Downloaded 546 times
Uploaded byHtet Wai Moe
Screening methods for Teratogenicity
This document discusses various screening methods for detecting teratogenicity or the capacity of drugs to cause fetal abnormalities. It describes animal models like rat and rabbit studies that are used to test drugs during key stages of pregnancy. In vitro techniques like whole embryo culture, micromass assays, and stem cell tests are summarized as alternatives that can reduce animal use. The mechanisms by which certain proven human teratogens cause defects are outlined. Regulatory guidelines for preclinical teratogenicity studies and testing categories for drug use in pregnancy are also summarized.
More Related Content
Similar to Screening methods for Teratogenicity
Screening methods for Teratogenicity
- 1. Screening methods for Teratogenicity Capt.Htet Wai Moe 6th July 2017 1
- 2. Teratogenicity • Capacity ofa drug to cause foetal abnormalities when administered to the pregnant mother • Teratogenicity testing came into being since the thalidomide tragedy of 1961 • High demand for a rapid, reliable and cost- effective method for detection of teratogenic toxicity 2
- 3. Drugs can affectthe foetus at 3 stages 1. Fertilization and implantation- conception to 17 days (unnoticed failure of pregnancy) 2. Organogenesis- 18-55 days of gestation (most vulnerable period) 3. Growth and development- 56 days onward (developmental and functional abnormalities can occur) Teratogenicity 3
- 4. Teratogenic mechanisms Folate antagonism •Inhibition of the folate methylation cycle • E.g. many antiepileptic drugs, metformin, methotrexate, sulfasalazine, trimethoprim Neural crest cell disruption • Interference in molecular pathways involved in migration, differentiation, and proliferation of neural crest cells • E.g. Bosentan, isotretinoin, ketoconazole 4
- 5. Teratogenic mechanisms Endocrine disruption(sex hormones) • Affecting the release, binding or metabolism of endogenous sex hormones, particularly in male development • E.g. Drugs used in fertility treatment, oral contraceptives, phthalates in coatings 5
- 6. Teratogenic mechanisms Oxidative stress •Imbalance between generation of reactive oxygen species and antioxidant defence mechanisms, causing irreversible oxidation of DNA, proteins and lipids and affecting gene expression • E.g. Class III antiarrhythmic drugs, iron supplements, phenytoin, terbutaline, tetracyclines, thalidomide, valproic acid 6
- 7. Teratogenic mechanisms Vascular disruption •Disturbances in blood circulation in the uterine–placental unit, placental–fetal unit or the fetus itself, interfering with prenatal development of vasculature • E.g. Antihypertensive medication, aspirin, ephedrine, NSAIDs 7
- 8. Teratogenic mechanisms Specific receptor-or enzyme-mediated teratogenesis ACE and AT II receptors • Disruption of fetal renin–angiotensin system, producing fetal hypotension, vascular disruption and decrease in fetal renal vascular tone • E.g. ACE inhibitors, AT II receptor inhibitors 8
- 9. Teratogenic mechanisms HMG-CoA reductase •Down-regulation of Hedgehog proteins, which regulate embryonic growth, patterning and morphogenesis of many structures • E.g. Statins Histone deacetylases (HDACs) • Interruption of cell proliferation, differentiation and apoptosis through disregulation of gene expression by chromatin remodelling • E.g. Boric acid as inactive ingredient, salicylates, valproic acid 9
- 10. Teratogenic mechanisms Cyclooxygenase-1 • Inhibitionof COX-1, which catalyzes the conversion of arachidonic acid to prostaglandins • E.g. NSAIDs NMDA receptors • Errors in migration of neuronal and glial elements in the developing brain • E.g. Amantadine, dextromethorphan, ketamine 10
- 11. Teratogenic mechanisms 5-HT receptorsand transporters • Increased stimulation or suppression of 5-HT receptors; inhibition of 5-HT uptake by 5-HT transporters • E.g. Risperidone, SSRIs, sumatriptan GABA receptors • Enhancements of the effects of GABA, the major inhibitory neurotransmitter • E.g. Barbiturates, benzodiazepines 11
- 12. Teratogenic mechanisms Carbonic anhydrase •Reduction in embryonic intracellular pH • E.g. Acetazolamide, topiramate 12
- 13. Proven Human Teratogensand Abnormality Drug Abnormality Thalidomide Phocomelia, multiple defects Anti-neoplastic drugs Multiple defects, foetal death Androgens Virilization, esophageal, cardiac defects Progestins Virilization of female foetus Stilboestrol Vaginal carcinoma Tetracyclines Discoloured teeth, bone defects Warfarin Nose, Eye, Hand defects, Growth retardation Phenytoin Cleft lip/palate, microcephaly, hypoplastic phalanges
- 14. Alcohol Low IQ,Growth Retardation, Foetal alcohol syndrome ACE inhibitors Hypoplasia of organs, growth retardation, foetal loss Lithium Foetal goiter, Cardiac abnormality Anti-thyroid Drugs Foetal goiter, hypothyroidism Indomethacin Premature closure of ductus arterious Isotretinoin Craniofacial, Heart & CNS defects Proven Human Teratogens and Abnormality
- 15. FDA category ofdrugs used in pregnancy Category A: • Adequate studies in human demonstrate no risk Category B: • Animal studies indicate no risk, but there are no adequate studies in human • Animal studies show adverse effects, but adequate studies in human have not demonstrated a risk
- 16. Category C: • Apotential risk, when: – Animal studies have been performed or – Animal studies indicated adverse effects and – There are no data from human studies • These drugs may be used when potential benefits outweigh the potential risks FDA category of drugs used in pregnancy
- 17. Category D: • Thereis evidence of human fetal risk, but the potential benefits to the mother may be acceptable Category X: • Studies in animals or humans show adverse reaction reports or both have demonstrated fetal abnormalities • The risk of use in a pregnant woman clearly outweighs any possible benefit FDA category of drugs used in pregnancy
- 18. Schedule Y • Requirementsand guidelines for permission to import and/or manufacture of new drugs for sale or to undertake clinical trials • Female reproduction and Developmental Toxicity Studies – Appendix I, Item 4.4 – Segment I – Female Fertility Study – Segment II – Teratogenicity Study – Segment III – Perinatal Study 18
- 19. Animal toxicity requirementsfor clinical trails and marketing of a new drug Toxicity study Human phase for which study is proposed to be conducted Segment II (Teratogenicity Study) Phase II, III involving female patients of child-bearing age Segment III (Perinatal Study) Phase III • Drugs to be given to pregnant or nursing mothers for long periods • Drugs with indications of possible adverse effects on foetal development
- 20. One rodent (preferablyrat) One non-rodent (rabbit) Drug administered throughout the period of organogenesis Three graded doses • Highest dose – minimum maternal toxicity • Lowest dose – proportional to the proposed dose for clinical use in humans • Intermediate dose – logarithmically between two other doses Teratogenicity Study (Segment II)
- 21. Route of administrationsame as intended for human therapeutic use Control and treated groups At least 20 pregnant rats (or mice) and 12 rabbits, on each dose level Teratogenicity Study (Segment II) All foetuses subjected to gross examination Skeletal and visceral abnormalities
- 22. Foetuses • Total number •Gender • Body length • Weight • Gross/ visceral/ skeletal abnormalities Teratogenicity Study (Segment II) Observation parameters Dams • Signs of intoxication • Effect on body weight • Effect on food intake • Examination of uterus, ovaries & uterine contents • Number of corpora lutea • Implantation sites • Resorption
- 23. One rodent species(preferably rat) Dosing at levels comparable to multiples of human dose by intended clinical route At least 4 groups (including control) each consisting 15 dams Perinatal study (Segment III)
- 24. Dams sacrificed andexamined Then, dose that causes low foetal loss should be continued throughout lactation and weaning Perinatal study (Segment III) Drug administered throughout the last trimester of pregnancy
- 25. Treated with vehicleor test substance throughout their periods of growth to • Sexual maturity • Pairing • Gestation • Parturition • Lactation Perinatal study (Segment III) One male and one female from each litter of F1 generation (total 15 males and 15 females in each group) selected at weaning
- 26. Mating performance andfertility of FI generation should be evaluated Perinatal study (Segment III) Obtain F2 generation whose growth parameters should be monitored till weaning
- 27. Pups • Clinical signs •Sex-wise distribution in dose groups, • Body weight • Growth parameters • Gross examination • Survival • Autopsy Perinatal Study (Segment III) Observation parameters Dams • Body weight • Food intake • General signs of intoxication • Progress of gestation/ parturition periods • Gross pathology
- 28. In vitro techniques •Simple and cost-efficient • Can reduce pressure imposed by society to decrease or replace the number of animals used in research and testing strategies 28
- 29. Features of anideal in vitro teratogenicity test system • Simple, easy to perform, yield of interpretable results • Rapid, usage of large numbers of samples • Giving few false negative • Having relevance to mechanisms of teratogenesis • Involving some aspects of progressive development • Usable with various types of agents • Usage of intact organisms capable to absorb, circulate and excrete chemicals 29
- 30. In vitro techniques •Whole embryo culture test • Micromass teratogen test • Embryonic stem cell test • Dictyostelium discoideum based assay 30
- 31. Whole embryo culturetest Rodent embryo culture (rat or mouse) • Culturing of whole embryos at early stage of organogenesis • Exposing of these to a potential teratogen • Endpoints generally used – Mortality – Malformation – Growth inhibition 31
- 32. Whole embryo culturetest Zebrafish embryo • Cover the whole period of organogenesis • Easy to maintain in large stocks due to their high fecundity • Develop rapidly ex utero, with most organs becoming functional between 3 and 5 days- post-fertilization (dpf) 32
- 33. Whole embryo culturetest Zebrafish embryo • Transparency of larval zebrafish • Reasonably tolerant to concentration of dimethylsulphoxide (DMSO) generally used as a solvent at drug screening • High concordance with mammalian data 33
- 34. Fig. Anomalies inducedby retinoic acid treatment. The notochord and tail were normally formed in control group (A). The treated group (A’) exhibited kinked notochord (arrow) and kinked tail (arrowhead) 34
- 35. Fig. Anomalies inducedby retinoic acid treatment. The eyes and otic vesicles were normally formed in control group (B). The treated group (B’) exhibited deficiencies of eyes (arrow) and deficiencies of otic vesicles (arrowhead) 35
- 36. Fig. Anomalies inducedby retinoic acid treatment. The treated group (C’) exhibited facial and abdominal edema (arrowhead) 36
- 37. Micromass Teratogen Test •Chick, mouse and rat embryo midbrain or limb cell culture • Exposed to test compounds for varying times and concentrations 37
- 38. In vitro culturingof embryo limb or rather central neural cells Differentiation of the cells into chondrocytes or neurons starting from numerous small aggregates or foci of cells Micromass Teratogen Test Observation of cell adhesion, movement, communication, division and differentiation involving the new synthesis of tissue specific patterns of enzymes and structural proteins
- 39. Micromass Teratogen Test •A phase of in vivo embryo exposure will act as a control for the effects of drug metabolism and pharmacokinetics • Using cells from different organs and species to reproduce the in vivo sensitivity of particular embryonic tissues or species to teratogenic agents 39
- 40. • Pluripotent embryonicstem cells (ESC) isolated from mouse blastocysts • ESC are able to differentiate into a variety of embryonal tissues, retain the euploid chromosome constitution, and proliferate very rapidly Embryonic stem cell test 40
- 41. Embryonic Stem CellTest • Based on the resemblance between early embryonic stages and the differentiation of embryonic stem cells in vitro • Cell lines are used to identify cytotoxic, mutagenic, embryo toxic and teratogenic effects of chemical compounds 41
- 42. Embryonic Stem CellTest • Evaluation of cell differentiation is performed both morphological and via molecular techniques • Identification of predictive marker genes for the major target tissues during organogenesis 42
- 43. Dictyostelium discoideum • Single-cell,eukaryotic microorganism competent to undergo both vegetative growth and development (fruiting body formation) • Cell differentiation leading to the formation of stalk and spore cells has been shown to be similar, to some extent, to the development of mammals 43
- 44. Dictyostelium discoideum • Reportergenes serve as highly specific indicators for the developmental fate of a certain cell at a given time point • Easier to handle and less expensive • Rapid large-scale screening of chemicals 44
- 45. Conclusions • Both invivo and in vitro tests have the possibility of generating false negative or positive data • Several types of test systems are required to securely identify different classes of teratogenic agents 45
- 46. • Anital Kotwaniet al., 1995. Methods for teratogenicity testing – Existing and future models. IJP; 27: 204-213 • Van Gelder MMHJ et al., 2010. Teratogenic mechanisms of medical drugs. Hum Reprod; 29: 168-183 • Julia Schumann. 2010. Teratogen Screening: State of the Art. Avicenna J Med Biotech; 2(3): 115-121 • Wayne R Cohen, Phyllis August. 2013. Obstetric medicine, Management of Medical Disorders in Pregnancy. 6th Ed. Shelton: PMPH-USA Ltd; p.51-86 • Akhito Yamashita et al., 2014. Improvement of the evaluation method for teratogenicity using zebrafish embryos. J. Toxicol. Sci. 39(3), 453-464 References 46
- 47.
Editor's Notes
- #4 Type of malformation depends upon the drug as well as the stage at which exposure to the teratogen occurred Foetal exposure depends on the blood level and duration for which the drugs remains in maternal circulation The teratogenic potential of a drug is to be considered against the background of congenital abnormalities Women to avoid all medications in the first 8 weeks after conception Effects of teratogens, during the developmental period, results in an “all or none effect” Any substance that can induce or increase the incidence of a congenital malformation Recognition of human teratogens offers the opportunity to prevent exposure Drugs are classified as to their teratogenic potential, based on anecdotal information or animal studies Less than 2% of congenital malformations are caused by drugs or chemicals Teratogenic drugs should be avoided either during or prior to conception Frequency of congenital malformations in women exposed is greater This data is sometimes not available for humans & is not unbiased There are clearly species differences between teratogenic effects Recognition of human teratogens offers the opportunity to prevent exposure
- #5 Nucleic acid metabolism Folate coenzymes play a vital role in DNA metabolism through two different pathways. (1) The synthesis of DNA from its precursors (thymidine and purines) is dependent on folate coenzymes. (2) A folate coenzyme is required for the synthesis of methionine from homocysteine, and methionine is required for the synthesis of S-adenosylmethionine (SAM). SAM is a methyl group (one-carbon unit) donor used in most biological methylation reactions, including the methylation of a number of sites within DNA, RNA, proteins, and phospholipids. The methylation of DNA plays a role in controlling gene expression and is critical during cell differentiation. Aberrations in DNA methylation have been linked to the development of cancer (see Cancer). Amino acid metabolism Folate coenzymes are required for the metabolism of several important amino acids, namely methionine, cysteine, serine, glycine, and histidine. The synthesis of methionine from homocysteine is catalyzed by methionine synthase, an enzyme that requires not only folate (as 5-methyltetrahydrofolate) but also vitamin B12. Thus, folate (and/or vitamin B12) deficiency can result in decreased synthesis of methionine and an accumulation of homocysteine. Elevated blood concentrations of homocysteine have been considered for many years to be a risk factor for some chronic diseases, including cardiovascular disease and dementia (see Disease Prevention). Nucleic acid metabolism Folate coenzymes play a vital role in DNA metabolism through two different pathways. (1) The synthesis of DNA from its precursors (thymidine and purines) is dependent on folate coenzymes. (2) A folate coenzyme is required for the synthesis of methionine from homocysteine, and methionine is required for the synthesis of S-adenosylmethionine (SAM). SAM is a methyl group (one-carbon unit) donor used in most biological methylation reactions, including the methylation of a number of sites within DNA, RNA, proteins, and phospholipids. The methylation of DNA plays a role in controlling gene expression and is critical during cell differentiation. Aberrations in DNA methylation have been linked to the development of cancer (see Cancer). Amino acid metabolism Folate coenzymes are required for the metabolism of several important amino acids, namely methionine, cysteine, serine, glycine, and histidine. The synthesis of methionine from homocysteine is catalyzed by methionine synthase, an enzyme that requires not only folate (as 5-methyltetrahydrofolate) but also vitamin B12. Thus, folate (and/or vitamin B12) deficiency can result in decreased synthesis of methionine and an accumulation of homocysteine. Elevated blood concentrations of homocysteine have been considered for many years to be a risk factor for some chronic diseases, including cardiovascular disease and dementia (see Disease Prevention). Neural crest cells are a temporary group of cells unique to vertebrates that arise from the embryonic ectoderm cell layer, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. Bosentan is a dual endothelin receptor antagonist used in the treatment of pulmonary artery hypertension (PAH) Sulfasalazine, sold under the trade name Azulfidine among others, is a medication used to treat rheumatoid arthritis, ulcerative colitis, and Crohn's disease. It is often considered as a first line treatment in rheumatoid arthritis.
- #7 Class III Amiodarone, Sotalol, Ibutilide , Dofetilide, Dronedarone
- #9 The decrease in fetal renal vascular tone may contribute to a human malformation syndrome that is typical for exposure to ACE inhibitors during the second and third trimesters of pregnancy, characterized by renal tubular dysgenesis and oligohydramnios, their sequelae, including limb contractures and pulmonary hypoplasia, and hypocalvaria
- #10 The mevalonate pathway is a complex pathway with cholesterol as an essential product. In embryonic tissues, cholesterol is needed for normal growth patterns, signaling domains in plasma membranes, syn- thesis of steroid hormones and activation of Hedgehog morphogens. Since Hedgehog pro- teins act as key regulators of embryonic growth, patterning and morphogenesis of many structures, down-regulation of the synthesis of these proteins may lead to birth defects. Statins inhibit (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate pathway which converts HMG-CoA to mevalonic acid.
- #11 COX-1,is expressed in most tissues, where it produces prostaglandins that are necessary for various physiologic processes, such as blood pressure regulation and platelet aggregation. COX-2 expression, on the other hand, is induced by inflammatory mediators, producing prostaglandins which are important in inflammation Recent epidemiologic studies indicate an increased risk of orofacial clefts and cardiovascular defects, especially cardiac septal defects
- #12 5HT It is involved in a wide range of processes during development, including morphogenesis of craniofacial structures SSRI Sertraline · Citalopram · Fluvoxamine · Paroxetine GABA is hypothesized to play a morphogenetic role during embryonic development. use of benzodiazepines in the first trimester has been associated with orofacial cleft, cardiovascular malformations and gastrointestinal tract atresia
- #13 Carbonic anhydrases are metalloenzymes that catalyze the reversible hydration of CO 2 into the bicarbonate ion and protons. This reaction is involved in many biological processes, including pH homeostasis, respiration, biosynthetic reactions and bone resorption. Several cytoplasmic and membrane-bound carbonic anhydrase isoenzymes are expressed in various tissues in developing human and mouse embryos. Acetazolamide, which is used in the treatment of epilepsy, altitude sickness, edema and sleep apnea, have been associated with birth defects, especially limb deformities
- #19 APPENDIXI A DATA REQUIRED TO BE SUBMITTED BY AN APPLICANT FOR GRANT OF PERMISSION TO IMPORT AND / OR MANUFACTURE A NEW DRUG ALREADY APPROVED IN THE COUNTRY 4. Special studies conducted with approval of Licensing Authority . 4.1 Bioavailability / Bioequivalence and comparative dissolution studies for oral dosage forms 4.2 Sub-acute animal toxicity studies for intravenous infusions and injectables
- #33 Transparency of larval zebrafish make it possible to easily examine the function or morphology of each organ.
- #34 Transparency of larval zebrafish make it possible to easily examine the function or morphology of each organ.
- #40 The test system can include a phase of in vivo embryo exposure that will act as a control for the effects of drug metabolism and pharmacokinetics.
- #41 Pluripotentan= (of immature cell or stem cell) capable of giving rise to several different cell types. Euploid =ˈhaving an equal number of all the chromosomes of the haploid set.
- #42 Based on the resemblance between early embryonic stages and the differentiation of embryonic stem cells in vitro, Cell lines are used to identify cytotoxic, mutagenic, embryo toxic and teratogenic effects of chemical compounds
- #43 Evaluation of cell differentiation is performed both morphological and via molecular techniques (analysis of tissue-specific genes via reporter gene expression; automated high-through put screening for changes in gene and protein expression patterns using microchip arrays for transcriptome and proteome analysis) Identification of predictive marker genes for the major target tissues during organogenesis is crucial to get precise information on the teratogenic action of a test compound
- #44 Dictyostelium discoideum is a species of soil-living amoeba belonging to the phylum Amoebozoa, infraphylum Mycetozoa. Commonly referred to as slime mold.
- #45 Dictyostelium discoideum is a species of soil-living amoeba belonging to the phylum Amoebozoa, infraphylum Mycetozoa. Commonly referred to as slime mold. So far, four transgenic Dictyostelium discoideum strains have been constructed. Each of these strains expresses the reporter gene β-galactosidase under the control of a distinct developmentally regulated Dictyostelium discoideum promoter. The promoters in use are (i) cprB: active in both stalk and spore, but not in undifferentiated vegetatively growing Dictyostelium discoideum cells, (ii)pspA: active in spore cells, (iii)ecmA: active in prestalk cells (iv) ecmB: active in stalk cells. Hence, the reporter genes serve as highly specific indicators for the developmental fate of a certain cell at a given time point.