HackTwat
MuscleChemistry Registered Member
Abstract
Athletes and bodybuilders have recognized for several decades that the use of anabolic steroids can promote muscle growth and strength but it is only relatively recently that these agents are being revisited for clinical purposes. Anabolic steroids are being considered for the treatment of cachexia associated with chronic disease states, and to address loss of muscle mass in the elderly, but nevertheless their efficacy still needs to be demonstrated in terms of improved physical function and quality of life. In sport, these agents are performance enhancers, this being particularly apparent in women, although there is a high risk of virilization despite the favourable myotrophic–androgenic dissociation that many xenobiotic steroids confer. Modulation of androgen receptor expression appears to be key to partial dissociation, with consideration of both intracellular steroid metabolism and the topology of the bound androgen receptor interacting with co-activators. An anticatabolic effect, by interfering with glucocorticoid receptor expression, remains an attractive hypothesis. Behavioural changes by non-genomic and genomic pathways probably help motivate training. Anabolic steroids continue to be the most common adverse finding in sport and, although apparently rare, designer steroids have been synthesized in an attempt to circumvent the dope test. Doping with anabolic steroids can result in damage to health, as recorded meticulously in the former German Democratic Republic. Even so, it is important not to exaggerate the medical risks associated with their administration for sporting or bodybuilding purposes but to emphasize to users that an attitude of personal invulnerability to their adverse effects is certainly misguided.
Androgens
Androgens exert their effects in many parts of the body, including reproductive tissues, muscle, bone, hair follicles in the skin, the liver and kidneys, and the haematopoietic, immune and central nervous systems. The androgenic effects of these hormones can be generally considered as those associated with masculanization and the anabolic effects as those associated with protein building in skeletal muscle and bone.
In the male foetus, androgens stimulate the development of the Wolffian ducts (epididymis, vas deferens, the seminal vesicles and ejaculatory duct) and the male external genitalia (penis, urethra and scrotum). During puberty, the androgenic effects resulting from increased testicular steroidogenesis are manifested by growth of the testes, external genitalia and the male accessory reproductive glands (prostate, seminal vesicles and bulbourethral), and secretory activity begins. Further, the secondary sexual characteristics manifested during puberty can be divided into those that are a result of androgenic and anabolic effects. The androgenic effects are the enlargement of the larynx causing a deepening of the voice, the growth of terminal hair (in the pubic, axillary and facial regions; in other regions such growth depends on a number of factors), an increase in sebaceous gland activity (can lead to acne), and CNS effects (libido and increased aggression). Anabolic effects are the growth of skeletal muscle and bone, the stimulation of linear growth eventually ceasing due to the closure of the epiphysis. In men, androgens are essential for sustaining reproductive function, and they play an important role in maintaining skeletal muscle and bone, cognitive function and a sense of well-being.
The most important androgen secreted is testosterone; in the eugonadal man, the Leydig cells in the testes produce ~95% of the testosterone in the body. The ovaries and the adrenal glands (in both sexes) produce very little testosterone but secrete weaker androgens; in particular, dehydroepiandrosterone (DHEA; and its sulpho-conjugate) and androstenedione are of physiological importance in the women, not least because they can undergo peripheral conversion to more potent androgens, for example to testosterone and 5α-dihydrotestosterone (DHT). Another weaker endogenous androgen, androstenediol, also binds to oestrogen receptors.
The effects of androgens are modulated at cellular level by the steroid-converting enzymes within the particular target tissue. In reproductive target tissues, testosterone can be considered to be a prohormone, being readily converted by 5α-reductase to the more potent androgen DHT. In other tissues, such as adipose tissue and parts of the brain, testosterone is converted by aromatase to the oestrogen, oestradiol. In bone, the mechanism of action of the anabolism of androgens has not been entirely elucidated but both a direct effect of testosterone and a mediated effect by aromatization to oestradiol are important. In the human skeletal muscle, 5α-reductase activity (either type 1 or 2) is not detectable, so testosterone itself is chiefly binding to the androgen receptor (as supported also by a number of animal studies, mainly in the rat). Aromatase expression and activity is significant in human skeletal muscle but whether the conversion of androgens to oestrogens within this tissue is physiologically important for mediating some of the myotrophic effect of androgens is yet to be determined.
<!--fig ft0--><!--fig mode=article f1-->Modulation of the effects of androgens may also occur at the molecular level due to differences in the distribution of androgen receptor coregulators in various tissues, these coregulators being proteins that affect the transcriptional activity of the androgen receptor. This is a developing field and the comparative importance of many of these coregulators is yet to be established for any particular cell type, let alone their relative in vivo importance in examining tissue differences in androgen action. It is envisaged that genetic manipulation of the mouse will assist in elucidating their physiological relevance.
With structural modifications to testosterone, the anabolic effects of androgens can be enhanced but, even so, these cannot be divorced entirely from their androgenic effects. Hence, a more accurate term for anabolic steroids is anabolic–androgenic steroids, but, for simplicity, the shorter term is used within this paper. The disassociation of anabolic from androgenic effects can be at cellular level, depending on the intracellular metabolism of the anabolic steroid in different tissues, with the activity of 5α-reductase being particularly important (see the section ‘Intracellular metabolism and the myotrophic–androgenic index'). An appealing hypothesis is that anabolic–androgenic dissociation can also occur as a result of anabolic steroids inducing specific conformational changes of the androgen receptor complex, which then affects subsequent interaction with various coregulators in different tissues (see the section ‘Androgen receptor expression and the importance of coregulators'). There is little data, as yet, to support such a hypothesis, but it is known that the androgen co-activator FHL2 is expressed predominantly in the heart, and it is possible that a number of other androgen receptor coregulators could be tissue specific. How an anabolic steroid may affect androgen receptor conformation and interaction with particular coregulators is of obvious interest, as such knowledge may eventually offer an additional mechanism for anabolic–androgenic dissociation.
The development of nonsteroidal selective androgen receptor modulators (SARMs) may offer better dissociation of biological effects than anabolic steroids and possibly even permit the therapeutic targeting of specific tissues and organs. Potential therapeutic modalities could then be specific agonists for restoration of fat-free muscle mass and strength in those with chronic illnesses such as HIV and specific antagonists for the treatment of prostate cancer in men or hirsutism in women. In anticipation of the potential of such agonists for performance enhancement in sport, SARMs have been added to the World Anti-Doping Agency's (WADA's) 2008 list of prohibited substances in sport, despite none yet being available on the market.
Control of anabolic steroids
Anabolic steroids are controlled substances in several countries, including Australia, Argentina, Brazil, Canada, the United Kingdom and the United States. Even so, there is a readily available supply of steroids worldwide for non-medicinal purposes, because, in most countries, anabolic steroids can be sold legally without a prescription. Thus, many foreign distributors do not violate the laws of their own country when they sell these substances to customers overseas via the Internet and by e-mail orders. The majority of the hormone products in the European market come from countries within the European Union and Russia, but also sometimes from Thailand, Turkey, Egypt, India and Pakistan. In the United States, significant quantities of anabolic steroids come from Mexico, as well as other countries such as Russia, Romania and Greece.
In the United Kingdom, anabolic steroids are controlled under Schedule IV Part 2 of the Misuse of Drugs Act; the Act includes most of the anabolic steroids, together with clenbuterol (adrenoreceptor stimulant) and human growth hormone. There is no restriction on the possession of these substances when they are part of a medicinal product and are for self-administration. However, prosecutions of intent to supply have been made of individuals found in possession of large quantities of these substances without a prescription for them. A Home Office licence is required for importation and exportation of anabolic steroids, except in cases of small quantities for legitimate purposes.
As to doping control in human sport, the International Olympic Committee (IOC) Medical Commission introduced anabolic steroids as a banned class in 1974. The name of this banned class was amended to anabolic agents in the 1990s to incorporate out-of-competition testing for clenbuterol and other β[SUB]2[/SUB]-agonists, which are also considered to have anabolic activity. In 1999, WADA was set up as a foundation under the initiative of the IOC with the support and participation of intergovernmental organizations, governments, public authorities, and other public and private bodies fighting against doping in human sport. Under WADA, the rules and technical documents concerning anabolic steroids (and other drugs) are constantly evolving and for up to date information the reader is strongly advised to access the WADA web site.
Misuse of anabolic steroids in sport and society
The use of anabolic steroids for cosmetic benefits among both adults and adolescents in society may be incorrectly regarded as a comparatively harmless pharmacological manipulation that can aid the development of bulging muscles and a well-toned figure. Surveys of anabolic steroid abuse by gymnasia users found that, overall, around 5% were using such drugs, whereas among people attending gyms equipped for competitive bodybuilding, the proportion of current or previous users was around 25–50%. Nevertheless, it is difficult to estimate the true number of anabolic steroid users in the whole of the United Kingdom but these drugs are used on a nationwide basis, as discussed in depth by the report from the British Medical Association. Similar surveys indicate a high prevalence of use in the United States.
For drug control in sport, anabolic steroids are regarded (correctly) as performance enhancers, as well as harmful to health. Of the 198,143 urine samples analysed in 2006 by 34 WADA-accredited laboratories, 4332 (2%) were found to contain a prohibited substance (‘A-sample'), of which 1966 (45% of all the adverse findings) were positive for anabolic steroids. Comparison of the adverse findings for worldwide testing for over a decade show that there has been little change year after year, the most common steroids being testosterone, nandrolone, stanozolol and methandienone. Testosterone has an unfavourable anabolic–androgenic dissociation compared with other anabolic steroids, but it is more difficult to prove its administration, as it is also produced endogenously. Some consider that the WADA statistics do not reflect the real extent of doping with anabolic steroids, particularly within top-level athletics but few would dispute that the urge to succeed and the rewards of success, both financial and otherwise, have provided powerful incentives to some competitors to look for every possible means of improving their performance, despite the risk of denunciation and penalties.
Chemical structures and activity
Common anabolic steroids
Some of the structural modifications that have been introduced into the testosterone in an attempt to maximize the anabolic effect and minimize the androgenic are shown in Figure 2, and examples of anabolic steroids are given in Figure 3. Many of these steroids have been withdrawn as licensed products in numerous countries worldwide but they continue to be available as pharmaceutical preparations in others, for example, methandienone, methyltestosterone, oxandrolone and stanozolol. The only preparations currently available as licensed products for human use within the United Kingdom are testosterone and its esters, nandrolone (as the decanoate ester), mesterolone and oxymetholone (named patient basis only). Boldenone and trenbolone are restricted to veterinary purposes only in some countries, but, nonetheless, sports competitors and bodybuilders have been known to administer these anabolic steroids.
<!--caption a4-->
<!--caption a4-->Oral activity can be conferred by substitution of the 17α-H on the steroid nucleus with a methyl or ethyl group to make the 17α-alkylated anabolic steroids. Alkyl substitution prevents deactivation of the steroid by first-pass metabolism by sterically hindering oxidation of the 17β-hydroxyl group. A methyl group attached to C-1 can also confer oral activity, as in methenolone or mesterolone, but these two anabolic steroids are considered to be relatively weak in pharmacological activity.
Parenteral preparations do not require a 17α-alkyl group but usually the 17β-hydroxyl group is esterified with an acid moiety to prevent rapid absorption from the oily vehicle, usually arachis oil plus a small amount of benzyl alcohol. Once in the circulation, hydrolysis rapidly occurs by the action of blood esterases to yield the active compound. The esters include cyclohexylpropionate, decanoate, laurate and phenylpropionate for nandrolone; acetate, cypionate, decanoate, enanthate, isocaproate, phenylpropionate, propionate and undecanoate for testosterone, undecylenate for boldenone and acetate for trenbolone. The mechanism of action of the nandrolone esters and other anabolic steroids, and the effect of drug delivery systems on their biological activity have been studied by van der Vies (1993). The duration of action of the esters depends upon the rate of absorption from the site of administration. This is dependent on the chain length of the acid moiety and also the formulation, being related to the partition coefficient of the derivatives between the oil used in the formulation and plasma. In general, the longer the chain length, the more slowly the preparation is released into circulation, thus prolonging the duration of action. Furthermore, testosterone undecanoate is also orally active, the 11 carbon chain ester making the molecule so lipophilic that its route of absorption is partially shifted from the hepatic portal vein to the lymph system, thus bypassing first-pass metabolism to some extent, it being released into the circulation via the thoracic duct.
Non-pharmaceutical water-based testosterone suspensions for injection are advertised on bodybuilding web sites and cheats in sport may find these attractive as, in theory, these should be relatively short acting. Non-pharmaceutical-based preparations, whether oil or water based, may be a particular hazard to health as the contents may not have been prepared under sterile conditions.
Transdermal formulations are invariably testosterone based, legitimately designed for replacement therapy, and include the ‘patch' and hydroalcoholic gels, to be applied on a daily basis. Other short-acting testosterone preparations include those that are designed to be administered by the sublingual or buccal route. Such short-acting formulations are of particular concern in sport, as the exogenous source of testosterone is rapidly eliminated following cessation of treatment. Increased out-of-competition testing helps to combat the cheat who is using short-acting preparations and ceasing administration prior to competition in anticipation of testing. It is of interest that an illicit preparation called ‘The Cream' was designed for transdermal application (see the section ‘Designer steroids').
Steroid dietary supplements
A current cause for concern is the recent manufacture of analogues of established anabolic steroids to tap into the bodybuilding market. To avoid the statutory controls of countries regarding the manufacture and supply of drugs, these compounds are often widely marketed as nutritional/dietary supplements, examples being DHEA, androstenedione, androstenediol, and their 19-nor equivalents (these steroids are prohormones), and analogues of testosterone and stanozolol called 1-testosterone and prostanozolol, respectively (Figure 4). It is a consequence of their widespread availability that a minority of athletes will also use these steroids in an attempt to improve sporting performance, and because they are structurally related to mainstream anabolic steroids, sports antidoping laboratories are made to incorporate such compounds into their drug screens under the WADA rules. These steroids are supplied for oral administration, and are therefore subject to first-pass metabolism, a very important factor as to the extent the steroid is deactivated or converted to a more active form. Some of the putative metabolites of dietary supplements have been identified by mass spectrometry, but not by other analytical techniques such as nuclear magnetic resonance spectroscopy to confirm configuration of the structure; the interested reader is referred to the extensive review by Van Eenoo and Delbeke (2006).
<!--caption a4-->With respect to prohormone supplements of testosterone, as recently reviewed by Brown et al. (2006), these are modelled on steroids that are endogenously produced, that is, androstenedione, androstenediol and DHEA. However, supplements of the weaker androgens DHEA or androstenedione may be of little or no benefit to healthy young men who wish to improve their strength and sporting performance if, as would be expected, any anabolic effect is primarily mitigated through peripheral conversion to testosterone. Ingestion of DHEA can result in an increase in circulating DHEA and androstenedione, but it is not resolved as to whether there is an increase in plasma testosterone. This is not surprising because in the adult men the overall peripheral contribution of these precursor steroids to circulating testosterone is small. Any contribution from exogenous DHEA or androstenedione will be largely moderated by the large amount of testosterone contributed by the testis. In women, an increase in performance may be possible following ingestion of these supplements, as circulating testosterone would be expected to increase. The plasma concentration of endogenous testosterone is approximately 1/10th that found in men and the relative proportion arising from peripheral conversion of weaker androgens is much greater. Even though only 12–14% of androstenedione is converted peripherally to testosterone, this amount accounts for about half the circulating testosterone in the women. As the peripheral contribution to blood testosterone is far greater in the young adult women than the men, ingestion of modest amounts of androstenedione, DHEA or androstenediol (the natural steroid or the Δ[SUB]4[/SUB] analogue) is likely to significantly raise circulating testosterone. There are modest-to-large increases in circulating testosterone following androstenedione administration to women. Women who chronically administer large doses of weaker androgens that can be converted to more potent steroids would be expected to suffer from virilizing effects. In 2004, the FDA (Food and Drug Administration), as part of its public health mission, sent warning letters to 23 companies in the United States requesting them to cease distributing androstenedione as dietary supplements.
Designer steroids
Designer anabolic steroids are considered as ones that are manufactured specifically to circumvent doping tests in human sport, and, therefore, for obvious reasons, they are supplied in a clandestine fashion. There are few examples to draw on. Classified documents saved after the collapse of the German Democratic Republic revealed that, since 1983, a pharmaceutical company had produced preparations of epitestosterone propionate exclusively for the governmental doping programme. Epitestosterone, an epimer of testosterone, is a steroid with no anabolic activity but its administration with testosterone simultaneously or sequentially enables an athlete to manipulate the test for testosterone administration if the test is based solely on determination of the urinary testosterone/epitestosterone (T/E) ratio. Recently, a company in California called BALCO (Bay Area Laboratory Co-operative; Burlingame, CA, USA) attracted much media attention due to the high profile of the athletes involved, not least because of the supply of a transdermal preparation coded as ‘The Cream' containing testosterone and epitestosterone, as well as a sublingual preparation of a new anabolic steroid coded as ‘The Clear', which was identified from the contents of a spent syringe as tetrahydrogestrinone (THG) by the WADA-accredited laboratory within the University of California, Los Angeles (UCLA).
Tetrahydrogestrinone can be easily manufactured by the catalytic hydrogenation of the ethynyl group of the progestogen gestrinone. This relatively simple synthetic step hides the thinking that probably lay behind the design of THG. Given the close homology of their receptors, there is an overlap between the activity of progestogens and androgens, especially those xenobiotic steroids that lack the C-19 methyl group, but which activity predominates depends on whether the alkyl substituent at carbon-17 is ethynyl or ethyl. Substitution of the 17α-H with an ethynyl group on nandrolone, a 19-nor anabolic steroid with some progestational activity, will result in a potent orally active progestogen, this being called norethisterone (norethindrone), a steroid that is still used in some contraceptives today. The synthetic route is described in a seminal paper by Djerassi et al. (1954). However, substitution with an ethyl group on nandrolone rather than ethynyl group results in another anabolic steroid known as norethandrolone, which also has oral activity. Gestrinone, is a pharmaceutically available progestogen that lacks the C-19 angular methyl group but has a 17α-ethynyl group, and it follows that reduction of this ethynyl group to the tetrahydro product should make THG a ‘potent' androgen. This is indeed the case, as subsequently THG was found to be a highly potent androgen (and progestogen) in an in vitro bioassay system expressing human steroid receptors, and it promotes muscle accretion in orchidectomized male rats. Despite the presence of the 17α-alkyl function, which should make the steroid resistant to first-pass metabolism, it is of interest that the instructions from BALCO Laboratories were to place a few drops of the liquid preparation under the tongue, that is, a sublingual route of administration. THG was invisible on the routine gas chromatography–mass spectrometry screen employed by the WADA-accredited laboratories and necessitated the development of a liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) screen for its detection; for a current and detailed review on the analysis of anabolic steroids see Kicman et al. (2008)
<!--caption a4-->Underground chemists appear also to be accessing information concerning other steroids that were synthesized several decades ago by pharmaceutical companies but were never marketed. Such steroids that have been detected until recently are norbolethone, which was reputed to have been the active ingredient of ‘The Clear' before being replaced by THG, and madol, which is also referred to as desoxymethyltestosterone (by the WADA-accredited laboratory in Montreal, who identified this steroid around the same time as the accredited laboratory at UCLA). Although the extent of this activity appears to be limited, as screening procedures rely on targeting selecting ions for monitoring by mass spectrometry, unknown steroids may escape detection. To demonstrate how this problem may be addressed, Thevis et al. (2005) developed an LC–MS/MS screening method based on common fragmentation pathways and Nielen et al. (2006) used a combination of androgen bioassay detection and electrospray quadrupole time-of-flight mass spectometric identification.
Athletes and bodybuilders have recognized for several decades that the use of anabolic steroids can promote muscle growth and strength but it is only relatively recently that these agents are being revisited for clinical purposes. Anabolic steroids are being considered for the treatment of cachexia associated with chronic disease states, and to address loss of muscle mass in the elderly, but nevertheless their efficacy still needs to be demonstrated in terms of improved physical function and quality of life. In sport, these agents are performance enhancers, this being particularly apparent in women, although there is a high risk of virilization despite the favourable myotrophic–androgenic dissociation that many xenobiotic steroids confer. Modulation of androgen receptor expression appears to be key to partial dissociation, with consideration of both intracellular steroid metabolism and the topology of the bound androgen receptor interacting with co-activators. An anticatabolic effect, by interfering with glucocorticoid receptor expression, remains an attractive hypothesis. Behavioural changes by non-genomic and genomic pathways probably help motivate training. Anabolic steroids continue to be the most common adverse finding in sport and, although apparently rare, designer steroids have been synthesized in an attempt to circumvent the dope test. Doping with anabolic steroids can result in damage to health, as recorded meticulously in the former German Democratic Republic. Even so, it is important not to exaggerate the medical risks associated with their administration for sporting or bodybuilding purposes but to emphasize to users that an attitude of personal invulnerability to their adverse effects is certainly misguided.
Androgens
Androgens exert their effects in many parts of the body, including reproductive tissues, muscle, bone, hair follicles in the skin, the liver and kidneys, and the haematopoietic, immune and central nervous systems. The androgenic effects of these hormones can be generally considered as those associated with masculanization and the anabolic effects as those associated with protein building in skeletal muscle and bone.
In the male foetus, androgens stimulate the development of the Wolffian ducts (epididymis, vas deferens, the seminal vesicles and ejaculatory duct) and the male external genitalia (penis, urethra and scrotum). During puberty, the androgenic effects resulting from increased testicular steroidogenesis are manifested by growth of the testes, external genitalia and the male accessory reproductive glands (prostate, seminal vesicles and bulbourethral), and secretory activity begins. Further, the secondary sexual characteristics manifested during puberty can be divided into those that are a result of androgenic and anabolic effects. The androgenic effects are the enlargement of the larynx causing a deepening of the voice, the growth of terminal hair (in the pubic, axillary and facial regions; in other regions such growth depends on a number of factors), an increase in sebaceous gland activity (can lead to acne), and CNS effects (libido and increased aggression). Anabolic effects are the growth of skeletal muscle and bone, the stimulation of linear growth eventually ceasing due to the closure of the epiphysis. In men, androgens are essential for sustaining reproductive function, and they play an important role in maintaining skeletal muscle and bone, cognitive function and a sense of well-being.
The most important androgen secreted is testosterone; in the eugonadal man, the Leydig cells in the testes produce ~95% of the testosterone in the body. The ovaries and the adrenal glands (in both sexes) produce very little testosterone but secrete weaker androgens; in particular, dehydroepiandrosterone (DHEA; and its sulpho-conjugate) and androstenedione are of physiological importance in the women, not least because they can undergo peripheral conversion to more potent androgens, for example to testosterone and 5α-dihydrotestosterone (DHT). Another weaker endogenous androgen, androstenediol, also binds to oestrogen receptors.
The effects of androgens are modulated at cellular level by the steroid-converting enzymes within the particular target tissue. In reproductive target tissues, testosterone can be considered to be a prohormone, being readily converted by 5α-reductase to the more potent androgen DHT. In other tissues, such as adipose tissue and parts of the brain, testosterone is converted by aromatase to the oestrogen, oestradiol. In bone, the mechanism of action of the anabolism of androgens has not been entirely elucidated but both a direct effect of testosterone and a mediated effect by aromatization to oestradiol are important. In the human skeletal muscle, 5α-reductase activity (either type 1 or 2) is not detectable, so testosterone itself is chiefly binding to the androgen receptor (as supported also by a number of animal studies, mainly in the rat). Aromatase expression and activity is significant in human skeletal muscle but whether the conversion of androgens to oestrogens within this tissue is physiologically important for mediating some of the myotrophic effect of androgens is yet to be determined.
<!--fig ft0--><!--fig mode=article f1-->Modulation of the effects of androgens may also occur at the molecular level due to differences in the distribution of androgen receptor coregulators in various tissues, these coregulators being proteins that affect the transcriptional activity of the androgen receptor. This is a developing field and the comparative importance of many of these coregulators is yet to be established for any particular cell type, let alone their relative in vivo importance in examining tissue differences in androgen action. It is envisaged that genetic manipulation of the mouse will assist in elucidating their physiological relevance.
With structural modifications to testosterone, the anabolic effects of androgens can be enhanced but, even so, these cannot be divorced entirely from their androgenic effects. Hence, a more accurate term for anabolic steroids is anabolic–androgenic steroids, but, for simplicity, the shorter term is used within this paper. The disassociation of anabolic from androgenic effects can be at cellular level, depending on the intracellular metabolism of the anabolic steroid in different tissues, with the activity of 5α-reductase being particularly important (see the section ‘Intracellular metabolism and the myotrophic–androgenic index'). An appealing hypothesis is that anabolic–androgenic dissociation can also occur as a result of anabolic steroids inducing specific conformational changes of the androgen receptor complex, which then affects subsequent interaction with various coregulators in different tissues (see the section ‘Androgen receptor expression and the importance of coregulators'). There is little data, as yet, to support such a hypothesis, but it is known that the androgen co-activator FHL2 is expressed predominantly in the heart, and it is possible that a number of other androgen receptor coregulators could be tissue specific. How an anabolic steroid may affect androgen receptor conformation and interaction with particular coregulators is of obvious interest, as such knowledge may eventually offer an additional mechanism for anabolic–androgenic dissociation.
The development of nonsteroidal selective androgen receptor modulators (SARMs) may offer better dissociation of biological effects than anabolic steroids and possibly even permit the therapeutic targeting of specific tissues and organs. Potential therapeutic modalities could then be specific agonists for restoration of fat-free muscle mass and strength in those with chronic illnesses such as HIV and specific antagonists for the treatment of prostate cancer in men or hirsutism in women. In anticipation of the potential of such agonists for performance enhancement in sport, SARMs have been added to the World Anti-Doping Agency's (WADA's) 2008 list of prohibited substances in sport, despite none yet being available on the market.
Control of anabolic steroids
Anabolic steroids are controlled substances in several countries, including Australia, Argentina, Brazil, Canada, the United Kingdom and the United States. Even so, there is a readily available supply of steroids worldwide for non-medicinal purposes, because, in most countries, anabolic steroids can be sold legally without a prescription. Thus, many foreign distributors do not violate the laws of their own country when they sell these substances to customers overseas via the Internet and by e-mail orders. The majority of the hormone products in the European market come from countries within the European Union and Russia, but also sometimes from Thailand, Turkey, Egypt, India and Pakistan. In the United States, significant quantities of anabolic steroids come from Mexico, as well as other countries such as Russia, Romania and Greece.
In the United Kingdom, anabolic steroids are controlled under Schedule IV Part 2 of the Misuse of Drugs Act; the Act includes most of the anabolic steroids, together with clenbuterol (adrenoreceptor stimulant) and human growth hormone. There is no restriction on the possession of these substances when they are part of a medicinal product and are for self-administration. However, prosecutions of intent to supply have been made of individuals found in possession of large quantities of these substances without a prescription for them. A Home Office licence is required for importation and exportation of anabolic steroids, except in cases of small quantities for legitimate purposes.
As to doping control in human sport, the International Olympic Committee (IOC) Medical Commission introduced anabolic steroids as a banned class in 1974. The name of this banned class was amended to anabolic agents in the 1990s to incorporate out-of-competition testing for clenbuterol and other β[SUB]2[/SUB]-agonists, which are also considered to have anabolic activity. In 1999, WADA was set up as a foundation under the initiative of the IOC with the support and participation of intergovernmental organizations, governments, public authorities, and other public and private bodies fighting against doping in human sport. Under WADA, the rules and technical documents concerning anabolic steroids (and other drugs) are constantly evolving and for up to date information the reader is strongly advised to access the WADA web site.
Misuse of anabolic steroids in sport and society
The use of anabolic steroids for cosmetic benefits among both adults and adolescents in society may be incorrectly regarded as a comparatively harmless pharmacological manipulation that can aid the development of bulging muscles and a well-toned figure. Surveys of anabolic steroid abuse by gymnasia users found that, overall, around 5% were using such drugs, whereas among people attending gyms equipped for competitive bodybuilding, the proportion of current or previous users was around 25–50%. Nevertheless, it is difficult to estimate the true number of anabolic steroid users in the whole of the United Kingdom but these drugs are used on a nationwide basis, as discussed in depth by the report from the British Medical Association. Similar surveys indicate a high prevalence of use in the United States.
For drug control in sport, anabolic steroids are regarded (correctly) as performance enhancers, as well as harmful to health. Of the 198,143 urine samples analysed in 2006 by 34 WADA-accredited laboratories, 4332 (2%) were found to contain a prohibited substance (‘A-sample'), of which 1966 (45% of all the adverse findings) were positive for anabolic steroids. Comparison of the adverse findings for worldwide testing for over a decade show that there has been little change year after year, the most common steroids being testosterone, nandrolone, stanozolol and methandienone. Testosterone has an unfavourable anabolic–androgenic dissociation compared with other anabolic steroids, but it is more difficult to prove its administration, as it is also produced endogenously. Some consider that the WADA statistics do not reflect the real extent of doping with anabolic steroids, particularly within top-level athletics but few would dispute that the urge to succeed and the rewards of success, both financial and otherwise, have provided powerful incentives to some competitors to look for every possible means of improving their performance, despite the risk of denunciation and penalties.
Chemical structures and activity
Common anabolic steroids
Some of the structural modifications that have been introduced into the testosterone in an attempt to maximize the anabolic effect and minimize the androgenic are shown in Figure 2, and examples of anabolic steroids are given in Figure 3. Many of these steroids have been withdrawn as licensed products in numerous countries worldwide but they continue to be available as pharmaceutical preparations in others, for example, methandienone, methyltestosterone, oxandrolone and stanozolol. The only preparations currently available as licensed products for human use within the United Kingdom are testosterone and its esters, nandrolone (as the decanoate ester), mesterolone and oxymetholone (named patient basis only). Boldenone and trenbolone are restricted to veterinary purposes only in some countries, but, nonetheless, sports competitors and bodybuilders have been known to administer these anabolic steroids.
<!--caption a4-->
<!--caption a4-->Oral activity can be conferred by substitution of the 17α-H on the steroid nucleus with a methyl or ethyl group to make the 17α-alkylated anabolic steroids. Alkyl substitution prevents deactivation of the steroid by first-pass metabolism by sterically hindering oxidation of the 17β-hydroxyl group. A methyl group attached to C-1 can also confer oral activity, as in methenolone or mesterolone, but these two anabolic steroids are considered to be relatively weak in pharmacological activity.
Parenteral preparations do not require a 17α-alkyl group but usually the 17β-hydroxyl group is esterified with an acid moiety to prevent rapid absorption from the oily vehicle, usually arachis oil plus a small amount of benzyl alcohol. Once in the circulation, hydrolysis rapidly occurs by the action of blood esterases to yield the active compound. The esters include cyclohexylpropionate, decanoate, laurate and phenylpropionate for nandrolone; acetate, cypionate, decanoate, enanthate, isocaproate, phenylpropionate, propionate and undecanoate for testosterone, undecylenate for boldenone and acetate for trenbolone. The mechanism of action of the nandrolone esters and other anabolic steroids, and the effect of drug delivery systems on their biological activity have been studied by van der Vies (1993). The duration of action of the esters depends upon the rate of absorption from the site of administration. This is dependent on the chain length of the acid moiety and also the formulation, being related to the partition coefficient of the derivatives between the oil used in the formulation and plasma. In general, the longer the chain length, the more slowly the preparation is released into circulation, thus prolonging the duration of action. Furthermore, testosterone undecanoate is also orally active, the 11 carbon chain ester making the molecule so lipophilic that its route of absorption is partially shifted from the hepatic portal vein to the lymph system, thus bypassing first-pass metabolism to some extent, it being released into the circulation via the thoracic duct.
Non-pharmaceutical water-based testosterone suspensions for injection are advertised on bodybuilding web sites and cheats in sport may find these attractive as, in theory, these should be relatively short acting. Non-pharmaceutical-based preparations, whether oil or water based, may be a particular hazard to health as the contents may not have been prepared under sterile conditions.
Transdermal formulations are invariably testosterone based, legitimately designed for replacement therapy, and include the ‘patch' and hydroalcoholic gels, to be applied on a daily basis. Other short-acting testosterone preparations include those that are designed to be administered by the sublingual or buccal route. Such short-acting formulations are of particular concern in sport, as the exogenous source of testosterone is rapidly eliminated following cessation of treatment. Increased out-of-competition testing helps to combat the cheat who is using short-acting preparations and ceasing administration prior to competition in anticipation of testing. It is of interest that an illicit preparation called ‘The Cream' was designed for transdermal application (see the section ‘Designer steroids').
Steroid dietary supplements
A current cause for concern is the recent manufacture of analogues of established anabolic steroids to tap into the bodybuilding market. To avoid the statutory controls of countries regarding the manufacture and supply of drugs, these compounds are often widely marketed as nutritional/dietary supplements, examples being DHEA, androstenedione, androstenediol, and their 19-nor equivalents (these steroids are prohormones), and analogues of testosterone and stanozolol called 1-testosterone and prostanozolol, respectively (Figure 4). It is a consequence of their widespread availability that a minority of athletes will also use these steroids in an attempt to improve sporting performance, and because they are structurally related to mainstream anabolic steroids, sports antidoping laboratories are made to incorporate such compounds into their drug screens under the WADA rules. These steroids are supplied for oral administration, and are therefore subject to first-pass metabolism, a very important factor as to the extent the steroid is deactivated or converted to a more active form. Some of the putative metabolites of dietary supplements have been identified by mass spectrometry, but not by other analytical techniques such as nuclear magnetic resonance spectroscopy to confirm configuration of the structure; the interested reader is referred to the extensive review by Van Eenoo and Delbeke (2006).
<!--caption a4-->With respect to prohormone supplements of testosterone, as recently reviewed by Brown et al. (2006), these are modelled on steroids that are endogenously produced, that is, androstenedione, androstenediol and DHEA. However, supplements of the weaker androgens DHEA or androstenedione may be of little or no benefit to healthy young men who wish to improve their strength and sporting performance if, as would be expected, any anabolic effect is primarily mitigated through peripheral conversion to testosterone. Ingestion of DHEA can result in an increase in circulating DHEA and androstenedione, but it is not resolved as to whether there is an increase in plasma testosterone. This is not surprising because in the adult men the overall peripheral contribution of these precursor steroids to circulating testosterone is small. Any contribution from exogenous DHEA or androstenedione will be largely moderated by the large amount of testosterone contributed by the testis. In women, an increase in performance may be possible following ingestion of these supplements, as circulating testosterone would be expected to increase. The plasma concentration of endogenous testosterone is approximately 1/10th that found in men and the relative proportion arising from peripheral conversion of weaker androgens is much greater. Even though only 12–14% of androstenedione is converted peripherally to testosterone, this amount accounts for about half the circulating testosterone in the women. As the peripheral contribution to blood testosterone is far greater in the young adult women than the men, ingestion of modest amounts of androstenedione, DHEA or androstenediol (the natural steroid or the Δ[SUB]4[/SUB] analogue) is likely to significantly raise circulating testosterone. There are modest-to-large increases in circulating testosterone following androstenedione administration to women. Women who chronically administer large doses of weaker androgens that can be converted to more potent steroids would be expected to suffer from virilizing effects. In 2004, the FDA (Food and Drug Administration), as part of its public health mission, sent warning letters to 23 companies in the United States requesting them to cease distributing androstenedione as dietary supplements.
Designer steroids
Designer anabolic steroids are considered as ones that are manufactured specifically to circumvent doping tests in human sport, and, therefore, for obvious reasons, they are supplied in a clandestine fashion. There are few examples to draw on. Classified documents saved after the collapse of the German Democratic Republic revealed that, since 1983, a pharmaceutical company had produced preparations of epitestosterone propionate exclusively for the governmental doping programme. Epitestosterone, an epimer of testosterone, is a steroid with no anabolic activity but its administration with testosterone simultaneously or sequentially enables an athlete to manipulate the test for testosterone administration if the test is based solely on determination of the urinary testosterone/epitestosterone (T/E) ratio. Recently, a company in California called BALCO (Bay Area Laboratory Co-operative; Burlingame, CA, USA) attracted much media attention due to the high profile of the athletes involved, not least because of the supply of a transdermal preparation coded as ‘The Cream' containing testosterone and epitestosterone, as well as a sublingual preparation of a new anabolic steroid coded as ‘The Clear', which was identified from the contents of a spent syringe as tetrahydrogestrinone (THG) by the WADA-accredited laboratory within the University of California, Los Angeles (UCLA).
Tetrahydrogestrinone can be easily manufactured by the catalytic hydrogenation of the ethynyl group of the progestogen gestrinone. This relatively simple synthetic step hides the thinking that probably lay behind the design of THG. Given the close homology of their receptors, there is an overlap between the activity of progestogens and androgens, especially those xenobiotic steroids that lack the C-19 methyl group, but which activity predominates depends on whether the alkyl substituent at carbon-17 is ethynyl or ethyl. Substitution of the 17α-H with an ethynyl group on nandrolone, a 19-nor anabolic steroid with some progestational activity, will result in a potent orally active progestogen, this being called norethisterone (norethindrone), a steroid that is still used in some contraceptives today. The synthetic route is described in a seminal paper by Djerassi et al. (1954). However, substitution with an ethyl group on nandrolone rather than ethynyl group results in another anabolic steroid known as norethandrolone, which also has oral activity. Gestrinone, is a pharmaceutically available progestogen that lacks the C-19 angular methyl group but has a 17α-ethynyl group, and it follows that reduction of this ethynyl group to the tetrahydro product should make THG a ‘potent' androgen. This is indeed the case, as subsequently THG was found to be a highly potent androgen (and progestogen) in an in vitro bioassay system expressing human steroid receptors, and it promotes muscle accretion in orchidectomized male rats. Despite the presence of the 17α-alkyl function, which should make the steroid resistant to first-pass metabolism, it is of interest that the instructions from BALCO Laboratories were to place a few drops of the liquid preparation under the tongue, that is, a sublingual route of administration. THG was invisible on the routine gas chromatography–mass spectrometry screen employed by the WADA-accredited laboratories and necessitated the development of a liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) screen for its detection; for a current and detailed review on the analysis of anabolic steroids see Kicman et al. (2008)
<!--caption a4-->Underground chemists appear also to be accessing information concerning other steroids that were synthesized several decades ago by pharmaceutical companies but were never marketed. Such steroids that have been detected until recently are norbolethone, which was reputed to have been the active ingredient of ‘The Clear' before being replaced by THG, and madol, which is also referred to as desoxymethyltestosterone (by the WADA-accredited laboratory in Montreal, who identified this steroid around the same time as the accredited laboratory at UCLA). Although the extent of this activity appears to be limited, as screening procedures rely on targeting selecting ions for monitoring by mass spectrometry, unknown steroids may escape detection. To demonstrate how this problem may be addressed, Thevis et al. (2005) developed an LC–MS/MS screening method based on common fragmentation pathways and Nielen et al. (2006) used a combination of androgen bioassay detection and electrospray quadrupole time-of-flight mass spectometric identification.
