Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates

Maria Eugenia Gallo Cantafio, Boye Schnack Nielsen, Chiara Mignogna, Mariamena Arbitrio, Cirino Botta, Niels M Frandsen, Christian Rolfo, Pierosandro Tagliaferri, Pierfrancesco Tassone, Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Boye Schnack Nielsen, Chiara Mignogna, Mariamena Arbitrio, Cirino Botta, Niels M Frandsen, Christian Rolfo, Pierosandro Tagliaferri, Pierfrancesco Tassone, Maria Teresa Di Martino

Abstract

Locked nucleic acid (LNA) oligonucleotides have been successfully used to efficiently inhibit endogenous small noncoding RNAs in vitro and in vivo. We previously demonstrated that the direct miR-221 inhibition by the novel 13-mer LNA-i-miR-221 induces significant antimyeloma activity and upregulates canonical miR-221 targets in vitro and in vivo. To evaluate the LNA-i-miR-221 pharmacokinetics and pharmacodynamics, novel assays for oligonucleotides quantification in NOD.SCID mice and Cynomolgus monkeys (Macaca fascicularis) plasma, urine and tissues were developed. To this aim, a liquid chromatography/mass spectrometry method, after solid-phase extraction, was used for the detection of LNA-i-miR-221 in plasma and urine, while a specific in situ hybridization assay for tissue uptake analysis was designed. Our analysis revealed short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. Up to 3 weeks, LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, together with p27 target upregulation. Importantly, no toxicity in the pilot monkey study was observed. Overall, our findings indicate the suitability of LNA-i-miR-221 for clinical use and we provide here pilot data for safety analysis and further development of LNA-miRNA-based therapeutics for human cancer.

Figures

Figure 1
Figure 1
Toxicity evaluation of Locked Nucleic Acid (LNA) LNA-i-miR-221 in mouse organs. Hematoxylin and eosin (H&E) staining of heart, liver, and kidney retrieved from mouse 1 week after one cycle (day 1, 4, 8, 15, and 22) of treatment. a, b, c, show representative areas of heart retrieved after saline (a), 25 mg/kg LNA-i-miR-221 (b) or 100 mg/kg course of treatments. d, e, f show representative areas of liver, while g, h, i kidney areas, after saline (d,g), 25 mg/kg (e,h) or 100 mg/kg (f,i) LNA-i-miR-221 treatment. Frames a, b, c, d, e, f are 100× while g, h, i are 200× magnification.
Figure 2
Figure 2
In situ hybridization (ISH) analysis of LNA-i-miR-221. Serial sections from formalin-fixed paraffin-embedded (FFPE) tissue samples of NOD.SCID mouse kidney after a single i.p. (intraperitoneal) injection of 25 mg/kg LNA-i-miR-221 (a–d) or saline (e), processed by ISH with Locked Nucleic Acid (LNA) probes for LNA-i-miR-221 (a, b, e), miR-126 (c), and miR-221 scramble (d). Intense LNA-i-miR-221 ISH signal is detectable in tubules at both low (5 nmol/l) and high (20 nmol/l) probe concentrations (a, b), whereas the probe results in no ISH signal in saline-treated kidney. The positive control probe, miR-126, stains endothelial cells including capillaries in the glomeruli (c), and only background staining is seen by the use of the scramble negative control probe (d).
Figure 3
Figure 3
In situ hybridization (ISH) detection of LNA-i-miR-221 in mouse tissues. LNA-i-miR-221 ISH on sections from organs retrieved from mice after 2 (I) and 7 (II) days of a single intraperitoneal (i.p.) injection of the LNA-i-miR-221 at therapeutic dose of 25 mg/kg or saline (III). Tissue sections from LNA-i-miR-221-treated (I and II) or saline-treated (III) liver (a), kidney (b), heart (c), bone marrow (d), and NCI-H929 xenograft tumor (e) samples after miR-221i probe hybridization. LNA-i-miR-221 staining is detectable in macrophages and/or vessels (liver and tumor), tubules (kidney), probably vascular cells (heart), subpopulation of hematopoietic cells (bone marrow). Bars: Liver, kidney, and heart: 100 µm; bone marrow: 12 µm; tumor: 50 µm.
Figure 4
Figure 4
Long-lasting LNA-i-miR-221 signal in mouse tissues. LNA-i-miR-221 in situ hybridization (ISH) in NOD.SCID mouse organs after injection of therapeutic doses of LNA-i-miR-221 at 25 mg/kg at days 1, 4, 8, 15, and 22. The organs were harvested at 1 week (I) or 3 weeks (II) after last injection. The staining intensity is high both after 1 and 3 weeks: (a) liver and (b) kidney maintain an intense signal throughout the period, while a slightly weaker signal is detectable in heart (c), bone marrow (d) and tumor tissue (e) 3 weeks after last dose. No ISH signal is seen in the organs from saline-treated animals (III). Bars: row I, II, and III (bone marrow and tumor) 50 µm, row III (liver, kidney, and heart) 25 µm.
Figure 5
Figure 5
LNA-i-miR-221 in situ hybridization (ISH) signal in mouse brain. LNA-i-miR-221 ISH in brain samples retrieved from mice after 2 (a) or 7 days (b) of a single intraperitoneal (i.p.) injection of the LNA-i-miR-221 at the therapeutic dose of 25 mg/kg or saline (c). Brain sections from LNA-i-miR-221 (a, b) or saline treated (c) animals using miR-221i probe (a, b, c), scramble negative control probe, (d) or positive control probe against miR-126 (e). Intense staining is seen in the liver (f) from the same mouse as in a. No nervous parenchyma showed detectable LNA-i-miR-221 ISH signal.
Figure 6
Figure 6
miR-221 inhibition by LNA-i-miR-221 induces p27 upregulation in mouse tissues. Immunohistochemistry (IHC) of p27 in liver and kidney mouse tissue from untreated mice (a, d) and from mice that underwent a course of 25 mg/kg LNA-i-miR-221 treatment retrieved after 1 (b,e) or 3 (c,f) weeks from last treatment. Increased p27 expression is showed in representative image of liver and kidney (80 and 90% of positive cells, respectively) from treated animals, compared with the same control tissue (60 and 50%) in liver and kidney, respectively. Heart tissue of untreated (g) and treated animals showing the same low expression of p27 (30%) at both time-points evaluated (h,i). MM xenograft tumors showed an increase of positive cells form 20% in the saline treated animals (l) to 40% after 1 week (m) and 60% after 3 weeks (n) from last injection. Positive cells were counted in 10 high-magnification random fields of the most representative areas; the degree of immunostaining for each antibody was expressed as the percentage of positive cells among the total number of cells. Representative image of different tissues are shown as 400× magnification.
Figure 7
Figure 7
Pharmacokinetics (PK) profile of LNA-i-miR-221 in mice. (a) Plot of plasma concentrations at different time-points for LNA-i-miR-221 indicates a rapid plasmatic clearance from 1.5 to 6 hours after single dose injection (25 mg/kg), followed by a distribution phase, till to almost no detectability 6 hours after injection; (b) plasma LNA-i-miR-221 concentration (ng/ml) at each time point from 1.5 to 24 hours; (c–d) ISH for LNA-i-miR-221 in xenograft tumors of untreated (c) and treated animals (d) retrieved after 12 hours of single injection of LNA-i-miR-221 (25 mg/kg). **: BLQ ≤ LLQ (LLQ = 25 ng/ml).
Figure 8
Figure 8
Pharmacokinetics (PK) profile of LNA-i-miR-221 in monkey study. (a) Plot of plasma concentration at different time-points for LNA-i-miR-221 indicates a rapid plasmatic clearance from 0.5 to 48 hours after single dose injection (875 mg/kg), followed by distribution phase, till to almost no detectablility after 48 hours; (b) plot of urine concentration versus four range of urine collection spanning from 2 hours to 48 hours, showing that most LNA-i-miR-221 excretion occurred within 6 hours; (c) plasma LNA-i-miR-221 concentration (ng/ml) at each time points (hours); (d) urine LNA-i-miR-221 concentration (ng/ml) at each time point. **: BLQ ≤ LLQ (LLQ = 50 ng/ml).

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