Changes in antioxidant enzymes and lipid peroxidation in extensor digitorum longus muscles of streptozotocin-diabetic rats may contribute to muscle atrophy.

We investigated muscle atrophy, major antioxidant enzymes and lipid peroxidation in the extensor digitorum longus (EDL, predominantly fast fibers) and soleus (predominantly slow fibers) muscle of streptozotocin-diabetic rats. Female Wistar rats were divided into a control (n = 5) and streptozotocin-induced diabetic group (n = 5). Eight weeks after diabetes induction the EDL and soleus muscles were removed and catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase activity (SOD), and thiobarbituric acid reactive substances (TBARS) levels measured. The CAT activity increased in both the EDL and soleus muscles of the diabetic rats (p < 0.01), whereas the GPX and SOD activities were increased only in the EDL muscle (p < 0.01 and p < 0.05). The TBARS levels were only increased in the EDL muscle of the diabetic rats (p < 0.01). Both muscles showed significant atrophy but the EDL muscle elicited the greatest atrophy. In conclusion, it appears that adaptive responses to oxidative stress were adequate in the soleus muscle, but not in the EDL muscle, of diabetic rats. Thus fast twitch muscle fibers may be more susceptible to oxidative stress than slow twitch muscle fibers and this may contribute to muscle atrophy under diabetic conditions.

5-HT2A promoter polymorphism is not associated with anorexia nervosa in Japanese patients.

Genetic factors have been implicated in playing a significant role in susceptibility to anorexia nervosa (AN). Among many candidate genes for AN, an association with the A allele of the -1438G/A polymorphism in the promoter region of the 5-HT2A receptor has been reported. However, these findings are controversial and all patients studied to date have been Caucasian. This study was designed to determine whether this association is reproducible in Japanese subjects. This case-control study of a cohort of 75 female Japanese AN sufferers and 127 normal female control subjects revealed no significant association between the 5-HT2A promoter polymorphism and AN. Thus, at least for Japanese subjects, the A-allele of the -1438G/A polymorphism in the promoter region of the 5-HT2A receptor gene does not contribute to a predisposition to AN.

Analysis of tumor necrosis factor-alpha gene promoter polymorphisms in anorexia nervosa.

Elevated plasma tumor necrosis factor-alpha (TNFalpha) levels and enhanced spontaneous TNFalpha release from peripheral blood mononuclear cells in patients with anorexia nervosa (AN) have been reported. TNFalpha activates the hypothalamic-pituitary-adrenal axis and reduces food intake, which is characteristic of eating disorders. Recently, three novel polymorphisms in the 5'-flanking region of the TNFalpha gene were reported at positions -1031 (T --> C substitution), -863 (C --> A) and -857 (C --> T). Differences in these alleles are reportedly related to altered TNFalpha-transcriptional promoter activity. Therefore, we performed a case-control association analysis to determine whether any of those three polymorphisms in the TNFalpha promoter region were involved in a predisposition to AN. The results of our analysis of a cohort of 79 female Japanese AN sufferers and 127 normal female control subjects provide no support for the hypothesis that -1031T/C, -863 C/A and -857C/T polymorphisms in the TNFalpha gene promoter region influence the susceptibility to AN.

Pharmacokinetic analysis and antitumor efficacy of MKT-077, a novel antitumor agent.

MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [14C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of 14C after i.v. administration of [14C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of 14C decreased with a T1/2 of approximately 5 min. In the second and terminal phase, the T1/2 of 14C was 2.8-4.6 h and 16.2 h, respectively. Cmax (1 min after injection) increased from 0.3 to 1.5 microg/ml linearly, but less than proportionately between the doses. The AUC(0-infinity) at 0.3, 1 and 3 mg/kg were 0.030 +/- 0.002, 0.60 +/- 0.12 and 1.73 +/- 0.25 microg x h/ml, respectively. Plasma clearance was approximately 1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), 14C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), 14C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [14C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors.

Determination of MKT-077, a novel antineoplastic agent, in plasma samples by high-performance liquid chromatography and its application to pharmacokinetics in rats.

A simple high-performance liquid chromatographic method was developed for determination of a novel antineoplastic agent MKT-077 in plasma. MKT-077 was extracted from 50 microl of plasma with acetonitrile containing 1 ml trifluoroacetic acid per liter. Chromatographic separation was achieved within 13.5 min using a reverse-phase Puresil C18 analytical column. A visible detector operated at 490 nm was used. The linearity of the calibration curve was obtained (r2 = 0.99986) over the analytical range of 10-500 ng/ml(-1). The intra- and inter-assay precision was in the range of 0.9-11.1 and 0.3-4.4%, respectively. The intra- and inter-assay bias ranged from -7.3 to 11.1% and from 0.4 to 11.6%, respectively. The utility of this assay was demonstrated after the administration of a single dose of MKT-077 to rats. The plasma elimination half-life of MKT-077 was 1.8-4 h.

Structure-activity of novel rhodacyanine dyes as antitumor agents.

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

Incrustation and uptake of skeletal imaging agent in transitional cell carcinoma.

We present a case of transitional cell carcinoma of the bladder visualized by 99mTc-HMDP bone scintigraphy and suggest possible uptake mechanisms. Pelvic CT demonstrated a sessile bladder tumor with punctate and curvilinear calcifications on the surface areas (incrustation). Technetium-99m-HMDP bone scintigraphy demonstrated intense uptake corresponding to the site of the bladder tumor. Chemisorption of urinary 99mTc-HMDP, rather than of blood-born 99mTc-HMDP, may have occurred at the tumor surface.

MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation.

MKT-077 (formerly known as FJ-776) is a newly synthesized, highly water-soluble ( > 200 mg/ml) rhodacyanine dye that exhibits significant antitumor activity in a variety of model systems. In culture, MKT-077 inhibits the growth of five human cancer cell lines (colon carcinoma CX-1, breast carcinoma MCF-7, pancreatic carcinoma (CRL 1420, bladder transitional cell carcinoma EJ, and melanoma LOX) but not monkey kidney CV-1, an indicator cell line for normal epithelial cells. In nude mice, MKT-077 inhibits the growth of s.c. implanted human renal carcinoma A498 and human prostate carcinoma DU145 and prolongs the survival of mice bearing i.p. implanted human melanoma LOX (tumor:control = 344%). Subcellular localization indicates that MKT-077 is taken up and retained by mitochondria, and flow cytometric analysis suggests that CX-1 cells take up MKT-077 to a much greater extent than CV-1 cells. Quantitation of MKT-077 uptake by ethanol extraction shows that CX-1 cells accumulate 65-fold more MKT-077 than do CV-1 cells. MKT-077 is the first delocalized lipophilic cation with a favorable pharmacological and toxicological profile in preclinical studies. MKT-077 is now being investigated in Phase I clinical trials.

A novel monoclonal antibody to the outer root sheath cells.

A novel monoclonal antibody to hair keratins (TYHF-1) which recognized a doublet of 43/44-kDa hair keratins in the immunoblot analysis was developed. TYHF-1 strongly reacted with the outer root sheath cells at the region of prekeratinization, the keratogeneous zone and the lower part of the isthmus portion in the indirect immunofluorescent examination. The cells of the inner root sheath and cuticle in the upper bulbar portion were also stained with this antibody. In addition, the tongue and nail matrix were also reacted with TYHF-1. However, TYHF-1 reacted with no epidermal keratins either in the indirect immunofluorescent examination or by immunoblot analysis using plantar epidermis. In addition, the cells of the eccrine and apocrine sweat glands, the epithelia of the buccal mucosa, larynx, tonsil, oesophagus, liver, intestine, gallbladder and urinary bladder demonstrated no positive reaction.

The location of Ted-R-1 antigen on the cell membrane of the stratum corneum: immunoelectron microscopic study.

As the Ted-R-1 monoclonal antibody yields fluorescence on the keratohyalin granules, in the cytoplasm of the lower stratum corneum cells and on the cell membrane region of the stratum corneum in indirect immunofluorescent examination, the location of the antigen of this Ted-R-1 monoclonal antibody was examined by the post-embedding, immunoelectron microscopic technique using gold labelled antibody. The number of gold particles located on the keratohyalin granules was 184.00 +/- 28.69 per micron2. In contrast, there were only a few gold particles in the nucleus and the cytoplasm of the stratum granulosum (3.85 +/- 1.34) and none were found in the intercellular spaces of these cells (< 1.00). On the other hand, they were diffusely located in the lower two to three cell layers of the stratum corneum (319.75 +/- 44.41) and on the inner surface of the marginal band of the upper cell layers of the stratum corneum, but only a few were located in the cytoplasm of the upper stratum corneum cells (12 +/- 7.00). These observations indicate that the Ted-R-1 antigen locates on both keratohyalin granules and the inner surface of the marginal band of the upper part of the stratum corneum cell layers.

[Phosphorus 31-magnetic resonance spectroscopic studies of animal liver after extracorporeal shock wave lithotripsy].

The technique of magnetic resonance spectroscopy has been developed, and the study of high-energy phosphate metabolites in the liver using phosphorus 31 magnetic resonance spectroscopy (31P-MRS) has been reported in humans and animals, but few studies have used 31P-MRS for the evaluation of extracorporeal shock wave lithotripsy (ESWL) of the liver. In this study, 31P-MRS was used to evaluate the metabolic changes in hamster liver after ESWL and histological correlation was performed. Syrian golden hamsters were anesthetized and shock waves were irradiated to the left side of the liver. Hamsters were irradiated by LITHOSTAR-PLUS (SIEMENS) at a voltage of 19 KV. 31P-MRS was studied by JNM-GSX model 270 (6.34 Tesla). Typical peaks of 31P-spectra of hamster liver showed a tendency for PDE/beta-ATP, alpha-ATP/beta-ATP and gamma-ATP/beta-ATP to decrease among the irradiated group compared with the control group. However, there were no significant differences in PME/Pi, beta-ATP/Pi or (alpha-ATP-beta-ATP)/beta-ATP between the control group and irradiated group. With regard to intracellular pH and PDE/beta-ATP, a decreasing tendency was noted in the irradiated groups (p less than 0.05). There was no difference in the signal intensity of T1WI and T2WI on 1H-MRI, between these two groups. Pathologically, the irradiated group showed minor hemorrhage and edema in the liver, and subcapsular hematoma. The results obtained from 31P-MRS clearly showed the metabolic changes and were correlated well with the histological findings, but MRI was not capable of providing close visualization of post-ESWL liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term results of aortic valvuloplasty for aortic regurgitation associated with ventricular septal defect.

Long-term results in 64 patients who underwent aortic valvuloplasty for aortic regurgitation associated with ventricular septal defect are presented. The average age of the patients was 10.2 years and the average cardiothoracic ratio was 0.57 at the time of operation. The average degree of aortic regurgitation, as classified by Sellers, was 2.7. The type of ventricular septal defect was subpulmonic in 31 patients, perimembranous in 20, and was a total conal defect in five patients. Valvuloplasty was performed at one end of an aortic cusp in 23 patients, at two ends in 33, at three ends in six, and at four ends in two patients. There was one operative death (1.6%). The follow-up period was 447.7 patient-years, and there were no late deaths. The actuarial survival rate was 98.3% at 5, 10, and 15 years. Postoperative cardiac catheterization was performed in 40 patients. Valvuloplasty failure was defined as the presence of a cardiothoracic ratio greater than 0.6, a loud regurgitant murmur (Levine grade 3/6 or greater), moderate or severe aortic regurgitation (Sellers grade 3 or 4), or the necessity of reoperation. There were 17 patients whose valvuloplasty failed. The freedom from valvuloplasty failure was 74.2% at 5 and 10 years and 55.3% at 15 years. Eight patients underwent reoperation because of residual aortic regurgitation, and all survived. The freedom from reoperation was 90.1% at 5 years, 80.5% at 10 years, and 63.7% at 15 years. Multiple regression analysis revealed that older age, a greater cardiothoracic ratio, perimembranous ventricular septal defect, and multiple valvuloplasties were significant contributing factors for residual regurgitation after aortic valvuloplasty.