Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment

Abstract

Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance ( 31 P-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of 31 P-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro . Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct 31 P-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature.

Smad7 Antisense Oligonucleotide-Based Therapy in Crohn's Disease: Is it Time to Re-Evaluate?

“…Solution 31P-NMR spectroscopy, which has the unique ability to provide information about the chemical environments of the phosphorus atoms, showed that each mongersen preparation had a distinct 31P-NMR profile, indicating that PS chirality was not homogeneous across the batches used in the clinical trials. Principal component analysis helped identify clusters of the batches with similar 31P-NMR spectra, and, interestingly, preparations with the same 31P-NMR spectrum profile had a similar in vitro inhibitory effect of Smad7 [16]. Post hoc analysis of 411 patients enrolled in phase III and receiving mongersen for at least 4 weeks allowed to identify 12 cohorts, each receiving either a single batch (n = 5) or two mixed batches (n = 7) of the active compound (Fig.…”

“…One-way ANOVA followed by Tukey's post hoc test; batch G vs. batch V/X, * p < 0.05. C Correlation analysis between the mean CDAI score reduction in patients treated with the indicated batches and the ability of such batches to inhibit Smad7 protein expression in vitro (as reported by Arrico et al [16]) [p = 0.0043, Pearson's test coefficient r = 0.8127]. For CD cohorts receiving a mix of two different batches, the in vitro activity was calculated as the mean value of the in vitro activity of the single batches (designated as the percentage of reduction of Smad7/β-actin protein expression vs. control).…”

“…During the advanced clinical program, many batches of mongersen were manufactured and then used in the clinical trial without controlling PS stereochemistry. By assessing Smad7 RNA and protein content in colonic epithelial cells, we have recently documented a marked difference in the ability of those batches to downregulate Smad7, with some of them exhibiting no inhibitory action and most of them exhibiting minimal inhibitory effect compared with the batch used in the previous phase I and II studies [16]. It is plausible that throughout the clinical development program, there was a lack of reproducibility of the drug substance (DS) manufacture, which could not be detected during quality control testing and by employing several techniques aimed at characterizing the composition and chemical/physical properties of the batches.…”

“…1b), and the greatest reductions in the Crohn's Disease Activity Index (CDAI) score, a clinical symptom score commonly used in CD drug trials [17], were seen in CD cohorts treated with batches showing the more powerful in vitro activity (see Fig. 1c and Arrico et al [16]). At this time point, neither biochemical parameters of inflammation nor endoscopy were available to support the clinical data.…”

“…For the phase I and II studies, batch size was quite similar and the DS was prepared in a very short time frame. All these batches efficiently inhibited Smad7 expression in vitro [11,16]. All 411 patients were included in the analysis (intention-totreat population; last observation carried forward analysis).…”