Medicinal Chemistry of Multiple Sclerosis: Focus on Cladribine

Background: In the recent years, many novel Disease-Modifying Drugs (DMD) have been introduced to the market in the treatment of multiple sclerosis. Objectives: To provide the reader with an up to date, compact review on the pharmacokinetic properties, mechanism of action, and clinical attributes of one of the most recently approved drugs in the therapy of multiple sclerosis, cladribine. Conclusion: Cladribine tablets proved to be a highly efficient treatment choice for Relapsing- Remitting Multiple Sclerosis (RRMS), especially for patients with high disease activity. It is the first DMD for MS with a complex mechanism of action, by inhibiting the adenosine-deaminase enzyme it increases the intracellular levels of deoxyadenosine triphosphate, which with relative selectivity depletes both T- and B-cells lines simultaneously. However long term follow-up safety and effectiveness data are still missing, and clear treatment protocols are lacking beyond the first two treatment years cladribine should prove to be a valuable addition to the therapeutic palette of RRMS, and potentially for Clinically Isolated Syndrome (CIS) as well.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca2+ sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched. RESULTS Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%–0.1% mutation abundance by use of Ion-Torrent and 0.1%–0.3% by use of Sanger sequencing. CONCLUSIONS Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.