Interfering ribonucleic acids that suppress expression of multiple unrelated genes

<p>Abstract</p> <p>Background</p> <p>Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of <it>VEGF-A </it>and <it>ICAM-1</it>; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy.</p> <p>Results</p> <p>Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated.</p> <p>Conclusion</p> <p>Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach.</p

Pharmacokinetic and pharmacogenetic determinants of the activity and toxicity of irinotecan in metastatic colorectal cancer patients

This study aims at establishing relationships between genetic and non-genetic factors of variation of the pharmacokinetics of irinotecan and its metabolites; and also at establishing relationships between the pharmacokinetic or metabolic parameters and the efficacy and toxicity of irinotecan. We included 49 patients treated for metastatic colorectal cancer with a combination of 5-fluorouracil and irinotecan; a polymorphism in the UGT1A1 gene (TA repeat in the TATA box) and one in the CES2 gene promoter (830C>G) were studied as potential markers for SN-38 glucuronidation and irinotecan activation, respectively; and the potential activity of CYP3A4 was estimated from cortisol biotransformation into 6β-hydroxycortisol. No pharmacokinetic parameter was directly predictive of clinical outcome or toxicity. The AUCs of three important metabolites of irinotecan, SN-38, SN-38 glucuronide and APC, were tentatively correlated with patients' pretreatment biological parameters related to drug metabolism (plasma creatinine, bilirubin and liver enzymes, and blood leukocytes). SN-38 AUC was significantly correlated with blood leukocytes number and SN-38G AUC was significantly correlated with plasma creatinine, whereas APC AUC was significantly correlated with plasma liver enzymes. The relative extent of irinotecan activation was inversely correlated with SN-38 glucuronidation. The TATA box polymorphism of UGT1A1 was significantly associated with plasma bilirubin levels and behaved as a significant predictor for neutropoenia. The level of cortisol 6β-hydroxylation predicted for the occurrence of diarrhoea. All these observations may improve the routine use of irinotecan in colorectal cancer patients. UGT1A1 genotyping plus cortisol 6β-hydroxylation determination could help to determine the optimal dose of irinotecan

A phase II trial of gefitinib with 5-fluorouracil, leucovorin, and irinotecan in patients with colorectal cancer

Inhibition of epidermal growth factor receptor (EGFR) signalling contributes to the therapy of colorectal cancer. Gefitinib, an oral EGFR tyrosine kinase inhibitor, shows supra-additive growth inhibition with irinotecan and fluoropyrimidines in xenograft models. We designed a study to determine the tolerability and efficacy of gefitinib in combination with irinotecan, infusional 5-fluorouracil (5-FU) and leucovorin (LV), on a 2-week schedule. Among 13 patients with advanced colorectal cancer, 10 required dose reductions of irinotecan and 5-FU because of dehydration, diarrhoea, and neutropenia, seven of whom required hospitalisation, three with neutropenic fever. One patient achieved partial response and seven had disease stabilisation. The combination of this standard chemotherapy regimen with gefitinib is associated with excessive toxicity, suggesting an interaction at a pharmacokinetic or pharmacodynamic level

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients

Phase I and pharmacokinetic (PK) study of MAG-CPT (PNU 166148): a polymeric derivative of camptothecin (CPT)

Polymeric cytotoxic conjugates are being developed with the aim of preferential delivery of the anticancer agent to tumour. MAG-CPT comprises the topoisomerase I inhibitor camptothecin linked to a water-soluble polymeric backbone methacryloylglycynamide ( average molecular weight 18 kDa, 10% CPT by weight). It was administered as a 30-min infusion once every 4 weeks to patients with advanced solid malignancies. The objectives of our study were to determine the maximum tolerated dose, dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document responses to this treatment. The starting dose was 30 mgm(-2) (dose expressed as mg equivalent camptothecin). In total, 23 patients received 47 courses at six dose levels, with a maximum dose of 240 mgm(-2). Dose-limiting toxicities were myelosuppression, neutropaenic sepsis, and diarrhoea. One patient died after cycle 1 MAG-CPT at the maximum dose. The maximum tolerated dose and dose recommended for further clinical study was 200 mgm(-2). The half-lives of both MAG-CPT and released CPT were prolonged (46 days) and measurable levels of MAG-CPT were retrieved from plasma and urine 4 weeks after treatment. However, subsequent pharmacodynamic studies of this agent have led to its withdrawal from clinical development

The presence of a systemic inflammatory response predicts poorer survival in patients receiving adjuvant 5-FU chemotherapy following potentially curative resection for colorectal cancer

There is increasing evidence that the presence of a systemic inflammatory response plays an important role in survival following curative resection for colorectal cancer. The present study evaluated the relationship between C-reactive protein concentrations and survival in a cohort of patients receiving adjuvant 5-fluorouracil (5-FU) chemotherapy following potentially curative resection for colorectal cancer. In all, 222 patients undergoing potentially curative resection for colorectal cancer were studied. Of these, 50 patients received adjuvant 5-FU-based chemotherapy. Circulating concentrations of C-reactive protein were measured prior to surgery. The minimum follow-up was 15 months; the median follow-up of the survivors was 38 months. During this period 61 patients died, 32 patients of their cancer and 29 of intercurrent disease. In those patients who did not receive adjuvant chemotherapy, age (P<0.001), Dukes stage (P<0.05) and an elevated C-reactive protein (P<0.01) were significantly associated with survival. In those patients who did receive adjuvant chemotherapy, an elevated C-reactive protein concentration (P<0.01) was significantly associated with survival. The presence of a systemic inflammatory response is an independent predictor of poor outcome in patients receiving adjuvant 5-FU-based chemotherapy following potentially curative resection for colorectal cancer

Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11

CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma

Secreted and tumour targeted human carboxylesterase for activation of irinotecan

Irinotecan (CPT-11) is an anticancer agent for the treatment of colon cancer. CPT-11 can be considered as a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. An approach to achieve tumour specific activation of CPT-11 is to transduce the cDNA encoding carboxylesterase into tumour cells. A secreted form of carboxylesterase may diffuse through a tumour mass and may activate CPT-11 extracellularly. This could enhance the antitumour efficacy by exerting a bystander effect on untransduced cells. In addition a secreted tumour-targeted form of carboxylesterase should prevent leakage of the enzyme from the site of the tumour into the circulation. We have constructed a secreted form of human liver carboxylesterase-2 by deletion of the cellular retention signal and by cloning the cDNA downstream of an Ig kappa leader sequence. The protein was secreted by transfected cells and showed both enzyme activity and efficient CPT-11 activation. To obtain a secreted, tumour-targeted form of carboxylesterase-2 the cDNA encoding the human scFv antibody C28 directed against the epithelial cell adhesion molecule EpCAM, was inserted between the leader sequence and carboxylesterase-2. This fusion protein showed CPT-11 activation and specific binding to EpCAM expressing cells. Importantly, in combination with CPT-11 both recombinant carboxylesterase proteins exerted strong antiproliferative effects on human colon cancer cells. They are, therefore, promising new tools for gene directed enzyme prodrug therapy approaches for the treatment of colon carcinoma with CPT-11

CYP2C19 pharmacogenetics in advanced cancer: compromised function independent of genotype

CYP2C19 is a drug-metabolising enzyme involved in the metabolism of a number of chemotherapeutic agents including cyclophosphamide. Variants of the CYP2C19 gene result in a loss of function polymorphism, which affects approximately 3% of the Caucasian population. These individuals are poor metabolisers (PM) of a wide range of medications including omeprazole (OMP). In healthy subjects PM can be identified through homozygous variant genotype. However, a discordance between CYP2C19 genotype and phenotype has been reported previously in a small study of cancer patients. To investigate whether CYP2C19 activity was decreased in patients with advanced cancer, CYP2C19 genotype was determined in 33 advanced cancer patients using PCR-RFLP analysis for the two important allelic variants (*2,681G>A and *3,636G>A) and the activity of the enzyme was evaluated using the CYP2C19 probe drug OMP. The activity of the drug-metabolising enzyme CYP2C19 was severely compromised in advanced cancer patients, resulting in a PM status in 37% of the patients who had normal genotype. This is significantly (P<0.0005) higher than that would be predicted from the genotypic status of these patients. There was no evidence of a correlation between compromised CYP2C19 activity and any of the proinflammatory cytokines or acute phase response proteins studied. However, there was preliminary evidence of an association between PM status and low body mass (P=0.03). There is increasing interest in using pharmacogenetics to ‘individualise medicine', however, the results of this study indicate that in a cancer population genotyping for CYP2C19 would significantly underestimate the number of phenotypic PM of drugs, such as cyclophosphamide, which may be metabolised by this enzyme