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Methods for determining the efficacy profile of a drug candidate

a drug candidate and efficacy profile technology, applied in the field of methods for determining the efficacy profile of a drug candidate, can solve the problems of inability to translatability the efficacy and toxicity profiles of drug candidates from rodent species to humans, and the use of nhp species in drug discovery remains a controversial matter

Pending Publication Date: 2019-05-23
F HOFFMANN LA ROCHE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While the in vitro testing of drug candidates allows to reduce the number of laboratory animals sacrificed for drug testing, it also posed challenges of outcome translatability from in vitro to in vivo and to the human physiology.
Indeed, the translatability of the efficacy and toxicity profiles of drug candidates from rodent species to humans can be challenging, due to non-close genetic relation between the species.
On the other hand, the use of NHP species in drug discovery remains a controversial matter.
However, there are formidable hurdles for gaining such primate inter-species transferable efficacy and toxicity data in vitro from stem cell-derived differentiated NCs: first, species specific cell culture protocols and non-transferability of cell culture conditions between primate species and, second, inhomogeneous distribution of the differentiating NPCs leading to non-optimal survival conditions or hampered differentiation effects due to local concentration of cells and autocrine and paracrine signalling leading to challenges in phenotypic assessment of drug effects.
Most notably, differentiated primate NCs are innate sensitive to cell culture conditions and do not tolerate harsh treatment which is an inherent obstacle to producing standardized assays with these cells.

Method used

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  • Methods for determining the efficacy profile of a drug candidate
  • Methods for determining the efficacy profile of a drug candidate
  • Methods for determining the efficacy profile of a drug candidate

Examples

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example 1

eotide Activity in Mouse Primary Neuronal Cell Cultures

[0310]Oligonucleotides targeting the part of SNHG14 long non-coding RNA which is antisense to the UBE3A pre-mRNA (position 55319 to 141053 of SEQ ID NO: 1) were tested for their ability to reduce the SNHG14 long non-coding RNA transcript preventing UBE3A expression (also termed UBE3A suppressor or UBE3A-SUP in the data table) and their ability to induce UBE3A mRNA re-expression in mouse primary cortical neuron cell cultures, obtained as described in the “Materials and methods” section above. The oligonucleotide concentration was 5 microM. The oligonucleotides were screened according to the protocol for screening in mouse cortical neuron cell cultures described in the section “Materials and methods”. The results are shown in table 4.

TABLE 4Oligonucleotide activity in primary mouse neuronal cell cultures.CMP% of Mock% of MockID NOoligonucleotideUBE3A_SUPsdUBE3Asd 95_1CTCAtacttgctttaAT 3.6 0.1154.115.1 95_2CTcatacttgctttaAT15.9 2.6...

example 2

eotide Activity in Human Neuronal Cell Cultures

[0311]Oligonucleotides targeting human SNHG14 in the region downstream of SNORD109B corresponding to position 25278410 to 25419462 on chromosome 15 (SEQ ID NO: 1) were tested in patient derived human neuronal cell cultures (see protocol in “Materials and methods” section). The oligonucleotides ability to reduce the SNHG14 transcript in the region downstream of SNORD109B (also termed UBE3A suppressor or UBE3A-SUP in the data table), without affecting expression of SNORD115 was analyzed. Furthermore, the ability to induce UBE3A mRNA re-expression was analyzed.

[0312]The oligonucleotides were screened according to the protocol for screening oligonucleotides in human neuronal cell cultures described in the section “Materials and methods” above.

[0313]The results are shown in table 5. The expression of UBE3A mRNA has been measured for all compounds, whereas the knock-down of the UBE3A suppressor and the maintenance of SNORD1115 levels have not...

example 3

n and Validation of Cyno Neuronal Differentiation In Vitro System

[0316]Cyno IPSCs were induced to differentiate into NPCs following the protocol as described in the Materials and Methods section and depicted in FIG. 4. The protocol allows to derive NPCs which can be maintained and expanded in basal medium supplemented with FGF, EGF and BDNF. To verify the efficient induction of cyno NPCs, expression of the neural stem cell markers SOX2 and NESTIN was evaluated by immunostaining (see FIG. 5A and FIG. 5B). Cyno NPCs express SOX2 and NESTIN and the expression pattern is highly comparable to human NPCs. To derive differentiated NCs, expanding NPCs are dissociated and plated in SFA medium for a week. Afterwards the cells are exposed to differentiation medium (BGAA). To evaluate the neuronal differentiation potential of cyno NPCs, a transcriptional analysis was performed and results were compared to the transcriptional profile of differentiated human NPCs (see FIG. 6A, FIG. 6B, FIG. 6C, F...

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Abstract

This patent application relates to a method for determining the in vitro efficacy profile of a drug candidate using standardized cell cultures of uniformly distributed differentiated neural cells (NCs) from at least two primate species, wherein the differentiated NC cultures are qualified for high throughput screening based on a dissociation and reseeding step performed on the differentiated NCs. The method includes differentiating human and / or non-human primate neuronal precursor cells (NPCs) to neuronal cells (NCs) followed by dissociating the differentiated NCs from its support and reseeding the differentiated NCs in a high-throughput cell culture format resulting in robust cultures suitable for high-throughput drug screening assays, in particular to screen antisense oligonucleotides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of PCT International Application No. PCT / EP2016 / 077435, filed on Nov. 11, 2016, the entire contents of which are incorporated herein in by reference, and which claims priority to European Patent Application No. 16189502.4 filed on Sep. 19, 2016 and European Patent Application No. 15194367.7 filed on Nov. 12, 2015.SEQUENCE LISTING[0002]This application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 20, 2018, is named P33890-US_SeqListing.txt, and is 554,456 bytes in size.FIELD OF THE INVENTION[0003]This patent application relates to a method for determining the in vitro efficacy profile of a drug candidate using standardized cell cultures of uniformly distributed differentiated neural cells (NCs) from at least two primate species, wherein the differentiated NC cultures are qualified for high throughput screening base...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50C12N5/0793C12N5/074
CPCG01N33/5014C12N5/0619C12N5/0696C12N2310/11G01N33/5058C12N2503/02C12N15/1137A61K31/7088A61P25/00C12N15/111C12N15/113A61P25/08A61P25/14A61P25/20A61P25/28C12N2310/20C12N2310/315C12N2310/321C12N2310/322C12N2310/3231C12N2310/33C12N2310/3341C12N2310/341C12N2310/346C12N2310/351C12N5/0623C12N2320/34C12N2500/38C12N2500/44C12N2501/01C12N2501/11C12N2501/115C12N2501/119C12N2501/13C12N2501/41C12N2501/60C12N2501/727C12N2501/999C12N2506/45C12N2533/52C12N5/0018C12N15/11
Inventor CUSULIN, CARLOCOSTA, VERONICAHOENER, MARIUSPATSCH, CHRISTOPHTHOMA, EVA CHRISTINA
Owner F HOFFMANN LA ROCHE INC
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