a new ketochlorin photosensitizer that must be formulated with an emulsifier because of its poor water solubility. In this report, we compare properties of Cremophor EL (CRM) and Tween 80 as delivery vehicles for C8KC. Unlike Tween 80, CRM altered the physical properties of both human and mouse plasma lipoproteins, resulting in decreased electrophoretic mobility of the individual lipoproteins along with the formation of a l ipoprotein degradation product: a phospholipid fraction of low buoyant density. In human plasma, where there was sufficient low-density lipoprotein (LDL) for a distinction to be made, CRM caused a shift in binding of a ketochlorin from albumin to LDL and the degraded lipoprotein fraction. In mice bearing the RIF tumor, the use of CRM for drug formulation was associated with longer plasma and tissue persistence of C8KC, and enhanced photodynamic therapy (PDT) efficacy. These results indicate the importance of both sensitizer and vehicle as determinants of PDT efficacy.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN II: THE EFFECTS OF INTERACTION WITH HUMAN PLASMA PROTEINS

The absorbance and fluorescence spectra of Photofrin II (PII) in the presence of albumin, globulins and lipoproteins from human plasma show that all of these proteins induce a degree of disaggregation of PII material. In addition, there are substantial rearrangements in the distribution of different fractions contained in PII and their binding to the protein. It is shown that these rearrangements have considerable impact on the uptake of PII by cultured cells and the ensuing retention of the drug in the cells. The information on the contribution of fluorescing and non-fluorescing components of PII in the cells was obtained by measuring first the PII fluorescence in suspensions of live cells, followed by chemical extraction of porphyrin material from the same cells. The interaction of PII with low density lipoproteins resulted in markedly lower levels of PII material retained in the cells, compared to protein-free drug exposure. Somewhat better but still inferior PII retention was observed with high density lipoproteins. The samples with very low density lipoproteins showed increased uptake of PII, but the subsequent retention of the drug was low, so that the remaining amount of the drug was not much different than in protein-free samples. The strongest inhibition of PII uptake was seen with albumin, with ensuing retention of PII not significantly different than in protein-free samples. The best retention of PII was observed with globulins, with approx. 25% higher total drug content retained in the cells after long-term clearance relative to protein-free samples.     

UPTAKE AND RETENTION OF PHOTOFRIN® BY CULTIVATED HUMAN LYMPHOBLASTIC CELLS (Reh6): PREFERENTIAL AFFINITY OF THE CELLS FOR A MINOR COMPONENT DEMONSTRATED BY NORMAL PHASE CHROMATOGRAPHY

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Synthesis,and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.     

Microseparation techniques for the study of the enantioselectivity of drug–plasma protein binding

Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer–protein interactions, enantiomer–enantiomer interactions as well as chiral drug–drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug–plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: II. THE EFFECTS OF HUMAN versus MOUSE AND BOVINE SERUM

The effects of human serum (HS), mouse serum (MS) and fetal bovine serum (FBS) on cellular delivery and retention of Photofrin were examined using human lung tumor cells (A549) cultured in vitro. The results show that these three kinds of sera exhibit substantial differences in: (i) degree of inhibition of Photofrin cellular uptake, (ii) retention capacity of Photofrin delivered to the cells in their presence and (iii) efficacy of promoting the clearance of Photofrin from the cells. It is suggested that these differences originate from unequal interaction of each of the sera with Photofrin material, which in turn is the consequence of variability in composition and in the levels of serum proteins in HS, MS and FBS. The highest degree of Photofrin disaggregation and and competitive binding of its constituents was attributed to HS. The lowest degree of Photofrin disaggregation, and the competitive binding limited mostly to monomeric porphyrin forms was implicated for FBS. For MS, the spectroscopic and cellular data indicated a lesser degree of Photofrin disaggregation than with HS, with little if any consequence in Photofrin retention characteristics. The implication of this comparative analysis is that in vitro studies using FBS may underestimate the extent of interaction of Photofrin with serum proteins in humans, and overestimate the retention capacity of the photosensitizer in human tissues. Studies in vivo using a mouse model may also underestimate the degree of disaggregation of Photofrin in human circulation, and give different photosensitizer tissue retention levels than in humans.     

Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE

The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.     

Pharmacokinetics of a tri-glucoconjugated 5,10,15-(meta)-trihydroxyphenyl-20-phenyl porphyrin photosensitizer for PDT. A single dose study in the rat

Photodynamic therapy (PDT) involves a non invasive treatment of small and superficial cancers using a photosensitive drug and light to kill tumoral cells. 5,10,15-meso-tri-(meta-O-beta-D-glucosyloxyphenyl)-20-phenylporphyrin [m-TPP(glu)3] is a new photosensitizer (PS) with more enhanced photocytotoxicity relative to 5,10,15,20-meso-tetra-(meta-hydroxyphenyl) chlorin [m-THPC] (Foscan). It was injected intravenously once to healthy rats at three different doses (0.25, 0.5 and 1 mg kg(-1)) and compared to m-THPC (0.3 mg kg(-1)). Pharmacokinetic parameters for both photosensitizers were derived from plasma concentration-time data using a non-compartmental analysis and a two-compartment pharmacokinetic model. m-TPP(glu)3 is more rapidly eliminated throughout the organism than m-THPC. Its mean plasma clearance is 19 mL h(-1) kg(-1) (6 mL h(-1) kg(-1) for m-THPC), and its mean residence time is 5h (20 h for m-THPC). The area under curve (AUC) and initial mean serum concentration (C0) were found to be proportional to the dose. As for Foscan, no metabolite of m-TPP(glu)3 was detected in plasma. The biodistribution study demonstrates that the most significant amount of m-TPP(glu)3 was concentrated in organs such as lung, liver and spleen which are rich in reticulo-endothelial cells. Maximum concentrations were reached in organs 14 h after IV administration. At 48 h, the photosensitizer was essentially eliminated from all organs. Because of its shorter elimination time, m-TPP(glu)3 is more attractive than m-THPC as a PDT agent since secondary side effects of shorter duration could be expected.     

The efficacy of certain anti-tuberculosis drugs is affected by binding to alpha-1-acid glycoprotein

One of the most ubiquitous plasma proteins, alpha-1-acid glycoprotein (AGP), has a high affinity, low capacity binding for basic drugs positively charged at physiological pH. Moreover, as an acute phase protein its level is increased in various disease states in a manner that is likely to influence the free plasma level of a drug, the ability to attain minimum effective concentration and overall in vivo effectiveness. AGP is a glycoprotein known to display disease specific changes in glycosylation and although this secondary modification is not directly involved in drug binding, it may influence the conformation of the binding site. Binding studies reveal that alpha-1-acid glycoprotein bind mainly to the tuberculosis drugs: rifampicin; isoniazid; pyrazinamide; p-aminosalicylic acid; capreomycin; ethionamide; levofloxacin and ofloxacin out with the therapeutic plasma range tested. These results are however still considered significant as not only are alpha-1-acid glycoprotein levels increased during the acute phase response but specific alpha-1-acid glycoprotein from tuberculosis samples are subject to glycosylation changes which can increase the binding affinity and cause binding to occur at the therapeutic concentration.     

THE TIME COURSE OF CUTANEOUS PORPHYRIN PHOTOSENSITIZATION IN THE MURINE EAR

This study was designed to investigate the time course of acute cutaneous photosensitivity following administration of Photofrin II using the murine ear swelling response (ESR) as an in vivo end-point. Ros:(ICR) mice were injected with 5 mg/kg Photofrin II and illuminated 7.5 h to 31 days later with 630-nm laser light; ESR was measured 24 h after illumination. There was a direct correlation between ESR and the concentration of [14C]Photofrin II in blood, while no relationship between ESR and the level of [14C]Photofrin II in the ear tissue of exsanguinated mice was evident. Photosensitivity in the mouse foot can be suppressed by preexposure to low doses of light via a photochemical destruction of tissue-bound sensitizer (Boyle and Potter, 1987, Photochem. Photobiol. 46, 997-1001). However, mouse ears pretreated with 84 J/cm2 of 630-nm light (28 J/cm2/day, given 2, 4 and 6 d after injection), a dose sufficient to reduce porphyrin fluorescence in ear tissue by about 75%, prior to the usual light dose (88.6 J/cm2, 630 nm, day 9 after injection) showed a mean ESR not significantly different (P less than 0.5) from that for ears which received only a single dose of 88.6 J/cm2 on day 9. It is concluded, for this animal model, that circulating porphyrin is the source of photoinduced ear-tissue edema and that photobleaching of tissue-bound sensitizer does not attenuate ear-tissue photosensitivity.