review article thalidomide embryopathy: an enigmatic challenge

Hindawi Publishing CorporationISRN Developmental BiologyVolume 2013 Article ID 241016 18 pageshttpdxdoiorg1011552013241016

Review ArticleThalidomide Embryopathy An Enigmatic Challenge

Neil Vargesson

School of Medical Sciences Institute of Medical Sciences University of Aberdeen Foresterhill Aberdeen AB25 2ZD UK

Correspondence should be addressed to Neil Vargesson nvargessonabdnacuk

Received 9 July 2013 Accepted 18 August 2013

Academic Editors J M Hurle and G Tettamanti

Copyright copy 2013 Neil Vargesson This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Thalidomide remains one of the worldrsquos most notorious drugs due to the severe birth defects it induced in children between1957 and 1962 Yet to some this drug is a lifesaver as it now enjoys renaissance in the treatment for a wide range of conditionsincluding leprosy multiple myeloma Behcetrsquos disease and some cancers However thalidomide has also been linked to causing anew generation of thalidomide survivors in Brazil where the drug is used to treat leprosy Surprisingly how thalidomide causesbirth defects and how it acts in the treatment of clinical conditions are still far from clear In the past decade great strides in ourunderstanding of the actions of the drug as well as molecular targets have been made The purpose of this review is to look atthe recent work carried out into understanding how thalidomide causes birth defects itrsquos molecular targets and the challenges thatremain to be elucidated These challenges include identifying clinically relevant but nonteratogenic forms of the drug and themechanisms underlying phocomelia and species specificity

1 Introduction

11 History Thalidomide was produced and released as anonaddictive nonbarbiturate sedative in 1957 by Chemie-Grunenthal Thalidomide was marketed as very safe withno untoward side effects [1] and quickly became somethingof a wonder drug between 1957 and 1961 in the treatmentof a range of conditions in particular morning sicknessThalidomide was marketed in 46 countries under differentnames (eg Distaval in the UK and Australia Isomin inJapan Contergan in Germany and Softenon in Europe) [1ndash4] However soon after the drugrsquos release in Europe reportslinked thalidomide to causing peripheral neuropathy in adultpatients (which prevented its licensing and general releasein the USA) as well as being behind the occurrence of ahigh and sudden increase of rare birth defects [3 5ndash8] Therange and type of birth defects seen were unprecedented withthe most striking and stereotypic feature being phocomeliawhere the handplate remains but the proximal elements aremissing or very short Children also exhibited amelia in somecases (no limb) forelimb anomalies handplate anomaliesand other damage to ears eyes internal organs genitaliaand the heart [7 9ndash20] The damage caused by thalidomidewas not mutually exclusive with the majority of children

exhibiting damage tomultiple organs and tissues [3 7] It tookuntil 1961 when reports and concerns from two independentclinicians Lenz in Germany and McBride in Australiaconfirmed that ingestion of thalidomide during pregnancyto relieve morning sickness was the cause of the birth defects[3 4 7 11 12 21] Thalidomide was finally withdrawn inNovember 1961 Thalidomide-damaged children were stillborn throughout 1962 but the number of children affecteddramatically declined following thalidomide withdrawal [722] In total over 10000 children were born with severedamage worldwide [3 23]

Thalidomide was not licensed for use in the USA between1957 and 1961 as Dr Frances Kelsey a physician workingfor the US Food and Drug Administration (FDA) hadconcerns about the drugrsquos safety [5 24] Dr Kelsey wasconcerned about the peripheral neuropathy side effect thathad been experienced and reported in some patients inEurope following thalidomide exposure [3 5 25 26] DrKelsey undoubtedly prevented an epidemic of thalidomide-induced birth defects in the USA and for her efforts wassubsequently given the Presidentrsquos Award for DistinguishedFederal Civilian Service by President John F Kennedy in 1962[4] Questions still remain about the testing carried out onThalidomide before it was marketed in 1957 and whether

2 ISRN Developmental Biology

the testing was stringent enough as well as whether the disas-ter was preventable Following the thalidomide disaster newlegislation was introduced around the world that changed theway drugs are tested and their approval for human use espe-cially for pregnant women Indeed Dr Frances Kelsey pub-lished several papers on improving drug testing and safetywhich form the basis of drug testing carried out today [5 6]

12 Thalidomide Today In 1965 thalidomide was found to bevery effective in reducing the lesions associated with leprosyand (ENL) erythema nodosum leprosum a complication ofleprosy involving a chronic skin and nerve infection causedby Mycobacterium leprae [27] This discovery led to thalido-midersquos actions being studied in detail for clinical purposesReports linked the drug to having anti-inflammatory actionsand being effective as a clinical treatment for Leprosy ENLand other inflammatory disorders Furthermore studies indi-cated thalidomide was antiangiogenic and potentially usefulfor the treatment of cancers [28 29] Today thalidomide islicensed around the world (including the USA since 1998)for the treatment of leprosy and multiple myeloma (whereit can prolong patients lives by up to 18 months) [30 31]Thalidomide and its analogs are also used to treat a broadrange of other conditions including in the gastrointestinalsystem (eg Behcetrsquos disease Crohnrsquos disease) rheumatologi-cal system (eg lupus arthritis and sarcoidosis) and cancers(eg renal cell cancer prostate cancer and Karposirsquos sarcoma)[1 32 33] Recently thalidomide has been shown to beeffective in relieving the clinical symptomsof hereditary hem-orrhagic telangiectasia (HHT) a bleeding disorder [34] andidiopathic pulmonary fibrosis [35 36] Clearly thalidomidehas some very beneficial clinical uses However the dark sideof thalidomide still remains Thalidomide still causes birthdefects today particularly in Brazilmdashwhere several childrenhave been identified with thalidomide embryopathy in thepast few years [37ndash40]

13 Brazil Tragedy In recent years tragically a newgenerationof thalidomide survivors have been born and identified inBrazil [37ndash40] This is primarily due to a culture of medi-cation sharing less-stringent methods of prescription mis-understanding of the drugrsquos labeling and pregnant womentaking the drug while suffering from leprosy Sadly manychildren have been identified with thalidomide embryopathy[39 40]This Brazilian cohort provides an opportunity to fol-low these victims from childhood to adulthood usingmoderndiagnostic techniques Such studies could help determine theprecise range and type of damage at birth as well as late onsetdamage So far the Brazilian thalidomide survivors appear toexhibit a similar spectrum of damage as seen in the 1957ndash61disaster [39 40]

2 Thalidomide Embryopathy(or the Thalidomide Syndrome)

Between 1957 and 1961 thalidomide caused severe birthdefects in at least 10000 children [1 3] Although any tissueof the forming body could be affected by thalidomide some

hallmark features associated with the drug were identifiedincluding damage to limbs eyes ears genitalia and internalorgans including kidney heart and gastrointestinal tractAlso associated are vertebral column problems and facialpalsy [1 3 7 9 11ndash15 18 20 41 42] In addition another com-mon feature was the appearance of a transient haemangiomaon the forehead which usually disappeared within the first12ndash24 months of life [7] Due to the wide range of conditionsthis drug caused the damage is usually referred to collectivelyas thalidomide embryopathy or thalidomide syndrome [714 15] Infant mortality in babies born with thalidomideembryopathy is estimated to be as high as 40 quite likelydue to the serious internal malformations such as heart andkidney defects [3 7] Furthermore many babies with theseserious internal malformations would have been miscarriedor died in utero or soon after birth Thus the true number ofbabies affected by thalidomide will never be known

21 Thalidomide Acts in a Time-Sensitive Window of Em-bryonic Development Thalidomide causes damage to theembryo in a short timewindow of development (also referredto as the ldquocritical periodrdquo) between 20 and 36 days afterfertilisation or 34 to 50 days after the last menstrual period[7 11 25] Exposure to thalidomide from 36 days afterfertilisation has no apparent outward morphological effectupon the embryofetus [7 14] In contrast thalidomideexposure before the time-sensitive window of developmentcan inducemiscarriage in humans and rats [9 43] Incrediblyreports indicate just one 50mg tablet of thalidomide duringthe time-sensitive window is sufficient to cause birth defectsin 50 of pregnancies underlining the high teratogenicpotency of thalidomide [7 14 15 25] Some reports go furtherand suggest it would be surprising if any pregnancy wentunharmed following thalidomide exposure during the timesensitive window [7] The time-sensitive window was deter-mined following interviews with mothers of thalidomide-affected children and their doctors From these interviewsthe dates of intake and the amount of drug exposure werecalculated and an accurate correlation could be made tothe period of exposure and relationship to anomalies inorder to determine the timing of the damage [3 11 25] Theoutward morphological birth defects seen in thalidomideembryopathy are caused and correlate with exposure withinthis time period Exposure to thalidomide in the first fewdays of the sensitive period (days 20ndash24) affected the earsand eyes followed by the upper limb (day 24ndash31) and thenthe lower limb (days 27ndash33) respectively [11 25] Effects uponthe nerves resulting in facial palsies hearing loss and autismand epilepsy could be induced from the start of the timesensitive window presumably as the brainrsquos neural wiring isundergoing great changes at this time The consequence ofsuch nerve damage however may not be diagnosed untilmuch later after birth [11 25 42] Thalidomide exposure inlate fetal ratrsquos indicates that brain damage in areas related toautism can be a late event [44]

The time-sensitive window coincides with a period ofrapid embryonic development (from 4 weeks onwards) withlots of cell movements organogenesis and many signalling

ISRN Developmental Biology 3

pathways active Embryogenesis typically lasts until aroundweeks 10-11 after conceptionThusThalidomide use to relievethe symptoms of morning sickness (which can occur fromweek 4 onwards) coincides with the major developmentalevents in the embryo underpinning the thalidomide tragedy[1 3]

Exposure to thalidomide after the time-sensitive windowwas thought to have no effect upon the embryo Recent workhas suggested late exposure of rat fetuses to thalidomide(after the documented time-sensitive window of action in thelate fetal stages) affects angiogenesis in the brain and causesmalformations of the brain in areas linked to epilepsy andautism with no obvious limb or other outward morphologi-cal damage [44] Epidemiology studies in Sweden have shownthat thalidomide survivors have an increased incidence ofautism when compared to the average population [42 45]Together this work suggests there is unlikely to be a safe timepoint in pregnancy for exposure to thalidomide Exposurein the time-sensitive window results in a range of outwardmorphological damage with the severity and complexitydepending on timing of exposure Exposure after the time-sensitive window could affect growth and maturation ofinternal organstissues particularly the brain

22 Limb Defects Phocomelia is the most striking limbdeformity caused by thalidomide and remains the stereo-typical image of thalidomide embryopathy Phocomelia isa severe shortening of the limbs where distal elements(handplate) remains but proximal elements (long bones) arereduced or missing [7 14 15 46 47] Phocomelia itself canrange in severity The most severe form is where long bonesare missing with just a flipper-like structure consisting pri-marily of a handplate (though digit number can vary betweenaffected individuals) articulating directly with the body Lesssevere forms show for example a shortening of the longbones [7 14 39 46] The majority of thalidomide survivorsexhibit some formof limb deformity for example phocomeliato radial dysplasia to triphalangeal thumb [1 7 20 39] Limbdeformities are usually reduction defects and most oftensymmetrical [7 9 14 48] Indeed severe symmetrical limbdefects remain one of the classical hallmarks for diagnosisof thalidomide embryopathy The range and type of limbdeformities following thalidomide exposure are describedin the literature (originating from the 1960s) as exhibitinga characteristic pattern The thumb is the first structure tobe affected followed by the radius humerus and lastly theulna [7 9 14 15 49] These descriptions were based onobservations of babies and children with severe forms ofthalidomide embryopathy usually exhibiting bilateral limbdamage [7 15 17]

The lower limb can also exhibit limb deformities How-ever abnormalities of the lower limbs are seen less commonlythan those of the upper limbs with lower limb deformitieson their own being very rare The reasons for this upperlimblower limb sensitivity difference to thalidomide remainsunclear [14 16 48]The femur is the bone most often affectedin the lower limb and like the ulna in the upper limb thefibula is usually the final bone to remain normal [7]

Embryologically the radiustibia (preaxial) and ulnafibula (postaxial) form in the developing limbs after thehumerusfemur has begun to form The digits of thehandfootplate are the last structures to form Initially thestructures form as cartilage condensations before ossifyinginto bone [50ndash52] This sequence of limb developmentoffers some explanation as to why proximal structures areshortened ormissing but distal structures are present in somethalidomide survivors that is early exposure causes loss ofproximal elements However how any distal structures thenform or remain is unclear (also see discussion on chondro-genesis and origins of phocomelia later in this review)

Why the radius is more sensitive to thalidomide-damagethan the ulna is unclear The radius is smaller and thinnerthan the ulna and so perhaps easier to damage Howevervariations in deficiencies longitudinally have been suggestedto be related to the timing and duration of a teratogenicinsult during early limb development [53 54] It has alsobeen reported that teratogenic insults could induce ulnardeficiencies earlier in gestation than radial deficiencies [53]

Unilateral limb damage is not considered to be part ofthe classical thalidomide embryopathy phenotype Howeverthere are reports in the literaturementioning cases of thalido-mide survivors with unilateral limb damage [7 11 16 17 2055] Unilateral defects in general are very rare and how theycome about remains a mystery

23 Shoulder and Hip Joints Thalidomide survivors also ex-hibit damage to the shoulder and hip joints where the limbsarticulate with the body and are often weaker than normalThe acromioclavicular joint of the shoulder is more promi-nent and sharpened in appearance when the shoulder isdeformed [14]Thehip jointmay be hypoplastic or completelyabsent as is true for the pubic bone [14] Damage to theshoulder and hip joints is a characteristic of thalidomideembryopathy and in some cases has helped physicians to dif-ferentiate thalidomide deformities from sporadic or geneticlimb defects Adult thalidomide survivors have commentedon ongoing joint problems pain and rotation difficulties andearly-onset osteoarthritis [56 57]

24 Eye and Ear Defects Damage to the eyes and ears(internal and external) is also seen in thalidomide survivorsThe eyes and ears form from week 45 until around week89 around the same time the limbs are rapidly grow-ing Thalidomide can cause small eyes (microphthalmia)anophthalmos (absence of the eyeball) and poor vision Eyedefects also include aberrant lacrimation (tear formation)coloboma and strabismus [7 14 17 20 25 45 58] Oculardefects can occur unilaterally although there may still bepoor vision in the unaffected eye [17] Thalidomide mayalso cause abnormalities in eye movement and usually occurin conjunction with ear defects and weakness of the facialmuscles [7]

Ear defects are usually symmetrical and range from com-plete absence of the outer ear or pinna (anotia) to part of theouter ear still remaining (microtia) [7 59 60] Anotia is alsolinked to abnormalities of the external auditory meatus and


such children affected are deaf [60]Thalidomide-induced eardefects have also been associated with cranial nerve palsiesresulting in facial palsies [13 14 25]

25 Facial Damage Another common feature seen inthalidomide survivors again used as a diagnostic hallmarkis the presence of an enlarged naevus at birth (also known asa ldquostorkmarkrdquo) The naevus is in the centre of the foreheadextending down over the nose and upper lip This is acapillary haemangioma and is temporary usually no longervisible within 2-3 years of birth [7 14 15] Facial palsy andfacial asymmetry have also been associated with thalidomideembryopathy [7 14 15 20 25] and are thought to be dueto weakened facial muscles and facial nerve damage [7 25]In addition a range of other effects on facial structures havebeen seen in thalidomide survivors including irregular teethnumbersspacing small jaw cleft palate and cleft lip andsmall noses [7 14]

26 Vertebral Column Thalidomide survivors can exhibitspinal problems including irregular vertebral spacing Fusionof vertebrae is also seen in the lower spinal column andprogressive kyphosis which can often require surgical inter-vention later in life [7 14] Thalidomide survivors have alsobeen reported to exhibit shorter stature than average with theshortness attributable to short leg bones being independentof vertebral defects [7 14 61] How these spinal defects occurfollowing thalidomide exposure is unclear

27 Internal Organ Defects All of the internal organs can beaffected by thalidomide exposure in utero Common damageseen includes malformations of the heart kidneys genitalsand bowel [7] The precise incidence of such deformitiesis unknown as such defects cannot be seen outwardly andsome problems do not always present until later in lifeDefects of the heart were thought to be the cause for manyof the intrauterine and postnatal deaths Many thalidomidesurvivors have heart problems as well as pulmonary stenosisand patent ductus arteriosus [7 59]

The urinary tract and kidneys are affected in manythalidomide survivors with horseshoe hypoplastic androtated and ectopic malformations of the kidney seen [714 15 17] Many children also suffered from genital defectsboth internal and external Absence of the testes testicularabnormalities and hypospadiasis were seen in males whilstin femalesmalformations of the uterus and reproductive tractdefects were observed [7 16 17 62] Thalidomide survivorsexhibited problems in the intestines including anorectalstenosis intestinal atresia pyloric stenosis and inguinalhernia [7 9 14ndash17]

28 Nerve and CNS Defects Children with thalidomideembryopathy did not appear at the time to have impairedlearning disabilities [63 64] There is evidence that somethalidomide-damaged children have facial palsies cranialnerve conduction problems and an increased incidenceof autism and epilepsy in later life [7 25 42] It has beenproposed that thalidomide could affect forming nerve

pathways during and outside the time sensitive window[42 44 45] possibly by preventing angiogenesis in the brain[44] resulting in cell death and loss of tissue and autism

29 Variability of Thalidomide-Induced Damage betweenIndividuals An outstanding question and perhaps ongoingchallenge is to understand the variability of thalidomide-induced damage between individuals [1 7] Some reportsindicate thalidomide exposure in utero could result in a50 risk of a birth defect [7 14 15] Given the potencyof the drug how could any pregnancy be unharmed Whyare some pregnancies apparently unharmed Aside fromdrug exposure in utero outside the time-sensitive window ofaction we know that individuals react differently to drugsand we know individuals have different metabolic ratesBirth defects can be multifactorial in origin resulting fromcombined genetic and environmental influences [65] Thusthese influences (as well as time of exposure) could explainwhy some pregnant women are affected by the drug andothers are not and also explain differences in the severity ofthe damage

210 Thalidomide Embryopathy Can Sometimes Be Difficultto Diagnose Reports have indicated that some thalidomidesurvivors may have been misdiagnosed and in fact sufferfrom mutations in the SALL4 gene or in the TBX5 gene [66ndash68] SALL4 and TBX5 are both expressed in limbs Mutationin SALL4 causes Okihiro syndrome characterized by limbreduction deformities Mutations in TBX5 causes Holt-Oramsyndrome where heart and limb reduction deficiencies areapparent Both Okihiro syndrome and Holt-Oram syndromecan be difficult to differentiate from thalidomide syndrome asthey can look similar

In addition another genetic condition that can be con-fused with thalidomide embryopathy is Roberts syndromeRoberts syndrome is also called pseudothalidomide syn-drome as it shares striking resemblance to Thalidomideembryopathy in that the main feature is tetraphocomelia[69] Roberts syndrome patients also suffer from facial andinternal organ damage [70] Roberts syndrome is caused by amutation in the ESCO2 gene (Establishment of Sister Chro-matid Cohesion 2) [71 72] involved in proper segregation ofchromatids in cell divisionThis gene has since been found tobe important in control of cell proliferation cell death andDNA repair [73 74] The advent of genetic screening nowallows better diagnosis of such conditions

Thalidomide can phenocopy these genetic defects andfurther highlights the difficulty in sometimes differentiatingthalidomide embryopathy from other disorders This alsounderlines the difficulties in pinpointing precise diagnosticparameters of thalidomide embryopathy given the range ofdamage and variability between individuals Furthermorewhether these genes may be involved in some of the down-stream tissue specific effects of the drug is unclear (see latersection on molecular targets)

211 Summary Thedamage caused by thalidomide is strikingand widespread The criteria describe above varied between


individuals the most severely affected children exhibitedlimb malformations facial genital and internal organ prob-lems These criteria are used to help diagnose thalidomideembryopathyThe criteria were determined through studyingthe most severely affected thalidomide babieschildren usu-ally those with bilateral limb damage [7] Damage that mayonly have observable effects in later life such as joint damageor reduced organtissue function could not be properlydeterminedThus some damage may not have been apparentat that young age and may not have been associated as partof the condition as patients got older Furthermore there arerecords of children with confirmed thalidomide exposure inutero with no apparent outward morphological damage [20]Whether such children have internal organ damagefunctionissues is unknownThe precise range of damagemay never beknown However the Brazilian thalidomide survivors manyof whom being children offer the possibility of studying thedamage caused its progression and late onset problems

Today we are aware of left-right asymmetry differencesin early embryonic formation asymmetric differences inter-nally and genomic variation between individuals whichcould influence susceptibility to a drugrsquos influence [65] Howthalidomide influences these processes remains unclear butcould shed further light on themolecular and tissue targets ofthe drug Understanding thalidomide and its targets remainsa priority due to its widespread use in clinical treatmentstoday

3 Thalidomide Has Side Effects in Adults

Thalidomide is not only toxic to the forming embryo Ithas been known since the original release of thalidomidethat long term use can cause peripheral neuropathy inadults [5 75ndash77] Peripheral neuropathy is a condition wherenerves are damaged resulting in pain and hypersensitivitytypically in the extremities [78] How the drug induces thiscondition is unclear however patients are advised to stoptaking thalidomide if symptoms begin In patients usingthalidomide to treat leprosy and multiple myeloma manysuffer from peripheral neuropathy [76 79]

In addition thalidomide use causes constipationdizziness drowsiness and skin rashes in some patientsRecent regulatory advice warns that thalidomide can alsocause blood clots in veins and arteries and has recently alsobeen linked to inducing secondarymalignancies (httpwwwthalomidcom httpwwwfdagov httpwwwmhragovukSafetyinformationDrugSafetyUpdateCON123111 httpwwwmhragovukSafetyinformationDrugSafetyUpdateCON282739)

4 Lenalidomide and Pomalidomide NewThalidomide Analogs

New analogs of thalidomide have been developed that arepurported to be safer and more clinically effective thanthalidomide in the treatment of inflammatory disordersThese analogs lenalidomide and pomalidomide are usedto effectively treat multiple myeloma [80] Pomalidomide

appears to be more effective in treating patients in whichlenalidomide and thalidomide have lost effectiveness [81 82](Figure 1)

However lenalidomide use has been linked to causingperipheral neuropathy in patients taking lenalidomidefor multiple myeloma treatment [83 84] In additionlenalidomide but not pomalidomide is teratogenic in chickand zebrafish embryos [85] Furthermore both lenalidomideand pomalidomide are associated with causing teratogenesisin some mammalian species [86] (also see FDA websitehttpwwwaccessdatafdagovdrugsatfda docslabel2013204026lblpdf)Thebasis for these species-specific differencesin action is far from understood (see Species Specificitysection of this review for further discussion)

Clearly these compounds including thalidomide remaina risk to the unborn child and are only administered undera strict program which is specifically designed to preventthalidomide embryopathy For example the System forThalidomide Education and Prescribing Safety (STEPS) is aprogram that educates clinicians and patients and carries outregular contraceptive counseling and pregnancy testing [87ndash89] However presently the other side effects (eg peripheralneuropathy) are sadly part of the risk-benefit decision asso-ciated with the drugrsquos health benefits It remains a primarygoal to ascertain if all such side effects can be prevented orminimized Whether new or alternative forms of the drugcan be made retaining the clinically beneficial aspects andwithout the side effect aspects is an ongoing challenge

5 Biochemistry of Thalidomide

Thalidomide is a derivative of glutamic acid a nonessentialamino acid with important functions in the brain and duringmuscle development Thalidomide exists as an enantiomerand can switch between its two chiral states in body fluidsand water [29] (Figure 1) It has long been assumed thatone form is the teratogenic causing agent and the other isthe sedative agent However the fact they can interchangeindicates just the safe form cannot be given clinically [29 9091] Thalidomide is metabolically broken down in the liverby cytochrome P450 (CYP2C19) enzymes and can also breakdown hydrolytically in body fluids Thalidomide has a half-life of 6ndash12 hrs [29] Furthermore there are species differencesin efficiency of breakdown of drug into byproducts forexample the rabbit is more effective at breaking downthalidomide than the rat [92 93] This could help explain thespecies differences in action of this drug (see later)

51 Inflammation Thalidomide has been shown to inhibitTNF120572 transcription directly through enhanced TNF120572mRNAdegradation [94] and COX2 inhibition [95] in monocytesand macrophage cells TNF120572 regulates and controls theinflammatory response through inducing cytokines suchas interleukins (eg IL-1 and IL-6) in response to injuryor stimuli Several autoimmune diseases arise from theoverproduction of unnecessary cytokines in healthy tissueleading to potentially debilitating conditions such as ery-thema nodosum leprosum (ENL) multiple myeloma (MM)


Thalidomide

N

NH

O

O

O

O

lowast

(a)

CPS49

F

F

F

F

F

F

O

O

N

(b)

Lenalidomide

O

O

ON

NH

NH2

(c)

Pomalidomide

O

O

OO

N

NH

NH2

(d)

Figure 1 Structures of thalidomide and its structural analogs Thalidomide (a) is enantiomeric and can exist in two chiral states (asteriskdenotes chiral centre) CPS49 (b) is a structural analog based on the antiangiogenic breakdown product of the thalidomide molecule and isfluorinated to enhance bioactivity and stability [1 29 54] Lenalidomide (c) and pomalidomide (d) are based on the thalidomide structureand used to treat multiple myeloma

and Crohnrsquos disease [96 97] The inhibition of TNF-120572 bythalidomide makes this drug excellent in the treatment ofthese autoinflammatory diseases

52 Angiogenesis Thalidomide was first demonstrated to beantiangiogenic in 1994 through destroying angiogenic vas-cularization in rat cornea induced by fibroblast growthfactor (FGF) protein [28] This finding led to the suggestionthat thalidomide may cause teratogenic damage by affectingblood vessels Indeed this discovery has made thalidomidea candidate for treatment of early-onset cancers to preventearly cancerous growthtumors acquiring a vasculature togrow [29 90 91] Thalidomide is also used to treat multiplemyeloma which is thought to be due to excessive vascular-ization of bone marrow tissue andor due to an overactiveinflammatory response [98] and also treat hereditary hem-orrhagic telangiectasia (HHT) [34]

Use of CPS49 an antiangiogenic analog of thalidomidedemonstrated that thalidomidersquos antiangiogenic action causesits teratogenic actions in a time-sensitive manner [54]

6 Angiogenesis and Mechanism ofThalidomide-Induced Teratogenesis

61 Role of Blood Vessels in Embryogenesis Blood vesselsare essential for normal embryonic formation Blood vesselssupply oxygen and nutrients to growing tissues and removeunwanted waste products Blood vessels form by two mainprocesses in the embryo vasculogenesis and angiogenesis(Figure 2) Vasculogenesis is where the first primitive bloodvessels form Endothelial cells migrate toward each other andcoalesce to form vascular tubes Angiogenesis is where these

primitive vascular tubes are built upon and elaborated to formthe complex networks of vessels throughout all tissues of theembryo Endothelial cells proliferate andmigrate to avascularareas in response to signals such as hypoxia or vascularendothelial growth factor Endothelial cells then quiesce andthe vascular tube recruits vascular smoothmuscle cells whichstabilise and strengthen the vessel In order to undergo laterangiogenesis to further elaborate the network or simply refinethe network the smooth muscle coating is lost to allowendothelial cells to migrate and form new vascular tubes(Figure 2) [99 100] As embryonic development proceedsblood vessels undergo constant rounds of angiogenesis andstabilisation to accommodate the rapid tissue changes andgrowth

Loss of or inhibition of blood vessels during embryoge-nesis can result in death or severe embryonic developmentconsequences [99ndash102] For example loss-of-function exper-iments of genes involved in vascular formation (eg VEGFand Notch) in mouse embryos cause lethality as the vascu-lature fails to form [99 100 103ndash105] in zebrafish Notchsignalling pathwaymutants display vascular disruption in theforming somites and spine of the embryo resulting in severespinal curving of the embryo [106 107] Blood vessels areimportant for normal embryonic development and their lossunsurprisingly results in death or birth defect [1 52 100 102]

62 Blood Vessels Are Targeted by Thalidomide in EmbryonicDevelopment Thalidomide has antiangiogenic actionswhichhas been proposed to play a role in thalidomide teratogenesis[1 2 28 108ndash110] Thalidomide can break down into manybyproducts Many stable structural analogs of thalidomideand its breakdown products have been synthesised and can


Vascular smooth musclerecruitment and stabilisation

ThalidomideAngiogenesis

Smooth muscle cell

Angiogenic endothelial cellsproliferate migrate form new

networksconnections and stabilise

Endothelial cell

Figure 2 Angiogenesis and the action of thalidomide uponangiogenesis Primitive vessels form through vasculogenesis whereendothelial cells coalesce to form primitive tubes through whichblood cells pass through Angiogenesis is the process which elabo-rates upon these early and newly formed vessels to form vascularnetworks throughout tissues and the embryo Angiogenesis isessential for formation of the embryo and continues into adult-hood for example wound healing As vessels form they recruitvascular smooth muscle which stabilises the vessel into a matureand quiescent state Angiogenesis is induced by signals such ashypoxia inducible factor-1 (HIF1) vascular endothelial growthfactor (VEGF) and transforming growth factor-120573 (TGF120573) Maturevessels will shed their vascular smoothmuscle coats and endothelialcells will proliferate and migrate to the source of the signals andform new vessels Thalidomide targets the immature unstablenewly formed vessels preventing proliferation and migration of theendothelial cells In rapidly growing tissues eg the forming limbvessel loss will be devastating as cell death will be induced andsignalling pathways will be disrupted

be screened to determine their function shedding light onwhich aspect of thalidomide action antiangiogenesis or anti-inflammation results in teratogenesis [29 54 109ndash111]

CPS49 is one such stable antiangiogenic analog ofThalidomide (Figure 1) CPS49 is a tetrafluorinated analogbased on the antiangiogenic breakdown product of thalido-mide 51015840-OH-thalidomide [1 29] Fluorination confers stabil-ity and increased biological activity [1 29 110 111]

Following CPS49 treatment in chick embryos as limbsare just forming (E25) blood vessels were destroyed within1 hr of exposure in the embryo and several hours before anychanges in limb signalling gene expression and cell deathwasobserved (Figure 3) Reduction in limb area was seen within6 hrs Truncated limbs were clearly observable by 24 hrswhere increased cell death was observed and loss of FGF andSonic hedgehog (Shh) signalling was seen FGF and Shh arekey signalling molecules in the developing limb controllingpatterning and outgrowth (Figure 3) Thalidomide has alsobeen reported to cause loss of FGF and Shh signaling inchick and rabbit embryonic limbs and in zebrafish embryos[112ndash114] In CPS49 treated embryos resulting limbs werephocomelia-like 7 days after just a single exposure (Figure 3)Even more interesting is that CPS49 acts in a time-sensitivewindow just likeThalidomide where early exposure resulted

Shh

Humerus

Radius

Digits

ZPAUlna

AER

Fgf8

(a) Normal Development

Cell deathBV

Lossreductionof

proximal structures

AER signalling recoveryafter thalidomide exposure allows distal structure formation

Shh

Fgf8

Thal

idom

ide

(b) Phocomelia

Figure 3 Limbdevelopment and thalidomide-induced phocomelia(a) Normal limb development The limb grows out from theembryonic flank under the control of several signalling regions theZone of Polarising Activity (ZPA) and the Apical Ectodermal Ridge(AER) These signalling regions release signalling molecules Shhand Fgf8 respectively which signal in a feedback loop and controllimb outgrowth and patterning Ultimately other genes are activatedincluding Hox genes which are involved in patterning the finedetail of the cartilage elements [51 52] (b) Model of thalidomide-induced phocomelia Thalidomide inhibits blood vessel formationand migration resulting in cell death and reduced signallingbetween the ZPA and AER Once thalidomide exposure has ceasedor limb has recovered AERZPA signalling could be reestablishedand remaining cells distalised to produce a phocomelic limb Ifthalidomide exposure was long term AERZPA signalling may becompletely abolished resulting in Amelia

in severely truncated limbs and later exposure caused lesssevere damage for example loss of a digit or digit tips onlySimilarly in zebrafish embryos blood vessels are stunted andlost rapidly following CPS49 treatment Zebrafish embryosoffer the advantage of being able to visualise vessel formationand migration ldquolive and in vivordquo and effects of drug uponvessel formation andmigration CPS49 was shown to preventendothelial cells from proliferating and migrating to formnew tubes [54] The action of CPS49 on the endothelial cellis through preventing cytoskeletal changes and inhibitionof filopodial tip cell formation and migration [54] It wasfurther demonstrated using in vitro rat andmouse aortic ringculture assays that blood vessels possessing smooth musclecoats are protected from the action of the drug but thosewithout smooth muscle are destroyed This indicates thatnewly formed and forming blood vessels are susceptible tothalidomide Established quiescent vessels are unharmed Inthe chick embryo the developing limb vasculature does notpossess smooth muscle until quite late in limb development[54 115] This observation correlates with the range of limbdefects seen depending on the timing of exposure Severedefects are seen after early exposure but by the time smoothmuscle has appeared on the limb vasculature defects arerestricted to digits [54] In contrast to the limb at the timeof exposure (E25) microvessels in the brain and vessels in


the neck and body of the embryo possess smooth musclecoats intimating that these vessels are quiescent and protectedat this time When the drug was applied to very youngembryos (E15) whenmost tissues are being vascularised andorganogenesis is occurring embryos died [54]

Several nonantiangiogenic analogs and hydrolysis prod-ucts tested using the same assays were found not to beteratogenic or harmful to embryos [54] This work indicatedthat the antiangiogenic action of the drug causes a wide rangeof limb defects in a time-dependent manner

A study using a different fluorinated analog of thethalidomide molecule (fluorothalidomide) in mouse andrabbit embryo cultures gave a range of damage that dif-fered from that exhibited following thalidomide exposure(eg no obvious or apparent limb defects) and also brokedown very rapidly [116] The work suggests fluorination ofcompounds changes their activity [116] Embryo culturesideally last around 2 days and as such it can be difficultto determine the precise range and type of damage causedby drugs in such embryos (certainly whether limbs exhibitlimb reduction phenotypes ormissing cartilage patterns)Thefluorothalidomide compound appears unstable and couldhave broken down before it could cause observable damageto limbs Whether there were any effects on limb signallingpathways (eg FGF Shh etc) changes in cell death orchanges in blood vessel patterning in the early limbs andembryos is unreported CPS49 is fluorinated and based on thestructure of the active antiangiogenic breakdown product ofthalidomide and induces a range of damage and molecularchanges in embryonic chick limbs and in vitro assays whichis consistent with reports from other groups and work usingthalidomide [112ndash114 117ndash120]

Several other studies have also demonstrated that bloodvessels are targeted and destroyed by thalidomide [28 44108 119 121] These include rat and rabbit corneal assayswhere blood vessels induced in response to an FGF soakedbead are destroyed following thalidomide application [28108] In rat fetuses exposed to thalidomide late in gestationblood vessels are disrupted which results in brain changesin areas relating to autism [44] In chick embryos nitricoxide a molecule required for endothelial cell function andthat appears to be protective of blood vessels is inhibitedby thalidomide causing vessel loss Overexpression of nitricoxide rescues andor prevents thalidomide-induced damage[119 121 122] In zebrafish embryos thalidomide causes bloodvessel defects [54 123] through VEGF receptor depletion[123]Thalidomide has also been shown to stabilise hereditaryhemorrhagic telangiectasia (HHT) in adult patients whereldquoleakyrdquo vessels stabilise through recruitment of smooth mus-cle and inhibition of angiogenesis [34]

Furthermore it is known that other antiangiogenic drugsare teratogenic for example sodium valproate [102 124]Moreover loss of blood vessels in early development is usuallylethal or causes serious deformitydamage [1 52 54 100 102115]

Many important molecules involved in embryonic vas-cular development have been reported to exhibit changedexpression patterns following thalidomide exposure forexample actin and tubulin integrins vascular endothelial

growth factor PDGF120573 nitric oxide ceramide angiopoietinsand reactive oxygen species [28 34 114 121ndash123 125ndash127]

Altogether there is a large body of evidence indicating arole for blood vessel damage leading to thalidomide embry-opathy (also see later Nitric Oxide sections)

63 Other Mechanisms of Thalidomide-Induced TeratogenesisMany models theories and hypotheses have previously beenpresented to try to explain thalidomide-induced embryopa-thy since the 1957ndash61 disaster More than 30 modelstheorieshave been described and include actions on nerves chondro-genesis cell death and DNAMany signaling molecules havebeen linked to causing thalidomide embryopathy includingFGFrsquos and IGFs [1 2 8 128 129]Themajority of thesemodelsdo not appear to be able to account for all the damage causedin the embryo by the drug nor the time sensitive nature of thedrug and the majority focus primarily on the limb damageSome of the popular models are described further belowHowever these models do not need to be mutually exclusiveand if put together to produce a framework in my opinioncan explain the primary and secondary consequences ofthalidomide embryopathy (see Figure 4)

631 Nerve Toxicity McCredie andMcBride (1973) [130 131]proposed that thalidomide could affect neural crestmigrationand nerve innervation of tissues resulting in limb outgrowthand other tissue formation failure However studies lookingat nerve functions in the developing chick and mouse limbindicate that following a loss of nerves limbs form a normalpattern although limbs and limb cartilage elements canbe shorter in length [132ndash136] Furthermore limbs are notinnervated until relatively late in limb development well afterlimb initiation and outgrowth [134 137] How embryonicnerve damage can explain the range of damage and thetime sensitive nature of the drug is unclear It is more likelythat nerve damage is secondary to the initial insult of tissueloss through loss of vessels causing misinnervation andexacerbating the damage

632 Cell Death and Reactive Oxygen Species (ROS) Induc-tion Thalidomide induces formation of reactive oxygenspecies (ROS) and also cell death in limb tissue [114 118 125138 139] and could explain the loss of skeletal elements in thelimb as well as other damage to tissues in the body Inductionof oxidative stress in embryonic tissue is required to causethalidomide embryopathy through cell death induction [114118 125 138 140] ROS induction is linked to causing terato-genesis by other drugs for example ethanol and phenytoin[65 140] However how thalidomide induces the cell deathand ROS in specific tissues and in a time sensitive manneris unclear Interestingly cell death induction has also beenobserved in the forming limb following application of CPS49an antiangiogenic analog of thalidomide suggesting theinhibition of blood vessels could be a trigger or involved ininduction of ROS and cell death [1 54]

633 Chrondrogenesis Chrondrogenesis is the process bywhich the cartilage condensations of the skeletal elements


ThalidomideAntiangiogenic

breakdownbyproduct

Blood vessel inhibition

Actin cytoskeletonchanges

Geneexpression

Mesenchymalcell death and ROS

Organtissue damage and function

Secondary damage(eg incorrect innervation)

Figure 4 Framework of thalidomide teratogenicity Thalidomide isbroken down into active byproducts The antiangiogenic byproductinhibits new blood vessel formation in forming tissues and organs(but not mature vascular smooth muscle coated vessels) Theendothelial cells are prevented from proliferating and migratingdue to actin cytoskeleton changes Cell death and localised reactiveoxygen species (ROS) are induced and gene signalling pathways areaffected resulting in further cell death In rapidly forming tissuesand organs such as a limb this is devastating causing tissue lossfor example of chondrogenic precursor cells and deformity Thedamaged and malformed tissues would then fail to recruit or resultin failed secondary cell population migrations such as nerves andmuscles Suchmigrating cell types would bemispositioned resultingin a worsening of the defect The framework could also be appliedto tissues and organs that have formed and been patterned beforethalidomide exposure In this case new vessel formation would beinhibited and localised cell deathgene changes would occur as thetissue tries to mature and grow resulting in reduced size andorreduced physiological function

form from mesenchyme and which later differentiate intobone Thalidomide and its antiangiogenic analog CPS49induce increased cell death in the early developing limbwhenproximal limb element precursors are likely forming causinglimb element reduction or loss [54 114]

It will be interesting to determine if other chondrogeniccondensations in the embryo are similarly affected by thalido-mide for example the vertebral column

Phocomelia The stereotypic limb anomaly associated withthalidomide embryopathy is phocomelia where proximallimb elements are missing or reduced but distal elementsremain How does phocomelia come about Phocomelia canbe induced experimentally by X-irradiating the early chicklimb bud resulting in loss of proximal skeletal elements[141 142] and also through physical removal of proximaland medial limb mesenchyme [143ndash145] Reduction of FGF

signaling in the forming mouse limb can also cause pho-comelia through loss of proximal elements by increased celldeath [146] We have previously proposed that phocomeliacould arise following thalidomide exposure in early limbdevelopment by loss of blood vessels inducing cell death andloss of or reduction of distal limb signalling which recoversto distalise remaining tissues (Figure 3) [1 54] We knowthalidomide and CPS49 induce increased cell death followedby loss of FGF signaling from the apical ridge in treatedlimbs soon after drug exposure [54 112ndash114] Could apicalridge signaling recover to signal to anddistalise the remainingmesenchymal cells In chick limbs where proximal andmedial tissues have been experimentally removed to inducephocomelia an upregulation of FGF signaling was observed24 hrs after tissue removal [145] In thalidomide exposedrabbit embryos FGF signalling was reduced in limbs budsbut recovered a fewdays later and phocomelic limbs can form[112]This suggests some distal limb signalling recovery couldbe a key to understanding thalidomide-induced phocomeliaAlternatively or in addition following thalidomide exposurecould the early loss of blood vessels and subsequent inducedcell death interfere with or prevent cartilage condensa-tionossification resulting in shortenedmissing elementsAndOr could other distally expressed genes be activatedprematurely at the expense of more proximal genes

634 DNA Interaction and Mutagenesis Based on the struc-ture of thalidomide it has been suggested that thalidomidecould intercalate into andor interferewithDNA function forexample gene activation [8 128 129 147 148] Thalidomidehas also been proposed to be able to form dimers which couldinteract transiently with DNA [147] It remains unclear howsuch intercalationinterference events would account for therange and severity of damage induced by thalidomide

Thalidomide is not mutagenic nor are defects hereditary[66ndash68 149 150] Reports suggesting thalidomide may be amutagen followed the birth of a child exhibiting thalidomide-like damage to a thalidomide-damaged parent caused obvi-ous concern [151] However several detailed and controlledstudies have concluded that thalidomide has no mutagenicactivity [66 149 150] As discussed earlier thalidomidecan phenocopy some human syndromes for example Holt-Oram syndrome and it can be difficult to differentiatebetween these conditions without genetic testing [67 68]Furthermore an epidemiological study in Sweden followedoffspring from thalidomide survivors and found no evidenceof transmission of thalidomide-damage in the offspring [150]

7 Molecular Targets of Thalidomide

71 Cereblon Cereblon is a candidate gene for mental retar-dation and has been identified as a binding target of thalido-mide leading to limb teratogenesis [113] Cereblon has alsobeen demonstrated to be required by thalidomide lenalido-mide and pomalidomide to exert their anti-inflammatoryactions [152ndash154] Cereblon clearly has multiple importantfunctions in the embryo and body involved in the inflam-matory response mental abilities teratogenesis and also


metabolism [155] but its full role in development remains tobe characterised

Cereblon is part of a ubiquitination complex involved inregulating the removal of proteins in the cell and tissues of theembryobody Thalidomide is proposed to bind to Cereblonin the forming limb which then causes a misregulationof signalling in the cells and limb bud due to the lossof ubiquitination activity and which results in thalidomideinduced limb defects [113] Overexpression in chick limbsand zebrafish embryos of a mutant form of Cereblon thatcannot bind thalidomide rescues the teratogenic effects ofthe drug [113] Several studies in chick rabbit and zebrafishfins show that thalidomide reduces FGF signalling fromthe apical ridge [54 112ndash114] This led to the proposal thatfollowing thalidomide binding to Cereblon perhaps FGFsignalling misregulation may occur in the limb [113 156]However the use of an antiangiogenic analog of thalidomideCPS49 demonstrated reduction and loss of FGF signallingfrom the limb apical ridge is secondary to the loss of bloodvessels and the induction of mesenchymal cell death [54]In addition the embryonic expression pattern of Cere-blon is unclear Presently reports suggest Cereblon may beubiquitously expressed in human and mouse tissues [113]How Cereblon binding to thalidomide can lead to the timesensitive and tissue specific defects associated with thalido-mide embryopathy remains unclear Furthermore a recentstudy investigating Cereblon function generated a loss-of-function mutant mouse line (where the gene was inactivatedthroughout the mouse) The study reports that Cereblonis a negative modulator of AMP-activated protein kinase(AMPK) leading to protection against enhanced weight gainin high fat diets [155] Strikingly the mice have no apparentchange in limb morphology or facial morphology in fact themice appear physically and morphologically normal thoughwhether the mice suffered from mental disabilities or lateonset morphological problems was not reported [155]

Further studies are required to determine Cereblonrsquos rolein teratogenesis

72 Actin Cytoskeleton Thalidomide lenalidomide andCPS49 have each been shown to affect the actin cytoskeletonand cell migratory ability of endothelial cells [54 85 121157 158] Actin is upregulated and tubulin is downregulatedindicating cells can not proliferate nor migrate Indeedfilopodial extensions from migrating endothelial cells areinhibited following CPS49 exposure preventing vasculartube formation [54] Could amolecular target of thalidomidein endothelial cells be the actin cytoskeleton A GeneChipArray screen of monkey fetuses exposed to thalidomideindicated a high number of vascular and cytoskeletal markerchanges [159] Whether these are primary or secondarytargets remains to be determined

73 Nitric Oxide Nitric oxide has a role in blood vessel pro-tection and has been shown to be able to rescue thalidomide-induced damage [119 122] A recent study looking at humanthalidomide survivors indicated survivors have a higherfrequency of polymorphisms associated with reduced Nitric

oxide expression suggesting individualswith such a genotypecould be more susceptible to thalidomide damage [160]perhaps due to reduced protection of blood vessels

74 Molecular Screens Recently proteomic and transcrip-tomic screens of thalidomide treated human embryonic stemcells and GeneChip Array and Bioinformatics screens ofthalidomide treated cynomolgusmonkey fetuses have lookedfor molecular targets of thalidomide [159 161] In the humancell lines changes in limb heart nerve and embryonic sig-naling molecules were uncovered [161] Strikingly more than2000 gene profiles were changed in the monkey tissue withthe majority being related to actin cytoskeleton the vascula-ture and inflammatory response [159] Together these screensindicate there are many potential targets of thalidomide

Altogether and as described earlier in this Review manygenes and molecular pathways have already been linked toor even shown to have changed gene expression patternsfollowing thalidomide exposure The challenge now is todetermine the precise order and number of the moleculartargets and downstream signaling events and how theylead to teratogenesis Determining the molecular targets ofthalidomide that result in teratogenesis could help developforms of the drug that retain clinical relevance without theteratogenic side-effect

8 Framework of Thalidomide Teratogenicity

Amodel underlying the mechanism of thalidomide-inducedteratogenesis needs to explain the time-sensitive action of thedrug on all tissues including limbs ears eyes facial internalorgan and genitalia

All of the models proposed to explain thalidomideembryopathy are not necessarily mutually exclusive and tosome extent each of these models can explain an aspect ofThalidomide embryopathy Effects upon the vasculature havebeen demonstrated to cause embryonic defect [34 54 119121 123] and molecules involved in vascular development arechanged following thalidomide exposure [102 119 121 123159ndash161] Furthermore the antiangiogenic analog of thalido-mide CPS49 acts in a time sensitive manner in the chickembryo [54] The action of thalidomide upon blood vesselstogether with evidence that birth defects can be caused bydamaging blood vessels either through drug exposure forexample sodium valproate [124] or by genetic ablation forexample in zebrafish [106] suggests the triggering event lead-ing to thalidomide embryopathy could well be blood vesselsThus at this point angiogenesis could be the triggering eventand by putting together the other models we can produce aframework fromexposure to defect and further consequences(Figure 4) What remains unclear is the molecular target(s)

Thalidomide breaks down into a range of byproductsthrough metabolism and hydrolysis [29] the antiangiogenicbyproduct causes teratogenesis [1 2 54] Blood vessels areessential for the development of tissues and organs [100]Exposure to thalidomide during periods of rapid angio-genesis and tissue formation results in vascular formationfailure induction of cell death and ROS in cells requiring


oxygen and nutrients and gene expression changes Suchexposure during early limb formation orwhen tissuesorgansare rapidly forming is devastating If the antiangiogenicinsult occurs earlier in development when all vessels areangiogenic and all the major tissues and organs are formingthis could result in cell death in the tissue concerned andmisexpression of signalling pathways The resulting loss ormisdevelopment of tissue may well be lethal or then resultin the failure of the correct recruitment and differentiation ofother cell types including nerves neural crest chondrocytesmuscle (particularly if the tissue was severely damagedresulting nerve innervationwould be severely compromised)This would also impact internal organ function as well asnervous system function exacerbating the defect in thealready damaged tissue (Figure 4)

The Framework would predict that late fetal exposureto thalidomide could damage newly formedforming bloodvessels in the internal organstissues as they are growing andmaturing preventing proper maturationgrowth of the organor proper function Such dysfunctionmay not be detected forsome time after birth depending on the amount of damage[1 2] Interestingly a study in rats following late fetal exposureto thalidomide found that the fetal rats had neurologicaldamage in areas of the brain associated with autism and thisappeared due to a loss of blood vessels [44]

9 Species Specificity

It is well known that thalidomide exhibits species-specificdifferences in its teratogenic actions [1 3 8 129]Many animalorganisms have been used to study thalidomidersquos actionsincluding chicks rabbits zebrafish marine fish armadillosmarsupials hydra and nonhuman primates [1 2 44 54121 123 159 162ndash165] One of the surprising aspects ofthalidomide is that rodent embryos are less sensitive tothalidomide with varying results of activity as well as beingmouse strain specific [166] and as such customarily arenot seen as a primary tool to study thalidomide inducedteratogenesis [165 167ndash174] In fact the thalidomide disasterof 1957ndash61 indicated for the first time that species differencesexist in the reaction to drugs

Rabbits and nonhuman primates though useful havesmall litter sizes (and rabbits also resorb malformed fetusesin utero making it difficult sometimes to determine the fullrange of damage etc) requiring large numbers of animalsto be used Furthermore the action of the drug (as well asthe timing and range of damage) cannot be followed ldquoinvivordquo in the living embryo of rabbits rodents marsupialsand nonhuman primates Embryo cultures of rabbit embryosindicate thalidomide and itrsquos breakdown products teratogenicactions can be studied in vitro [175] In addition despiterodents being less sensitive to thalidomide mouse embryocultures following thalidomide exposure have been carriedout and typically such embryos survive up to 2 days andexhibit thalidomide-like damage [116 166] Although theseembryo culture techniques are very useful and informativeit can be difficult to determine the precise range of damageto the limbs and other tissues as the cultures do not develop

consistently further than around 2 days Efforts to improvethe length of time mouse and rabbit embryos can be culturedin vitro would be a major advance Specifically allowing thegenetic tractability of the mouse to be used to understandmolecular targets of thalidomide in more detail Chick andzebrafish embryos offer goodmodels to study drug action andtoxicity on limb and embryonic development and to followthe effects ldquoliverdquo and have been widely used [54 85 113 114117 121 123 124 176 177]

Species-specific differences in teratogenic action are alsoapparent for lenalidomide and pomalidomide Lenalidomideis nonteratogenic in rabbits [178] but is in chick and zebrafishembryos [85] and in monkeys In contrast pomalidomide isnot teratogenic in chick and zebrafish embryos [85] yet isteratogenic in rats and rabbits [86] (httpwwwaccessdatafdagovdrugsatfda docslabel2013204026lblpdf)

What are the reasons for these species differences inactivity Many theories have been proposed includingmetabolism differences between the species Thalidomidehas more antiangiogenic activity following incubation withhuman and rabbit liver microsomes than rat microsomes[92 93] Whether the teratogenic breakdown products ofthalidomide are made in the mouse embryo or are presentlong enough is also unclear as clearance of the drug isextremely rapid in mice compared with humans [179 180]To add further complexity to the issue thalidomide treatedwith rat liver homogenate can cause embryotoxicity in chickembryos suggesting that even though rat liver is not aseffective as human and rabbit in thalidomide metabolismthe enzymes are present [181] Metabolic clearance rates maydiffer between species meaning teratogenic forms of the drugmay not be present for long Placental transport differencescould also exist between species It remains unclear how thedrugs gain access to cells whether through a receptor ortransport channel

91 Glutathione and ROS Differences A study reported thatmouse embryonic fibroblasts have high levels of glutathionewhich inhibit superoxide formation and thus prevent ROSformation and prevent cell death induction [182] In contrasthuman cell lines have low levels of glutathione and aresuggested to be more able to produce ROS following thalido-mide exposure [120 182] Likewise mice embryos suffer fromthalidomide-induced embryopathies when glutathione isdepleted [140] As discussed earlier production of ROS leadsto cell death and embryopathy but how the time sensitiveand tissue specific actions come about requires further studyAs depleting glutathione makes mouse embryos sensitive tothalidomide perhaps this is one method to potentially openthe mouse embryo up for thalidomide teratogenesis studies[140 182]

Understanding the basis of species specificity may helpunderstand how to make improved or alternative drugs thatcannot cause teratogenesis or perhapsmake forms of the drugthat cant break down into teratogenic byproducts

92 Screening for Safer and Nonteratogenic Forms of Thalido-mide Clearly thalidomide and its licensed sister compounds


lenalidomide and pomalidomide are clinically useful par-ticularly for treatment of multiple myeloma However theyretain nasty and unwanted side-effects We still do notfully understand how these side effects are caused or comeabout Moreover administering a compound that exerts anti-inflammatory and antiangiogenic effects for an inflammatorycondition is not desirable What would be therapeuticallymore effective would be to retain the clinically relevantaspects of the drug and lose the bad aspects and also to havemore targeted and tissue specific versions Can this be done

The antiangiogenic action of thalidomide has beendemonstrated to cause teratogenesis in chick and zebrafishembryos [1 54] It is also known that lenalidomide andpomalidomide also have antiangiogenic properties in rodentassays and myeloma cell lines [157 158 183 184] Lenalido-mide is also antiangiogenic (and teratogenic) in chickenand zebrafish embryos whereas pomalidomide is not [85]Precisely what the molecular targets of these compounds areremains unclearThe nature of the species-specific differencesin action is also unclear However this indicates that theantiangiogenic action of these compounds likely underliestheir teratogenic activities Producing versions of the drugor alternative drugs without the antiangiogenic actions butretaining anti-inflammatory actions could ultimately follow-ing testing in higher species and clinical trials produce acompound retaining clinical benefit for treatment of inflam-matory disorders with reduced side effect

Indeed some non-antiangiogenic analogs and hydrolyticbreak down products of thalidomide have been shown tobe nonteratogenic in chicken embryos [54] This indicatesthe possibility that screening other structural analogs ofthalidomide could lead to identifying compounds with spe-cific actions with reduced or lessened side effects Furtherwork is required to elucidate analogsderivatives that arepurely anti-inflammatory or purely antiangiogenic Suchanalogsderivatives could initially be screened in embry-onic systems as well as in vitro assays to investigate theireffectsactions upon angiogenesis inflammation terato-genicity and nerve function Identification of such com-pounds could mean after further testing and screeningin mammalian and human model assays potentially moretargeted compounds to treat inflammatory disorders withoutthe spectre of birth defect or peripheral neuropathy but alsoto treat cancers without the side effect of dampening downthe inflammatory system

10 Conclusion

How thalidomide causes birth defects has puzzled scientistsand clinicians alike since the first cases of thalidomide embry-opathy were described between 1958 and 1961 Thalidomideremains an enigma in many respects Thalidomide causedthe biggest medical disaster of all time founded the field ofmodern toxicology and changed forever the ways drugs aretested Yet we still do not fully understand how this drugcaused the range and severity of birth defects nor do weunderstand the drugrsquos species specific differences in actionThalidomide has important functions in the inflammatory

and vascular tissues and it is also unclear how precisely thedrug acts in these tissues

In the past decade great progress has been made in eluci-dating themechanismsunderlying thalidomide embryopathyand its targets and making safer forms of the drug Thalido-mide and its analogs lenalidomide and pomalidomide areused to treat multiple myeloma and leprosy Thalidomideis also used to treat Crohnrsquos disease Behcetrsquos disease HIVand cancers But they still have serious side effects such asperipheral neuropathy And tragically thalidomide embry-opathy still occurs particularly in Brazil Understandinghow thalidomide causes birth defect remains important todetermine as does how it treats the conditions it is successfulin Ultimately this information will help to make safer formsof the drug targeted for specific conditions without the sideeffects

Thalidomidersquos action on the forming vasculature causeslimb defects and likely damages other tissues throughdestroying vessels as tissues are forming growing and requir-ing a vasculature By uniting the many models suggestedfor thalidomide embryopathy a framework of thalidomideteratogenesis can be produced which acts as a template foreffects on all the other tissues affected by thalidomide

Yet significant challenges remain specifically under-standing the molecular targets andor sequence of eventsresulting in thalidomide embryopathy and determining thebasis of species specificity differences in the action of drugFurthermore given the actions of the drug on different path-ways understanding how to modulate these for the benefit ofthe patient to make safer and tissue specifictargeted versionsof the drug remains an important goal

Finally how does thalidomide induce phocomelia (loss ofproximal elements) Does it arise through a recovery of apicalridge signalling after loss of limb mesenchyme distalisingthe remaining mesenchyme Or could the early loss ofblood vessels and cell death induction directly interfere withcartilage condensationossification processes resulting inshortenedmissing proximal elements Or could phocomeliaarise due to changes in limb patterning signallingwhere distalmarkers come on earlier than normal in the damaged limbUnderstanding the mechanisms underlying phocomelia willalso help further understanding on normal mechanismscontrolling proximodistal patterning of the limb bud

Conflict of Interests

The author declares there is no conflict of interests

Acknowledgments

Thanks go to Dr Chris Mahony Prof Martin CollinsonShaunna-Leigh Beedie and Alexandra Diamond for criticalcomments on the paper Thanks also go to Prof LyndaErskine Dr W D Figg Dr Lavinia Schuler-Faccini DrRobert L Smith Prof Nigel Brown Prof Ruth Ross ProfCheryll Tickle and Prof Lewis Wolpert for fantastic discus-sions on thalidomide This work is dedicated to C G V andC W M V


References

[1] N Vargesson ldquoThalidomide-induced limb defects resolving a50-year-old puzzlerdquo BioEssays vol 31 no 12 pp 1327ndash13362009

[2] N Vargesson ldquoThalidomiderdquo in Reproductive and Developmen-tal Toxicology R Gupta Ed pp 395ndash403 Academic PressElsevier Amsterdam The Netherlands 2011

[3] W Lenz ldquoA short history of thalidomide embryopathyrdquo Teratol-ogy vol 38 no 3 pp 203ndash215 1988

[4] SV Rajkumar ldquoThalidomide tragic past andpromising futurerdquoMayo Clinic Proceedings vol 79 no 7 pp 899ndash903 2004

[5] F O Kelsey ldquoEvents after thalidomiderdquo Journal of DentalResearch vol 46 no 6 pp 1201ndash1205 1967

[6] F O Kelsey ldquoThalidomide update regulatory aspectsrdquo Teratol-ogy vol 38 no 3 pp 221ndash226 1988

[7] R W Smithells and C G H Newman ldquoRecognition ofthalidomide defectsrdquo Journal of Medical Genetics vol 29 no 10pp 716ndash723 1992

[8] T D Stephens C J W Bunde and B J Fillmore ldquoMechanismof action in thalidomide teratogenesisrdquo Biochemical Pharmacol-ogy vol 59 no 12 pp 1489ndash1499 2000

[9] T Kajii M Kida and K Takahashi ldquoThe effect of thalidomideintake during 113 human pregnanciesrdquo Teratology vol 8 no 2pp 163ndash166 1973

[10] T Kajii and M Shinohara ldquoThalidomide in JapanrdquoThe Lancetvol 1 no 7279 pp 501ndash502 1963

[11] W Lenz and K Knapp ldquoThalidomide embryopathyrdquoArchives ofEnvironmental Health vol 5 pp 100ndash105 1962

[12] W G McBride ldquoStudies of the etiology of thalidomide dysmor-phogenesisrdquo Teratology vol 14 no 1 pp 71ndash87 1976

[13] C G H Newman ldquoClinical observations on the thalidomidesyndromerdquo Proceedings of the Royal Society of Medicine vol 70no 4 pp 225ndash227 1977

[14] C G H Newman ldquoTeratogen update clinical aspects ofthalidomide embryopathymdasha continuing preoccupationrdquo Ter-atology vol 32 no 1 pp 133ndash144 1985

[15] C G H Newman ldquoThe thalidomide syndrome risks of expo-sure and spectrum of malformationsrdquo Clinics in Perinatologyvol 13 no 3 pp 555ndash573 1986

[16] RW Smithells ldquoThalidomide andmalformations in liverpoolrdquoThe Lancet vol 279 no 7242 pp 1270ndash1273 1962

[17] R W Smithells ldquoDefects and disabilities of thalidomide chil-drenrdquoBritishmedical journal vol 1 no 5848 pp 269ndash272 1973

[18] I M Leck and E L Millar ldquoIncidence of malformations sincethe introduction of thalidomiderdquo BritishMedical Journal vol 2no 5296 pp 16ndash20 1962

[19] H B Taussig ldquoA study of the German outbreak of phocomeliaThe thalidomide syndromerdquoThe journal of the American Medi-cal Association vol 180 pp 1106ndash1114 1962

[20] U K Government Report ldquoDeformities caused by Thalido-miderdquo Reports on Public Health and Medical Subjects 112Ministry of Health HMSO London UK 1964

[21] G F Somers ldquoThalidomide and congenital abnormalitiesrdquoTheLancet vol 1 no 7235 pp 912ndash913 1962

[22] R W Smithells and I Leck ldquoThe incidence of limb and eardefects since the withdrawal of thalidomiderdquo The Lancet vol1 no 7290 pp 1095ndash1097 1963

[23] B M Ances ldquoNew concerns about thalidomiderdquoObstetrics andGynecology vol 99 no 1 pp 125ndash128 2002

[24] F O Kelsey ldquoDrug embryopathy the thalidomide storyrdquoMaryland State Medical Journal vol 12 pp 594ndash597 1963

[25] M TMiller andK Stromland ldquoTeratogen update thalidomidea review with a focus on ocular findings and new potentialusesrdquo Teratology vol 60 pp 306ndash321 1999

[26] H B TAUSSIG ldquoThe thalidomide syndromerdquo Scientific Ameri-can vol 207 pp 29ndash35 1962

[27] J Sheskin ldquoThalidomide in the Treatment of Lepra ReactionsrdquoClinical Pharmacology and Therapeutics vol 6 pp 303ndash3061965

[28] R J DrsquoAmato M S Loughnan E Flynn and J FolkmanldquoThalidomide is an inhibitor of angiogenesisrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol91 no 9 pp 4082ndash4085 1994

[29] M E Franks G RMacpherson andWD Figg ldquoThalidomiderdquoThe Lancet vol 363 no 9423 pp 1802ndash1811 2004

[30] T Facon J Y Mary C Hulin et al ldquoMelphalan and pred-nisone plus thalidomide versus melphalan and prednisonealone or reduced-intensity autologous stem cell transplantationin elderly patients with multiple myeloma (IFM 99-06) arandomised trialrdquoThe Lancet vol 370 no 9594 pp 1209ndash12182007

[31] A Palumbo and M Boccadoro ldquoA new standard of care forelderly patients with myelomardquo The Lancet vol 370 no 9594pp 1191ndash1192 2007

[32] B Ladizinski E J Shannon M R Sanchez and W R LevisldquoThalidomide and analogues potential for immunomodulationof inflammatory and neoplastic dermatologic disordersrdquo Jour-nal of Drugs in Dermatology vol 9 no 7 pp 814ndash826 2010

[33] J J Wu D B Huang K R Pang S Hsu and S KTyring ldquoThalidomide dermatological indications mechanismsof action and side-effectsrdquo British Journal of Dermatology vol153 no 2 pp 254ndash273 2005

[34] F Lebrin S Srun K Raymond et al ldquoThalidomide stimulatesvessel maturation and reduces epistaxis in individuals withhereditary hemorrhagic telangiectasiardquo Nature Medicine vol16 no 4 pp 420ndash428 2010

[35] M R Horton and R W Hallowell ldquoRevisiting thalidomidefighting with caution against idiopathic pulmonary fibrosisrdquoDrugs Today vol 48 pp 661ndash671 2012

[36] J Knobloch D Jungck and A Koch ldquoApoptosis inductionby thalidomide critical for limb teratogenicity but therapeuticpotential in idiopathic pulmonary fibrosisrdquo Current MolecularPharmacology vol 4 no 1 pp 26ndash61 2011

[37] E E Castilla PAshton-Prolla E Barreda-Mejia et al ldquoThalido-mide a current teratogen in SouthAmericardquoTeratology vol 54pp 273ndash277 1996

[38] L Schuler-Faccini R C F Soares A C M de Sousa et alldquoNew cases of thalidomide embryopathy in BrazilrdquoBirthDefectsResearch A vol 79 no 9 pp 671ndash672 2007

[39] F S L Vianna J S Lopez-Camelo J C L Leite et alldquoEpidemiological surveillance of birth defects compatible withthalidomide embryopathy in Brazilrdquo PLoS ONE vol 6 no 7Article ID e21735 2011

[40] F S Vianna L Schuler-Faccini J C Leite et al ldquoRecognitionof the phenotype of thalidomide embryopathy in countriesendemic for leprosy new cases and review of the main dysmor-phological findingsrdquoClinical Dysmorphology vol 22 pp 59ndash632013

[41] W GMcBride ldquoThalidomide embryopathyrdquo Teratology vol 16no 1 pp 79ndash82 1977


[42] M T Miller K Stromland L Ventura M Johansson J MBandim and C Gillberg ldquoAutism associated with conditionscharacterized by developmental errors in early embryogenesisa mini reviewrdquo International Journal of Developmental Neuro-science vol 23 no 2-3 pp 201ndash219 2005

[43] W H James ldquoTeratogenetic properties of thalidomiderdquo BritishMedical Journal vol 2 no 5469 p 1064 1965

[44] K L Hallene E Oby B J Lee et al ldquoPrenatal exposure tothalidomide altered vasculogenesis and CNS malformationsrdquoNeuroscience vol 142 no 1 pp 267ndash283 2006

[45] M T Miller and K K Stromland ldquoWhat can we learn fromthe thalidomide experience an ophthalmologic perspectiverdquoCurrent Opinion in Ophthalmology vol 22 no 5 pp 356ndash3642011

[46] L Henkel and H G Willert ldquoDysmelia classification and apattern of malformation in a group of congenital defects of thelimbsrdquo Journal of Bone and Joint Surgery B vol 51 no 3 pp399ndash414 1969

[47] A L Speirs ldquoThalidomide and congenital abnormalitiesrdquo TheLancet vol 1 no 7224 pp 303ndash305 1962

[48] I Leck and R W Smithells ldquoThe ascertainment of malforma-tionsrdquo Lancet vol 1 pp 101ndash103 1963

[49] J McCredie and H-G Willert ldquoLongitudinal limb deficienciesand the sclerotomes An analysis of 378 dysmelicmalformationsinduced by thalidomiderdquo Journal of Bone and Joint Surgery Bvol 81 no 1 pp 9ndash23 1999

[50] K C Oberg J M Feenstra P R Manske and M ATonkin ldquoDevelopmental biology and classification of congeni-tal anomalies of the hand and upper extremityrdquo Journal of HandSurgery vol 35 no 12 pp 2066ndash2076 2010

[51] M Towers and C Tickle ldquoGeneration of pattern and form inthe developing limbrdquo International Journal of DevelopmentalBiology vol 53 no 5-6 pp 805ndash812 2009

[52] A Zuniga R Zeller and S Probst ldquoThe molecular basis ofhuman congenital limb malformationsrdquoWiley InterdisciplinaryReviews Developmental Biology vol 1 pp 803ndash822 2012

[53] K C Oberg T E Harris M D Wongworawat and V EWood ldquoCombined congenital radial and ulnar longitudinaldeficiencies report of 2 casesrdquo Journal of Hand Surgery vol 34no 7 pp 1298ndash1302 2009

[54] C Therapontos L Erskine E R Gardner W D Figg and NVargesson ldquoThalidomide induces limb defects by preventingangiogenic outgrowth during early limb formationrdquoProceedingsof the National Academy of Sciences of the United States ofAmerica vol 106 no 21 pp 8573ndash8578 2009

[55] M Schmidt and F M Salzano ldquoDissimilar effects of thalido-mide in dizygotic twinsrdquo Acta Geneticae Medicae et Gemellolo-giae vol 29 no 4 pp 295ndash297 1980

[56] R J Newman ldquoShoulder joint replacement for osteoarthrosisin association with thalidomide-induced phocomeliardquo ClinicalRehabilitation vol 13 no 3 pp 250ndash252 1999

[57] L Ruffing ldquoEvaluation of thalidomide childrenrdquo Birth Defectsvol 13 no 1 pp 287ndash300 1977

[58] J F Cullen ldquoOcular defects in thalidomide babiesrdquo BritishJournal of Ophthalmology vol 48 pp 151ndash153 1964

[59] R Cuthbert and A L Speirs ldquoThalidomide induced malforma-tionsmdasha radiological surveyrdquo Clinical Radiology vol 14 no 2pp 163ndash169 1963

[60] G Livingstone ldquoCongenital ear abnormalities due to thalido-miderdquo Proceedings of the Royal Society of Medicine vol 58 pp493ndash497 1965

[61] C G D Brook S N Jarvis and C G H Newman ldquoLineargrowth of children with limb deformities following exposureto thalidomide in uterordquo Acta Paediatrica Scandinavica vol 66no 6 pp 673ndash675 1977

[62] G Chamberlain ldquoThe obstetric problems of the thalidomidechildrenrdquo British Medical Journal vol 298 no 6665 p 6 1989

[63] I Fletcher ldquoReview of the treatment of thalidomide childrenwith limb deficiency in great Britainrdquo Clinical Orthopaedics andRelated Research vol 148 pp 18ndash25 1980

[64] B J Soules ldquoThalidomide victims in a rehabilitation centerrdquoHa-Ahot be-Yisrael vol 12 no 60 pp 43ndash45 1966

[65] J M Hansen and C Harris ldquoRedox control of teratogenesisrdquoReproductive Toxicology vol 35 pp 165ndash179 2013

[66] J Ashby and H Tinwell ldquoThalidomide is not a human muta-genrdquo British Medical Journal vol 325 no 7374 p 1245 2002

[67] J Kohlhase and L B Holmes ldquoMutations in SALL4 in mal-formed father and daughter postulated previously due to reflectmutagenesis by thalidomiderdquo Birth Defects Research A vol 70no 8 pp 550ndash551 2004

[68] J Kohlhase L Schubert M Liebers et al ldquoMutations at theSALL4 locus on chromosome 20 result in a range of clinicallyoverlapping phenotypes including Okihiro syndrome Holt-Oram syndrome acro-renal-ocular syndrome and patientspreviously reported to represent thalidomide embryopathyrdquoJournal of Medical Genetics vol 40 no 7 pp 473ndash478 2003

[69] A W Bates ldquoA case of Roberts syndrome described in 1737rdquoJournal of Medical Genetics vol 38 no 8 pp 565ndash567 2001

[70] E S-Y Goh C Li S Horsburgh Y Kasai E Kolomietz and CF Morel ldquoThe Roberts syndromeSC phocomelia spectrummdashacase report of an adult with review of the literaturerdquo AmericanJournal of Medical Genetics A vol 152 no 2 pp 472ndash478 2010

[71] B Schule A Oviedo K Johnston S Pai and U FranckeldquoInactivating mutations in ESCO2 cause SC phocomelia andRoberts syndrome no phenotype-genotype correlationrdquo Amer-ican Journal of Human Genetics vol 77 no 6 pp 1117ndash11282005

[72] H Vega Q Waisfisz M Gordillo et al ldquoRoberts syndromeis caused by mutations in ESCO2 a human homolog of yeastECO1 that is essential for the establishment of sister chromatidcohesionrdquo Nature Genetics vol 37 no 5 pp 468ndash470 2005

[73] M Monnich Z Kuriger C G Print and J A HorsfieldldquoA zebrafish model of roberts syndrome reveals that Esco2depletion interferes with development by disrupting the cellcyclerdquo PLoS ONE vol 6 no 5 Article ID e20051 2011

[74] G Whelan E Kreidl J M Peters and G Eichele ldquoThe non-redundant function of cohesin acetyltransferase Esco2 someanswers and new questionsrdquo Nucleus vol 3 pp 330ndash334 2012

[75] S Cundari and G Cavaletti ldquoThalidomide chemotherapy-induced peripheral neuropathy actual status and new perspec-tives with thalidomide analogues derivativesrdquo Mini-Reviews inMedicinal Chemistry vol 9 no 7 pp 760ndash768 2009

[76] M Delforge J Blade M A Dimopoulos et al ldquoTreatment-related peripheral neuropathy in multiple myeloma the chal-lenge continuesrdquo The Lancet Oncology vol 11 no 11 pp 1086ndash1095 2010

[77] F O Kelsey ldquoProblems raised for the FDA by the occurrenceof thalidomide embryopathy in Germany 1960-1961rdquoAmericanJournal of PublicHealth and theNationrsquoSHealth vol 55 pp 703ndash707 1965

[78] AC Peltier and JWRussell ldquoAdvances in understanding drug-induced neuropathiesrdquo Drug Safety vol 29 no 1 pp 23ndash302006


[79] P G Richardson M Delforge M Beksac et al ldquoManage-ment of treatment-emergent peripheral neuropathy in multiplemyelomardquo Leukemia vol 26 no 4 pp 595ndash608 2012

[80] M S Raab K Podar I Breitkreutz P G Richardson and K CAnderson ldquoMultiple myelomardquo The Lancet vol 374 no 9686pp 324ndash339 2009

[81] M Q Lacy S R Hayman M A Gertz et al ldquoPomalidomide(CC4047) plus low-dose dexamethasone as therapy for relapsedmultiple myelomardquo Journal of Clinical Oncology vol 27 no 30pp 5008ndash5014 2009

[82] P G Richardson D Siegel R Baz et al ldquoPhase 1 studyof pomalidomide MTD safety and efficacy in patients withrefractory multiple myeloma who have received lenalidomideand bortezomibrdquo Blood vol 121 pp 1961ndash1967 2013

[83] A Palumbo J Freeman L Weiss and P Fenaux ldquoThe clinicalsafety of lenalidomide in multiple myeloma and myelodysplas-tic syndromesrdquo Expert Opinion on Drug Safety vol 11 no 1 pp107ndash120 2012

[84] P M Voorhees J Laubach K C Anderson and P GRichardson ldquoPeripheral neuropathy in multiple myelomapatients receiving lenalidomide bortezomib and dexametha-sone (RVD) therapyrdquo Blood vol 121 article 858 2013

[85] C Mahony L Erskine J Niven et al ldquoPomalidomide is nonter-atogenic in chicken and zebrafish embryos and nonneurotoxicinvitrordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 110 pp 12703ndash12708 2013

[86] R L Smith S Fabro H Schumacher and R T WilliamsldquoStudies on the relationship between the chemical structure andembryotoxic activity of thalidomide and related compoundsrdquo inEmbryopathic Activity of Drugs J M Robson F Sullivan and RL Smith Eds pp 194ndash209 Churchill London UK 1965

[87] C P Castaneda J B Zeldis J Freeman C Quigley N ABrandenburg and R Bwire ldquoRevAssist a comprehensive riskminimization programme for preventing fetal exposure tolenalidomiderdquo Drug Safety vol 31 no 9 pp 743ndash752 2008

[88] K Uhl E Cox R Rogan et al ldquoThalidomide use in the USexperience with pregnancy testing in the STEPS programmerdquoDrug Safety vol 29 no 4 pp 321ndash329 2006

[89] J B Zeldis B A Williams S D Thomas and M E ElsayedldquoSTEPS a comprehensive program for controlling and mon-itoring access to thalidomiderdquo Clinical Therapeutics vol 21 no2 pp 319ndash330 1999

[90] J B Bartlett K Dredge and A G Dalgleish ldquoThe evolutionof thalidomide and its IMiD derivatives as anticancer agentsrdquoNature Reviews Cancer vol 4 no 4 pp 314ndash322 2004

[91] C Galustian M-C Labarthe J B Bartlett and A G DalgleishldquoThalidomide-derived immunomodulatory drugs as therapeu-tic agentsrdquo Expert Opinion on Biological Therapy vol 4 no 12pp 1963ndash1970 2004

[92] K S Bauer S C Dixon and W D Figg ldquoInhibition of angio-genesis by thalidomide requires metabolic activation which isspecies-dependentrdquo Biochemical Pharmacology vol 55 no 11pp 1827ndash1834 1998

[93] M G Marks J Shi M O Fry et al ldquoEffects of putative hydrox-ylated thalidomide metabolites on blood vessel density in thechorioallantoic membrane (CAM) assay and on tumor andendothelial cell proliferationrdquo Biological and PharmaceuticalBulletin vol 25 no 5 pp 597ndash604 2002

[94] A L Moreira E P Sampaio A Zmuidzinas P Frindt K ASmith and G Kaplan ldquoThalidomide exerts its inhibitory actionon tumor necrosis factor 120572 by enhancing mRNA degradationrdquo

Journal of Experimental Medicine vol 177 no 6 pp 1675ndash16801993

[95] F Payvandi L Wu M Haley et al ldquoImmunomodulatory drugsinhibit expression of cyclooxygenase-2 from TNF-120572 IL-1120573 andLPS-stimulated human PBMC in a partially IL-10-dependentmannerrdquo Cellular Immunology vol 230 no 2 pp 81ndash88 2004

[96] T Hideshima D Chauhan Y Shima et al ldquoThalidomide and itsanalogs overcome drug resistance of human multiple myelomacells to conventional therapyrdquo Blood vol 96 no 9 pp 2943ndash2950 2000

[97] W Luo Q-S Yu I Salcedo et al ldquoDesign synthesis and biolog-ical assessment of novel N-substituted 3-(phthalimidin-2-yl)-26-dioxopiperidines and 3-substituted 26-dioxopiperidinesfor TNF-120572 inhibitory activityrdquo Bioorganic andMedicinal Chem-istry vol 19 no 13 pp 3965ndash3972 2011

[98] D Ribatti G Mangialardi and A Vacca ldquoAntiangiogenictherapeutic approaches in multiple myelomardquo Current CancerDrug Targets vol 12 pp 768ndash775 2012

[99] P Carmeliet NMackman LMoons et al ldquoRole of tissue factorin embryonic blood vessel developmentrdquo Nature vol 383 no6595 pp 73ndash75 1996

[100] N Vargesson ldquoVascularization of the developing chick limbbud role ofThe TGF120573 signalling pathwayrdquo Journal of Anatomyvol 202 no 1 pp 93ndash103 2003

[101] E Crivellato ldquoThe role of angiogenic growth factors in organo-genesisrdquo International Journal of Developmental Biology vol 55no 4-5 pp 365ndash375 2011

[102] T B Knudsen andN C Kleinstreuer ldquoDisruption of embryonicvascular development in predictive toxicologyrdquo Birth DefectsResearch C vol 93 no 4 pp 312ndash323 2012

[103] H Chang D Huylebroeck K Verschueren Q Guo MM Matzuk and A Zwijsen ldquoSmad5 knockout mice die atmid-gestation due to multiple embryonic and extraembryonicdefectsrdquo Development vol 126 no 8 pp 1631ndash1642 1999

[104] N Ferrara K Carver-Moore H Chen et al ldquoHeterozygousembryonic lethality induced by targeted inactivation of theVEGF generdquo Nature vol 380 no 6573 pp 439ndash442 1996

[105] F Shalaby J Rossant T P Yamaguchi et al ldquoFailure of blood-island formation and vasculogenesis in Flk-1 deficient micerdquoNature vol 376 no 6535 pp 62ndash66 1995

[106] J D Leslie L Ariza-McNaughton A L Bermange RMcAdowS L Johnson and J Lewis ldquoEndothelial signalling by the Notchligand Delta-like 4 restricts angiogenesisrdquo Development vol134 no 5 pp 839ndash844 2007

[107] C Therapontos and N Vargesson ldquoZebrafish notch signallingpathway mutants exhibit trunk vessel patterning anomaliesthat are secondary to somite misregulationrdquo DevelopmentalDynamics vol 239 no 10 pp 2761ndash2768 2010

[108] B M Kenyon F Browne and R J DrsquoAmato ldquoEffects ofthalidomide and related metabolites in a mouse corneal modelof neovascularizationrdquo Experimental Eye Research vol 64 no6 pp 971ndash978 1997

[109] E R Lepper N F Smith M C Cox C D Scripture and WD Figg ldquoThalidomidemetabolism and hydrolysis mechanismsand implicationsrdquo Current Drug Metabolism vol 7 no 6 pp677ndash685 2006

[110] N A Warfel E R Lepper C Zhang W D Figg and P ADennis ldquoImportance of the stress kinase p38120572 in mediating thedirect cytotoxic effects of the Thalidomide analogue CPS49 incancer cells and endothelial cellsrdquo Clinical Cancer Research vol12 pp 3502ndash3509 2006


[111] S S W Ng M Gutschow M Weiss et al ldquoAntiangiogenicactivity of N-substituted and tetrafluorinated thalidomide ana-loguesrdquo Cancer Research vol 63 no 12 pp 3189ndash3194 2003

[112] J M Hansen S-G Gong M Philbert and C Harris ldquoMis-regulation of gene expression in the redox-sensitive NF-120581B-dependent limb outgrowth pathway by thalidomiderdquo Develop-mental Dynamics vol 225 no 2 pp 186ndash194 2002

[113] T Ito H Ando T Suzuki et al ldquoIdentification of a primarytarget of thalidomide teratogenicityrdquo Science vol 327 no 5971pp 1345ndash1350 2010

[114] J Knobloch J D Shaughnessy Jr and U Ruther ldquoThalidomideinduces limb deformities by perturbing the BmpDkk1Wntsignaling pathwayrdquo The FASEB Journal vol 21 no 7 pp 1410ndash1421 2007

[115] N Vargesson and E Laufer ldquoSmad7 misexpression duringembryonic angiogenesis causes vascular dilation and malfor-mations independently of vascular smooth muscle cell func-tionrdquo Developmental Biology vol 240 no 2 pp 499ndash516 2001

[116] C J J Lee N Shibata M J Wiley and P G Wells ldquoFluo-rothalidomide a characterization ofmaternal and developmen-tal toxicity in rabbits and micerdquo Toxicological Sciences vol 122no 1 pp 157ndash169 2011

[117] J Boylen H Horne and W Johnson ldquoTeratogenic effects ofthalidomide and related substancesrdquo The Lancet vol 1 p 5521963

[118] J Knobloch I Schmitz K Gotz K Schulze-Osthoff andU Ruther ldquoThalidomide induces limb anomalies by PTENstabilization Akt suppression and stimulation of caspase-dependent cell deathrdquo Molecular and Cellular Biology vol 28no 2 pp 529ndash538 2008

[119] J H Siamwala V Veeriah M K Priya et al ldquoNitric oxiderescues thalidomidemediated teratogenicityrdquo Scientific Reportsvol 2 article 679 2012

[120] J M Hansen K K Harris M A Philbert and C HarrisldquoThalidomidemodulates nuclear redox status and preferentiallydepletes glutathione in rabbit limb versus rat limbrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 768ndash776 2002

[121] K P Tamilarasan G K Kolluru M Rajaram M IndhumathyR Saranya and S Chatterjee ldquoThalidomide attenuates nitricoxidemediated angiogenesis by blockingmigration of endothe-lial cellsrdquo BMC Cell Biology vol 7 article 17 2006

[122] S Majumder M Rajaram A Muley et al ldquoThalidomideattenuates nitric oxide-driven angiogenesis by interacting withsoluble guanylyl cyclaserdquo British Journal of Pharmacology vol158 no 7 pp 1720ndash1734 2009

[123] T Yabu H Tomimoto Y Taguchi S Yamaoka Y Igarashiand T Okazaki ldquoThalidomide-induced antiangiogenic actionis mediated by ceramide through depletion of VEGF receptorsand is antagonized by sphingosine-1-phosphaterdquoBlood vol 106no 1 pp 125ndash134 2005

[124] A I Whitsel C B Johnson and C J Forehand ldquoAn in ovochicken model to study the systemic and localized teratogeniceffects of valproic acidrdquo Teratology vol 66 no 4 pp 153ndash1632002

[125] J M Hansen and C Harris ldquoA novel hypothesis for thalid-omide-induced limb teratogenesis redox misregulation of theNF-120581B pathwayrdquo Antioxidants and Redox Signaling vol 6 no 1pp 1ndash14 2004

[126] R Neubert N Hinz R Thiel and D Neubert ldquoDown-regulation of adhesion receptors on cells of primate embryos as

a probablemechanismof the teratogenic action of thalidomiderdquoLife Sciences vol 58 no 4 pp 295ndash316 1995

[127] A Vacca C Scavelli V Montefusco et al ldquoThalidomidedownregulates angiogenic genes in bone marrow endothelialcells of patients with active multiple myelomardquo Journal ofClinical Oncology vol 23 no 23 pp 5334ndash5346 2005

[128] T D Stephens ldquoProposedmechanisms of action in thalidomideembryopathyrdquo Teratology vol 38 no 3 pp 229ndash239 1988

[129] T D Stephens and B J Fillmore ldquoHypothesis thalidomideembryopathy-proposed mechanism of actionrdquo Teratology vol61 pp 189ndash195 2000

[130] J McCredie ldquoHistory heresy and radiology in scientific discov-eryrdquo Journal ofMedical Imaging and RadiationOncology vol 53no 5 pp 433ndash441 2009

[131] J McCredie and W G McBride ldquoSome congenital abnormali-ties possibly due to embryonic peripheral neuropathyrdquo ClinicalRadiology vol 24 no 2 pp 204ndash211 1973

[132] F Edom-Vovard B Schuler M-A Bonnin M-A Teillet andD Duprez ldquoFgf4 positively regulates scleraxis and tenascinexpression in chick limb tendonsrdquo Developmental Biology vol247 no 2 pp 351ndash366 2002

[133] T Fukuda S Takeda R Xu et al ldquoSema3A regulates bone-mass accrual through sensory innervationsrdquo Nature vol 497pp 490ndash493 2013

[134] S Harsum J DW Clarke and PMartin ldquoA reciprocal relation-ship between cutaneous nerves and repairing skin wounds inthe developing chick embryordquo Developmental Biology vol 238no 1 pp 27ndash39 2001

[135] T R Strecker and T D Stephens ldquoPeripheral nerves do not playa trophic role in limb skeletal morphogenesisrdquo Teratology vol27 no 2 pp 159ndash167 1983

[136] G J Swanson ldquoPaths taken by sensory nerve fibres in aneuralchick wing budsrdquo Journal of Embryology and ExperimentalMorphology vol 86 pp 109ndash124 1985

[137] P Martin and J Lewis ldquoOrigins of the neurovascular bundleinteractions between developing nerves and blood vessels inembryonic chick skinrdquo International Journal of DevelopmentalBiology vol 33 no 3 pp 379ndash387 1989

[138] T Parman M J Wiley and P G Wells ldquoFree radical-mediatedoxidative dna damage in the mechanism of thalidomide terato-genicityrdquo Nature Medicine vol 5 no 5 pp 582ndash585 1999

[139] P GWells G PMccallum C S Chen et al ldquoOxidative stress indevelopmental origins of disease teratogenesis neurodevelop-mental deficits and cancerrdquo Toxicological Sciences vol 108 no1 pp 4ndash18 2009

[140] P G Wells Y Bhuller C S Chen et al ldquoMolecular and bio-chemical mechanisms in teratogenesis involving reactive oxy-gen speciesrdquo Toxicology and Applied Pharmacology vol 207 no2 pp S354ndashS366 2005

[141] J L Galloway I Delgado M A Ros and C J Tabin ldquoA reeval-uation of X-irradiation-induced phocomelia and proximodistallimb patterningrdquo Nature vol 460 no 7253 pp 400ndash404 2009

[142] LWolpert C Tickle andM Sampford ldquoThe effect of cell killingbyX-irradiation onpattern formation in the chick limbrdquo Journalof Embryology and Experimental Morphology vol 50 pp 175ndash198 1979

[143] A Barasa ldquoOn the regulative capacity of the chick embryo limbbudrdquo Experientia vol 20 no 8 p 443 1964

[144] A Hornbruch Abnormalities Along the Proximo-Distal Axis ofthe ChickWing Budthe Effect of Surgical Intervention Walter deGruyter Berlin Germany 1980


[145] C Mahony ldquoVargesson N Molecular analysis of regulativeevents in the developing chick limbrdquo Journal of Anatomy vol223 pp 1ndash13 2013

[146] F V Mariani C P Ahn and G R Martin ldquoGenetic evidencethat FGFs have an instructive role in limb proximal-distalpatterningrdquo Nature vol 453 no 7193 pp 401ndash405 2008

[147] D E Hague J R Idle S C Mitchell and R L Smith ldquoRace-mates revisited heterochiral assemblies and the example of DL-thalidomiderdquo Xenobiotica vol 41 no 10 pp 837ndash843 2011

[148] N A Jonsson ldquoChemical structure and teratogenic propertiesIV An outline of a chemical hypothesis for the teratogenicaction of thalidomiderdquo Acta Pharmaceutica Suecica vol 9 no6 pp 543ndash562 1972

[149] J Ashby H Tinwell R D Callander et al ldquoThalidomidelack of mutagenic activity across phyla and genetic endpointsrdquoMutation Research vol 396 no 1-2 pp 45ndash64 1997

[150] K Stromland E Philipson and M A Gronlund ldquoOffspring ofmale and female parents with thalidomide embryopathy birthdefects and functional anomaliesrdquo Teratology vol 66 no 3 pp115ndash121 2002

[151] W G McBride and A P Read ldquoThalidomide may be a muta-genrdquo British Medical Journal vol 308 no 6944 pp 1635ndash16361994

[152] A Broyl R Kuiper M van Duin et al ldquoHigh cereblon expres-sion is associated with better survival in patients with newlydiagnosed multiple myeloma treated with thalidomide mainte-nancerdquo Blood vol 121 pp 624ndash627 2012

[153] A Lopez-Girona D Mendy T Ito et al ldquoCereblon is a directprotein target for immunomodulatory and antiproliferativeactivities of lenalidomide and pomalidomiderdquo Leukemia vol26 pp 2326ndash2335 2012

[154] Y X Zhu E Braggio C-X Shi et al ldquoCereblon expression isrequired for the antimyeloma activity of lenalidomide andpomalidomiderdquo Blood vol 118 no 18 pp 4771ndash4779 2011

[155] KM Lee S J Yang Y D Kim et al ldquoDisruption of the cereblongene enhances hepatic AMPK activity and prevents high-fatdiet-Induced obesity and insulin resistance in micerdquo Diabetesvol 62 pp 1855ndash1864 2013

[156] T Ito H Ando and H Handa ldquoTeratogenic effects of thalido-mide molecular mechanismsrdquo Cellular and Molecular LifeSciences vol 68 no 9 pp 1569ndash1579 2011

[157] K Dredge R Horsfall S P Robinson et al ldquoOrally admin-istered lenalidomide (CC-5013) is anti-angiogenic in vivo andinhibits endothelial cell migration and Akt phosphorylation invitrordquoMicrovascular Research vol 69 no 1-2 pp 56ndash63 2005

[158] L Lu F Payvandi L Wu et al ldquoThe anti-cancer drug lena-lidomide inhibits angiogenesis and metastasis via multipleinhibitory effects on endothelial cell function in normoxic andhypoxic conditionsrdquo Microvascular Research vol 77 no 2 pp78ndash86 2009

[159] M Ema R Ise H Kato et al ldquoFetal malformations and earlyembryonic gene expression response in cynomolgus monkeysmaternally exposed to thalidomiderdquo Reproductive Toxicologyvol 29 no 1 pp 49ndash56 2010

[160] F S Vianna L Fraga L Tovo-Rodrigues et al ldquoPolymorphismsin the nitric oxide synthase gene in thalidomide embryopathyrdquoNitric Oxide vol 30 pp 89ndash92 2013

[161] KMeganathan S Jagtap VWagh et al ldquoIdentification of thali-domide-specific transcriptomics and proteomics signaturesduring differentiation of human embryonic stem cellsrdquo PLoSOne vol 7 no 8 Article ID e44228 2012

[162] M Brooun A Manoukian H Shimizu H R Bode andH McNeill ldquoOrganizer formation in Hydra is disrupted bythalidomide treatmentrdquoDevelopmental Biology vol 378 pp 51ndash63 2013

[163] B E Hagstrom and S Lonning ldquoThe teratogenic action ofthalidomide on marine fish larvaerdquo Experientia vol 33 no 9pp 1227ndash1228 1977

[164] M Marin-Padilla ldquoThalidomide injury to an implantedarmadillo blastocystrdquoThe Anatomical Record vol 149 pp 359ndash361 1964

[165] H-J Merker W Heger K Sames H Sturje and D NeubertldquoEmbryotoxic effects of thalidomide-derivatives in the non-human primate callithrix jacchus I Effects of 3-(13-dihydro-1-oxo-2H-isoindol-2-yl)-26-dioxopiperidine (EM 12) on skeletaldevelopmentrdquo Archives of Toxicology vol 61 no 3 pp 165ndash1791988

[166] C J J Lee L L Goncxalves and P G Wells ldquoResistanceof CD-1 and ogg1 DNA repair-deficient mice to thalidomideand hydrolysis product embryopathies in embryo culturerdquoToxicological Sciences vol 122 no 1 pp 146ndash156 2011

[167] J A Dipaolo H Gatzek and J Pickren ldquoMalformationsinduced in the mouse by thalidomiderdquoThe Anatomical Recordvol 149 pp 149ndash155 1964

[168] S Fabro and R L Smith ldquoThe teratogenic activity of thalido-mide in the rabbitrdquo The Journal of Pathology and Bacteriologyvol 91 no 2 pp 511ndash519 1966

[169] S Fabro R L Smith and R T Williams ldquoToxicity and terato-genicity of optical isomers of thalidomiderdquo Nature vol 215 no98 p 296 1967

[170] I D Fratta E B Sigg and K Maiorana ldquoTeratogenic effects ofthalidomide in rabbits rats hamsters andmicerdquoToxicology andApplied Pharmacology vol 7 no 2 pp 268ndash286 1965

[171] L M Newman E M Johnson and R E Staples ldquoAssessmentof the effectiveness of animal developmental toxicity testing forhuman safetyrdquo Reproductive Toxicology vol 7 no 4 pp 359ndash390 1993

[172] M Parkhie andMWebb ldquoEmbryotoxicity and teratogenicity ofthalidomide in ratsrdquo Teratology vol 27 no 3 pp 327ndash332 1983

[173] K T Szabo and R L Steelman ldquoEffects of thalidomide treat-ment of inbred female mice on pregnancy fetal developmentand mortality of offspringrdquo American Journal of VeterinaryResearch vol 28 no 127 pp 1829ndash1835 1967

[174] K T Szabo andR L Steelman ldquoEffects ofmaternal thalidomidetreatment on pregnancy fetal development andmortality of theoffspring in random-bredmicerdquoAmerican Journal of VeterinaryResearch vol 28 no 127 pp 1823ndash1828 1967

[175] C J J Lee L L Goncalves and P G Wells ldquoEmbryopathiceffects of thalidomide and its hydrolysis products in rabbitembryo culture evidence for a prostaglandin H synthase(PHS)-dependent reactive oxygen species (ROS)-mediatedmechanismrdquo The FASEB Journal vol 25 no 7 pp 2468ndash24832011

[176] K T Al-Jamal W T Al-Jamal S Akerman et al ldquoSystemicantiangiogenic activity of cationic poly-L-lysine dendrimerdelays tumor growthrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 9 pp 3966ndash3971 2010

[177] A Jurand ldquoEarly changes in limb buds of chick embryos afterthalidomide treatmentrdquo Journal of Embryology and Experimen-tal Morphology vol 16 no 2 pp 289ndash300 1966


[178] M S Christian O L Laskin V Sharper A Hoberman DI Stirling and L Latriano ldquoEvaluation of the developmentaltoxicity of lenalidomide in rabbitsrdquoBirthDefects Research B vol80 no 3 pp 188ndash207 2007

[179] F Chung J Lu B D Palmer et al ldquoThalidomide pharmacoki-netics and metabolite formation in mice rabbits and multiplemyeloma patientsrdquo Clinical Cancer Research vol 10 no 17 pp5949ndash5956 2004

[180] J Lu N Helsby B D Palmer et al ldquoMetabolism of thalidomidein liver microsomes of mice rabbits and humansrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 310 no 2 pp571ndash577 2004

[181] D Vesela D Vesely and R Jelınek ldquoEmbryotoxicity in chickembryo of thalidomide hydrolysis products followingmetabolicactivation by rat liver homogenaterdquo Functional and Develop-mental Morphology vol 4 no 4 pp 313ndash316 1994

[182] J Knobloch K Reimann L-O Klotz and U Ruther ldquoThalido-mide resistance is based on the capacity of the glutathione-dependent antioxidant defenserdquo Molecular Pharmaceutics vol5 no 6 pp 1138ndash1144 2008

[183] R J DrsquoAmato S Lentzsch K C Anderson and M S RogersldquoMechanism of action of thalidomide and 3-aminothalidomidein multiple myelomardquo Seminars in Oncology vol 28 no 6 pp597ndash601 2001

[184] S Lentzsch M S Rogers R LeBlanc et al ldquoS-3-amino-phthalimido-glutarimide inhibits angiogenesis and growth ofB-cell neoplasias in micerdquo Cancer Research vol 62 no 8 pp2300ndash2305 2002

Submit your manuscripts athttpwwwhindawicom

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Zoology


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Microbiology


the testing was stringent enough as well as whether the disas-ter was preventable Following the thalidomide disaster newlegislation was introduced around the world that changed theway drugs are tested and their approval for human use espe-cially for pregnant women Indeed Dr Frances Kelsey pub-lished several papers on improving drug testing and safetywhich form the basis of drug testing carried out today [5 6]

12 Thalidomide Today In 1965 thalidomide was found to bevery effective in reducing the lesions associated with leprosyand (ENL) erythema nodosum leprosum a complication ofleprosy involving a chronic skin and nerve infection causedby Mycobacterium leprae [27] This discovery led to thalido-midersquos actions being studied in detail for clinical purposesReports linked the drug to having anti-inflammatory actionsand being effective as a clinical treatment for Leprosy ENLand other inflammatory disorders Furthermore studies indi-cated thalidomide was antiangiogenic and potentially usefulfor the treatment of cancers [28 29] Today thalidomide islicensed around the world (including the USA since 1998)for the treatment of leprosy and multiple myeloma (whereit can prolong patients lives by up to 18 months) [30 31]Thalidomide and its analogs are also used to treat a broadrange of other conditions including in the gastrointestinalsystem (eg Behcetrsquos disease Crohnrsquos disease) rheumatologi-cal system (eg lupus arthritis and sarcoidosis) and cancers(eg renal cell cancer prostate cancer and Karposirsquos sarcoma)[1 32 33] Recently thalidomide has been shown to beeffective in relieving the clinical symptomsof hereditary hem-orrhagic telangiectasia (HHT) a bleeding disorder [34] andidiopathic pulmonary fibrosis [35 36] Clearly thalidomidehas some very beneficial clinical uses However the dark sideof thalidomide still remains Thalidomide still causes birthdefects today particularly in Brazilmdashwhere several childrenhave been identified with thalidomide embryopathy in thepast few years [37ndash40]

13 Brazil Tragedy In recent years tragically a newgenerationof thalidomide survivors have been born and identified inBrazil [37ndash40] This is primarily due to a culture of medi-cation sharing less-stringent methods of prescription mis-understanding of the drugrsquos labeling and pregnant womentaking the drug while suffering from leprosy Sadly manychildren have been identified with thalidomide embryopathy[39 40]This Brazilian cohort provides an opportunity to fol-low these victims from childhood to adulthood usingmoderndiagnostic techniques Such studies could help determine theprecise range and type of damage at birth as well as late onsetdamage So far the Brazilian thalidomide survivors appear toexhibit a similar spectrum of damage as seen in the 1957ndash61disaster [39 40]

2 Thalidomide Embryopathy(or the Thalidomide Syndrome)

Between 1957 and 1961 thalidomide caused severe birthdefects in at least 10000 children [1 3] Although any tissueof the forming body could be affected by thalidomide some

hallmark features associated with the drug were identifiedincluding damage to limbs eyes ears genitalia and internalorgans including kidney heart and gastrointestinal tractAlso associated are vertebral column problems and facialpalsy [1 3 7 9 11ndash15 18 20 41 42] In addition another com-mon feature was the appearance of a transient haemangiomaon the forehead which usually disappeared within the first12ndash24 months of life [7] Due to the wide range of conditionsthis drug caused the damage is usually referred to collectivelyas thalidomide embryopathy or thalidomide syndrome [714 15] Infant mortality in babies born with thalidomideembryopathy is estimated to be as high as 40 quite likelydue to the serious internal malformations such as heart andkidney defects [3 7] Furthermore many babies with theseserious internal malformations would have been miscarriedor died in utero or soon after birth Thus the true number ofbabies affected by thalidomide will never be known

21 Thalidomide Acts in a Time-Sensitive Window of Em-bryonic Development Thalidomide causes damage to theembryo in a short timewindow of development (also referredto as the ldquocritical periodrdquo) between 20 and 36 days afterfertilisation or 34 to 50 days after the last menstrual period[7 11 25] Exposure to thalidomide from 36 days afterfertilisation has no apparent outward morphological effectupon the embryofetus [7 14] In contrast thalidomideexposure before the time-sensitive window of developmentcan inducemiscarriage in humans and rats [9 43] Incrediblyreports indicate just one 50mg tablet of thalidomide duringthe time-sensitive window is sufficient to cause birth defectsin 50 of pregnancies underlining the high teratogenicpotency of thalidomide [7 14 15 25] Some reports go furtherand suggest it would be surprising if any pregnancy wentunharmed following thalidomide exposure during the timesensitive window [7] The time-sensitive window was deter-mined following interviews with mothers of thalidomide-affected children and their doctors From these interviewsthe dates of intake and the amount of drug exposure werecalculated and an accurate correlation could be made tothe period of exposure and relationship to anomalies inorder to determine the timing of the damage [3 11 25] Theoutward morphological birth defects seen in thalidomideembryopathy are caused and correlate with exposure withinthis time period Exposure to thalidomide in the first fewdays of the sensitive period (days 20ndash24) affected the earsand eyes followed by the upper limb (day 24ndash31) and thenthe lower limb (days 27ndash33) respectively [11 25] Effects uponthe nerves resulting in facial palsies hearing loss and autismand epilepsy could be induced from the start of the timesensitive window presumably as the brainrsquos neural wiring isundergoing great changes at this time The consequence ofsuch nerve damage however may not be diagnosed untilmuch later after birth [11 25 42] Thalidomide exposure inlate fetal ratrsquos indicates that brain damage in areas related toautism can be a late event [44]

The time-sensitive window coincides with a period ofrapid embryonic development (from 4 weeks onwards) withlots of cell movements organogenesis and many signalling








































Thalidomide

N

NH

O

O

O

O

lowast

(a)

CPS49

F

F

F

F

F

F

O

O

N

(b)

Lenalidomide

O

O

ON

NH

NH2

(c)

Pomalidomide

O

O

OO

N

NH

NH2

(d)













Smooth muscle cell



Endothelial cell





Shh

Humerus

Radius

Digits

ZPAUlna

AER

Fgf8


Cell deathBV

Lossreductionof

proximal structures


Shh

Fgf8

Thal

idom

ide

(b) Phocomelia


















breakdownbyproduct



Geneexpression










































10 Conclusion









Acknowledgments



References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Thalidomide

N

NH

O

O

O

O

lowast

(a)

CPS49

F

F

F

F

F

F

O

O

N

(b)

Lenalidomide

O

O

ON

NH

NH2

(c)

Pomalidomide

O

O

OO

N

NH

NH2

(d)













Smooth muscle cell



Endothelial cell





Shh

Humerus

Radius

Digits

ZPAUlna

AER

Fgf8


Cell deathBV

Lossreductionof

proximal structures


Shh

Fgf8

Thal

idom

ide

(b) Phocomelia


















breakdownbyproduct



Geneexpression










































10 Conclusion









Acknowledgments



References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Smooth muscle cell



Endothelial cell





Shh

Humerus

Radius

Digits

ZPAUlna

AER

Fgf8


Cell deathBV

Lossreductionof

proximal structures


Shh

Fgf8

Thal

idom

ide

(b) Phocomelia


















breakdownbyproduct



Geneexpression










































10 Conclusion









Acknowledgments



References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















breakdownbyproduct



Geneexpression










































10 Conclusion









Acknowledgments



References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























10 Conclusion









Acknowledgments



References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References







































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article thalidomide embryopathy: an enigmatic challenge

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