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

Sequence determinants of innate immune activation by short interfering RNAs

BACKGROUND: Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated. RESULTS: A total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity. CONCLUSION: The data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes. Differential hydrolysis for the lactone and carboxylate forms

Irinotecan (CPT-11) is a new camptothecine derivative presently in development for the treatment of several advanced malignancies. It is converted in vivo to a highly potent metabolite, SN-38, by carboxylesterases. All camptothecine derivatives undergo lactonolysis in a pH-dependent reversible manner, generating inactive carboxylate forms. We have investigated in vitro the kinetics of transformation of CPT-11 to SN-38 by human liver microsomes originating from several donors. Microsomes from seven livers were studied individually or as a pooled preparation. CPT-11, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 formed was measured by HPLC with fluorometric detection. In the deacylation-limited carboxylesterase reaction, the linear steady-state kinetics between 10 and 60 min were determined. At all concentrations of CPT-11, the steady-state velocity of SN-38 formation as well as the intercept concentrations of SN-38 were about 2-fold higher when the substrate was under the lactone form than under the carboxylate form. We estimated the values (±SD) of K'(m) and V(max) to be 23.3±5.3 μM and 1.43±0.15 pmol/min/mg for the lactone and 48.9±5.5 μM and 1.09±0.06 pmol/min/mg for the carboxylate form of CPT-11, respectively. We conclude that the greater rate of conversion of CPT-11 lactone may contribute to the plasma predominance of SN-38 lactone observed in vivo. The inter-individual variation of SN-38 formation was relatively high (ratio of 4 between extreme values) but no large age- or gender-related differences were seen. The effect of twelve drugs of different therapeutic classes (antibiotics, antiemetics, antineoplastics, antidiarrhoeics, analgesics), which could be administered in association with irinotecan in the clinical setting, was evaluated in this system (drug concentration: 100 μM; CPT-11 lactone concentration: 10 μM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38

Conversion of irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38) by human liver carboxylesterase

We have investigated the conversion of the novel anti-topoisomerase I agent CPT-11 (irinotecan; 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), by human liver carboxylesterase (HLC). Production of SN-38 was relatively inefficient and was enzyme deacylation rate-limited with a steady-state phase occurring after 15-20 min of incubation. This later phase followed Michaelis-Menten kinetics with an apparent K(m) of 52.9 ± 5.9 μM and a specific activity of 200 ± 10 μmol/sec/mol. However, the total enzyme concentration estimated from the intercept concentrations of SN-38 was much lower than that estimated directly from the titration of active sites with paraoxon (0.65 vs. 2.0 μM, respectively). Because deacylation rate-limiting kinetics result in the accumulation of inactive acyl-enzyme complex, we postulated that incubation of CPT-11 with HLC would result in an inhibition of the HLC-catalysed hydrolysis of p-nitrophenylacetate (p-NPA), an excellent substrate for this enzyme. Indeed, this was found to be the case although complete inhibition could not be attained. Analysis of possible kinetic schemes revealed that the most likely explanation for the disparity in estimated enzyme concentrations and the incomplete inhibition of p-NPA hydrolysis is that CPT-11 also interacts at a modulator site on the enzyme, which profoundly reduces substrate hydrolysis. Furthermore, loperamide, a drug often used for the treatment of CFT-11-associated diarrhea, was found to inhibit both CPT-11 and p-NPA HLC-catalysed hydrolysis, most likely by a similar interaction. These observations have direct implications for the clinical use of CPT-11

Aging, acute oxidative injury and hepatocellular glucose transport in the rat

We studied the effects of aging and acute exposure to hydrogen peroxide on hepatocellular glucose transport to determine whether (1) acute oxidative stress impairs glucose transport, (2) aging is associated with reduced glucose transport and (3) there are similarities between these changes that may provide insight into the aging process. Glucose transport was measured in the perfused livers of young and aged rats using the multiple indicator-dilution method. There were significant reductions in the rate constants for glucose influx (P < 0.001) and a significant increase in the extracellular volume in the livers from aged rats (aged: 0.39 ± 0.05 ml/g, young: 0.27 ± 0.04 ml/g). However, the K and V for glucose influx in the livers from aged rats (44 ± 22 mM, 7.1 ± 1.4 μmol.sg, respectively) were not significantly different from the values in young rats (64 ± 20 mM, 8.8 ± 1.3 μmol.sg). In the livers of young rats, treatment with hydrogen peroxide caused a significant reduction in glucose transport from 1.18 ± 0.22 to 0.49 ± 0.25 μmol.sg. This was partly restored to 0.69 ± 0.20 μmol.sg by occluding the outflow catheter and expanding the extracellular space. Thus, although aging did not influence the rate of glucose transport in the perfused rat liver, this may be due to a compensatory age-related increase in the extracellular volume. In conclusion, the changes observed in hepatocellular glucose transport in the aged liver could be simulated by oxidative injury in the young liver, suggesting a role for oxidative injury in the aging process

Semisynthesis of RPR 121056A, a major metabolite of irinotecan (CPT-11)

The semisynthesis of RPR 121056A (4), a major metabolite of irinotecan (CPT-11, 2) is reported starting from SN-38 (3) and an appropriate side-chain precursor, and using a 2-step sequence. This semisynthesis is based on the 10-O-acylation of SN-38 with the conveniently protected carbamoylchloride derivative 10 followed by cleavage of the benzylic protecting groups by hydrogenolysis. Preliminary in vitro results show that RPR 121056A displays no cytotoxicity

Identification of a new metabolite of CPT-11 (irinotecan): Pharmacological properties and activation to SN-38

Irinotecan, or CPT-11 (7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecine), is a water-soluble derivative of camptothecine with promising activity against several types of malignancies. In addition to 7-ethyl-10-hydroxycamptothecine (SN-38), its active metabolite, we were able to identify several metabolites in the plasma of patients treated with this drug, especially an oxidative metabolite, 7- ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine. During our study of the biosynthesis of 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine from CPT-11 by human liver microsomes, we were able to detect another quantitatively important polar metabolite, which was also present in the plasma and urine of patients treated with CPT-11. On the basis of preliminary experiments, the structure of this compound was postulated to be 7-ethyl-10(4-amino-1- piperidino)carbonyloxycamptothecine, and this structure was synthesized by Rhone-Poulenc Rorer. Urine samples and human liver microsomal extracts were studied by high-performance liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry to identify its structure formally. The identification of the metabolite was supported by identical retention time, mass-to-charge ratio and tandem mass spectrometry fragmentation as a synthetic standard. Like irinotecan, 7-ethyl-10-(4-amino- 1-piperidino) carbonyloxycamptothecine was a weak inhibitor of cell growth of P388 cells in culture (IC = 3.4 μg/ml vs. 2.8 μg/ml for irinotecan and 0.001 μg/ml for SN-38). It was also a poor inducer of topoisomerase I- DNA cleavable complexes (100-fold less potent than SN-38). However, unlike 7- ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine, this new metabolite could be hydrolyzed to SN-38 by human liver microsomes and purified human liver carboxylesterase, though to a lesser extent than irinotecan. This compound can therefore contribute to the activity and toxicity profile of irinotecan in vivo