GB2421730A - Method for evaluating pharmacological target related toxicity - Google Patents

Method for evaluating pharmacological target related toxicity Download PDF

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GB2421730A
GB2421730A GB0525618A GB0525618A GB2421730A GB 2421730 A GB2421730 A GB 2421730A GB 0525618 A GB0525618 A GB 0525618A GB 0525618 A GB0525618 A GB 0525618A GB 2421730 A GB2421730 A GB 2421730A
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Ian Timothy Guy Pyrah
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AstraZeneca AB
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Abstract

Method for evaluating pharmacological target related toxicity in a mammal or in vitro based cell system wherein small interfering RNA (IRNA or RNAi or siRNA) is supplied to the mammal or cell to reduce target gene expression and detecting any change in at least one parameter of toxicity. The mammal may be a rat, mouse, guinea pig, hamster, pig, rabbit, dog, non-human primate or a human. The parameter of toxicity measured may include clinical observations, pathology, histopathology, chemistry, haematology, safety pharmacology or genotoxicity. The target may be a kinase, GPCR, ion channel or protease. Preferably the target is VEGFR-1, VEGFR-2 or VEGFR-3. Specific siRNA molecules are also claimed.

Description

1 2421730
METHOD
The invention relates to a method for the evaluation of target toxicity liability for use as a target-screening tool, particularly in pharmaceutical research and development.
Toxicity is a major cause of project failure and delay in the pharmaceutical industry. Some toxicities are related to target, whilst others are related to drug chemistry.
Understanding target liability allows a developer of pharmaceuticals to incorporate a major cause of failure in target selection, and also allows project teams to make rapid decisions as to whether toxicity can be avoided by altering chemistry. These factors are expected to reduce both attrition rates and project timelines.
RNA interference (RNAi) was first discovered in 1998 as a mechanism of post- transcriptional gene silencing in the nematode Caenorhabditis elegans (Nencioni). Since then, RNAi has been found to operate in diverse plant and animal species, including mammals (Nencioni). In some biological systems RNAi serves as a defence against viruses and transposons (Wilson).
Several groups have demonstrated that it is possible to promote RNAi in mammalian cells using short interfering RNA (siRNA) (Wilson, Wadhwa). Techniques for the introduction of siRNAs into cells have recently been established thereby allowing the sequence-specific inhibition of gene transcription; see for example Breakthrough; Small RNAs Make Big Splash, Jennifer Couzin, Science, Vol 298, pages 2296 - 2297, December 2002.
The induction of RNAi activity has quickly become an essential research tool for functional genomic analyses, for example large-scale RNAi-based loss-of-function screens have been reported in C elegans and Drosophila melanogaster and, more recently, in human cells. The second major application of RNAi of significance to the pharmaceutical industry is its potential as a candidate approach for development of highly specific dsRNA- based gene-silencing therapeutics (Nencioni, Dorsett & Tuschl, Scherr, Tuschl, Wadhwa, Wilson). To illustrate, RNAi has been used in identifying the key steps in the P53 pathway (Berns), protecting the liver against hepatitis virus infection through Fas inhibition (Radhakrishnan, Song) and as a model therapeutic agent to treat neurodegenerative disease (Caplen, Xia). * III * q**
* * . ** , S * * * * * * * S * * S S S * *a* .55 * Models in which to study target toxicity are challenging to prepare. For example, the production of a gene knockout animal is time consuming, expensive and impossible where gene knockout is embryolethal. For genes which are embryolethal, organ-specific conditional knockouts may be prepared, however this technique is even more time consuming and expensive. Thus, there is a need for more convenient, efficient and less costly methods of studying target toxicity.
Despite the massive interest in RNA1 among scientists, there has been no disclosure whatsoever regarding the use of RNAi in studying drug target toxicity liability.
The present invention is based on the discovery that RNAi technology can be applied in evaluation of target toxicity liability.
One aspect of the invention provides a method for evaluation of pharmacological target related toxicity in a mammal or in an in vitro cell-based model assay thereof comprising: i) administering RNAi in an amount sufficient to reduce target expression in the mammal or in an the vitro cell-based assay and ii) detecting whether there has been a statistically significant change in at least one parameter of toxicity, whereby to evaluate the pharmacological target related toxicity.
The specific inhibition of a gene using RNAi is anticipated to produce clinical and pathological changes similar to those seen with drug inhibitors of the gene products, and is analogous to the production of a gene knock out animal. The advantage of siRNA over gene knockout is the potential rapid turnaround with relatively lower costs, and the ability to dose adult animals in a mimic of a toxicity study (especially important for genes which are embryolethal). For example, the administration of siRNA specific for a kinase target with a well characterised pharmacological toxicity, with the growth plate disturbance associated with VEGFR-2 inhibition being a suitable example. The correlation between gene inhibition in a variety of tissues and the development of the expected histopathological changes would provide a convenient model of target related toxicity.
Preferably the method is carried out using a non-diseased or normal mammal or cell thereof. Preferably the method is carried out using a mammal in vivo. Preferably sufficient RINAi is administered to reduce target expression in at least one tissue of the mammal. Preferably the mammal is selected from any one of rat, mouse, guinea pig, e *.
* q** * * * 1 * : *.
I I * * I I * I * * I I I I * II' II. * * hamster, non-human primate, pig, rabbit, dog and human. Preferably the mammal is a human. Preferably the parameter is selected from any one of clinical observations, gross pathology, histopathology, clinical chemistry, haematology, safety pharmacology or genotoxieity. Preferably the target is selected from any one of a kinase, a GPCR, an ion channel or a protease. More preferably the target is a vascular endothelial growth factor receptor (VEGFR). More preferably the target is a kinase selected from any one of VEGFR-I VEGFR-2 or VEGFR-3. Preferably the parameter of toxicity is selected from any one of bone growth plate histopathology, reproductive organ histopathology or changes in a cardiovascular parameter. Preferably the cardiovascular parameter comprises measurement of heart rate or blood pressure, for example to measure hypertension.
Preferably the RNAi is selected from any one of siRNA or a vector-based siRNA expression system.
Preferably the siRNA administered is selected from the double-stranded RNA molecule arising from hybridisation of any one of; i) SEQ IDNO:l and SEQ IDNO:2, ii) SEQ ID NO:3 and SEQ ID NO:4, iii) SEQ ID NO:5 and SEQ ID NO:6, iv) SEQ ID NO:7 and SEQ ID NO:8, v) SEQ ID NO:9 and SEQ ID NO:l0, vi) SEQ ID NO: 11 and SEQ ID NO:12.
In an alternative embodiment RNAi may be substituted by antisense in the method as described hereinbefore.
There is also provided use of a target selected according to the above method in a drug screen. There is also provided a method of drug screening comprising; evaluation of pharmacological target related toxicity according to the method as described hereinbefore and screening potential pharmaceutical agents for activity against a pharmacological target evaluated according to said method.
The invention will now be described in the following non-limiting Examples.
General molecular biology techniques are described in "Current Protocols in Molecular Biology Volumes 1-3, edited by F M Asubel, R Brent and RE Kingston; published by John Wiley, 1998 and Sambrook, J. and Russell, D.W. , Molecular Cloning: A Laboratory * *q.
* ,* . . * : : : * * . * . * a. I I * * I I IS* I **. *. a Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Example 1
Typical Protocol for a 14 day In Vivo Rat study for VEGFR inhibition 1. Objective The aim of this study is to investigate the toxicity caused by daily oral, intravenous or other administration of RNAi for a period of 14 days in the rat.
2. Study Design The study is designed using current guidelines for repeat dose toxicity studies issued by the European Commission, the Japanese MHW and the US FDA.
The frequency and duration of dosing, and the recovery period, are described in section 5.
Main study animals; 5 male and 5 female controls, 5 male and 5 female dosed.
Recovery animals; 5 male and 5 female controls, 5 male and 5 female dosed.
3. Animals And Dosing Animals The rat is used in this study as this species has a well-established use in safety assessment studies and is accepted by regulatory authorities.
Strain: Han:Wistar substrain Crl:WI(Glx/BRIJHan)BR BrlHan:WIST@Mol Supplier: Charles River, UK M&B A/S, Denmark Age range at start of dosing = within 6 to 8 weeks.
Acclimatisation period: At least Iweek before first dose.
Randomisation: Animals are randomised by a method appropriate to the study design.
An animal number and related marking uniquely identify each animal within the study.
Housing, environmental conditions, enrichment, water and diet: Animals are multiple-housed in standard conditions. Water from the site drinking water supply and food (R&M No.1, SQC, pelleted diet, supplied by Special Diets Services Ltd., England) is freely available except during urine collection, when the latter is temporarily removed.
* * * * , : * II *: ; : * I * I $ * I * *1. I *a. .11 Dosing Intravenous (via a tail vein) or intraperitoneal. Frequency of administration: * Single low volume bolus injection repeated as necessary over a period of two weeks * One high volume bolus administered hydrodynamically into the tail vein at 0 hours and then repeated as necessary every three days for two weeks * Three high volume boluses administered hydrodynamically into the tail vein at 0, 8 and 24 hours and repeated as necessary every three days for two weeks * Continuous infusion of siRNA into the tail vein over a 24 hour period repeated as necessary every three days * Single/multiple intraperitoneal administration of siRNA It is likely for bolus intravenous techniques that the total volume of solution in ml will equal 10% of the animal's bodyweight, with the fluid introduced over a period of 7-10 seconds in phosphate buffered saline solution.
Main study animals will be dosed to the day prior to scheduled termination, which is completed during a 1 day period. Time of dosing: In the morning between 08.00 and 12.00 hours. Control animals are dosed with a dsRNA cocktail with no sequence homology to known rat genes.
RNAi molecules RNAi molecules are likely to be si (short interfering) RNA molecules, designed by reference to the sequence of the desired target gene. They are obtained either using internal company resource or via a commercial source e.g. Invitrogen/DharmaconlQiagen by requesting siRNA to target the Accession Number of the target gene. Algorithms are used to design a number of alternative siRNA sequences, which in use will exceed a minimum percentage gene knockdown. The set of empirical guidelines devised to aid effective siRNA design aim to ensure efficient binding, RISC (RNA interfering silencing complex) activation and accurate identification of the specific mRNA target prior to cleavage at a single site. They are: * Each strand must have 2-nt 3' overhangs **, , a * a I: I f * * : * al a * AIU at the 5' end of the sense strand and G/C at the 5' end of the sense strand * AU rich in the 5' terminal third of the antisense strand * Targeting of certain mRNA regions must be avoided. These are introns, 5' and 3' untranslated regions, regions within 75 bases of the start codon and sequences with >50% G and C content * Non-specific effects are avoided by performing a BLAST search of a genome sequence database to ensure that the chosen siRNA targets a single gene Examples of siRNAs targeting the VEGFR-2 gene are given below.
Stealth siRNA 1) GCCAAGUCCGAAUCCCUGUGAAGUA (SEQ ID NO:l) UACUUCACAGGGAUUCGGACUUGGC (SEQ ID NO:2) 2) GCAAGAGCAGAGACACUCUTJCAUAA (SEQ ID NO:3) UUAUGAAGAGUGUCUCUGCUCUUGC (SEQ ID NO:4) Quiagen 4 for I I) CGUUAAGCGGGCCAAUGAAUU (SEQ ID NO:5) UUCAUUGGCCCGCUUAACGGU (SEQ ID NO:6) 2) AAUGAUCGUUGGUGAUGAAUU (SEQ ID NO:7) UUCAUCACCAACGAUCAUUGU (SEQ ID NO:8) 3) CAUUGGUGAGACCAUCGAAUU (SEQ ID NO:9) UUCGAUGGUCUCACCAAUGGU (SEQ ID NO:1O) 4) ACGUGUCCGCAUUAUACAAUU (SEQ ID NO:1 1) UUGUAUAAUGCGGACACGUAG (SEQ ID NO:12) 4. Observations Clinical observations: All animals are thoroughly examined after arrival and before the start of dosing. Thereafter, the main study animals and recovery animals are observed daily in connection with dosing. Clinical observations at appropriate time intervals after dosing are performed on main study animals. A thorough examination for more permanent observations is performed once weekly before dosing. A check for moribund or dead animals is done twice on working days and at least once on weekends/national holidays.
Ophthalmology Group: animals are examined using an indirect ophthalmoscope, after * .** a * a *** * I I *I a S S S I I a. . a a.
* S I * S S S * S S a S S * S a.. . ... a,. a application of a mydriatic agent, once pre-test, mid term and during the last week of dosing. Group 2 animals are examined as required during the recovery period. Body weight: Body weights of all animals are recorded, starting the week before the start of dosing on day 1 and then twice weekly. Food consumption: Food consumption is measured for the animals in each cage of rats in all groups. Measurements are taken from the week before dosing starts until termination. The mean daily food consumption is calculated for each group and sex. Water consumption: Water consumption is measured for the animals in each cage of rats in all groups. Measurements are taken from the week before dosing starts until termination. The mean daily water consumption is calculated for each group and sex.
5. Clinical Pathology 5.1 Blood and urine sample collection schedules Animals to be bled: All animals as scheduled. Animals killed prematurely because of toxicity, are bled terminally for haematology and blood chemistry. Blood collection site and schedule: Blood for scheduled haematology and blood chemistry is taken from a tail vein during the last week of dosing and at the end of the recovery period. Blood for coagulation is taken from the heart at necropsy. Blood for immunotoxicology is taken from the vena cava. Blood sample volume and anticoagulant Blood chemistry: Interim bleed 0.6 ml (lithium heparin) or Terminal bleed 2.0 ml (lithium heparin) Haematology: 0.5 ml (EDTA) Coagulation: 1.0 ml (citrate). Animals for urine collection: All groups: Urine collection schedule and procedure: Individual urine samples are collected for approximately 6 hours, starting immediately post-dose, once mid term, during the last week of dosing and at the end of the recovery period. Collections may be taken on separate days for each sex. Water is freely available during the collection period but food will be withdrawn.
5.2 Sample analysis The following parameters are analysed. Other additional analyses may be included on a case-by-case basis.
5.2.1 Haematology Erythrocytes Platelets Haemoglobin Leucocytes Haematocrit Neutrophils Mean corpuscular haemoglobin Lymphocytes Mean corpuscular haemoglobin concentration * SIS * * S *** * . S IS I * *
S I S I S S IS
* I S S S S * S * I I S S SSS S 551 *I* S S Monocytes Mean red cell volume Basophils Red cell distribution width Eosinophils UK: Reticulocytes Large unstained cells.
5.2.2 Blood chemistry Albumin Glucose, Albuminlglobulin ratio UK: Glutamate dehydrogenase, Alanine Aminotransferase Potassium,Alkaline Phosphatase UK: Phosphate (inorganic), Aspartate Aminotransferase Sodium, Bilirubin (Total) Total protein, Calcium Triglycerides, Cholesterol Urea, Creatinine UK: Abnormal colour and lipaemia to be noted.
5.2.3 Coagulation Prothrombin time Activated partial thromboplastin time.
5.2.4 Immunotoxicology (as required) Lymphocyte subsets, NK cell activity.
5.2.5 Urine analysis Volume Ketones, Specific gravity Bilirubin, Osmolality, Blood pH, Cytological examination of urinary sediment, Protein Abnormal colour to be noted, Glucose.
6. Tissue inhibition of gene and protein expression Tissues collected for histopathological examination are studied for evidence of a reduction in target expression, either mRNA or protein, using such techniques as quantitative RT- PCR, Western blotting, or gel electrophoresis.
7. Pathology 7.1 Gross pathology Any premature decedents are necropsied. Necropsy procedure: Body weight is recorded at scheduled necropsy only. For all necropsies, external features are inspected and the cranial, thoracic and abdominal cavities and contents examined. Macroscopic abnormalities are recorded, a bone marrow smear will be prepared, and a full range of tissues will be weighed, as appropriate, fixed and preserved. Wherever possible, the likely cause of any premature death is established.
7.2 Organ weights The following organs are weighed intact and unfixed at scheduled necropsy; adrenal glands, brain, heart, kidneys, liver, lungs, ovaries, pituitary gland, prostate gland, spleen, * II* * I * *e* * I S II I I I I S
S I I S S S SI
* I S S S I S * I I I S * S S SII I *IS *I* * testes, epididymides, thymus and uterus for immunotoxicity assessment in draining and distant lymph nodes.
7.3 Microscopic pathology Samples of the following tissues are taken, as appropriate, at necropsy; adrenal glands, aorta (thoracic), bone and marrow - sternum, bone marrow smear, brain, cervix, epididymides, eyes, femur/femoro-tibial joint, harderian glands, heart, intestine - jejunum, intestine - ileum, intestine - colon, intestine - caecum, intestine rectum, kidneys, larynx, liver, lungs, lymph node - mandibular, lymph node - mesenteric, muscle - skeletal, nerve - sciatic, skin/site of mammary gland, oesophagus/thyroid and parathyroid glands/trachea, optic nerves, ovaries, pancreas, pituitary gland, prostate gland - ventral, salivary gland - parotid, salivary gland - sub-max/ling, seminal vesicles, spinal cord (mid-lumbar and cervical), spleen, stomach/duodenum, testes, thymus (or tissue from area), tongue, urinary bladder, uterus, vagina or abnormal tissues.
For parenteral studies sites of administration: Fixatives: Tissues are fixed and preserved according to SOP. Tissues are processed and examined by light microscopy. All preserved tissues will be processed to wax blocks. Blocks from all groups and any premature decedents are sectioned, stained, and examined according to SOP. Additional sections and stains: These may be requested by the study pathologist to determine the significance of any observed findings.
8. Statistical Analysis Routine analysis of data: Numerical data is analysed automatically by computer using Jonckheere's trend test and Wilcoxon Rank Sum Test. A statistician performs any additional analyses and methods used are recorded. Body weights, food consumption and clinical pathology parameters of main and recovery study animals are compared with those of the control group using the Dunnett's test generated by Artemis II. P-values reaching the 0.05 or 0.01 level are indicated. Calculation of Group mean values for final body weights and organ weights (absolute and relative organ weights) is performed by the PathData weighing programme. The statistical methods are presented in the PathData System Users Reference Manual.
* II* I I * *I* * S * ti I S * S S
S S I S S I US
* I I S I S S * . S S S S S S a.. S *I* Its I S
Example 2
In Vitro Study 1. Objective The objective is to inhibit target gene expression in a rat cell line using RNAi. Effective inhibition is demonstrated by a reduction in the expression of encoded mRNA andlor protein levels.
2. Study Design A rat cell line is used in order to mimic the corresponding in vivo experiment as closely as possible. The cell line is shown to express sufficient mRNA of the gene of interest to facilitate measurement of inhibition using RTPCR (Real Time Polymerase Chain Reaction). For example Rinm5F cell line expresses high quantities of VEGFR-2 mRNA.
These cells are grown in appropriate media in multiwell plates (for example 24 well) at an even confluency suitable for transfection with RNAi oligonucleotides. e.g. siRNA molecules. These molecules are obtained from a commercial (e.g. Dharmacon, Qiagen, Invitrogen) or in house source and are complexed with an effective transfection reagent to aid transfection of RNAi into said cells. Reagents used may be selected from lipid formulations (e.g. Lipofectamine 2000, RNAifect, Atugen lipids) and other related and non-related compounds (e.g. Transit TKO, Membrane permeant peptides). Additional transfection aids e.g. electroporation, magnetic assisted transfection may be employed as required.
Efficiency of transfection of the RNAi into the cells is assessed using various methods e.g. fluorescent-labelled oligonucleotides (siGLO, Dharmacon) or cytotoxic oligonucleotides (e.g. siTOX, Dharmacon) and transfection conditions are optimised using these gauges of transfection efficiency. Throughout the study numerous control experiments are also run using already optimised oligonucleotides targeting control genes e.g. GAPDH and scrambled oligonucicotides which target no known rat gene.
3. Analysis of results Measuring the levels of encoded mRNA and protein assesses the effectiveness of RNAi in reducing expression of the target gene. Parallel experiments are run; some cells for mRNA extraction measured by RTPCR and other cells for protein extraction measured using, for * ass * . * a..
a * s a. S * * S S a * a * S a 55 a a a a S a a * a * * a * a S aa. * as. as. * * -ii - examples, western blotting and immunohistochemistry. The RNAi oligonucleotide giving the greatest reduction in mRNA and protein levels of the gene of interest is chosen for use in future in vivo experiments.
References Berns, K., 2004, Nature, 428(698 1), 43 1-7.
Caplen, N., 2004, Gene Therapy, 11(16), 1241-8.
Couzin, J., 2002, Science, 298, 2296 - 2297.
Dorsett & Tuschl, Y., 2004, Nature Reviews; Drug Disc, 3,319.
Nencioni, A., 2004, Curr Opin Mol Thcrap, 6(2), 136-140.
Radhakrishnan, 2004, Virology, 323(2), 173-8 1.
Scherr, M., 2004, Cun Opin Mol Therap, 6(2), 129.
Song, E., 2003, Nature Medicine, 9(3), 347-5 1.
Tuschl, T., 2002, Molecular Interventions, 2(3), 158.
Wadhwa, R., 2004, Cun Opin Mol Therap, 6(4), 367.
Wilson, J., 2003, Curr Opin Mol Therap, 5(4), 389.
Xia, H., 2004, Nature Medicine, 10(8), 816.

Claims (13)

* II* * * S SS* * S S ** I I I I * I I * I I S SI * I S I I I * * I S S S S * I.. * **S SS* I * CLAIMS
1. A method for evaluation of pharmacological target related toxicity in a mammal or in an in vitro cell-based model assay thereof comprising: i) administering RNAi in an amount sufficient to reduce target expression in the mammal or in the in vitro cell-based model assay and ii) detecting whether there has been a statistically significant change in at least one parameter of toxicity, whereby to evaluate the pharmacological target related toxicity.
2. A method according to claim 1 wherein the mammal is non-diseased.
3. A method according to claim 2 wherein the method is carried out in a mammal in vivo.
4. A method according to claim 3 using a mammal in which sufficient RNAi is administered to reduce target expression in at least one tissue of the mammal.
5. A method according to claim 4 in which the mammal is selected from any one of rat, mouse, guinea pig, hamster, non-human primate, human, pig, rabbit and dog.
6. A method according to claim 5 in which the mammal is human.
7. A method according any preceding claim wherein the parameter is selected from any one of clinical observations, gross pathology, histopathology, clinical chemistry, haematology, safety pharmacology or genotoxicity.
8. A method according to any preceding claim wherein the target is selected from any one of a kinase, a GPCR, an ion channel or a protease.
* I*S * * * S** * * * IS * S S I S I * * * S I IS * * S a S S S * S S S S S S S SSS I 5(5 5(5 * S
9. A method according to claim 8 wherein the target is a kinase selected from any one of VEGFR-1, VEGFR-2 or VEGFR-3.
10. A method according to any preceding claim wherein the parameter of toxicity is selected from any one of bone growth plate histopathology, reproductive organ histopathology or a cardiovascular parameter.
11. A method according to any preceding claim wherein the RNAi is selected from any one of siRNA or a vector-based siRNA expression system.
12. A method according to claim 10 wherein the siRNA administered is selected from the double-stranded RNA molecule arising from hybridisation of any one of; i) SEQ IDNO:l and SEQ ID NO:2, ii) SEQ ID NO:3 and SEQ ID NO:4, iii) SEQ ID NO:5 and SEQ ID NO:6, iv) SEQ ID NO:7 and SEQ ID NO:8, v) SEQ ID NO:9 and SEQ ID NO:l0, vi) SEQ IDNO:11 and SEQ IDNO:12.
13. A method of drug screening comprising; i) evaluation of pharmacological target related toxicity according to the method of any one of claims 1-12, and; ii) screening potential pharmaceutical agents for activity against a pharmacological target evaluated according to i).
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