Anthony H. Cincotta

NOVEL RED ABSORBING BENZO[a]PHENOXAZINIUM and BENZO[a]PHENOTHIAZINIUM PHOTOSENSITIZERS: in vitro EVALUATION

Abstract Taking advantage of the‘heavy atom’effect, we have recently prepared a series of nine novel halogenated and sulfur‐substituted benzophenoxazines which have enhanced intersystem crossing to the triplet state as measured by singlet oxygen photobleaching of 1,3‐diphenylisobenzofuran. The dyes were evaluated for their dark and light‐induced toxicities towards several carcinomata and normal primary cell lines. One of these days, 5‐amino‐6‐iodo‐9‐diethylaminobenzol[a]phenoxazinium chloride, was found to be a potent photosensitizer for a murine sarcoma 180 and four human carcinomata cell lines (larynx Hep2, cervical HeLa, rectal tumor cell HRT, and transitional‐cell bladder BTC). Several dyes caused marked light dependent killing of two tumor cell lines (Hep2 and HRT) but were minimally toxic to a normal bovine fetal kidney (BFK) line. Sulfur substitutuion into the benzophenoxazine nucleus results, under the conditions studied, in the largest enhancement of phototoxicity both to normal and cancer cells. Comparisons between appropriate dyes show a correlation between the efficiency of singlet oxygen generation and phototoxicity. These results suggest that the photosensitizing efficacy of certain cationic benzophenoxazinium chromophores, such as Nile Blue A, can be greatly increased by appropriate structural modification. The demonstrated selectivity for carcinoma cells by some of these new photosensitizers may be useful therapeutically.

Bromocriptine/SKF38393 treatment ameliorates obesity and associated metabolic dysfunctions in obese (ob/ob) mice

It has been postulated that dopaminergic activities comprise a major functional component of a central regulatory system for metabolism which can be manipulated by dopamine modulating drugs. The present study is aimed at delineating the role and importance of pharmacological dopaminergic activation in the regulation of metabolism during obesity and diabetes. We treated C57BL/6J ob/ob mice for 2 weeks with bromocriptine (dopamine D2 agonist), SKF38393 (dopamine D1 agonist), both drugs combined or vehicle and monitored the effects of such treatment on body composition, food consumption, and serum metabolites. Bromocriptine and SKF38393 individually produced moderate improvements in obesity, hyperglycemia, and hyperinsulinemia. However, a combination of bromocriptine plus SKF38393 resulted in major reductions in body weight (7.5 g), body fat (40%), food consumption (42%), and serum concentrations of glucose (59%), triglyceride (37%), free fatty acid (45%) and insulin (49%) while increasing protein mass (8%). These results indicate that regulatory components of metabolism in the ob/ob mouse are modulated by and/or are comprised of dopaminergic activities. Importantly, dopaminergic D1/D2 receptor coactivation maximizes this dopaminergic response (i.e., improvement of metabolic abnormalities) in these mice.

Reductions of body fat stores and total plasma cholesterol and triglyceride concentrations in several species by bromocriptine treatment

Administrations (injections and in feed) of bromocriptine, a dopamine agonist that inhibits prolactin secretion, reduced body fat stores and plasma total cholesterol and triglyceride concentrations in several rodent species (Syrian hamsters, Djungarian hamsters, Swiss Webster mice and obese Zucker rats). Body fat indices were reduced by at least 50% in the hamsters and mice within 10-15 days of treatment and by 29% in 8 weeks in the rats. Bromocriptine reduced total plasma cholesterol concentration by 17% in Syrian hamsters, 41% in mice and 30% in rats fasted before blood sampling. In nonfasted obese rats, bromocriptine dramatically reduced both cholesterol (from 440 to 130 mg/dl) and triglyceride (from 1570 to 540 mg/dl) levels compared with controls. These findings offer further evidence for a primary role of prolactin in lipid metabolism and indicate that bromocriptine may be useful for treating obesity and lipid-based cardiovascular disorders.

Novel Phenothiazinium Photosensitizers For Photodynamic Therapy

The replament of the oxygen atom in benzophenoxazines with sulfur leads to the corresponding phenothiazinium ehromophorus The effect that this change, as well as other structural alterations, has on photosensitized singlet oxygen formation partitioning coefficient, and in-vitro phototoxicity is presented. A brief discussion concerning how structural modifications can bring about changes in these parameters is also presented. Preliminary results suggest that some phenothiazines could be promising photosensitizers for photodynamic therapy.

Hepatic Vagotomy Abolishes the Circadian Rhythm of Lipogenic Responsiveness to Insulin and Reduces Fat Stores in Hamsters

The effects of hepatic vagotomy upon the circadian rhythm of lipogenesis and body fat store levels were tested in seasonally obese Syrian hamsters. Lipogenesis was studied 8 weeks after surgery by measuring the incorporation of label into epididymal and retroperitoneal fat pad lipid in animals killed 30 min after intraperitoneal [3H]-glucose injection and 2 h after bovine insulin injections. A marked circadian variation in insulin-stimulated lipogenesis was present in sham-operated controls. The peak of lipogenic activity occurred at light onset and was 3-fold greater than during the middle of the photophase. The peak in the circadian variation of plasma insulin coincided with the peak of lipogenic responsiveness to insulin in these animals. Hepatic vagotomy completely abolished the circadian variation in insulin-stimulated lipogenesis and reduced the magnitude of the daily lipogenic peak at light onset by 65%, but only slightly altered the phase of the circadian variation in plasma insulin by 4 h. Hepatic vagotomy did not influence plasma glucose levels, which did not vary as a function of time of day in either group. Body fat stores were severely reduced (40%) by hepatic vagotomy when examined 8 weeks after surgery. The present study demonstrates important roles for interactions of the circadian rhythms of insulin and tissue lipogenic responses to insulin in the regulation of body fat stores. Furthermore, this study demonstrates that hepatic vagal activity regulates the daily interval of lipogenic responsiveness to insulin and thereby directly influences body fat stores in the Syrian hamster.

Structure And Properties Of Novel Benzo[a]phenoxazinium Photochemotherapeutic Agents

The approach used for the development of some novel benzophenoxazinium photosensitizers is presented. Considerations that have influenced the design of these dyes include structure/function relationships pertaining to such properties as selective tumor staining, fluorescent quantum yields and photosensitized singlet oxygen formation (1O2). General trends in the relative photodynamic behavior exhibited by these new dyes in cell culture are discussed. The results indicate that several of the dyes have significant photosensitizing efficacy.

IDENTIFICATION OF HEPATIC, NON-MONOAMINE, DIHYDROERGOCRYPTINE BINDING SITES WITH SIGNIFICANT GENDER DIFFERENCES

High affinity [3H] dihydroergocryptine binding sites different from alpha1/alpha2-adreno, dopamine or serotonin receptors were detected in a crude membrane fraction from hamster liver by radioligand binding filtration assay. The binding was saturable and reversible, as well as time and protein dependent. Scatchard analysis revealed a single population of binding sites with Kd 3.8 +/- 0.9 nM and Bmax = 675 +/- 130 pmol/g tissue (mean +/- S.E.M., n=6) in the male hamster crude liver membrane fraction. In the female liver membranes the Kd value was 4.4 + 1.2 nM and Bmax = 1025 +/- 190 pmol/g tissue (mean + S.E.M., n = 6). Differences between males and females in Bmax values were significant (P < 0.01). The most potent inhibitors of [3H] dihydroergocryptine binding were bromocriptine > ergotamine > dihydroergocryptine > dihydroergocristine > alpha ergocristine > dihidroergotamine > ergocornine > ergocristine > nicardipine > (+) butaclamol > PK 11195 > nitrendipine > domperidone > (-)butaclamol (in order of decreasing affinity). The described type of dihydroergocryptine binding sites was not detected in hamster brain, kidney, spleen or lungs. Obtained data support the concept that some ergot-derivatives may induce metabolic effects in the liver through peripheral mechanisms other than those, mediated by alpha-adrenoreceptors.