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Pharmacodynamic assays to facilitate preclinical and clinical development of pre-mRNA splicing modulatory drug candidates.

Shi Y, Joyner AS, Shadrick W, Palacios G, Lagisetti C, Potter PM, Sambucetti LC, Stamm S, Webb TR - Pharmacol Res Perspect (2015)

Bottom Line: We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression.Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments.The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, SRI International Menlo Park, California, 94025.

ABSTRACT
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

No MeSH data available.


Related in: MedlinePlus

In vivo measurement of spliceosome modulation by bioluminescent imaging BLI. (A) Athymic (nude) mice were injected s.c. with SK-MEL-2/Luc-MDM2 cells in both left and right flanks. Representative pictures of one animal treatment at different time points are shown. Either vehicle control (V) on the left frank or 12 mg/kg SD6 (S) on the right flank were intratumorally injected into mice. Mice were imaged at 0, 3, 6, and 24 h after injection. (B) Regions of interest (ROIs – red circles) were quantified with ImageQuant software. (C) SK-MEL-2/Luc-MDM2 cells were injected s.c. in right flanks of nude mice. Vehicle control or three dose levels of SD6 were injected intratumorally into established tumors. Mice were imaged 0, 3, and 6 h after injection of SD6 and ROIs were quantified, and dosed once daily for four more consecutive days. After 3 weeks, mice were sacrificed, tumors excised and weighed. P-values determined by Student’s t-test versus vehicle control are shown. * p = 0.02, ** p = 0.002, *** p = 0.001 (D) H&E stained sections of tumor tissues. Images were taken at 200× magnification.
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fig04: In vivo measurement of spliceosome modulation by bioluminescent imaging BLI. (A) Athymic (nude) mice were injected s.c. with SK-MEL-2/Luc-MDM2 cells in both left and right flanks. Representative pictures of one animal treatment at different time points are shown. Either vehicle control (V) on the left frank or 12 mg/kg SD6 (S) on the right flank were intratumorally injected into mice. Mice were imaged at 0, 3, 6, and 24 h after injection. (B) Regions of interest (ROIs – red circles) were quantified with ImageQuant software. (C) SK-MEL-2/Luc-MDM2 cells were injected s.c. in right flanks of nude mice. Vehicle control or three dose levels of SD6 were injected intratumorally into established tumors. Mice were imaged 0, 3, and 6 h after injection of SD6 and ROIs were quantified, and dosed once daily for four more consecutive days. After 3 weeks, mice were sacrificed, tumors excised and weighed. P-values determined by Student’s t-test versus vehicle control are shown. * p = 0.02, ** p = 0.002, *** p = 0.001 (D) H&E stained sections of tumor tissues. Images were taken at 200× magnification.

Mentions: We recognized an intriguing advantage of the SK-MEL-2/Luc-MDM2 stable reporter cell line in that it can be used for in vivo bioluminescent imaging (BLI) to rapidly and specifically assess the in vivo PD of splicing modulatory drugs. In conjunction with administration of the luciferase substrate d-luciferin, this enables real time non-invasive measurement of alternative splicing in an animal model. We report here that the SK-MEL-2/Luc-MDM2 stable cell line can form subcutaneous (s.c.) tumors in immunocompromised mice, and that these tumors respond to SD6, as measured by induction of the luciferase reporter compared to a vehicle control (Fig.4A). Using this model we established that an intratumoral injection of 12 mg/kg SD6 produced a marked increase in Luc activity, with 32-fold induction of signal observed 6 h post dosing (Fig.4B). We further observed increasing luciferase activity with increasing doses of SD6 by intratumoral injection (Fig.4C). No adverse effects were noted on the behavior or health of the animals during the study.


Pharmacodynamic assays to facilitate preclinical and clinical development of pre-mRNA splicing modulatory drug candidates.

Shi Y, Joyner AS, Shadrick W, Palacios G, Lagisetti C, Potter PM, Sambucetti LC, Stamm S, Webb TR - Pharmacol Res Perspect (2015)

In vivo measurement of spliceosome modulation by bioluminescent imaging BLI. (A) Athymic (nude) mice were injected s.c. with SK-MEL-2/Luc-MDM2 cells in both left and right flanks. Representative pictures of one animal treatment at different time points are shown. Either vehicle control (V) on the left frank or 12 mg/kg SD6 (S) on the right flank were intratumorally injected into mice. Mice were imaged at 0, 3, 6, and 24 h after injection. (B) Regions of interest (ROIs – red circles) were quantified with ImageQuant software. (C) SK-MEL-2/Luc-MDM2 cells were injected s.c. in right flanks of nude mice. Vehicle control or three dose levels of SD6 were injected intratumorally into established tumors. Mice were imaged 0, 3, and 6 h after injection of SD6 and ROIs were quantified, and dosed once daily for four more consecutive days. After 3 weeks, mice were sacrificed, tumors excised and weighed. P-values determined by Student’s t-test versus vehicle control are shown. * p = 0.02, ** p = 0.002, *** p = 0.001 (D) H&E stained sections of tumor tissues. Images were taken at 200× magnification.
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Related In: Results  -  Collection

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fig04: In vivo measurement of spliceosome modulation by bioluminescent imaging BLI. (A) Athymic (nude) mice were injected s.c. with SK-MEL-2/Luc-MDM2 cells in both left and right flanks. Representative pictures of one animal treatment at different time points are shown. Either vehicle control (V) on the left frank or 12 mg/kg SD6 (S) on the right flank were intratumorally injected into mice. Mice were imaged at 0, 3, 6, and 24 h after injection. (B) Regions of interest (ROIs – red circles) were quantified with ImageQuant software. (C) SK-MEL-2/Luc-MDM2 cells were injected s.c. in right flanks of nude mice. Vehicle control or three dose levels of SD6 were injected intratumorally into established tumors. Mice were imaged 0, 3, and 6 h after injection of SD6 and ROIs were quantified, and dosed once daily for four more consecutive days. After 3 weeks, mice were sacrificed, tumors excised and weighed. P-values determined by Student’s t-test versus vehicle control are shown. * p = 0.02, ** p = 0.002, *** p = 0.001 (D) H&E stained sections of tumor tissues. Images were taken at 200× magnification.
Mentions: We recognized an intriguing advantage of the SK-MEL-2/Luc-MDM2 stable reporter cell line in that it can be used for in vivo bioluminescent imaging (BLI) to rapidly and specifically assess the in vivo PD of splicing modulatory drugs. In conjunction with administration of the luciferase substrate d-luciferin, this enables real time non-invasive measurement of alternative splicing in an animal model. We report here that the SK-MEL-2/Luc-MDM2 stable cell line can form subcutaneous (s.c.) tumors in immunocompromised mice, and that these tumors respond to SD6, as measured by induction of the luciferase reporter compared to a vehicle control (Fig.4A). Using this model we established that an intratumoral injection of 12 mg/kg SD6 produced a marked increase in Luc activity, with 32-fold induction of signal observed 6 h post dosing (Fig.4B). We further observed increasing luciferase activity with increasing doses of SD6 by intratumoral injection (Fig.4C). No adverse effects were noted on the behavior or health of the animals during the study.

Bottom Line: We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression.Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments.The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, SRI International Menlo Park, California, 94025.

ABSTRACT
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

No MeSH data available.


Related in: MedlinePlus