Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins

New negatively charged water-soluble bacteriochlorophyll (Bchl) derivatives were developed in our laboratory for vascular-targeted photodynamic therapy (VTP). Here we focused on the synthesis, characterization and interaction of the new candidates with serum proteins and particularly on the effect of serum albumin on the photocytotoxicity of WST11, a representative compound of the new derivatives. Using several approaches, we found that aminolysis of the isocyclic ring with negatively charged residues markedly increases the hydrophilicity of the Bchl sensitizers, decreases their self-association constant and selectively increases their affinity to serum albumin, compared with other serum proteins. The photocytotoxicity of the new candidates in endothelial cell culture largely depends on the concentration of the serum albumin. Importantly, after incubation with physiological concentrations of serum albumin (500-600 microM), WST11 was found to be poorly photocytotoxic (>80% endothelial cell survival in cell cultures). However, in a recent publication (Mazor, O. et al. [2005] Photochem. Photobiol. 81, 342-351) we showed that VTP of M2R melanoma xenografts with a similar WST11 concentration resulted in approximately 100% tumor flattening and >70% cure rate. We therefore propose that the two studies collectively suggest that the antitumor activity of WST11 and probably of other similar candidates does not depend on direct photointoxication of individual endothelial cells but on the vascular tissue response to the VTP insult.     

Prompt assessment of WST11-VTP outcome using luciferase transfected tumors enables second treatment and increase in overall therapeutic rate

Abstract This study hypothesized that success rate assessment of vascular targeted photodynamic therapy (VTP) of solid tumors 24 h post-treatment may allow prompt administration of a second treatment in case of failure, increasing the overall success rate. Here, we show that treatment of luciferase transfected CT26-luc mouse colon carcinoma tumors in BALB/c mice by VTP with WST11 (a Pd-bacteriochlorophyll-based photosensitizer) allows fast assessment of treatment success 24 h post-treatment, using bioluminescence imaging (BLI). WST11-VTP was found to abolish luciferin bioluminescence in the treated tumors resulting in two types of responses. One, comprising 75% of the mice, signified successful outcome, presenting neither BLI signal nor tumor regrowth (24 h-90 days post-VTP). The second (the remaining 25% of the mice) signified treatment failure, presenting various levels of BLI signal with subsequent tumor regrowth (24 h-90 days). Consequently, the mice that failed the first treatment were treated again. We show that treatment success rate in both VTP sessions was identical and that the cumulative success rate of the treatment increased from 75% to over 90%. These results therefore, present a fast method of assessing VTP outcome and support the feasibility of successive multiple treatments with these sensitizers in the clinical arena. The presented methodology can also be helpful in future preclinical studies, and expedite the development of VTP drugs.     

Positron-emitting N-[18F]fluoroalkyl and [18F]fluoropyrrolidinyl analogues of eticlopride as potential in vivo radioligands for dopamine D2 receptors.

N-Fluoroalkyl and 4-fluoropyrrolidinyl eticlopride analogues with high affinity toward central nervous system dopamine D2 receptors in vitro were labelled with positron emitting fluorine-18 (t1/2 = 110 min), and their in vivo biodistribution was investigated in rats. N-[18F]Fluoro-ethyl and -propyl eticlopride derivatives showed poor in vivo selectivity in the rat brain. On the other hand, 4-[18F]fluoropyrrolidinyl eticlopride exhibited almost constant and relatively high striatal concentration. The striatal/cerebellar radioactivity ratio, which corresponds to the ratio of a brain D2 receptor-rich to poor region, gradually increased to 5.2-6.4, 90 min after the injection. The striatal accumulation was selectively inhibited by pre-injection of haloperidol, a dopamine D2 antagonist, without affecting accumulation in other tissues. Thus, the selective striatal accumulation of 4-[18F]fluoropyrrolidinyl eticlopride in striatal tissue appears to be due to the specific binding to dopamine D2 receptors.     

Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer, in normal and tumor-bearing mice

The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (mastocytoma) or M1 (rhabdomyosarcoma) tumor-bearing DBA/2J mice. A dose of 80 micrograms of 3H-BPD-MA was determined at 3, 24, 48, 72, 96 and 168 h post injection. The following tissues were tested: blood, brain, heart, intestine, kidney, lung, liver, muscle, skin, stomach, spleen, thymus and tumor. The biodistribution of 3H-BPD-MA in normal and tumor-bearing mice was comparable overall. 3H-BPD-MA localized in tumors better than in other tissues except kidney, liver and spleen. The tumor to tissue ratios were in the range 1.5-3 at 24 h post injection and increased further during the next 72 h. The highest levels of 3H-BPD-MA were observed in all tissues at 3 h post injection and decreased rapidly during the first 24 h. After 24 h the clearance from tissues was rather slow. The preliminary clearance data obtained in a group of five normal mice indicated that the majority of the injected dose (60%) cleared from the body via the bile and feces, while only about 4% cleared via kidneys and urine. Studies in which 3H-BPD-MA was extracted from tumor, kidney and liver 3 and 24 h after injection showed that, at 3 h, all the photosensitizing activity in tumor was retained. At 24 h only 39% of the activity was retained and considerably less active material was present in liver and kidney.     

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.     

Serine Conjugates of Chlorophyll and Bacteriochlorophyll: Photocytotoxicity in vitro and Tissue Distribution in Mice Bearing Melanoma Tumors

Chlorophyll (Chl) and bacteriochlorophyll (Bchl) have been made water soluble by transesterfication with serine (Ser) at the propionyl residue and tested as potential reagents for photodynamic therapy (PDT). Photocytotoxicity of the conjugates Chl-Ser and Bchl-Ser in M2R mouse melanoma was tested in cell cultures. Tissue uptake and clearance of the photosensitizers in CD1 nude and C57B1 mice implanted with M2R tumors are described. Photocytotoxicity in cell cultures was determined microscopically and by [³H]thymidine incorporation. The LD₅₀ values in vitro were 0.05-0.1 μM for both sensitizers while that of the commercially available hematoporphyrin derivative (HPD, Photosan) was over 100 times higher for the same light intensity (45 mW/cm²). Pigment concentrations were determined fluorometrically in acetone extracts of the tissues of interest at different times after intraperitoneal injection of 20 mg pigment/kg body weight. The distribution pattern of Chl-Ser in the different tissues resembled that reported for Photofrin, chlorin and bacteriochlorin derivatives. Clearance from normal tissues was essentially completed within 16 h for Bchl-Ser and 72 h for Chl-Ser with mean half-lives (t_1/2) of about 2 and 7 h, respectively. In contrast, the clearance rates of these pigments and their metabolites from melanoma tumor tissue were significantly longer: t_1/2= 20 h for Chl-Ser and 15 h for Bchl-Ser and metabolites. The clearance rates showed biphasic or single exponential decay patterns in normal tissues and in tumors, respectively. Cumulatively the high phototoxicity, simple mode of delivery and fast tissue clearance rates reported here suggest that polar conjugates of Chl and Bchl promise to be highly effective PDT reagents.     

Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.     

Applications of high throughput microsomal stability assay in drug discovery

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.     

Determination of isoliquiritigenin and its distribution in mice by synchronous fluorescence spectrometry

The aim of the present work was to develop a new method using synchronous fluorescence spectrometry (SFS) to determine the concentration of isoliquiritigenin (ISL) in mouse blood and tissues, and to investigate ISL's distribution among organs after an intraperitoneal (IP) dose of ISL. The synchronous fluorescence method was optimized with the sample pH, stability, metal ions, concentration of Al(3+), and surfactants. The proposed method was used to determine the ISL concentration in mouse blood, brain, heart, kidney, liver, spleen and lung after an IP injection of ISL. The optimal conditions for the determination of ISL using SFS were found to be: excitation and emission wavelengths of 469 and 557 nm, respectively; the use of 3% AlCl(3) as a fluorescence intensity enhancer; measuring samples within 1 h of collection, sample pH 7-8, isolation of samples from surfactants; and wavelength interval (Δλ) = 70 nm. After IP injection, the distribution of ISL in mouse organs was: liver > kidney > spleen > blood > lung > brain > heart. The blood concentration of ISL peaked at 60 min; concentrations of ISL in liver, kidney and spleen achieved maxima at 120 min. SFS provides a simple, but effective analytical method that will benefit the study of in vivo biological effects of ISL, including absorption, distribution, metabolism, and excretion.     

Automated-extraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine, I) is a highly A2-selective (A2/A1 = 140), high-affinity adenosine agonist. A method has been devised to extract the compound from biological matrices with automated solid-phase extraction using C18 bonded silica columns. This is followed by reversed-phase, paired-ion chromatography on a Supelco LC-18-S column with fluorescence detection. The limit of quantitation is 5 ng/ml, but 1 ng/ml (five times the signal-to-noise ratio) can readily be detected. Tritium-labeled compound was used to study the pharmacokinetics in rats. After an intravenous dose of 0.3 mg/kg, biphasic elimination kinetics were observed for parent I, characterized by half-lives of 1.8 min (distribution) and 15 min (elimination). The volume of distribution in the terminal phase (V beta) was low (0.27 l/kg) and plasma clearance was moderate (0.83 l/kg/h). Although the compound was rapidly absorbed (mean Tmax = 13 min), low concentrations (mean Cmax = 94 ng/ml) were observed after an oral dose of 3.0 mg/kg, and bioavailability was only approximately 1.4%. Radioactivity persisted in plasma longer than parent compound after either dose, but levels were too low for isolation and structure identification of drug-derived compounds.     

人参皂苷Rb2在大鼠体内的药代动力学行为及代谢产物研究

建立快速高分离度液相色谱-四极杆飞行时间质谱联用方法(RRLC-Q-TOF-MS),分析人参皂苷Rb2在大鼠体内的药代动力学行为,并探索人参皂苷Rb2在大鼠体内的代谢过程.采用Agilent SB-C18色谱柱,流动相A为0.1％甲酸溶液,B为乙腈,流速为0.2 mL/min,进样量为5μL,二元线性梯度洗脱分离,采用电喷雾负离子模式进行质谱检测.方法的检出限(S/N=3)和定量限(S/N=10)分别为0.08 μg/mL和0.1 μg/mL,线性范围为0.10～ 1.26 μg/mL.结果表明,人参皂苷Rb2静脉注射后的体内代谢过程符合二室模型特征,血药浓度半衰期的α相(t1/2α)和β相(t1/2β)分别为(23.58±1.10)和(1306.55±147.23) min.通过对静脉注射人参皂苷Rb2的大鼠尿液和口服后的粪便样本进行分析,发现Rb2的代谢产物为M6,M2(C-Y),F2,C-K.     

Pharmacokinetics and tissue distribution of pefloxacin mesylate in chickens

A specific, sensitive and stable high‐performance liquid chromatography (HPLC)‐based analytical method was established to determine the level of pefloxacin mesylate (PM) in the plasma and various tissues of chickens. Chickens were randomly assigned to 12 equal experiment groups, including 11 treatment groups and one control group. The chickens in the treatment groups received oral administration of PM and were sacrificed at different pre‐determined time points, with their blood and various organs harvested, extracted and analyzed by HPLC to quantify the level of the residual antibiotic. Method validation studies indicated that the HPLC measurement showed excellent precision, reproducibility, stability and robustness. The obtained pharmacokinetic parameters suggested that PM reached peak levels in various tissues within 1–2 h after its oral administration, and was mainly concentrated in liver and kidney. The antibiotic was also found to be cleared from chicken crureus, brain, testes, ovaries and pancreas at higher rates compared with other organs. Overall, the rapid accumulation of PM could at least be partially attributed to its relatively slow organ clearance. These results could serve as a useful guidance for the rational use of PM and other quinolone‐derived antimicrobials in the treatment of infectious diseases in chickens and other animals.     

Chlorin e6 Derivative Radachlorin Mainly Accumulates in Mitochondria,Lysosome and Endoplasmic Reticulum and Shows High Affinity toward Tumors in Nude Mice in Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) depends upon the amount of photosensitizer accumulated in the malignant tissues. Radachlorin is a popular photosensitizer used in photodynamic therapy to treat various types of cancer. In this study, we have studied the main organelles responsible for the accumulation of radachlorin in human anaplastic thyroid cancer in vitro and in vivo. The optimal time window for uptake and clearance of radachlorin also was studied. Confocal microscopic images confirmed that the radachlorin is mainly acquired by mitochondria and partially by lysosome and endoplasmic reticulum. Studies also showed that the maximum amount of radachlorin was accumulated within 3–6 h after the treatment. Radachlorin also showed a higher affinity toward malignant tumors compared to the other organs in mice xenograft model. Uptake of radachlorin reached an optimum amount within 6 h and most of the radachlorins were also cleared from the body in next 48 h. Therefore, detailed information regarding exact accumulation sites and a time window in which maximum amount of drug is accumulated and cleared were obtained by this study. Hence, not only the efficacy of the treatment can be increased but the phototoxicity after the treatment also can be controlled.     

Improved pharmacokinetics, biodistribution and necrosis in vivo using a new near infra-red photosensitizer: tetrahydroporphyrin tetratosylat

The search for better photosensitizers for photodynamic therapy of malignancies has led to the investigation of a new water-soluble, positively charged, and chemical stable tetrahydroporphyrin tetratosylat (THPTS) with a strong absorption at 760.5 nm, belonging to the bacteriochlorophyll family. THPTS undergoes a rapid uptake by human choroidal melanoma (CM) cells with a weak dark toxicity after a 24-h incubation (LD10 = 150 microM, LD50 = 6.0 mM). In response to laser light at 760+/-3 nm and doses of 10, 15 and 30 J/cm2, around 71%, 76%, and 92% of the CM cells were killed, respectively. Studies of pharmacokinetics and biodistribution in vivo (living mice) and ex vivo (excised organs) were made in a Balb/c mice bearing subcutaneously inoculated C26 colon carcinoma using fiber-optic spectrofluorimetry (FOS). Tumours were irradiated 3 h after intraperitoneal (i.p.) injection of 5.0 mg/kg THPTS with an incoherent light source at 750+/-20 nm and an intensity of 100 mW/cm2 and fluences of 60, 90 and 120 J/cm2. THPTS demonstrated preferential accumulation in C26 colon carcinoma in comparison with most normal tissues except kidneys. For the tissues of liver, colon, muscle, and spleen the tumour/normal tissue ratio (TNTR) ranged from 8.0 to 50. After irradiation with 120 J/cm2 the depth of tumour necrosis reached 15 mm. Histological examination of the tumour samples 24 h after THPTS-PDT, revealed severe stasis in the blood vessels and coagulative necrosis. These results suggest that THPTS-PDT may be of particular importance in the treatment of accessible malignancies.     

Analysis of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane in rat plasma. II. Pharmacokinetic profile in male and female Sprague-Dawley rats evaluated by capillary electrophoresis

This paper describes a pharmacokinetic study performed in Sprague-Dawley rats after i.v. administration of a single 6-mg/kg dose of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane (Altropane). Plasma samples were collected from the retro-orbital sinus at times up to 3 h after drug administration, extracted by solid-phase extraction, and the drug levels determined by capillary electrophoresis (CE). Pharmacokinetic parameters were determined by a standard noncompartmental model using WinNonlin version 1.5. The maximum plasma concentrations, clearances of the drug, and areas under the curve for male and female rats were 5.74 and 7.26 microg/ml, 135.7 and 98.5 ml/kg x min, and 44.23 and 60.92 microg x min/ml, respectively. The drug was cleared very rapidly from the systemic circulation, with a terminal t(1/2) of 7 to 10 min and a mean residence time of about 11 min for both sexes. The volume of distribution was approximately 1 l/kg. No metabolites were detected when the samples were analyzed individually. However, after samples were pooled and concentrated, traces of two unknown peaks that may represent metabolites were detected in concentrates from the last two timepoints. Part I of this work [J. Chromatogr. A, 895 (2000) 87] describes validation of CE methods for the analysis of aqueous and plasma samples of Altropane, including its solid-phase extraction from rat plasma.     

Combined OX40 Agonist and PD-1 Inhibitor Immunotherapy Improves the Efficacy of Vascular Targeted Photodynamic Therapy in a Urothelial Tumor Model

Purpose: Vascular targeted photodynamic therapy (VTP) is a nonsurgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for upper tract urothelial cancer (UTUC) based on preclinical data. Toward increasing response rates to VTP, we evaluated its efficacy in combination with concurrent PD-1 inhibitor/OX40 agonist immunotherapy in a urothelial tumor-bearing model. Experimental design: In mice allografted with MB-49 UTUC cells, we compared the effects of combined VTP with PD-1 inhibitor/OX40 agonist with those of the component treatments on tumor growth, survival, lung metastasis, and antitumor immune responses. Results: The combination of VTP with both PD-1 inhibitor and OX40 agonist inhibited tumor growth and prolonged survival to a greater degree than VTP with either immunotherapeutic individually. These effects result from increased tumor infiltration and intratumoral proliferation of cytotoxic and helper T cells, depletion of Treg cells, and suppression of myeloid-derived suppressor cells. Conclusions: Our findings suggest that VTP synergizes with PD-1 blockade and OX40 agonist to promote strong antitumor immune responses, yielding therapeutic efficacy in an animal model of urothelial cancer.     

Characterization of quantum dots using capillary zone electrophoresis

Commercially available quantum dots (QDs) were characterized using CE. The CE instruments were laboratory-built, each being capable of both electrokinetic and hydrodynamic injection. Modes of detection include UV absorption and LIF. The CE-LIF system was further modified to handle microliter sample volumes during injection. Sodium phosphate (5-25 mM, pH 7.5-11) was found to be a good buffer electrolyte. Sodium mercaptoproprionate CdTe/CdS (ADS620) QDs and carboxylic acid CdSe/ZnS (T2-Evitag) QDs yielded high separation efficiencies of N = 1.5x10(6) plates at t(M) = 10 min and N = 1.0x10(5) plates at t(M) = 3.8 min, respectively. Apparently the EDC/sulfo-NHS bioconjugation chemistry worked well with the neutral T2-Evitag QDs, but not so well with the negatively charged ADS620 QDs. This preliminary knowledge will serve as a basis for new CE immunoassay studies of QD-biomolecule conjugates and their immunocomplexes with target analytes.     

Synthesis of C-labeled imipramine and its biodistribution in mice: a potential tracer for positron emission tomography

A tricyclic antidepressant, C-labeled imipramine was synthesized by N-methylation of desipramine with 11CH3I to assist in the imaging of the human imipramine receptor by positron emission tomography. The radiochemical yield after purification of 11C-imipramine by high performance liquid chromatography was 28-63% at a specific activity of 26-53 Ci/mmol. The time required for synthesis, including purification was 30 min from the end of 11CH3I trapping. The organ distribution of 11C-imipramine was investigated in mice at various times after i.v. injection. The main accumulation of radioactivity was in the kidney, followed by the lung and the heart. In the brain, the radioactivity levels in the hypothalamus and striatum were the highest and remained constant, differentiating them from other portions of the brain. Furthermore, the result of a binding assay with 3H-labeled imipramine suggested that the regional distribution of 11C-imipramine in the same mouse brain correlated to that of the high affinity imipramine binding site.     

Design, synthesis and biological evaluation of a novel series of potent, orally active adenosine A1 receptor antagonists with high blood-brain barrier permeability

A novel series of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines (5-38) were synthesized and evaluated for their in vitro adenosine A1 and A(2A) receptor binding activities, and in vitro metabolism by rat liver in order to search for orally active compounds. Most of the test compounds were potent adenosine A1 receptor antagonists with high A1 selectivity and the A1 affinity and A1 selectivity of carbonyl derivatives (5-11) was particularly high. In particular, compound 7 was an extremely potent and selective adenosine A1 antagonist with high A1 selectivity (Ki=0.026 nM, A(2A)/A1=5400). In terms of metabolic stability, 2-oxopropyl (5), 2-hydroxypropyl (12), N-methylacetamide (16), 2-(piperidin-1-yl)ethyl (28) and 1-methylpiperidin-4-yl (32, FR194921) were the most stable compounds in this series of analogues. Further in vivo evaluation indicated that compounds 5, 13, 17, 28 and 32 were detected in both plasma and brain after oral administration in rats. In particular, 32 displayed good plasma and brain concentrations (dose: 32 mg/kg (n=3); after 30 min, plasma conc.=3390+/-651nM, brain conc.=3670+/-496nM; after 60min, plasma conc.=1580+/-348nM, brain conc.=2143+/-434nM), and a good brain/plasma ratio (1.11+/-0.060 (30min), 1.39+/-0.172 (60min)). As a result, we could show that 32 is a good candidate for an orally active adenosine A1 receptor antagonist with high blood-brain barrier permeability and good bioavailability (Ki=6.6nM, A(2A)/A1=820, BA=60.6+/-4.9% (32 mg/kg)).     

AN ULTRASTRUCTURAL COMPARATIVE EVALUATION OF TUMORS PHOTOSENSITIZED BY PORPHYRINS ADMINISTERED IN AQUEOUS SOLUTION, BOUND TO LIPOSOMES OR TO LIPOPROTEINS

Abstract— Balb/c mice bearing a transplantedMS–2 fibrosarcoma have been injected with 2 mg kg^-1 hematoporphyrin either dissolved in phosphate-buffered saline (Hp-aq), or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (Hp-lip), or precomplexed with low density lipoproteins (Hp-LDL). At 24 h after injection, the mice received 150 J cm^-2 of600–680 nm light irradiation. Electron microscopic studies performed on tumor tissues taken from mice sacrificed at different times after the phototreatment showed that, in the presence of Hp-aq. tumor necrosis is largely the consequence of vascular damage. On the other hand, in the presence of Hp-lip and Hp-LDL, the response of the tumor to the phototreatment occurs at a faster rate and is mainly determined by direct damage of neoplastic cells. These findings are correlated with the different distribution of the various hematoporphyrin forms (Hp-aq, Hp-lip, Hp-LDL) among the serum proteins and the modalities of hematoporphyrin delivery to tissues by the possible carriers.