Total synthesis and antitubulin activity of c10 analogues of cryptophycin-24

The unsubstituted, 3'-Cl, 4'-C1, and 3',4'-diCl C10 analogues of cryptophycin-24 were prepared via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED(50) values ranged from 7.2 to 15.8 microM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence of a 3'-C1 and/or 4'-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7 cell line compared to the unsubstituted phenyl ring. The most potent compound in this series possessed a 3'-C1 substituent on the C10 phenyl ring. The 3'-C1 analogue had ED(50) values of 50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very similar to the parent compound cryptophycin-24. Substitution of the 4'-MeO group in cryptophycin-24 with a 4'-C1 moiety did not significantly affect cytotoxicity against MCF-7 and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the 4'-MeO group in cryptophycin-24 is not essential and can be replaced with 3'-C1 or 4'-C1 substituents.     

Vaccination with hybrids of tumor and dendritic cells induces tumor-specific T-cell and clinical responses in melanoma stage III and IV patients

Hybrid cell vaccination was developed as therapeutic approach that aims at stimulating tumor-specific cytotoxic T-cell responses in cancer patients using hybrids of autologous tumor and allogeneic dendritic cells. We tested this concept and the efficacy of the vaccines in inducing clinical and immunologic responses in a clinical trial with melanoma stage III and IV patients. Of the 17 patients evaluated, 1 experienced a complete response, 1 a partial response and 6 stable disease with remarkably long survival times. In 11 of 14 patients analyzed, high-frequency T-cell responses to various tumor-associated T-cell epitope were induced and detectable in the peripheral blood. These immune responses were detected in clinical response patients as well as nonresponders. Failures of clinical responses in all the cases investigated correlated with loss of antigen expression and presentation. Hybrid cell vaccination thus proves effective in inducing tumor-specific T-cell responses in cancer patients.     

Developmental toxicity of thiodiglycol in Sprague-Dawley rats

Thiodiglycol (TG), a hydrolysis product of sulfur mustard (HD), is a potential contaminant of soil and water at certain military sites. To establish developmental toxicity criteria for TG, an oral developmental toxicity study was conducted in Sprague-Dawley rats. Neat thiodiglycol (99.9 %) was administered orally to mated female rats from gestation days (GDs) 5 through 19. The day of positive mating was considered day 0. A pilot study was conducted with TG at dose levels 250, 500, 1,000, 2,000, or 5,000 mg/kg to select suitable doses for the main study. In the main study, three groups of rats (25/group) received TG by gavage at dose levels of 430, 1,290, or 3,870 mg/kg/day. A fourth group served as a sham control. On day 20 of gestation, all females were euthanized and a cesarean section performed. Litters were examined for soft tissue and skeletal alterations. Maternal toxicity was limited to dams receiving TG at 3,870 mg/kg/day. At this dose, body weights and food consumption were reduced during certain periods of gestation. Fetuses derived from those dams exhibited a nonstatistically significant increased incidence of variations when compared to controls. Fetal body weights in the 3,870 mg/kg/day group were significantly lower than controls. There was no increased incidence of anomalies when thiodiglycol-treated fetuses were compared to controls. It was concluded that TG did not produce terata. Developmental toxicity (decreased fetal weights and associated delays in development) occurred only at the maternally toxic dose of 3,870 mg/kg. It appears that 1,290 mg/kg/day could be considered no observed adverse effect level (NOAEL) for oral developmental toxicity. The lowest observed adverse effect level (LOAEL) was 3,870 mg/kg for maternal toxicity.     

Does video-mediastinoscopy improve the results of conventional mediastinoscopy?

OBJECTIVE: Despite new technologies, mediastinoscopy remains the gold standard for mediastinal staging of lung cancer even though the procedure is not standardised. Introduction of video-mediastinoscopy (VM) may help to overcome this problem as it better visualises the anatomy and allows a more uniform dissection than conventional mediastinoscopy (CM). Does the use of VM result in more lymph node tissue, higher accuracy and lower complication rates as compared to CM? METHODS: All mediastinoscopies from June 2003 to December 2005 were analysed. In a protocol surgeons documented location of lymph node stations, number of lymph nodes resected or biopsied and technique (VM or CM). Two groups were created for analysis: group 1 (n=366) consisting of all mediastinoscopies was reviewed for complication rates; group 2 included all patients with lung cancer who had a pN0 status by mediastinoscopy and underwent subsequent thoracotomy (n=171). This group was studied for the number of lymph nodes resected or biopsied according to the technique (VM or CM), on accuracy and negative predictive value. RESULTS: Of 366 mediastinoscopies, 132 were CM (36.1%) and 234 VM (63.9%). Complications occurred in 17 patients (4.6%): 9 recurrent laryngeal nerve palsies (VM 2.1%, CM 3.0%), 5 mediastinal enlargement on routine chest radiography interpreted as postoperative bleeding (VM 0.9%, CM 2.3%), pneumonia (1), intraoperative laceration of the pleura (1) and main bronchus (1), both corrected during the procedure (all VM 1.3%). No intraoperative haemorrhage or death occurred. VM resected more lymph nodes (mean 8.1, range 3-25) then CM (mean 6.0, range 3-11), for all mediastinoscopies the mean lymph node yield was 7.6 (range 3-25). Comparison of lymphadenectomy via thoracotomy in patients classified pN0 by mediastinoscopy (n=171) showed an accuracy of 87.9% for VM versus 83.8% for CM (85.8% for all mediastinoscopies) with a negative predictive value of 0.83 for VM and 0.81 for CM (0.82 for all mediastinoscopies). CONCLUSION: This study demonstrates that in comparison with CM, VM routinely yields more lymph nodes with fewer complications with a tendency towards better accuracy and negative predictive value. For these reasons, we believe that VM should replace CM as the method of choice. Furthermore VM would allow standardisation, thereby having an advantage in comparison to the less invasive newer staging techniques. This way mediastinoscopy could remain the gold standard despite its invasiveness.     

Video-mediastinoscopic resection of a long bronchial stump and reclosure of bronchial insufficiency after pneumonectomy

Bronchial stump insufficiency after pneumonectomy is a severe problem and there is still debate about the appropriate method (transthoracic or transsternal) for reclosure. Access through a sterile operative field for a successful redo-procedure seems to be important so an alternative to the open methods could be the video-mediastinoscopy as it allows approaching the bronchial stump via the mediastinum. Previously in 1996 Azorin performed the first mediastinoscopic reclosure by stapling an early insufficiency after left pneumonectomy. We report the first case to our knowledge of resection and reclosure in bronchial stump insufficiency via mediastinoscopy. An HIV-positive man presented with late bronchial stump insufficiency after left pneumonectomy for lung cancer. The cause was a long bronchial stump and there was no sign of tumour recurrence. Decision was made for a video-mediastinoscopy and resection and reclosure successfully performed by using an endostapler device. Postoperative bronchoscopy at six months revealed a well-healed stump and two years postoperatively the patient is doing well. The mediastinoscopic approach is a novel option in highly selected patients. It warrants minimal surgical trauma; however, one has to be prepared to convert to an open technique immediately.     

Liver-homing of purified glucose oxidase: a novel in vivo model of physiological hepatic oxidative stress (H2O2)

BACKGROUND/AIMS: Reactive oxygen species (ROS), such as H2O2, are implicated in normal and pathological liver function. However, due to the lack of suitable in vivo models of ROS generation the (patho) physiological relevance of H2O2 remains controversial. METHODS: We established a novel model of sustained hepatic H2O2 release using intravenous administration of purified Aspergillus niger glucose oxidase (GOX) in rats. RESULTS: GOX is rapidly cleared from the blood stream and almost exclusively localizes to Kupffer cells. GOX maintained its ability to generate H2O2 over 24h. While sublethal GOX doses induced hepatocellular necrosis, our morphological and functional studies show that lower levels of GOX which generate H2O2 comparable to release by inflammatory cells are non-toxic and do not induce histological inflammation. However, these non-toxic H2O2 levels increased oxidized glutathione and induced heme oxygenase 1 in the liver. In addition, several hepatocyte transporters were downregulated, while no decrease of bile formation, a sensitive marker of liver function, was observed. CONCLUSIONS: Our in vivo model allows to study the effects of extracellular H2O2 in the liver as is released by inflammatory cells. Thus analysis of the role of this major ROS in the absence of confounding inflammatory cofactors will be possible.     

Physiologically based pharmacokinetic modeling of cyclotrimethylenetrinitramine in male rats

A physiologically based pharmacokinetic (PBPK) model for simulating the kinetics of cyclotrimethylene trinitramine (RDX) in male rats was developed. The model consisted of five compartments interconnected by systemic circulation. The tissue uptake of RDX was described as a perfusion‐limited process whereas hepatic clearance and gastrointestinal absorption were described as first‐order processes. The physiological parameters for the rat were obtained from the literature whereas the tissue : blood partition coefficients were estimated on the basis of the tissue and blood composition as well as the lipophilicity characteristics of RDX (logP = 0.87). The tissue : blood partition coefficients (brain, 1.4; muscle, 1; fat, 7.55; liver, 1.2) obtained with this algorithmic approach were used without any adjustment, since a focused in vitro study indicated that the relative concentration of RDX in whole blood and plasma is about 1 : 1. An initial estimate of metabolic clearance of RDX (2.2 h^?1 kg^?1) was obtained by fitting PBPK model simulations to the data on plasma kinetics in rats administered 5.5 mg kg^?1 i.v. The rat PBPK model without any further change in parameter values adequately simulated the blood kinetic data for RDX at much lower doses (0.77 and 1.04 mg ^?1 i.v.), collected in this study. The same model, with the incorporation of a first order oral absorption rate constant (K_a 0.75 h^?1), reproduced the blood kinetics of RDX in rats receiving a single gavage dose of 1.53 or 2.02 mg kg^?1. Additionally, the model simulated the plasma and blood kinetics of orally administered RDX at a higher dose (100 mg kg^?1) or lower doses (0.2 or 1.24 mg kg^?1) in male rats. Overall, the rat PBPK model for RDX with its parameters adequately simulates the blood and plasma kinetic data, obtained following i.v. doses ranging from 0.77 to 5.5 mg kg^?1 as well as oral doses ranging from 0.2 to 100 mg kg^?1. Published in 2009 by John Wiley & Sons, Ltd.     

Antenatal mental representations about the child and mother–infant interaction at three months post partum

OBJECTIVE: To study the predictive value of antenatal maternal mental representations about the child for mother-infant interaction at three months after birth. PROBANDS: A total of 73 pregnant women attending a routine ultrasound examination during the third trimester, who agreed to return with their baby for the interaction study. METHODS: Antenatally a questionnaire for assessing maternal representations about the child was given to the mothers. Also, depression and pregnancy risk were assessed. At three months, mother and infant were video-taped during a 4-phase interaction. Interaction analysis was performed using the Munich Communication Diagnostic Scale. RESULTS: Overall maternal regulatory ability was predicted by a compound factor comprising the antenatal representations. Maternal interactive behavior was not predicted by antenatal representations about the child. Infant overall eye contact was predicted by maternal representations as was interaction readiness during the still-face period. Significant covariates were maternal age, education, parity and the Pregnancy Optimality Score as well as neurologic optimality of the infant. CONCLUSION: Antenatal maternal representations about the child predicted parental regulatory ability and infant interaction behavior especially during the still-face period.     

Mitochondria-targeted metformin (mitomet) inhibits lung cancer in cellular models and in mice by enhancing the generation of reactive oxygen species

Lung cancer is the leading cause of cancer-related mortality in the United States. Although some epidemiological studies have shown an inverse relationship between the use of metformin, a widely used antidiabetic drug, and the incidence of lung cancer, the real benefits of the drug are unclear as the efficacy is low and the outcomes are quite heterogeneous. To develop a more potent form of metformin, we synthesized mitochondria-targeted metformin (mitomet) and tested its efficacy in in vitro and in vivo models of lung cancer. Mitomet was cytotoxic to transformed bronchial cells and several non-small cell lung cancer (NSCLC) cell lines but relatively safe to normal bronchial cells, and these effects were mediated mainly via induction of mitochondrial reactive oxygen species. Studies using isogenic A549 cells showed that mitomet was selectively toxic to those cells deficient in the tumor suppressor gene LKB1, which is widely mutated in NSCLC. Mitomet also significantly reduced the multiplicity and size of lung tumors induced by a tobacco smoke carcinogen in mice. Overall, our findings showed that mitomet, which was about 1000 and 100 times more potent than metformin, in killing NSCLC cells and reducing the multiplicity and size of lung tumors in mice, respectively, is a promising candidate for the chemoprevention and treatment of lung cancer, in particular against LKB1-deficient lung cancers which are known to be highly aggressive.     

Role of tagged SNPs of the AGT gene in causing susceptibility to essential hypertension

Aim: Angiotensinogen (AGT) is one of the candidate genes that has been extensively investigated for association of its variants with essential hypertension. Studies focusing on the contribution of tagged single nucleotide polymorphisms (SNPs) in the AGT gene are limited and lacking from Indian population. Hence, the present study was carried out to examine the role of five tagged SNPs viz., g.6147G>A (rs7539020), g.5978A>G (rs2493134); g.6241T>C (rs1078499), g.7781G>T (rs11122577), and g.5855G>A (rs3789678) in the development of hypertension. Materials and Methods: 202 hypertensives and 222 normotensives were screened for five tagged SNPs using the method of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: The present study revealed significant association of g.5855G>A polymorphism with essential hypertension in different logistic regression models wherein protection was conferred by g.5855G>A against developing the condition. The polymorphism led to the creation of new exonic splicing enhancer and destruction of exonic splicing silencer site thereby enhancing the process of mRNA splicing. The haplotypes AGTG and GACG were found to have a significant protective effect. Other polymorphisms did not show any significant association with hypertension. Conclusion: The present study is the first one to report the protective role of g.5855G>A polymorphism in the development of essential hypertension. The results reflect possibility of ethnic variation in the contribution of g.5855G>A polymorphism of the AGT gene to essential hypertension.