Studies on curcumin and curcuminoids. XLVI. Photophysical properties of dimethoxycurcumin and bis-dehydroxycurcumin.

The steady-state absorption and fluorescence, as well as the time-resolved fluorescence properties of dimethoxycurcumin and bis-dehydroxycurcumin dissolved in several solvents differing in polarity and H-bonding capability are presented. The singlet oxygen generation efficiency of the two compounds relative to curcumin is estimated. The photodegradation quantum yield of the former compound in acetonitrile and methanol is determined. The dimethoxycurcumin and bis-dehydroxycurcumin decay mechanisms from the S (1) state are discussed and compared with those of curcumin, dicinnamoylmethane and bis-demethoxycurcumin.

Ground reaction force profiles after partial and pancarpal arthrodesis in dogs.

OBJECTIVES: To evaluate and compare long-term functional outcome after partial carpal arthrodesis and pancarpal arthrodesis in dogs using kinetic gait analysis. METHODS: Fourteen dogs with 19 partial carpal or pancarpal arthrodeses were retrospectively examined and underwent force-plate gait analysis. Mean times since surgery were 29.4 and 24.4 months for pancarpal and partial carpal arthrodesis respectively. Vertical and braking-propulsive ground reaction force profiles were compared between treatment groups, and to those of normal dogs (control group) using Kruskal-Wallis one-way analysis of variance. RESULTS: With the exception of time to vertical peak that occurred earlier in dogs with pancarpal than in dogs with partial carpal arthrodesis (p <0.01), there was no difference between the two treatment groups. Several parameters differed significantly between operated and healthy dogs (p <0.01): vertical impulses were significantly lower in both treatment groups, braking forces and impulses were also reduced after both techniques. Propulsive forces and impulses were only reduced in dogs with pancarpal arthrodesis. When comparing gait parameters of sound limbs of unilateral operated dogs to those of control dogs, braking forces and impulses (p <0.01; p <0.05) were significantly higher in the sound legs of unilateral operated dogs. CLINICAL SIGNIFICANCE: Long-term outcome after partial carpal and pancarpal arthrodesis is good and comparable to each other. Propulsive action may be altered more in dogs with pancarpal arthrodesis.

Revision of an unstable HELICA endoprosthesis with a Zurich cementless total hip replacement.

A six-year-old, female, spayed Labrador Retriever was evaluated for progressive lameness of one year duration, ending in non-weight-bearing of the right hindlimb. The dog had a history of severe coxarthrosis of both hip joints, and had a HELICA hip prosthesis implanted in the right hip 18 months before. On survey radiographs, the acetabular and femoral components appeared unstable, with a large void in the proximal femur and a lacy periosteal reaction on the trochanter. Arthrocentesis was performed to rule out septic loosening. As culture samples were negative, the dog underwent surgery. We report the successful revision of an unstable HELICA screw hip prosthesis with a Zurich cementless total hip replacement. The patient had a good clinical and radiological outcome seven months postoperatively.

Effects of laser irradiation on hematoporphyrin-treated normal and transformed thyroid cells in culture.

Laser irradiation of tissues treated in vivo with the hematoporphyrin derivative (HPD) is known to result in a cytocidal effect, reportedly more pronounced in the tumor than in the surrounding normal tissues. In order to ascertain if this phenomenon had a clear cellular basis, it has been now reproduced in vitro in a model system consisting of normal and transformed cell lines. Epithelial rat thyroid cells were infected and transformed with a RNA oncogenic virus. Both the original (normal) and the viral-transformed (tumorigenic) cells were incubated with HPD and exposed to two types of laser irradiation: 631 nm, continuous wave; and 337.1 nm, pulsed. Under the conditions tested, the percentage survival of the transformed cells was found to be lower (up to approximately 3 times) than that of the normal cells. The cytocidal effect was greater using the pulsed than using the continuous-wave irradiation. The difference between normal and tumor cells was more evident at 30 micrograms than at 50 micrograms of HPD per ml. The HPD not followed by laser irradiation had no effect on the cell growth rate. The findings of a significant difference in the sensitivity to photoactivated HPD between normal and tumor cells under strictly controlled and highly comparable conditions opens new possibilities to the study of the cellular and molecular mechanisms involved in the phototherapy of tumors. Furthermore, studies in vitro on the active components of the photosensitizer and on their selectivity towards the tumor cells, explained at a cellular level, will lead to better approaches to photochemotherapy in vivo.

Triplet state characteristics and singlet oxygen generation properties of anthracyclines.

The triplet states of adriamycin (Ad), daunomycin (D) and two daunomycin analogues, daunomycinone (Dc) and daunomycin N-trifluoroacetamide (DAc), have been studied using laser flash photolysis and pulse radiolysis techniques. Triplet lifetimes, molar absorption coefficients, energy levels and quantum yields have been obtained for Dc and DAc, and estimated for D and Ad. Time-resolved near-infrared singlet oxygen luminescence measurements have been carried out on D, Ad and 5-iminodaunomycin (5-ID) in 2H2O solution and Dc in benzene solution at room temperature. Singlet oxygen quenching by the water-soluble anthracyclines was observed and a second-order rate constant of approx. 10(8) M-1.s-1 obtained. Electron spin resonance experiments have demonstrated that D photoexcited at lambda less than or 365 nm gives rise to singlet oxygen as shown by its reaction with 2,2,6,6-tetramethyl-4-piperidone to give the corresponding nitroxyl radical. Although all the anthracyclines studied have the ability to photosensitize the formation of singlet oxygen, the quantum yields are very low (phi delta approximately 0.02-0.03), suggesting that these anthracyclines would be poor photodynamic sensitisers.

Cell photosensitization by 5-iminodaunomycin activated with red light.

5-Iminodaunomycin, an anthracycline antitumor drug exhibiting an absorption peak at 595 nm, is shown to photosensitize in vitro cell kill. The photoactivation is performed irradiating the culture dishes during the incubation with the drug for 2 h with 34 mW/cm2 intensity, that is with light doses of up to 245 J/cm2. Long-term effects of administering 50 ng/ml and light for 2 h are studied in terms of growth curves. We show that photoactivation enhances the dark toxicity by a factor of about 10. Immediate cell death is produced by irradiating the cells in the presence of higher drug concentrations (e.g., 1000 ng/ml) which, however, are not toxic in the short term if administered in the dark. The viable cell percentage decreases at increasing light doses, being about 0.6% at the maximum dosage. Administering lower light doses, such as 30 J/cm2, which corresponds to an exposure duration of 15 min, has a short-term effect on the cell survival that strongly depends on the timing of the exposures within the incubation period.

Failure of glucose infusion to suppress the exaggerated GH response to GHRH in patients with anorexia nervosa.

The growth hormone (GH) response to GH-releasing hormone (GHRH) is characteristically exaggerated in anorexia nervosa (AN). Hyperglycemia suppresses the GH response to GHRH in normal subjects. To test whether this inhibitory action of hyperglycemia is preserved in AN, we performed a GHRH (GHRH 1-40, 1 micrograms/kg) test under basal conditions (saline infusion) and during steady-state hyperglycemia (200 mg/dl, induced by the intravenous administration of 8 mg/min.kg of glucose) in 6 adolescent girls with acute-stage AN (as diagnosed by psychopathological, hormonal, and nutritional criteria) and in 5 age-matched female controls. In control subjects, GHRH stimulated GH release during saline, but not glucose, infusion. In the anorectic patients, the GH response to GHRH was exaggerated during both saline infusion (2.97 +/- 0.79 versus 0.52 +/- 0.22 micrograms.120 min.ml-1, p less than 0.02) and under hyperglycemic conditions (4.61 +/- 0.56 versus 0.33 +/- 0.10, p less than 0.001). We conclude that the inhibitory action of hyperglycemia on GHRH-induced GH release is lost in the acute phase of AN.

Blockade of cholinergic muscarinic receptors by pirenzepine and GHRH-induced GH secretion in the acute and recovery phase of anorexia nervosa and atypical eating disorders.

In view of the important role played by the cholinergic system in the neural regulation of growth hormone (GH) secretion, the ability of pirenzepine, a selective antagonist of muscarinic cholinergic receptors, to blunt the GH response to GH-releasing hormone (GHRH) was studied in adolescent females with anorexia nervosa in the acute (AN-AP) five AN-AP patients, administration of GHRH 1-40 (1 microgram/kg IV) evoked a significantly higher GH response than in controls at established intervals, whereas in eight AN-RP and seven AED patients it was higher than in controls at only one (150-min) and two (150-min, 180-min) time intervals, respectively. In the AN-AP patients, pretreatment with pirenzepine (0.6 mg/kg IV) only partially blocked the GH response to GHRH, whereas in the same AN-AP patients tested during recovery, and in AN-RP and AED patients, the drug completely suppressed the GH response to GHRH, as it did in controls. In view of pirenzepine's mechanism of action, these findings are best explained by the existence in the hypothalamus of AN-AP patients of a cholinergic hypertone and/or a diminished somatostatinergic function. Evaluation of the clinical and hormonal characteristics of the anorectic patients studied would indicate that factors other than undernutrition and its biological consequences, which subside in the recovery stage of the disease and are not present in AED patients, contribute to the anomalous GH response pattern of AN-AP patients.

B16 melanoma response in vivo to photochemotherapy with mitoxantrone and red light.

The cytocidal activity of light-activated mitoxantrone in mice bearing B16 melanoma was investigated. Mice inoculated with 10(6) tumor cells on day 0 were i.p. injected with 1 mg/kg body weight of mitoxantrone on days 1, 5 and 9 and exposed to 108 J/cm2 of suitably filtered red light from a halogen lamp on days 2, 6 and 10. The treatment significantly prolonged the median survival time compared to both therapy with mitoxantrone and with red light alone.

Hematoporphyrin derivative photochemotherapy of spontaneous animal tumors: clinical results with optimized drug dose.

Hematoporphyrin derivative (HpD) photochemotherapy was performed on 13 primary spontaneous tumors in dog and cat. The animals received an optimized drug dose of 5 mg/kg body wt i.v. 48 h before the first treatment with laser light at 631 nm. An evaluation of the clinical results is presented and discussed. Complete disappearance of the primary tumors was obtained in all cases with one or more light irradiations. Five cases presented recurrences that were cured with a further treatment. In 4 cases treated after surgical exeresis of the primary tumors, this therapy resulted in complete cure.

Hematoporphyrin derivative photoradiation therapy in murine solid tumors.

The cytocidal activity of light-activated hematoporphyrin derivative (Hpd) in experimental and human tumors is under investigation in many laboratories. This activity is based upon preferential incorporation of Hpd in malignant tissues and its photosensibilization by red light. Treatment of mice bearing MS-2 fibrosarcoma and B16 melanoma, a metastastic tumor, with Hpd and laser light, externally or delivered through a quartz fiber optic imbedded directly into the tumor, significantly prolonged the median survival time. This therapy was compared with surgical excision of primary tumors, and preliminary results on metastatic neoplasm suggest that the photoradiation therapy is more effective than surgery.

Corticotrophin-releasing hormone does not inhibit growth hormone-releasing hormone-induced release of growth hormone in control subjects but is effective in patients with eating disorders.

Previous studies have shown that corticotrophin-releasing hormone (CRH) inhibits GH secretion in response to GH-releasing hormone (GHRH) in normal women and men, and animal studies suggest that this effect is mediated by an increased release of somatostatin from the hypothalamus. It has been reported that there are abnormalities in the neuroendocrine regulation of the hypothalamo-pituitary-somatotrophic axis and the hypothalamo-pituitary-adrenocortical axis in patients with eating disorders. The present study therefore investigated the ability of CRH to inhibit the GH response to GHRH in eight young women with anorexia nervosa (AN) and in seven young women with eating disorders which were not otherwise specified (NOS). We also compared the effect of CRH in the patients with the response it caused in ten control women. In contrast to a previous report, combined i.v. administration of 50 micrograms human CRH (hCRH) and 50 micrograms GHRH(1-29) caused a GH response in control women which was higher, although not significantly so, than that induced by GHRH alone (area under the curve (AUC) 988.5 +/- 506.0 compared with 1568.4 +/- 795.6 (S.E.M.) ng/ml per 120 min for GHRH alone and GHRH plus hCRH respectively). Conversely, the administration of hCRH given together with GHRH markedly inhibited the GH response induced by the latter in both AN patients (AUC 2253.0 +/- 385.7 compared with 1224.4 +/- 265.7 ng/ml per 120 min for GHRH and GHRH plus hCRH respectively; P < 0.005 and NOS patients (AUC 2827.4 +/- 281.1 compared with 308.5 +/- 183.4 ng/ml per 120 min for GHRH and GHRH plus hCRH respectively; P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of brain catecholamines and acetylcholine in growth hormone hypersecretory states. Pathophysiological, diagnostic and therapeutic implications.

Secretion of growth hormone (GH) is excessive in acromegaly, but also in a number of other pathological states such as anorexia nervosa, insulin-dependent diabetes mellitus (IDDM), liver cirrhosis, depression, renal failure and GH-insensitivity syndrome. Abnormalities in the neuroendocrine control of GH secretion and/or a state of insensitivity to GH contribute to hypersecretion of GH in these states, with the possible exception of acromegaly, which appears to be a primary pituitary disease. GH hypersecretion may also occur in neonates or adolescents with tall stature, thus reflecting particular physiological or paraphysiological conditions. In the cohort of brain neurotransmitters, catecholamines and acetylcholine reportedly play a major role in the control of neurosecretory GH-releasing hormone (GHRH) and somatostatin (SS)-producing neurons, and hence GH secretion. Activation of alpha 2-adrenoceptors or of muscarinic cholinergic receptors in the hypothalamus stimulates GH release, probably through stimulation of GHRH and inhibition of SS release, respectively. Activation of dopamine receptors likewise stimulates GH release, while activation of beta-receptors inhibits GH release through stimulation of hypothalamic SS function. This review discusses the involvement of brain catecholamines and acetylcholine in GH hypersecretory states, including anorexia nervosa, acromegaly, IDDM, liver cirrhosis, depression, renal failure and GH insensitivity syndrome, with a view to providing a fuller understanding of their pathophysiology and, whenever possible, diagnostic and therapeutic implications.

Photocytotoxicity of anthracyclines upon laser excitation in their long-wavelength absorption bands.

Newly synthesized daunomycin derivatives with red-shifted absorption compared to the parent molecule are shown to be able to photosensitize cells in vitro upon excitation with either argon or argon-pumped dye laser. Administering 86 J/cm2 total fluence (1 h irradiation) to Fisher rat thyroid cells during 2 h incubation with either daunomycin (excitation wavelength: 488 nm) or 5-iminodaunomycin (595 nm) produced cell killing at doses (about 2.7 X 10(-7) M for 50% cell survival) which were not toxic if administered in the dark. Greater photocytotoxicity (about 7 X 10(-8) M for 50% cell survival) was obtained with 4-demethoxydaunomycin as well as with its 6- and 11-amino derivatives (514 nm) while no cell killing as a result of photosensitization was observed for either Adriamycin or its 4'-iodo derivative. Our results suggest that the photosensitizing efficacy correlates with the absence of the methoxy group in the anthraquinone chromophore but is rather independent of the occurrence of triplet-mediated photoreactions. Finally, the fact that the imino- or amino-substituted 4-demethoxy compounds exhibit red-shifted absorption spectra compared to the parent molecule might be exploited for in vivo applications of the photoactivated cytotoxicity reported in this work.

Antivenom and biological effects of ar-turmerone isolated from Curcuma longa (Zingiberaceae)

A potent antivenom against snakebite was isolated from Curcuma longa, a plant commonly used in traditional Brazilian medicine. The fraction consisting of ar-turmerone neutralized both the hemorrhagic activity present in Bothrops jararaca venom, and the lethal effect of Crotalus durissus terrificus venom in mice. Immunological studies demonstrated that this fraction also inhibited the proliferation and the natural killer activity of human lymphocytes.

A new bradykinin-potentiating peptide (peptide P) isolated from the venom of Bothrops jararacussu (jararacuçu tapete, urutu dourado).

Several bradykinin potentiators were identified in the venom of Bothrops jararacussu by chromatographic techniques and biological assays. One of them which was isolated inhibited the angiotensin-converting enzyme in vitro and potentiated the bradykinin-induced lowering of the arterial pressure in the rat.

Time-resolved luminescence spectroscopy of photosensitizers of biomedical interest.

Studying the fluorescence decay of chromophores, either used as fluorescent labels to stain specific biomolecules or as photosensitizers to produce irreversible chemical or physico-chemical modifications on biological substrates, is being demonstrated to be a valuable method of investigating the interactions underlying a variety of phenomena. In fact, all possible primary steps in a photosensitized biological system are phenomena that may occur during the chromophore S1 lifetime and act as quenching mechanisms of the S1 state. Thus they can be identified, and the relative importance of the corresponding transient species quantitatively determined, with suitable techniques of time-resolved fluorescence spectroscopy. The examples discussed in this paper concern both tumor photosensitizing drugs, such as anthracyclines and porphyrins, and skin sensitizers (e.g. furocoumarins).

Enhancement of antitumor drug cytotoxicity via laser photoactivation.

We investigate the efficacy of daunomycin, some imino- and amino-substituted daunomycin analogues and the disubstituted aminoanthracenedione, mitoxantrone, in photosensitizing short-term cell kill upon irradiation in the long wavelength visible range, during incubation of Fisher rat thyroid cells with the drugs. While all compounds exhibit similar cytocidal effects on our cell line, in the absence of irradiation, administering 86 J/cm2 at wavelengths either coincident or close to drug absorption peaks causes greater enhancement in cell mortality for the 4-demethoxydaunomycin analogues than either the parent drug or its 5-imino-derivative. A lower enhancement is observed with mitoxantrone. In particular, C50 doses (i.e. concentrations that would kill 50% cells) as low as approximately 10(-9) M are found for both 6- and 11-amino 4-demethoxydaunomycin, compared with the values obtained in the absence of light, which are 2.59 x 10(-4) and 0.43 x 10(-4) M, respectively. Our previous studies of the photophysical and photochemical properties of the excited states of these drugs, and ESR and spin trapping studies of photosensitized generation of singlet oxygen, which were extended in this work to include mitoxantrone, indicate that the cytocidal effects proceed via type I rather than type II mechanisms.

Laser photosensitization of cells by hypericin.

Administering a light dose of 90 J/cm2 at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line (FRTL-5), derived from Fisher rat thyroid, and to a neoplastic cell line (MPTK-6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.

Kinetic transport analysis of daunorubicin by LoVo and LoVo/DX cells.

We describe a fluorometric technique for the measurement of transport parameters of fluorescent drugs through cellular membranes. Unlike other procedures, this method gives an accurate measure of drug accumulated in the cells and measures the fraction of free and bound drug in the cell. The kinetic parameters of transport through cellular membranes are determined using a simple three-compartment model combined with fluorescence measurements performed on the extracellular medium and on Triton-permeabilized cells during daunorubicin incorporation. With this technique we found that LoVo cells have a greater daunorubicin uptake, a similar input rate constant and a lower output rate constant than the drug-resistant LoVo/DX cells.