Nishan Guha

The development of decision limits for the implementation of the GH-2000 detection methodology using current commercial insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays

BACKGROUND The GH-2000 project developed a method for detecting GH misuse based on the measurement of insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objective of this study was to develop decision limits for the GH-2000 score to detect GH misuse in elite athletes using two currently available commercial assays for each analyte. STUDY DESIGN SUBJECTS 404 male (mean age 23.9 yrs, range 12-37 yrs) and 94 female elite athletes (mean age 24.5 yrs, range 18-34 yrs) participated. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including 238 samples collected as part of the UK Anti-Doping Testing Programme. Laboratory analysis: IGF-I was measured by Siemens Immulite IGF-I assay and Immunotech A15729 IGF-I IRMA. P-III-NP was measured by RIA-gnost P-III-P and the UniQ™ PIIINP RIA. STATISTICAL ANALYSIS The GH-2000 score decision limits were developed through the analysis of the elite athlete samples. RESULTS For males and females separately, the distributions of GH-2000 scores were consistent with Normal distributions. Using a specificity of 99.99% new decision limits were determined which included an allowance for uncertainty associated with calculations based on a finite sample size. One outlier was identified with results incompatible with normal physiology and tested positive with the current isoform GH test. CONCLUSIONS We have developed decision limits using currently available commercial assays to measure IGF-I and P-III-NP in elite athletes. This should allow the introduction of a test for GH misuse based on the measurement of these GH sensitive biomarkers.

IGF-I abuse in sport: Current knowledge and future prospects for detection

As the tests for detecting growth hormone (GH) abuse develop further, it is likely that athletes will turn to doping with insulin-like growth factor-I (IGF-I). IGF-I mediates many of the anabolic actions of growth hormone. It stimulates muscle protein synthesis, promotes glycogen storage and enhances lipolysis, all of which make IGF-I attractive as a potential performance-enhancing agent. Pharmaceutical companies have developed commercial preparations of recombinant human IGF-I (rhIGF-I) for use in disorders of growth. The increased availability of rhIGF-I increases the opportunity for athletes to acquire supplies of the drug on the black market. The long-term effects of IGF-I administration are currently unknown but it is likely that these will be similar to the adverse effects of chronic GH abuse. The detection of IGF-I abuse is a challenge for anti-doping organisations. Research has commenced into the development of a test for IGF-I abuse based on the measurement of markers of GH action. Simultaneously, the effects of rhIGF-I on physical fitness, body composition and substrate utilisation in healthy volunteers are being investigated.

Serum Insulin-Like Growth Factor-I and Pro-Collagen Type III N-Terminal Peptide in Adolescent Elite Athletes: Implications for the Detection of Growth Hormone Abuse in Sport

CONTEXT A method based on two GH-dependent markers, IGF-I and pro-collagen type III N-terminal peptide (P-III-P), has been devised to detect exogenously administered GH. Because previous studies on the detection of GH abuse involved predominantly adult athletes, the method must be validated in adolescent athletes. OBJECTIVE The aim of the study was to examine serum IGF-I and P-III-P concentrations in elite adolescent athletes and to determine whether the method developed in adults is appropriate to detect GH abuse in this population. DESIGN AND SETTING We conducted a cross-sectional observational study at national sporting organization training events. SUBJECTS A total of 157 (85 males, 72 females) elite athletes between 12 and 20 yr of age participated in the study. INTERVENTION Serum IGF-I and P-III-P were each measured by two commercially available immunoassays. GH-2000 discriminant function scores were calculated. RESULTS Both IGF-I and P-III-P rose to a peak during adolescence, which was earlier in girls than in boys. All GH-2000 scores lay below the proposed cutoff limit of 3.7 (although some scores were close to this value), indicating that none of these athletes would be accused of GH doping if the GH-2000 discriminant formulae were used. The results between the two immunoassays for IGF-I and P-III-P were closely aligned. CONCLUSIONS The GH-2000 score rises in early adolescence, reaches a peak in athletes aged 13-16 yr, and then falls. We have found no evidence that the proposed GH-2000 score developed in adults would lead to an unacceptable rate of false-positive results in adolescent athletes, but caution may be required around the time of peak growth velocity.

IGF-I abuse in sport

It is widely believed that growth hormone (GH) is abused by athletes for its anabolic and lipolytic effects. Many of the physiological effects of GH are mediated by the production of insulin-like growth factor-I (IGF-I). Both GH and IGF-I appear on the World Anti-Doping Agency list of prohibited substances. Little is known, however, about the prevalence of abuse with exogenous IGF-I. IGF-I has effects on carbohydrate, lipid and protein metabolism and some of these actions could prove beneficial to competitive athletes. No studies have demonstrated a positive effect of IGF-I on physical performance in healthy individuals but this has not yet been studied in appropriately designed trials. Two pharmaceutical preparations of IGF-I have recently become available for the treatment of growth disorders in children. This availability is likely to increase the prevalence of IGF-I abuse. Combining IGF-I with its binding protein IGFBP-3 in one preparation has the potential to reduce the side-effect profile but the adverse effects of long term IGF-I abuse are currently unknown. Detection of abuse with IGF-I is a major challenge for anti-doping authorities. It is extremely difficult to distinguish the exogenous recombinant form of the hormone from endogenously-produced IGF-I. One approach currently being investigated is based on measuring markers of GH and IGF-I action. This has already proved successful in the fight against GH abuse and, it is hoped, will subsequently lead to a similar test for detection of IGF-I abuse.

The development of decision limits for the GH-2000 detection methodology using additional insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

The GH-2000 and GH-2004 projects have developed a method for detecting GH misuse based on measuring insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non-radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF-I was measured by the Immunotech A15729 IGF-I IRMA, the Immunodiagnostic Systems iSYS IGF-I assay and a recently developed mass spectrometry (LC-MS/MS) method. P-III-NP was measured by the Cisbio RIA-gnost P-III-P, Orion UniQ™ PIIINP RIA and Siemens ADVIA Centaur P-III-NP assays. The GH-2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF-I - Orion P-III-NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non-radioisotopic assays to measure IGF-I and P-III-NP in elite athletes, which should allow wider flexibility to implement the GH-2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays.

Insulin-like growth factor-I (IGF-I) misuse in athletes and potential methods for detection

AbstractTo athletes, insulin-like growth factor-I (IGF-I) is an attractive performance-enhancing drug, particularly as an alternative to growth hormone (GH) because IGF-I mediates many of the anabolic actions of GH. IGF-I has beneficial effects on muscle protein synthesis and glycogen storage that could enhance performance in several sporting disciplines. Recombinant human IGF-I (rhIGF-I) is used in clinical practice, but a variety of IGF-I compounds and IGF-I analogues are also advertised on the internet and many have been available on the black market for several years. Although methods for detecting GH misuse are now well established and there have been several cases in which athletes have tested positive for GH, no test is yet in place for detecting IGF-I misuse. The GH-2004 research group has been investigating methods for detection of IGF-I misuse and a test is being developed on the basis of the principles of the successful GH-2000 marker method, in which markers from the IGF axis and markers of collagen and bone turnover are used to detect GH misuse. Commercial immunoassays for these markers have been validated for anti-doping purposes but new methods, including IGF-I measurement by use of mass spectrometry, should improve the performance of the tests and help in the detection of athletes who are doping with these peptide hormones. FigurePotential serum markers of IGF-I misuse. rhIGF-I/rhIGFBP-3 administration for 28 days caused an increase in serum IGF-I, P-III-NP and IGFBP-2 and decrease in serum IGF-II and ALS in recreational athletes

The effects of a freeze-thaw cycle and pre-analytical storage temperature on the stability of insulin-like growth factor-I and pro-collagen type III N-terminal propeptide concentrations: Implications for the detection of growth hormone misuse in athletes

A method based on two serum biomarkers - insulin-like growth factor-I (IGF-I) and pro-collagen type III N-terminal propeptide (P-III-NP) - has been devised to detect growth hormone (GH) misuse. The aims of this study were to determine the stability of IGF-I and P-III-NP concentrations in serum stored at -20°C and to establish the effects of one freeze-thaw cycle. Blood was collected from 20 healthy volunteers. Serum aliquots were analyzed after storage for one day at 4°C and one day, one week, five weeks, and three months at -20°C. IGF-I and P-III-NP results were combined to calculate a GH-2000 discriminant function score for each volunteer. Inter-assay precision was determined by analysing one quality control sample at each time-point. A single freeze-thaw cycle, storage of serum at 4°C for one day and at -20°C for up to three months had no significant effect on IGF-I or P-III-NP concentration. Intra-sample variability for IGF-I was 6.8% (Immunotech assay) and 12.9% (DSL assay). Intra-sample variability for P-III-NP was 10.9% (Cisbio assay) and 13.7% (Orion assay). When IGF-I and P-III-NP results were combined, intra-sample variability of the GH-2000 score expressed as a standard deviation varied between 0.31 and 0.50 depending on the assay combination used. Variability in IGF-I and P--III-NP results of stored samples is largely determined by the characteristics of the assays. A single freeze-thaw cycle, storage of serum at 4°C for one day or at -20°C for up to 3 months does not result in a significant change in GH-2000 score.

Biochemical markers of insulin-like growth factor-I misuse in athletes: the response of serum IGF-I, procollagen type III amino-terminal propeptide, and the GH-2000 score to the administration of rhIGF-I/rhIGF binding protein-3 complex.

CONTEXT The GH-2000 and GH-2004 research groups developed a method for detecting GH misuse in athletes based on the measurement of serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). There are reports that IGF-I is also misused by athletes, but currently there is no internationally recognized test designed to detect recombinant human IGF-I misuse. OBJECTIVE The objective of the study was to examine the response of serum IGF-I, P-III-NP, and the GH-2000 score to recombinant human (rh) IGF-I/rhIGF binding protein-3 (IGFBP-3) administration in recreational athletes. DESIGN AND SETTING This was a randomized, double-blind, placebo-controlled rhIGF-I/rhIGFBP-3 administration study at Southampton General Hospital (Southampton, United Kingdom). PARTICIPANTS Fifty-six recreational athletes (26 women, 30 men) participated in the study. INTERVENTION Participants were randomized to treatment with low-dose (30 mg/d) or high-dose (60 mg/d) rhIGF-I/rhIGFBP-3 complex or placebo for 28 days. Blood was collected throughout the drug administration and washout periods. Serum IGF-I and P-III-NP were measured using commercial immunoassays and GH-2000 scores were calculated. RESULTS IGF-I, P-III-NP, and the GH-2000 score rose in response to both low- and high-dose rhIGF-I/rhIGFBP-3 administration. The relative maximum response of IGF-I (approximately 4-fold increase in women and men) was greater than that of P-III-NP (40%-50% increase in women, 35%-50% increase in men). The GH-2000 formulae, which incorporate IGF-I and P-III-NP results, detected up to 61% of women and 80% of men in the rhIGF-I/rhIGFBP-3 groups but, using IGF-I concentrations alone, the sensitivity increased to 94% in both women and men during the administration period. CONCLUSIONS The rise in P-III-NP after rhIGF-I/rhIGFBP-3 administration is small compared with that after rhGH administration. Although rhIGF-I/rhIGFBP-3 administration can be detected using the GH-2000 score method, a test based on serum IGF-I alone provides better sensitivity.

Therapeutic drug monitoring in the era of precision medicine: opportunities!

Bioanalytical assays are available for virtually all drugs used in humans, partly because of the regulatory requirements to characterize a drug’s pharmacokinetic properties during its preclinical and clinical development. However, only a few drugs are subject to routine therapeutic drug monitoring (TDM) in patients, including several immunosuppressive drugs, antibiotics, antiepileptics, antidepressants, digoxin and methotrexate. Major reasons for this relatively small number of drugs include a lack of a straightforward relationship between serum/blood levels and effect, a wide therapeutic window and an unfavourable balance between intraand interpatient and intraand interoccasion variability in pharmacokinetics [1, 2]. Moreover, there are surprisingly few prospective randomized data available to demonstrate a true beneficial effect of routine TDM, especially when looking at defined outcome parameters. Instead, most published data rather suggest that TDM might benefit patients [2–8]. Another important reason for the relatively low number of drugs for which levels are monitored routinely might be that interpretation of drug levels may be perceived to be complicated. TDM data are often used to adjust dose regimens using fairly challenging pharmacokinetic calculations, or even using population pharmacokinetic models and Bayesian forecasting embedded in sophisticated software packages [9, 10]. Although deemed useful by most clinical pharmacologists, and for some even a ‘raison d’être’, this relatively complicated use of data tends to scare off clinicians, minimizing the use of TDM. In our opinion, straightforward, easy-to-use TDM will result in its much broader use by the average clinician, which can be achieved by implementing user-friendly information technology tools, but can also be achieved by returning to user-friendly sampling strategies, such as the use of trough levels wherever possible and by considering alternative matrices such as saliva or dried blood spots [11, 12]. Broader application is further supported by the rapidly developing field of pharmacogenetics, whichmakes it possible to identify patients who might benefit from higher or lower doses of some drugs without even having to determine a drug level [13]. However, despite being able to explain some variability in the pharmacokinetics of some drugs, some aspects relevant for drug exposure are simply not covered by pharmacogenetics such as ontogeny in paediatric patients, poor adherence, and drug–drug and drug–food interactions, which can easily be monitored adequately by measuring trough levels for most drugs [14–17]. A more practical but also important reason for the relatively small number of drugs for which levels are measured routinely is the limited availability of drug assays with turnaround times adequate for patient care. Most drug assays available in routine clinical chemistry and toxicology laboratories are automated immunoassays, which are fairly easy to perform and have a relatively short turnaround time. Other methodologies to determine drug levels are mostly chromatography based, such as high-performance liquid chromatography combined with ultraviolet detection (HPLC-UV) and liquid chromatography combined with mass spectrometry (LC-MS). While usually having a longer turnaround time and needing specialized technologists to perform them, these methods are much more versatile than automated assays and allow the development of assays for individual drugs by clinical laboratories themselves, so-called ‘laboratory-developed tests’ (LDTs) or ‘in-house’ assays. The recent growth in the number of LC-MS instruments in many clinical laboratories around the world could, therefore, produce enormous growth in quantitative ‘in house’ assays for TDM but this has not happened yet. The discrepancy between the increased availability of instruments and the relatively modest number of TDM assays may be because, for most drugs, levels are requested only rarely, which makes it difficult to cover the costs of developing, validating and maintaining a clinical assay for such a drug, even for large reference laboratories, and even though British Journal of Clinical Pharmacology Br J Clin Pharmacol (2016) 82 900–902 900

Statistical methodology for age-adjustment of the GH-2000 score detecting growth hormone misuse

BackgroundThe GH-2000 score has been developed as a powerful and unique technique for the detection of growth hormone misuse by sportsmen and women. The score depends upon the measurement of two growth hormone (GH) sensitive markers, insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). With the collection and establishment of an increasingly large database it has become apparent that the score shows a positive age effect in the male athlete population, which could potentially place older male athletes at a disadvantage.MethodsWe have used results from residual analysis of the general linear model to show that the residual of the GH-2000 score when regressed on the mean-age centred age is an appropriate way to proceed to correct this bias. As six GH-2000 scores are possible depending on the assays used for determining IGF-I and P-III-NP, methodology had to be explored for including six different age effects into a unique residual. Meta-analytic techniques have been utilized to find a summary age effect.ResultsThe age-adjusted GH-2000 score, a form of residual, has similar mean and variance as the original GH-2000 score and, hence, the developed decision limits show negligible change when compared to the decision limits based on the original score. We also show that any further scale-transformation will not change the adjusted score. Hence the suggested adjustment is optimal for the given data. The summary age effect is homogeneous across the six scores, and so the generic adjustment of the GH-2000 score formula is justified.ConclusionsA final revised GH-2000 score formula is provided which is independent of the age of the athlete under consideration.