Single-dose pharmacokinetic and toxicity analysis of pyrrole–imidazole polyamides in mice

Purpose: Pyrrole–imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove–binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. Methods: Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. Results: The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. Conclusions: The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models

Prevention of early renal disease, dyslipidaemia and lipid peroxidation in STZ-diabetic rats by LR-9 and LR-74, novel AGE inhibitors

Background: Increased formation of advanced glycation/lipoxidation end-products (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several compounds have been developed as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR-9 and LR-74, on the development of early renal disease and lipid metabolism in streptozotocin (STZ)-induced diabetic rats. Methods: Diabetic Sprague-Dawley rats were treated with either of the LR compounds for 32 weeks. Progression of renal disease was evaluated by measurements of urinary albumin and plasma creatinine concentrations. AGE/ALE and nitrotyrosine levels in kidneys were determined by immunohistochemistry. AGE-induced chemical modification of the tail tendon collagen and levels of Nε-(carboxymethyl) and (carboxyethyl)- lysines (CML and CEL) in skin collagen were measured. Plasma lipids and their lipid hydroperoxide concentrations were also determined. In vitro, both compounds were tested for inhibiting lipid peroxidation reactions. Results: Treatment of either LR compounds significantly inhibited the increase in albuminuria, creatinaemia, hyperlipidaemia and lipid peroxidation in diabetic rats without any effect on hyperglycaemia. Both compounds also reduced CML-AGE and nitrotyrosine accumulation in kidney glomeruli and tubules, AGE-linked fluorescence and cross-linking of tail collagen, and levels of CML and CEL in skin collagen. In vitro, LR compounds inhibited the oxidation of human low-density lipoprotein (LDL). Conclusion: Both compounds can inhibit the progression of renal disease and also prevent dyslipidaemia in type-1 diabetic animals. These compounds may have an additional beneficial effect as an antioxidant against lipid peroxidation, and thus may provide alternative therapeutic options for the treatment of various diabetic macrovascular complications. Copyright © 2005 John Wiley & Sons, Ltd

Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

Abstract Background Epigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer. Methods The expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model. Results DOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo. Conclusions Our findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer

A noninvasive surfactant adsorption test predicting the need for surfactant therapy in preterm infants treated with continuous positive airway pressure

Objective: To determine the diagnostic accuracy of the surfactant adsorption test (SAT) as a predictor for the need for surfactant replacement therapy in neonates with respiratory distress syndrome (RDS). Study design Amniotic fluid samples were collected from 41 preterm neonates with RDS treated with continuous positive airway pressure (CPAP) and 15 healthy control term neonates. Purified porcine surfactant served as a further control. Lamellar bodies and lung ultrasound score were also measured in a subset of the neonates treated with CPAP. Surfactant was administered according to the European guidelines, and clinical data were collected prospectively. Surfactant activity was measured as adsorption at the air/liquid interface and given in relative fluorescent units (RFU). Results: Surfactant activity differed among native porcine surfactant (median, 4863 RFU; IQR, 4405-5081 RFU), healthy term neonates (median, 2680 RFU; IQR, 2069-3050 RFU), and preterm neonates with RDS (median, 442 RFU; IQR, 92-920 RFU; P < .0001). The neonates who failed CPAP had lower surfactant activity compared with those who did not fail CPAP (median, 92 RFU; IQR, 0-315 RFU vs 749 RFU; IQR, 360-974 RFU; P = .0002). Differences between groups were more evident beyond 20-30 minutes of fluorescence; the 30-minute time point showed the highest area under the curve (0.84; P < .001) and the best cutoff level (170 RFU; specificity, 72%; sensitivity, 96%) for the prediction of CPAP failure. Surfactant activity at 30 minutes was significantly correlated with lamellar bodies (r = 0.51, P = .006) and lung ultrasound score (r = -0.39, P = .013). Conclusion: This technique has the potential to be developed into a fast, simple-to-interpret clinical test. The SAT can reliably identify preterm infants with subsequent CPAP failure and shows promise as a screening test for surfactant replacement in preterm neonates

Additional file 1: Table S1. of Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

The primer sequences used for PCR. (DOCX 15 kb

Additional file 6: Figure S4. of Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer

DOT1L regulates the transcription of G1 arrest genes CDK6 and CCND3 through H3K79 dimethylation. (a) and (b) RT-PCR detecting the change of primary G1 arrest genes, D-type cyclins (D1, D2, and D3), cyclin E, Cdks (2, 4, and 6) in mRNA levels in TOV21G cells knocking down of DOT1L with shDOT1L shRNAs lentiviral particles (a) and DOT1L inhibitors, EPZ004777 and SGC0946 (b) (P < 0.05, Student’s t test, data represented as mean±SD). (c) and (d) DOT1L transcriptionally regulates the expression of CDK6 (c) and CCND3 (d) through induction of H3K79 dimethylation in the binding regions of these genes. Chromatin immunoprecipitation (ChIP) PCR assays were performed in a loss-of-function system. TOV21G cells were transfected with shDOT1L shRNAs and shCON lentiviral particles, and ChIP assays using anti-DOT1L and anti-H3K79me2 antibodies were performed with ChIP primers targeting the binding regions of CDK6 and CCND3 (P < 0.05, Student’s t test, data represented as mean±SD). (TIF 815 kb

Phase I study of nelfinavir in liposarcoma

PURPOSE: HIV protease inhibitors are associated with HIV protease inhibitor–related lipodystrophy syndrome. We hypothesized that liposarcomas would be similarly susceptible to the apoptotic effects of an HIV protease inhibitor, nelfinavir. METHODS: We conducted a phase I trial of nelfinavir for liposarcomas. There was no limit to prior chemotherapy. The starting dose was 1,250 mg twice daily (Level 1). Doses were escalated in cohorts of three to a maximally evaluated dose of 4,250 mg (Level 5). One cycle was 28 days. Steady-state pharmacokinetics (PKs) for nelfinavir and its primary active metabolite, M8, were determined at Levels 4 (3,000 mg) and 5. RESULTS: Twenty subjects (13 males) were enrolled. Median (range) age was 64 years (37–81). One subject at Level 1 experienced reversible, grade 3 pancreatitis after 1 week and was replaced. No other dose-limiting toxicities were observed. Median (range) number of cycles was 3 (0.6–13.5). Overall best responses observed were 1 partial response, 1 minor response, 4 stable disease, and 13 progressive disease. Mean peak plasma levels and AUCs for nelfinavir were higher at Level 4 (7.3 mg/L; 60.9 mg/L × h) than 5 (6.3 mg/L; 37.7 mg/L × h). The mean ratio of M8:nelfinavir AUCs for both levels was ~1:3. CONCLUSIONS: PKs demonstrate auto-induction of nelfinavir clearance at the doses studied, although the mechanism remains unclear. Peak plasma concentrations were within range where anticancer activity was demonstrated in vitro. M8 metabolite is present at ~1/3 the level of nelfinavir and may also contribute to the anticancer activity observed