Investigation of human serum albumin (HSA) binding specificity of certain photosensitizers related to pyropheophorbide-a and bacteriopurpurinimide by circular dichroism spectroscopy and its correlation with in vivo photosensitizing efficacy

A series of pyropheophorbide-a and bacteriopurpurinimides were investigated to understand the correlation between HSA (site II) binding affinity and in vivo photosensitizing activity. In our study, photosensitizers that bound to site II of HSA produced a significant difference in the circular dichroism spectra of the corresponding complexes, especially at Soret band region of the photosensitizers. Our results suggest that CD spectroscopy of the photosensitizer-HSA complexes could be a valuable tool in screening new photosensitizers before evaluating them for in vivo efficacy.     

Photosensitizers related to purpurin-18-N-alkylimides: a comparative in vivo tumoricidal ability of ester versus amide functionalities

For a comparative study, 3-(alkyloxyethyl)-3-devinylpurpurin-18-N-hexylimides with ester and amide functionalities were investigated for tumor selectivity and in vivo photosensitizing efficacy. Compared to amide analogues, the related photosensitizers with ester functionalities were found to be more effective. Among these compounds the 3-devinyl-(3-hexyloxyethyl)-purpurin-18-N-hexylimide as methyl ester 12 showed excellent tumor uptake (tumor versus muscle ratio: 8:1), and produced 100% tumor cure on day 30 at a dose of 1.0 micromol/kg. The mice were treated with light (135 J/cm2, 705 nm) at 24 h post injection of the drug.     

Photodynamic therapy and some clinical applications in oncology

Photodynamic therapy (PDT), a new treatment modality for localized cancers involving the selective interaction of visible light with photosensitizers, such as hematoporphyrin derivatives (HpD) or dihematoporphyrin ether/ester (DHE) (Photofrin II). Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (i) absorption of photosensitizing agent; (ii) selective retention of photosensitizer in tumors and (iii) irradiation of sensitized tumor by laser irradiation. This paper provides a review of photosensitizers, photochemistry, subcellular targets, side effects and lasers involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vivo and in vitro mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.     

Synthesis and receptor binding studies of halogenated N,N-dialkylel-(2-phenyl-1H-indol-3-yl)glyoxylamides to visualize peripheral benzodiazepine receptors with SPECT or PET

A library of halogenated 2-arylindolyl-3-oxocarboxamides was prepared to develop radioligands to visualize cerebral PBR by SPECT and PET imaging. In vitro evaluation showed that most of the synthesized compounds were selective,high-affinity PBR ligands with adequate lipophilicity (log D7.4 in the range of 1.6-2.4). The iodinated derivative 11 (Ki = 2.6 nM) and the fluorinated analog 26 (Ki = 6.2 nM) displayed higher affinity than reference compounds.     

Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.     

Identification, optimisation and in vivo evaluation of oxadiazole DGAT-1 inhibitors for the treatment of obesity and diabetes

A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening (HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid containing clinical candidate 53 (AZD3988), which demonstrated excellent DGAT-1 potency (0.6 nM), good pharmacokinetics and pre-clinical in vivo efficacy that could be rationalised through a PK/PD relationship.     

Determination of the antibacterial efficacy of a new porphyrin-based photosensitizer against MRSA ex vivo.

Following extensive in vitro screening of new photosensitizers the purpose of the present study was to examine penetration as well as antibacterial efficacy of a lead photosensitizer against MRSA using an ex vivo porcine skin model. Two different applications were performed: (i) preincubation of bacteria in solution with a porphyrin-based photosensitizer XF73 and subsequent application on the ex vivo porcine skin; (ii) application of pure bacteria on the explants followed by an incubation with XF73 in a water-ethanol formulation for up to 60 min under occlusion. The localisation of XF73 was restricted to the stratum corneum. Different concentrations (0-10 microM) of XF73 and different incubation times (5-60 min) were used to determine phototoxicity against methicillin-resistant and methicillin-sensitive S. aureus, which was applied on the explants. Preincubation of S. aureus with 0.1 microM XF73 in solution prior to the application of these XF73-incubated bacteria on the skin demonstrates a higher efficacy (>3 log10) after irradiation. Antibacterial photodynamic inactivation resulted in a approximately 1 log10 (0.1 microM)-3.64+/-0.035 (10 microM) log10 growth reduction independently of the antibiotic resistance pattern of used S. aureus strains. Irradiation of applied bacteria without photosensitizer incubation did not show any marked decrease (<1 log10) of bacteria cell number, indicating a significant phototoxicity of the XF73. Histological evaluations of untreated and treated skin areas upon irradiation within 24 h showed no significant degree of necrosis or apoptosis determined by TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates that this XF73 porphyrin-based photosensitizer had concentration-dependent differences in killing efficacy of MRSA in comparison to skin cells using an ex vivo porcine skin model. The results described here imply that topical delivery of XF73 may be considered as a possible treatment in patients with superficial infections of the skin.     

Discovery of 3-OH-3-methylpipecolic hydroxamates: potent orally active inhibitors of aggrecanase and MMP-13

A series of 3-hydroxy-3-methylpipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was designed based on the observation of increased aggrecanase activity with substitution at the 3-position of the piperidine ring. Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group that binds in the S1' pocket. These compounds also possess markedly improved bioavailability and lower metabolic clearance compared to analogous 3,3-dimethyl-5-hydroxypipecolic hydroxamates. These improvements are attributed to lowered lipophilicity proximal to the metabolically labile hydroxamic acid. Synthesis, structure activity relationships, and in vivo efficacy data are described.     

Photosensitizing action of isomeric zinc N-methylpyridylporphyrins in human carcinoma cells

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N-alkylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺ and ZnTE-2-PyP⁴⁺) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP⁴⁺ (ZnTM-2(3,4)-PyP⁴⁺) and respective ligands on a human colon adenocarcinoma cell line. At 10 μM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 μM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP⁴⁺ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD).     

Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.     

Investigation of substituted 6-aminohexanoates as skin penetration enhancers

Skin penetration enhancers are compounds used to facilitate the transdermal delivery of drugs that are otherwise not sufficiently permeable. Through a synthetic route implementing two series of esters, we generated transdermal penetration enhancers by a multi-step reaction with substituted 6-aminohexanoic acid. We present the synthesis of all newly prepared compounds here with structural confirmation accomplished by (1)H NMR, (13)C NMR, IR and mass spectroscopy (MS). The lipophilicity (logk) of all compounds was determined via RP-HPLC and their hydrophobicity (logP/ClogP) was also calculated using two commercially available programs. Ab initio calculations of geometry and molecular dynamic simulations were employed to investigate the 3-dimensional structures of selected compounds. The transdermal penetration-enhancing activity of all the synthesized esters were examined in vitro and demonstrated higher enhancement ratios than oleic acid. Compounds 2e (C(10) ester chain) and 2f (C(11) ester chain) exhibited the highest enhancement ratios. It can be concluded that the series non-substituted at the C((2)) position by a ω-lactam ring showed significantly higher activity than those with azepan-2-one. None of the prepared compounds penetrated through the skin. All of the investigated agents demonstrated minimal anti-proliferative activity using the SK-N-MC neuroepithelioma cell line (IC(50)>6.25μM), suggesting these analogs would have a low cytotoxic profile when administered in vivo as chemical penetration enhancers. The correlation between the chemical structure of the studied compounds and their lipophilicity is discussed in regards to transdermal penetration-enhancing activity.     

Synthesis and in vivo influenza virus-inhibitory effect of ester prodrug of 4-guanidino-7-O-methyl-Neu5Ac2en

A series of ester prodrugs of 7-O-methyl derivative of Zanamivir (compound 3) was synthesized and their efficacy was evaluated in an influenza infected mice model by intranasal administration. Compound 7c (CS-8958), octanoyl ester prodrug of the C-9 alcohol of compound 3, was found to be much longer-acting than Zanamivir. Furthermore, the in vivo efficacies of compounds 12a, 12b, and 12c, the linear alkyl ester prodrug of the carboxylic acid, were comparable to that exerted by compound 7c.     

Peripheral benzodiazepine receptor antisense knockout increases tumorigenicity of MA-10 Leydig cells in vivo and in vitro

Peripheral benzodiazepine receptors (PBR), first described more than 20 years ago, have been attributed with many putative functions including ones in cellular proliferation and cellular respiration. Hence, it is quite conceivable that deregulation of this receptor could lead to pathology. We and others have reported the existence of PBR overexpression in different human and nonhuman malignancies, but it has never been made clear whether this aberrant malignant PBR expression is a cause or consequence of the cancer. In the current study we induced PBR underexpression by downregulating one critical subunit of the PBR complex, the isoquinoline-binding protein (IBP), using the stable antisense knockout approach, in the MA-10 Leydig cell line. Resultant clones, showing PBR deregulation, also demonstrated increased tumorigenicity, using both in vitro (loss of contact inhibition and growth in soft agar) and in vivo (increased mortality on grafting back into isogenic mice) assays. We suggest that this type of deregulation could be a later event in natural tumor progression. Consequently, PBR deregulation should be more closely studied in human malignancy.     

Discovery and in vivo evaluation of alcohol-containing benzothiazoles as potent dual-targeting bacterial DNA supercoiling inhibitors

A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C⁵, as well as incorporating solubilizing groups at the C⁷ position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo.     

Process engineering and optimization of glycerol separation in a packed-bed reactor for enzymatic biodiesel production

A process model for efficient glycerol separation during methanolysis in an enzymatic packed-bed reactor (PBR) was developed. A theoretical glycerol removal efficiency from the reaction mixture containing over 30% methyl esters was achieved at a high flow rate of 540 ml/h. To facilitate a stable operation of the PBR system, a batch reaction prior to continuous methanolysis was conducted using oils with different acid values and immobilized lipases pretreated with methyl esters. The reaction system successfully attained the methyl ester content of over 30% along with reduced viscosity and water content. Furthermore, to obtain a high methyl ester content above 96% continuously, long-term lipase stability was confirmed by operating a bench-scale PBR system for 550 h, in which the intermediates containing methyl esters and residual glycerides were fed into the enzyme-packed columns connected in series. Therefore, the developed process model is considered useful for industrial biodiesel production.     

Novel lipophilic 7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid derivatives as potential antitumor agents: improved synthesis and in vitro evaluation

A convenient route for the synthesis of some acyloxymethyl esters and carboxamides of levofloxacin (LV) with modulated lipophilicity is described. The synthesized compounds were evaluated in vitro for their growth inhibitory effect in five human cancer cell lines. The most efficient LV derivatives (ester 2e and amide 4d) displayed IC(50) values in the 0.2-2.2 μM range, while IC(50) values for parent LV ranged between 70 and 622 μM depending on the cell line. The esters displayed no in vivo toxicity up to 80 mg/kg when administered intraperitoneally. This study thus shows that LV analogs displayed antitumor efficacy, at least in vitro, a feature that appeared to be independent from the lipophilicity of the grafted substituent.     

Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.     

New anti-malarial peroxides with in vivo potency derived from spongean metabolites

The structure-activity relationship of the anti-malarial substance 3 having a 6-carbomethoxymethyl-3-methoxy-1,2-dioxane structure was studied. The ester portion of the peroxide 3, showing little in vivo efficacy in malaria-infected mice in spite of the potent in vitro activity, was hydrolyzed in serum to afford an inactive free acid 4. The amide analogues (8 and 9) robust to mouse serum were disclosed to exhibit in vivo anti-malarial potency.     

In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.     

The use of N-methylprotoporphyrin dimethyl ester to inhibit ferrochelatase in Rhodopseudomonas sphaeroides and its effect in promoting biosynthesis of magnesium tetrapyrroles.

N-Methylprotoporphyrin dimethyl ester inhibits ferrochelatase in isolated membranes of Rhodopseudomonas sphaeroides at low concentrations (around 10 nm). Full inhibition developed after a short lag phase. The inhibition was non-competitive with porphyrin substrate. Addition of inhibitor to growing cultures of Rps. sphaeroides caused a decrease (near 40%) in cytochrome content and a severe inhibition of ferrochelatase; the excretion of haem into the medium by cell suspensions was also severely inhibited. The addition of N-methylprotoporphyrin dimethyl ester to suspensions of photosynthetically competent Rps. sphaeroides Ga caused excretion of Mg-protoporphyrin monomethyl ester. When added to mutants V3 and O1, magnesium divinylphaeoporphyrin a5 monomethyl ester and 2-devinyl-2-hydroxyethylphaeophorbide a were excreted, with maximum effect at around 3 microM-inhibitor in the medium. The results are interpreted to suggest that the inhibitor decreases concentration of intracellular haem, which normally controls the activity of 5-aminolaevulinate synthetase. Unregulated activity of this enzyme leads to overproduction of protoporphyrin, which is diverted to the bacteriochlorophyll pathway. Further control operates at magnesium protoporphyrin ester conversion in normal cells.