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Keywords:

  • antiangiogenesis;
  • chick limb development;
  • CPS49;
  • phocomelia;
  • thalidomide analogue;
  • therapeutics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References

Despite the recent discovery that thalidomide causes limb defects by targeting highly angiogenic, immature blood vessels, several challenges still remain and new ones have arisen. These include understanding the drug's species specificity, determining molecular target(s) in the endothelial cell, shedding light on the molecular basis of phocomelia and producing a form of the drug that is clinically effective without having side effects. Now that the trigger of thalidomide-induced teratogenesis has been uncovered, a framework is proposed, incorporating and uniting previous models of thalidomide action, explaining how thalidomide causes not just limb defects, but also all the other defects it induces.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References

Historical background

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

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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).

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Table 1. Thalidomide-induced birth defects and incidences
  1. Summary of published data from the UK (1958–1962),4, 5, 66 Germany (1956–1962)9 and South America (1969–1995)21 indicating the most commonly seen defects and their incidences at birth (following exposure during pregnancy). The nature and incidence of the internal organ defects may be under-represented, as it was difficult to determine accurately such problems in the 1950s/1960s.

Limb malformations79–89%
Where described in detail: 
  Four limb phocomelia16/165 = 9.7%
  Upper limb defects + lower limb defects19/165 = 11.5%
  Upper limb phocomelia + normal lower limb61/165 = 37%
  Lower limb defects14/165 = 8.4%
  Other forelimb defects (e.g. thumb loss)38/165 = 23%
 
Other organ defects11–21%
Where described in detail: 
  ear 
  genitals 
  kidney 
  gut 
  neurological 
Other organ defects occurring together with limb defects are:18%
Infant mortality

5, 7

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.

Biochemistry

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.

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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

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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.

Nerve toxicity

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.

Requirements to explain embryopathy

In order to be considered an explanation or model of thalidomide-induced limb defects and embryopathy, several parameters need to be addressed (a similar set has previously been outlined)62, 63:

  • explain limb specificity and cause of the other defects – any mechanism must be able to explain the range of effects observed, i.e. all defects are caused by a common mechanism,

  • explain the time-sensitive window within which thalidomide causes defects,

  • explain how the action of the drug is primarily restricted to the embryo – mothers were not harmed by the drug.

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).

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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.

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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.

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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.

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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.

Challenges

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References

Despite our understanding of how thalidomide induces and triggers limb defects in embryos, challenges remain, and some previously postulated models for its teratogenic action may help provide some answers. Some of these challenges are outlined in this section.

Species specificity

Thalidomide is a teratogen in humans and primate species, as well as chickens, but has no effect on rodents6, 76 or hamsters.39, 77, 78 The reason for this species specificity remains unclear. CPS49, and thalidomide, can inhibit angiogenesis in mouse and rat aortic ring cultures and mouse cornea models,29, 34, 79 highlighting that angiogenic events in mice and rats are not insensitive to the action of the drug. So why were mouse and rat embryos not affected in utero?

Evidence exists for differential ways of thalidomide metabolism between species in which the drug after incubation with human or rabbit liver microsomes, shows greater antiangiogenic activity than after incubation with rat liver microsomes.76, 80 Species differences exist in the metabolism of the drug and the resulting half-life activities of such metabolites but it remains unclear whether the teratogenic, antiangiogenic products are present in mice/rats.81 Alternatively, species differences in receptors preventing transport of specific forms may exist at the placental surface or indeed placental structural differences may play a role. Indeed, it is unclear how thalidomide gains entry into cells – whether through a transport protein or channel – and species differences in the presence of such transport proteins or channels may exist. If the basis of the species specificity or protection can be identified, this mechanism could possibly be used to prevent transfer of the drug to the embryo, or prevent the drug from being broken down into the teratogenic metabolites.

Is thalidomide embryopathy due to a common mechanism – blocking angiogenesis?

Thalidomide causes a wide range of effects on multiple tissues, including limb, eye, ear, internal organs, genitalia and nerves4, 5, 7, 9, 11 (Table 1). Are these effects all caused by one action or mechanism of the drug? We have demonstrated that an antiangiogenic-only analogue of thalidomide, CPS49, blocks angiogenesis and causes severe limb malformations in chick embryos.34 Earlier application of CPS49 to embryos, as the body plan and organ formation require a rapidly changing angiogenic network, results in lethality; later application leads to minor limb defects. This suggests that the defects caused by thalidomide are induced through one mechanism that blocks angiogenesis.

Indeed, there is evidence that blocking angiogenesis whilst tissues/organs are forming, results in defect. For example, micropthalmia is induced in mouse embryos lacking VEGF164, a receptor involved in the regulation of angiogenesis.82 Defects in eye vasculature cause eye disorders, including retinopathy.83 Valproic acid, another human teratogen, which is used to treat epilepsy and shows antiangiogenic activity, causes a range of morphological defects in chick embryos, including the limb.84–86 In addition, development of the internal organs, including the kidney and the genitalia, requires angiogenesis for normal formation and function.87, 88 Finally, developmental cues that are essential for normal blood vessel formation are also required for neuronal guidance and development.89 Thus, a loss of blood vessels or failure of angiogenesis following thalidomide exposure could then, as a consequence, lead to neuronal innervation failure as well as neuropathies.

Assuming vessels are the target and trigger of all thalidomide-induced birth defects4, 9, 11 (Table 1), then a form of the drug that treats inflammatory disorders, such as leprosy, but does not contain the antiangiogenic part of the drug, could be made and used. This would prevent any more children from being born with thalidomide-induced limb defects in South America and Africa.

Molecular targets of thalidomide in the endothelial cell

CPS49, when applied to human umbilical vein endothelial cells (HUVEC) or cancer cell lines, induces the stress response kinase p38α, leading to endothelial cell death.32 This suggests that CPS49 induces cellular stress to the endothelial cells, resulting in death. Indeed, CPS49, and thalidomide, induce actin stress fibres and prevent endothelial cell proliferation.34, 37 Both CPS49 and thalidomide exposure causes massive cell death in the chick limb mesenchyme,34, 38, 45 as a result of the blood vessel inhibition.34 Cell survival pathways, including Akt and Wnt signalling, are down-regulated in thalidomide-treated chick limb buds,38, 45 likely leading to a localised up-regulation of reactive oxygen species and cell death. This increased mesenchymal cell death could then reduce or antagonise growth factor signalling, in turn affecting cell differentiation, tissue formation and development.34 This mechanism could provide a template of action for all thalidomide-sensitive tissues targeted by the drug (see Conclusion).

However, the expression of many molecules involved in the stimulation and regulation of angiogenesis has been shown to change following thalidomide treatment. These molecules include integrins, vascular endothelial growth factor, ceramide/sphingosine 1-phosphate, hepatocyte growth factor, angiopoietins and insulin growth factor-1.6, 19, 37, 41, 43, 90, 91

The key is now to study the detailed molecular events in the endothelial cell and the resulting downstream consequences. How do they fit together? What is the sequence or order of events? Are the events the same in all tissues? This would open up the possibility of targeted forms or uses of the drug in specific conditions.

Phocomelia

How do distal limb structures form at the expense of proximal ones, resulting in the rare conditon, phocomelia?

CPS49 has an active half life of 6–12 hours92 and prevents angiogenesis, which in the forming, outgrowing limb is devastating, inducing cell death, shutting down limb signalling pathways and resulting in a range of severe limb defects in a time-sensitive manner.34 We have proposed that phocomelia could arise, following thalidomide exposure in early limb development, blocking angiogenesis and resulting in the almost complete loss of mesenchyme.34 If some signalling remained in the AER, this would allow the re-establishment of FGF signalling and limb outgrowth and specification of distal fate, in the remaining mesenchymal cells. Indeed in limbs of rabbit embryos, Fgf8 and Fgf10 expression is reduced after thalidomide exposure, followed by both gene expression patterns, recovering within several days, and phocomelic limbs can form.93 In addition, mouse limbs with severely reduced FGF signalling capacity lack proximal elements, due to increased, localised proximal cell death.73 This highlights the key role that FGF signalling plays in the proximo-distal specification of limb pattern.

It has been demonstrated that when proximal chick limb tissue is X-irradiated, only distal limb elements form.60, 61 This finding has been attributed directly to a destruction of proximal limb element precursor cells, resulting in phocomelia.61 But what is the trigger for such an event in thalidomide-treated limbs? We have shown that blood vessels are targeted by thalidomide, leading to limb defects. Taken together, following thalidomide-induced vessel inhibition during early limb development, cell populations, destined to form proximal elements, could be lost due to reduced AER/FGF signalling, which results in cell death. Once the drug effect has worn off, the remaining cell populations expand in response to re-covered FGF signalling from the AER and form distal structures, thus producing phocomelia34 (Fig. 3B).

The exact mechanism underlying phocomelia remains unclear and a challenge. However, understanding the molecular basis of phocomelia will help understand normal proximo-distal limb outgrowth and also provide a basis for understanding the aetiology of genetic forms of human phocomelia.94–96

Therapeutic potential and applications

We have shown that analogues and specific metabolites of thalidomide can be used, in in vivo embryo assays, to determine what part of the drug causes limb defects, and to gain insights into its mechanism of action.34 Over 100 analogues of thalidomide have been produced, each with a different range of actions.1 These analogues/metabolites could now also be used as tools to (i) investigate their mechanism(s) of action in detail to further understand the action of thalidomide; (ii) investigate their potential as early anti-tumour agents; e.g. CPS49 prevents angiogenesis and new vessel formation, suggesting that such metabolites/analogues could prevent early tumours from becoming vascularised; (iii) investigate their potential use as safer alternatives in current treatments as, for example, prolonged clinical use of thalidomide causes peripheral neuropathy.97, 98 Can a thalidomide analogue be found or produced that does not induce peripheral neuropathy, but retains clinical benefits? This in turn would shed light on how side effects such as peripheral neuropathy are actually caused; (iv) identify forms or engineer new forms of the drug for targeting and treatment of a specific condition without side effects, e.g. to treat leprosy without the side effects caused by the antiangiogenic actions of the drug; (v) determine how exactly the drug treats some of the conditions for which it is used today, for example, multiple myeloma.

Multiple myeloma is a condition in which the bone marrow overproduces white blood cells. The primary cause of this condition is unclear. Excessive blood vessels in the bone marrow have been reported.17, 90, 99, 100 An immunomodulatory analogue of thalidomide, lenalidomide, has gained FDA approval2, 17 to successfully treat multiple myeloma in the US and Europe. However, it is unclear how lenalidomide successfully treats this condition as the drug possesses immunomodulatory and antiangiogenic actions.27, 101 Its antiangiogenic action means that it could also exert teratogenic effects. Its use and administration is very carefully controlled.102 What is required are metabolites or analogues with only immunomodulatory or antiangiogenic actions. Such substances could be used in models of the multiple myeloma condition to determine which component, or indeed whether both components are required, to treat multiple myeloma. Such research will help understand the condition and perhaps produce further therapeutic options for the treatment of multiple myeloma.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References

How thalidomide causes limb defects is a question which medicine and science have been asking ever since the first cases of thalidomide embryopathy were described between 1958 and 1961.

The isolation and synthesis of thalidomide metabolites, byproducts and analogues has opened up the field of thalidomide and some of these byproducts and analogues are now being used as clinical therapeutic drugs to treat a very wide range of diseases including leprosy, multiple myeloma, cancer, HIV, Crohn's disease etc.1, 2, 6, 17, 101 However, due to the global renaissance that thalidomide is now enjoying, there have been new cases of thalidomide embryopathy reported in South America and Africa.21, 22 This further highlights the need to understand how this drug acts and produces its teratogenic effects. Indeed, the large number of isolated metabolic byproducts and analogues of thalidomide now finally allow the elucidation of how the drug causes limb defects. This raises the possibility of producing a form of the drug with clinical benefits but without the teratogenic side effects. Using such an approach, we have finally demonstrated that the antiangiogenic action of the drug is responsible for the development of limb defects (and likely also for all thalidomide-induced birth defects), and shown how the action of thalidomide was restricted to such a small developmental time window.34

Understanding how thalidomide causes limb defects, resolves a 50-year-old puzzle. This finding, in turn, provides an opportunity to produce a framework to understand the teratogenic mechanism of action of the drug, uniting some previously postulated models of action in the limb. This framework also acts as a template for effects on all the other tissues affected by thalidomide, although the precise nature of the downstream molecular and morphological consequences in each tissue/organ may differ (Fig. 5). In essence, tissues/organs are most sensitive to thalidomide when they are undergoing growth and expansion, which requires rapid angiogenesis and new vessels to supply nutrients, etc. Exposure to thalidomide at this stage of tissue development inhibits angiogenesis, which causes death of cells and disruption of growth factor signalling pathways, resulting in tissue failure/loss of cell types (i.e. chrondrocytes, nerves, etc.) and birth defect.

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Figure 5. Framework of thalidomide teratogenesis. Thalidomide inhibits angiogenesis in endothelial cells, causing localised cell death and resulting in tissue/mesenchymal loss and disruption of growth factor signalling pathways, which will likely also contribute to the increase in cell death, as cells lose maintenance and protective signals, resulting in defect. In the developing limb, one effect would be the loss of cell populations that may contribute to chrondrogenesis.

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However, questions remain on how thalidomide affects the developing limb, particularly with respect to the induction of phocomelia (loss of proximal elements). Does it arise through a recovery of apical ridge signalling, after loss of limb mesenchyme? Understanding this process will also help further understand the normal mechanisms that control the proximo-distal patterning of the limb bud.

Several key questions and challenges remain in the quest to produce a ‘safer’ form of the drug – one that retains clinical benefits but has no side effects and is not teratogenic. These include confirming that all birth defects caused by thalidomide are through effects on blood vessels (i.e. a common mechanism), understanding why rodent embryos are resistant to the drug, and understanding the detailed tissue-specific molecular events following thalidomide exposure and vascular inhibition. Understanding each of these challenges will not only shed light on the molecular action of thalidomide, but also provide insights into the use of the drug for new clinical treatments or to treat specific conditions. Such an approach may contribute to understand the basis of the clinical conditions thalidomide is used to treat (e.g. multiple myeloma) as well as gain a handle on some of the side effects that are caused by thalidomide (e.g. peripheral neuropathy). Together with advances in molecular biology and pharmacology, a huge range of thalidomide derivatives and analogues already exist, each of which contains different actions of the drug. Thus the possibility of producing specific analogues of the drug targeted to a specific condition, without having side effects, is becoming a real prospect.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References

Thanks to Martin Collinson for critical comments on the manuscript and to Lynda Erskine for helpful discussion. I am most grateful to Gary Linney, (Ellon, Scotland) for lending to me and for permission to photograph his Distaval tablets and packaging. Thanks also to Christina Therapontos, Erin R. Gardner and William D. Figg for helpful discussions throughout this project. I also thank Robert L. Smith, Juergen Knobloch and John Shaughnessy Jr. for useful discussions about thalidomide. Dedicated to C.G.V and C.W.M.V.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Challenges
  5. Conclusions
  6. Acknowledgements
  7. References