The mere mention of thalidomide instils fear in some people to this day, although 50 years have passed since it caused the biggest medical disaster in history and even though it currently raises hopes for the treatment of leprosy and multiple myeloma.1–4 Originally marketed as a sedative and anti-hypnotic in 1957 by the German company Chemie-Grunenthal, it was also used to give effective relief from morning sickness during early pregnancy.4–7 It was used in over 46 countries and was advertised and marketed as being entirely safe (Fig. 1). By 1961 it had become clear that thalidomide was the reason for a huge increase in birth defects, principally to the limbs of babies8–10 (Table 1). Other birth defects comprised ear and eye problems, including micropthalmia and coloboma, genital abnormalities, peripheral neuropathies and internal organ defects, particularly to the kidneys, lungs, intestinal tract and heart (Table 1). Over 10,000 children were born with severe birth defects.4, 5, 7, 9, 11
Figure 1. Distaval was the trade name of thalidomide in the UK. (A–C) Original Distaval packet with tablets. A: Front of the packet. B: Rear of the packet. Instructions highlight how the drug was thought to be safe and very effective, without side effects. C: Inside packet explaining dosage and showing three original tablets. This packet was kindly lent to me by Mr Gary Linney (Ellon, Scotland).
Download figure to PowerPoint
Table 1. Thalidomide-induced birth defects and incidences
|Where described in detail:|| |
| Four limb phocomelia||16/165 = 9.7%|
| Upper limb defects + lower limb defects||19/165 = 11.5%|
| Upper limb phocomelia + normal lower limb||61/165 = 37%|
| Lower limb defects||14/165 = 8.4%|
| Other forelimb defects (e.g. thumb loss)||38/165 = 23%|
|Other organ defects||11–21%|
|Where described in detail:|| |
| ear|| |
| genitals|| |
| kidney|| |
| gut|| |
| neurological|| |
|Other organ defects occurring together with limb defects are:||18%|
|Infant mortality within the first year of life, increased by:||40%|
Defects occurred when drug exposure took place within a short time-sensitive window ranging from day 20 to 36 after fertilisation (days 34–50 after last menstrual cycle).4, 9, 12, 13 Surprisingly, just a single tablet of the drug during the time-sensitive window was enough to cause a limb defect.4, 12, 13 Sadly, the mortality rate for babies born with thalidomide-induced embryopathy was very high, estimated to be up to 40% before their first birthday, and likely due to internal organ defects, particularly heart and kidney defects.4, 5 An unknown number of miscarriages may also have been caused by thalidomide.5 Indeed, there is evidence that exposure of thalidomide before and during the embryological time-sensitive window, leads to spontaneous abortion in humans and rats.14, 15 As many babies with such internal organ conditions would have been miscarried or died soon after birth, this is likely to be the reason why the majority of survivors (up to 90%) exhibit limb defects, and less suffer from severe, internal problems. The drug was subsequently banned in 1962.
However, thalidomide has enjoyed a renaissance in recent years. It is now used to successfully treat, under strict and carefully controlled guidelines, leprosy and multiple myeloma.1–3, 16, 17 Thalidomide is also being used to treat other ailments, ranging from Crohn's disease to AIDS and some cancers.1, 2, 6, 18–20 Yet, tragically, there are still children being born today with thalidomide-induced limb defects in Africa and South America, where the drug is the preferred treatment for leprosy, due to poor patient understanding of the drug and contraceptive administration.21, 22 Despite this, the mechanism(s) underlying thalidomide-induced teratogenesis have, until recently, proven elusive.
One reason for the difficulty in determining the mechanism(s) of action of thalidomide is due to the complex chemistry and the multiple actions of this drug.1, 2, 6 Structurally, thalidomide is a glutamic acid derivative and consists of a glutarimide ring and pthalimido ring (Fig. 2). Thalidomide exists as two isomeric forms, S(−) and R(+), which can interchange under physiological conditions.1, 2 A racemate mix of these two isomers is used clinically and the two isomers are believed to have different biological properties. The S(−) isomer is thought to be responsible for the teratogenic actions of thalidomide, the R(+) isomer appears to be an effective sedative.1 Due to the ability of the isomers to interchange under physiological conditions, it is not possible to isolate one form from the other for clinical applications.
Figure 2. Structures of thalidomide and CPS49. Thalidomide (A) is metabolised by cytochrome p450 enzymes (Cyp2C19) into 5′-OH thalidomide (B), which has antiangiogenic actions.1, 31–34 (C) CPS49, a synthetic analogue, is based on the structure of 5′-OH thalidomide.1, 29 The fluorination of the compound markedly enhances its bioactivity.1, 32, 34
Download figure to PowerPoint
Thalidomide requires metabolic breakdown to be active, which occurs in the liver through the cytochrome P450 group of enzymes, and results in over 20 metabolic byproducts (Fig. 2).1, 23–26 Thalidomide exerts anti-inflammatory, immunomodulatory and antiangiogenic actions.1, 2 Analogues based on the parent thalidomide structure and retaining its immunomodulatory and antiangiogenic actions, e.g. lenalidomide, have been produced and are currently used to treat multiple myeloma.2, 17, 27 Synthetic analogues based on the structures of antiangiogenic metabolites of thalidomide have also been synthesised for use as improved anti-cancer agents.1, 28–32 Such analogues differ in structure from thalidomide as they are either fluorinated or have substitutions of amino groups29, 31, 33 (Fig. 2). Such substitutions confer greater biological activity in vitro and in vivo and, in particular, the tetrafluorinated CPS49 has been shown to possess only antiangiogenic activities in vitro and in vivo29, 32, 34 (Fig. 2). Furthermore, over 100 analogues of the breakdown products of thalidomide have been synthesised,1 which allows a full analysis of their respective functions in vivo, to determine which of thalidomide's breakdown products cause defect, and more importantly, study their potential for therapeutic uses.
Mechanism of limb teratogenesis
Most research into thaliomide's teratogenic mode of action has focused on how thalidomide caused limb defects, as limbs are the structures most often seen to be affected, in survivors of thalidomide embryopathy. Thus, many models of thalidomide action focus entirely on the limb but cannot explain other defects seen in thalidomide embryopathy. A number of animal embryo model systems have been used to study its actions, the most popular being chick,35–38 rabbit39, 40 and marmoset.41 Over 30 models/hypotheses have been postulated over the years.6, 19, 42 However, only a few are based on experimental evidence or are plausible, and even these have difficulties to explain all aspects of thalidomide teratogenesis. The models can basically be grouped into seven categories, which are briefly outlined below:
Inhibitor of angiogenesis
Several studies have shown that thalidomide blocks angiogenesis in the chick limb36 and in the rat eye.43 However, how blood vessels would be targeted in specific tissues has neither been demonstrated nor discussed.
Induction of cell death
Thalidomide can induce cell death and formation of reactive oxygen species in limb tissue,38, 44, 45 which could explain the loss of skeletal elements in thalidomide-treated limbs. However, why such cell death and generation of reactive oxygen species would be specific to the limb only, as well as what the actual trigger inducing cell death is, remains unclear (for further discussion, see Challenge 3).
Antagonism of integrin/cell adhesion signalling
Integrins are essential for cell adhesion and migration, through controlling the actin cytoskeleton, in many developing tissues.46 Thalidomide antagonises integrin expression in marmoset embryos41 and can bind to N-cadherin.47 In addition, specific vascular integrins such as αVß3 have been proposed to be inhibited by thalidomide through antagonising FG2/IGF1 signalling, although this was not demonstrated in vivo.6, 19 Importantly, the mechanism underlying the tissue specificity of thalidomide is neither described nor discussed.
Effects on growth factor signalling
An interesting theory by Tabin (1998)48 suggested that thalidomide could cause distalisation of the limb bud by blocking or reducing growth factor signalling during limb development, causing loss of proximal tissue, but allowing remaining tissue to be distalised, thus producing phocomelia. However, the mechanism(s) causing or triggering this effect was not demonstrated, neither was how this model can account for the other tissue defects induced by thalidomide.
McCredie and McBride (1973)49 suggested that neural tissues are involved in inductive events in the limb and that thalidomide damages neural tissue, resulting in limb outgrowth failure and potential loss of cartilage elements. This was postulated to be also the cause of all other defects.50 However, recent work has shown that chick and mouse limbs are not innervated until relatively late in limb development51, 52 and nerveless limbs form normally without any apparent limb deformity.51, 53, 54
Intercalation with DNA
Based on the structure of thalidomide, it has been suggested that thalidomide could intercalate into, and interfere with, DNA function, i.e. gene promoter activation.42, 55 However, it remains unclear how such intercalation events would account for the specificity and range of defects induced by thalidomide.19, 42 Moreover, thalidomide is not mutagenic and defects are not hereditary.56–59
Effects on chrondrogenesis
Could thalidomide affect chrondrogenesis directly and block skeletal element formation? Phocomelia can be experimentally induced by X-irradiating the early chick limb bud. This results in a loss of proximal skeletal elements.60, 61 However, if thalidomide acts this way, why would such an effect be specific to just limb skeletal elements, and not other skeletal elements? How would skeletal elements be targeted directly by thalidomide?
The number of models/hypotheses published, with varying levels of experimental evidence, highlights the continuing and ongoing controversy in the field. It seems that the above models can, to some extent, each explain certain aspects of thalidomide action. However, what is still fundamentally missing in our understanding is the determination of the actual triggering event induced by thalidomide, followed by the establishment of the order of the resulting secondary effects.
Why does the limb represent the main target?
Surprisingly, 90% of surviving children, born to mothers, who took thalidomide during pregnancy, had a limb defect. Limb development is a complicated, yet carefully controlled process. The limbs form relatively late during embryonic development, i.e. after the body plan has been established and the major tissues and organs have been formed. In humans, limb development starts at day 23 after fertilisation (week 4) and lasts until day 55 (week 9). The time-sensitive window of thalidomide action ranges from day 20 to 36 post-fertilisation.4, 9, 12, 13, 64 Thalidomide was typically taken to relieve the symptoms of morning sickness (around 4–10 weeks), a time period in which the limbs are rapidly developing. Limb development proceeds from proximal (humerus) to distal (digits). Thalidomide exposure before and after the start of this time window resulted in the most severe forms of limb defect, and later exposure resulted in digit loss.4, 11–13, 64–66
Through an interplay between specialised signalling regions, the zone of polarising activity (ZPA) and the apical ectodermal ridge (AER), the limb grows out from the flank and is patterned into the final form, with correctly positioned bone, muscle, nerve, blood vessel and tendon formation.67–70 Essentially, the AER signals (Fgf8) to the underlying mesenchyme (Fgf10).71 FGF signalling maintains cell proliferation and survival and hence limb outgrowth, and through induction of gene networks, including Hox genes, patterns the limb67, 68, 71–73 (Fig. 3A).
Figure 3. Limb development and origins of phocomelia. A: Normal limb development. The limb bud grows out from the flank of the embryo at day 2.5 in chicks and day 23 in humans. Under the control of the ZPA and the AER, the limb is patterned and carefully regulated to produce the fully formed limb. The ZPA releases sonic hedgehog (Shh), which exists in a feedback loop with FGFs in the AER. The FGFs in the AER in turn exist in a feedback loop with FGFs in the mesenchyme. These loops control the outgrowth and patterning of the limb through activation of genes including Hox genes. B: Model of the origins of phocomelia: CPS49 inhibits vessel formation, resulting in cell death and changes in gene expression, leading to limb defect. Prolonged exposure to the drug results in amelia. Short exposure results in phocomelia or limb deformity, depending on timing of exposure, allowing AER function to recover.
Download figure to PowerPoint
As limb development is a carefully regulated process, there are many events that can fail, thereby causing limb defects. Indeed, thalidomide induced a range of limb defects ranging from amelia (the whole limb is missing) to phocomelia (loss of or severe shortening of the proximal elements) to ‘just’ a loss of thumb or digits4, 64–66 (Table 1). In the majority of cases, thalidomide affected both forelimbs; in some cases all four limbs were affected.4, 11, 64–66 Abnormalities of the lower limbs are rarely seen alone74 (Table 1). This may be due to the fact that the lower limbs develop after the upper limbs. How do these limb defects come about? And how can such a range be induced? Why are such defects induced in such a small developmental time window?
Thalidomide destroys immature blood vessels
Thalidomide can induce limb defects when applied to chick embryos, which are ideal for testing drug action as the embryos are large, develop rapidly and are easy to manipulate and image.34–36, 38 However, due to the complicated nature of the biochemistry and metabolism of thalidomide, it is very difficult to determine which of its actions and byproducts are actually responsible for inducing defects. Breakdown products and analogues of the drug, with specific actions, have recently been isolated, allowing dissection of the drug action in vivo, to finally determine which aspect of thalidomide is responsible for teratogenesis.1, 29–34 This approach also raises the possibility of producing a ‘safer’ form of the drug.
Taking advantage of these new reagents, we recently demonstrated, using the chick embryo, that the application of an antiangiogenic-only analogue of thalidomide, CPS49, at the time when the limbs are forming, specifically causes limb reduction defects; the rest of the embryo developed normally34 (Fig. 4). Anti-inflammatory metabolites and other hydrolysis products do not cause limb defects and the embryos are perfectly normal.34 Thus, it is the antiangiogenic nature of the drug that causes limb defects. Although it has previously been suggested that blood vessels/angiogenesis could be a target of thalidomide to cause limb teratogenesis,6, 19, 36, 43, 75 this work is the first to conclusively demonstrate, in vivo, that blood vessels are the primary target. Gene expression changes, including the loss of Fgf8 and Fgf10 signalling, and an increase in cell death are all secondary to the effect on the vessels.
Figure 4. CPS49 induces limb defects. Chick limbs 4 days after treatment at day 2.5, when limbs are just starting to grow out from the embryonic flank, are shown. A: Untreated control limb. B: CPS49-treated limb. Note that the limb bud is truncated and is just a stump (black asterisk). Other parts of the embryo appear normal. This is due to vessel immaturity in the limb at the time of drug application. Scale bar, 1,000 µm.
Download figure to PowerPoint
A combination of in vivo (using chick and zebrafish embryos) and in vitro studies (using mouse, rat and human cell cultures) demonstrated that CPS49 selectively destroys newly formed, angiogenic vessels and temporarily stunts outgrowth of mature, stable vessels by preventing filopodial extensions from the endothelial tip cell.34 This work further showed that mouse and rat tissues are in fact sensitive to thalidomide derivatives, and has implications for understanding the species specificity of the drug (see Challenge 1). The loss of newly formed vessels and prevention of new vascular network formation is devastating in highly angiogenic tissues such as the limb bud. Indeed, a 50% decrease in limb vessel density is observed within 6 h of CPS49 application, without decrease in limb area.34 The time at which the drug was taken by pregnant mothers, corresponds to a period in which the limb vasculature is highly angiogenic, and to the period of the most rapid limb outgrowth. This is in contrast to the rest of the embryo, which has less angiogenic and more mature blood vessels at this time period.34 However, addition of the drug earlier in embryogenesis, when all vessels are angiogenic, results in lethality and multi-organ defect, addition much later in development, when most vessels in the embryo and limb are stable and mature, results in ‘only’ minor defects to digit tips.34 This further suggests that thalidomide would likely have little effects in adults/pregnant mothers taking the drug for short-term periods.
These data explain the time-sensitive window of action on the limb, the increase in miscarriage rates and reasons underlying the increased infant mortality rates, and can also explain the range of other birth defects, all dependent on the timing of exposure and state of tissue vascularisation, e.g. eye, ear, internal organs.34 These data further explain why limb defects are the most common defect seen in live-born thalidomide-damaged children, because internal organ defects induced through effects on vascularisation, as the organ was forming, were likely lethal in utero.34
The major outcomes of the work are:
identification of the antiangiogenic action of thalidomide as the triggering event which then causes limb defects, and explanation of the range of limb defects induced by thalidomide, through timing of exposure;
the antiangiogenic action of the drug could be the underlying cause of all defects associated with thalidomide embryopathy;
demonstration of the cell biological basis of the antiangiogenic nature of thalidomide;
consideration of the requirements to explain embryonic thalidomide embryopathy.