Pre-clinical compartmental pharmacokinetic modeling of 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) as a photosensitizer in rat plasma by validated HPLC method.

A second-generation chlorin-based photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) has shown tremendous therapeutic potential in clinical trials in the treatment of esophageal cancer. Herein, we have developed and validated a bioanalytical method for estimation of HPPH in rat plasma using High Performance Liquid Chromatography (HPLC) with a photo diode array (PDA) detector. The method was applied for carrying out pharmacokinetic study of HPPH. Further pharmacokinetic modeling was carried out to understand the compartment kinetics of HPPH. The developed method was fully validated as per the United States Food and Drug Administration (US-FDA) guidelines for bioanalytical method validation. The linearity of the method was in the range of 250-8000 ng mL-1, and the plasma recovery was found to be 70%. Pharmacokinetic parameters were evaluated and compared via non-compartment analysis and compartment modeling after the intravenous (i.v.) bolus administration in rats using Phoenix WinNonlin 8.0 (Certara™, USA). From the obtained results, we hypothesize that the HPPH complies with two compartmental pharmacokinetic model. Furthermore, it was observed that HPPH has the rapid distribution from the central compartment to peripheral compartment along with slow elimination from peripheral compartment.

Population pharmacokinetics of the photodynamic therapy agent 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a in cancer patients.

Photodynamic therapy is an effective and often curative treatment for certain solid tumors. The porphyrin-based photosensitizer Photofrin, the only Food and Drug Administration-approved drug for this therapy, suffers from certain disadvantages: its complex chemical nature; retention by skin (leading to protracted cutaneous photosensitivity); and less than optimal photophysical properties. In this study, we examine the population pharmacokinetics and cutaneous phototoxicity of 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), a chlorin-type photosensitizer with more favorable photophysical properties. HPPH plasma concentration-time data were obtained in 25 patients enrolled in Phase I-II clinical trials for the treatment of partially obstructive esophageal carcinoma, high-grade dysplasia associated with Barrett's esophagus, carcinoma of the lung, or multiple basal cell carcinomas. Doses of 3, 4, 5, or 6 mg/m(2) were administered as 1-h i.v. infusions. The pharmacokinetic data for each patient were fitted with a standard two-compartment (biexponential) model with continuous infusion. The model fitting approach was iteratively reweighted nonlinear regression, with weights equal to the reciprocal of the square of the predicted HPPH plasma concentrations. The complete set of data for all 25 patients was then fitted simultaneously with nonlinear mixed effects modeling. Cutaneous phototoxicity responses were determined, as a function of time after HPPH infusion, following exposure to various doses of light from a solar simulator. The estimates of the population mean (variance) for each parameter were as follows: volume of distribution (V(C)), 2.40 liters/m(2) (0.259); steady-state volume (V(SS)), 9.58 liters/m(2) (11.6); systemic clearance (CL), 0.0296 liter/h/m(2) (0.000094); and distributional clearance (CL(D)), 0.144 liter/h/m(2) (0.00166). These parameters were independent of dose. Clearance increased with age. A relative error model was used for the difference in the raw and fitted data, and the overall coefficient of variation estimate across all of the data was 14.5%. The estimated mean population alpha and beta half-lives (95% confidence interval) were 7.77 h (3.46-17.6 h) and 596 h (120-2951 h), respectively. High-performance liquid chromatography analysis of serum showed no circulating HPPH metabolites, and in vitro incubation of HPPH with human liver microsomal preparations resulted in no metabolite or glucuronic acid-HPPH conjugate production. A minimal skin response to the solar simulator was observed, mostly in patients treated with the highest dose of HPPH, 6 mg/m(2). All of the HPPH pharmacokinetic parameters were consistent with a highly lipophilic agent that is concentrated in plasma and is nearly 100% bound to plasma proteins; this was verified by plasma protein binding studies. Whereas low concentrations of HPPH can be detected in plasma several months after a single infusion, no instances of cutaneous photosensitivity have been noted in these patients. In general, HPPH pharmacokinetic profiles are readily predictable from the global population model. This is the first comprehensive human population pharmacokinetic/pharmacodynamic study of a clinical anticancer photodynamic therapy agent.

Isolation and initial characterization of 13(2)-hydroxychlorophyll a induced by cyclohexanedione derivatives in tobacco cell suspension cultures.

This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene-5-5-dim ethyl- cyclohexane-1,3-dione (RWH-21), stimulates accumulation of 13(2)-hydroxychlorophyll a in cultured tobacco cells. This was shown based on isolation of 13(2)-hydroxychlorophyll a from pigment extracts of cultured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis. 13(2)-Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH-21 (50 microM). Analysis of 13(2)-hydroxychlorophyll a formation in tobacco cells indicates that 13(2)-hydroxychlorophyll a is rapidly accumulated within 20 h incubation time both in the dark and light. Although the amount of 13(2)-hydroxychlorophyll a is continuously increased in the dark, the amount of 13(2)-hydroxychlorophyll a decreased remarkably in the light after 20 h incubation. Analysis of 13(2)-hydroxychlorophyll a formation and lipid peroxidation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 13(2)-hydroxychlorophyll a has the potential to cause a photodynamic action in cultured tobacco cells.

Parabolic quantitative structure-activity relationships and photodynamic therapy: application of a three-compartment model with clearance to the in vivo quantitative structure-activity relationships of a congeneric series of pyropheophorbide derivatives used as photosensitizers for photodynamic therapy.

An open three-compartment pharmacokinetic model was applied to the in vivo quantitative structure-activity relationship (QSAR) data of a homologous series of pyropheophorbide photosensitizers for photodynamic therapy (PDT). The physical model was a lipid compartment sandwiched between two identical aqueous compartments. The first compartment was assumed to clear irreversibly at a rate K0. The measured octanol-water partition coefficients, P(i) (where i is the number of carbons in the alkyl chain) and the clearance rate K0 determined the clearance kinetics of the drugs. Solving the coupled differential equations of the three-compartment model produced clearance kinetics for each of the sensitizers in each of the compartments. The third compartment was found to contain the target of PDT. This series of compounds is quite lipophilic. Therefore these drugs are found mainly in the second compartment. The drug level in the third compartment represents a small fraction of the tissue level and is thus not accessible to direct measurement by extraction. The second compartment of the model accurately predicted the clearance from the serum of mice of the hexyl ether of pyropheophorbide a, one member of this series of compounds. The diffusion and clearance rate constants were those found by fitting the pharmacokinetics of the third compartment to the QSAR data. This result validated the magnitude and mechanistic significance of the rate constants used to model the QSAR data. The PDT response to dose theory was applied to the kinetic behavior of the target compartment drug concentration. This produced a pharmacokinetic-based function connecting PDT response to dose as a function of time postinjection. This mechanistic dose-response function was fitted to published, single time point QSAR data for the pheophorbides. As a result, the PDT target threshold dose together with the predicted QSAR as a function of time postinjection was found.

Photodynamic chlorophyll a metabolites, including phytoporphyrin (phylloerythrin), in the blood of photosensitive livestock: overview and measurement.

AIM: To validate a spectrofluorometric method for measuring chlorophyll a metabolites, specifically phytoporphyrin (= phylloerythrin), as well as the chlorins, pheophorbide a and pyropheophorbide a, in the blood of photosensitive cattle and sheep. METHODS: Standard methanolic solutions of pheophorbide a (25 microM), pyropheophorbide a (25 microM), and phytoporphyrin (<3.7 microM) were prepared. Serum and plasma samples were obtained from cattle (n=5), sheep (n=3), and one alpaca, with clinical facial eczema (i.e. photosensitive), as well as from clinically normal (n=2 of each) adult cows, recently weaned calves, and sheep (controls). Standard solutions of the three metabolites were characterised using high-performance liquid chromatography (HPLC), with mass spectrometry, in conjunction with absorption and emission spectral data, and were compared with sera from photosensitive animals. In the latter, phytoporphyrin was the only metabolite detected. Calibration curves were prepared by adding different ratios of methanol and standard solutions of phytoporphyrin in methanol to diluted serum from control animals. Peak areas of fluorescence spectra were determined in samples from photosensitive animals. RESULTS: Pheophorbide a and pyropheophorbide a produced typical chlorin spectra, and had excitation/emission maxima of 408/669 nm and 409/669 nm, respectively. Phytoporphyrin showed a typical porphyrin fluorescence spectrum, with excitation/ emission maxima of 425/644 nm. Pyropheophorbide a and phytoporphyrin had very similar chromatographic retention times, the same chemical formula and same mass, but were distinguishable by differences in their absorption spectra. In sera from photosensitive animals, the fluorescence emission at 644 nm was shown to arise solely from phytoporphyrin and not from any other chlorophyll a metabolites. Calibration curves using sera and plasma from control animals gave reliable measurements of phytoporphyrin in the range 0.4-6 microM. The sera of facial eczema-affected cattle and sheep had concentrations of phytoporphyrin ranging from 0.4 to 1.8 and 0.9 to 2.8 microM, respectively. Haemolysed serum samples were not suitable for determination of phytoporphyrin with this method. CONCLUSIONS AND CLINICAL RELEVANCE: A spectrofluorometric method for the quantification of phytoporphyrin in the blood of photosensitive animals has been validated, and can be applied to the measurement of other chlorophyll a metabolites in blood. This will be a useful tool in the further investigation of the cause and pathogenesis of idiopathic photosensitivities of farm animals.

Detection of singlet oxygen in blood serum samples of clinically healthy lambs and lambs suffering from alveld disease.

Alveld is a disease in lambs of domestic sheep (Ovis aries L.), characterized by a combination of photosensitivity and liver damage. Generation of singlet oxygen play a major role in phototoxicity reactions. The compound phylloerythrin (phytoporphyrin) is so far assumed to be the main photodynamic agent in hepatogenous photosensitivity diseases in sheep. Phylloerythrin is a potent photosensitizer and an efficient source of singlet oxygen. The compound accumulates in the peripheral circualtion upon liver damage. Liver dysfunction is also likely to cause an increase in the blood level of bilirubin. Formation of singlet oxygen by bilirubin is reported. In the present work the photosensitizing potential of serum has been measured and related to the bilirubin- and phylloerythrin levels in lambs suffering from alveld and in clinically healthy controls. The singlet oxygen level of the serum was taken as a measure of the photosensitizing potential. The observed singlet oxygen values in serum from alveld lambs were significantly higher than the corresponding values observed in clinically healthy control lambs. This indicates that the serum of the alveld lambs contains an elevated concentration of photosensitizer. The singlet oxygen level was not correlated to the concentration of bilirubin or phylloerythrin. The results indicate that the photosensitizing mechanism is quite complex and may involve other sensitizer(s) than phylloerythrin.

Photosensitized inactivation of Plasmodium falciparum- and Babesia divergens-infected erythrocytes in whole blood by lipophilic pheophorbide derivatives.

BACKGROUND AND OBJECTIVES: Blood transfusions can transmit parasitic infections, such as those caused by Plasmodium (malaria), Trypanosoma cruzi (Chagas' disease), and Babesia (babesiosis). A higher degree of blood transfusion safety would be reached if methods were available for inactivating such parasites. MATERIALS AND METHODS: We evaluated the effectiveness of photosensitization using lipophilic pheophorbide and red light illumination to eradicate red blood cells infected with Plasmodium falciparum, and with Babesia divergens, in whole blood. Fluorescence microscopy and conventional fluorometry showed the specific accumulation of pheophorbide derivatives in the RBC infected with either parasite, compared with uninfected RBC. The effectiveness of different derivatives in eradicating infected RBC was first estimated in parasite cultures. RESULTS: The best photosensitizer was the N-(4-butanol) pheophorbide derivative (Ph4-OH) at 0.2 microM concentration and 5-min illumination. In whole blood, the eradication of RBC infected with B. divergens and P. falciparum was obtained with 2 microM Ph4-OH and 10 and 20 min illumination, respectively. Under these conditions of photosensitization, low levels of RBC hemolysis were noted even after 2 weeks of storage at 4 degrees C and a subsequent 48-hour incubation at 37 degrees C. No reduction of negative charges on treated RBC was noted and no increase in methemoglobin content. CONCLUSIONS: In plasma, Ph4-OH is mainly transported by high-density lipoproteins (HDL). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives in the intracellular parasites. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites and viruses in blood bank units.

Phenytoin metabolism to 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) in man, cat and rat in vitro and in vivo, and susceptibility to phenytoin-induced gingival overgrowth.

Interspecies differences in phenytoin (PHT) metabolism to 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were examined in human, cat and rat hepatic microsomes in vitro. Rat liver microsomes were 25 and 650 times more efficient at the conversion of PHT to HPPH than human and cat liver microsomes, respectively. Sulphaphenazole (83%) and tolbutamide (TOL) (64%) were the most potent inhibitors of HPPH formation in human liver microsomes, while ciprofloxacin (27%), enoxacin (27%) and TOL (26%) produced the greatest inhibition in cat liver microsomes. TOL was tested for its effect on HPPH formation and gingival overgrowth in cats in vivo. Eight cats received PHT sodium (4 mg/kg/d) and another 8 cats received PHT sodium together with TOL (20 mg/kg/d) for 10 wk. Six cats (75%) in the PHT group and 4 cats (50%) in the PHT & TOL group developed significant gingival overgrowth by the end of the study. However, the extent and incidence of the overgrowth were similar in the 2 groups. There were no significant differences in mean AUC 0-10 weeks for plasma PHT (552.90 +/- 29.6 micrograms.d/mL [PHT alone] vs. 582.41 +/- 24.49 micrograms.d/mL [PHT & TOL]) and unconjugated HPPH (1016.4 +/- 295.5 ng.d/mL [PHT alone] vs. 1174.5 +/- 397.2 ng.d/mL [PHT & TOL]) concentrations between the 2 groups of cats. Neither PHT nor HPPH were detectable in the plasma of 8 rats which received PHT (4 mg/kg/d) over a 10-wk period. The rats showed no sign of gingival inflammation (mean gingival index = 0) or gingival overgrowth (mean gingival overgrowth index = 0). Thirty-six adult epileptic patients on chronic PHT therapy were examined; 17 (47%) of the patients demonstrated clinically significant overgrowth. The mean steady-state plasma PHT concentration was comparable to, and the mean plasma unconjugated HPPH concentration 5-fold greater than, that observed in the cats. The results suggest that the rapid metabolism and elimination of PHT and HPPH in the rat may enable it to become more resistant towards developing gingival overgrowth, compared to the cat and man.