Review Article Indian J Med Res 117, May 2003, pp 185-197 Genetics of asthma: current research paving the way for development of personalized drugs Balaram Ghosh, Shilpy Sharma & Rana Nagarkatti Molecular Immunogenetics Laboratory, Institute of Genomics & Integrative Biology, Delhi, India Received May 27, 2003 Asthma is a complex genetic disorder involving the interplay between various environmental and genetic factors. In this review, efforts have been made to provide information on the recent advances in these areas and to discuss the future perspective of research in the area of developing personalized drugs using pharmacogenomic approach. Atopic asthma is found to be strongly familial, however the mode of inheritance is controversial. A large number of studies have been carried out and a number of candidate genes have been identified. In addition, a number of chromosomal regions have been identified using genome-wide scans, which might contain important unknown genes. It has been shown in studies carried out in different populations that the genetic predisposition varies with ethnicity. In other words, genes that are associated with asthma in one population may not be associated with asthma in another population. In addition to the involvement of multiple genes, gene-gene interactions play a significant role in asthma. The importance of environmental factors in asthma is beyond doubt. However, it remains controversial whether a cleaner environment or increased pollution is a trigger for asthma. Despite the increasing prevalence of the disorder, only a limited number of therapeutic modalities are available for the treatment. A number of novel therapeutic targets have been identified and drugs are being developed for better efficacy with less side-effects. With the rapid progress in the identification of genes involved in various ethnic populations combined with the availability in future of well-targeted drugs, it will be possible to have appropriate medicine as per the genetic make-up of an individual. Key words Asthma - chemokines - immunoglobulin E - interleukins - pharmacogenomics Asthma is a chronic inflammatory disorder of the airways of the lungs. Many cells and cellular elements, including mast cells, eosinophils, T-lymphocytes, macrophages, neutrophils and epithelial cells are involved in the process. The inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and coughing in susceptible individuals, particularly in the night or early in the morning1. These episodes are usually associated with widespread but variable airflow obstruction that is reversible either spontaneously or with treatment. The infiltration of leukocytes, particularly eosinophils, into the lungs and release of vasoactive mediators from mast cells set the stage for asthmatic inflammation. Two functional alterations are typically associated with asthma. These include variable airway obstruction and bronchial hyperresponsiveness. The narrowing of the airways is associated with smooth muscle contraction, airway wall thickening, oedema and increased mucus secretion1 , 2. Along with these, there is denudation of the airway epithelium and collagen deposition beneath the basement membrane 3. Several quantitative traits are associated 185 186 INDIAN J MED RES, MAY 2003 with the asthma phenotype. These include forced expiratory volume in one second (FEV1), forced vital capacity (FVC), airway hyperresponsiveness by methacholine challenge, serum total immunoglobulin E (IgE), serum immunoglobulin E specific to certain allergens, eosinophil counts in the peripheral blood, and skin prick test to a panel of locally predominant environmental allergens4-6. Studies over the last 25 years have clearly demonstrated that both genetic and environmental factors determine the phenotypic expression of asthma 7. It affects nearly 155 million individuals the world-over8. In an epidemiological study conducted in India, approximately 10-15 per cent of the Indian population, particularly women and children (under 5 yr of age), were found to be affected by atopic asthma 9. It has been estimated that around 34 per cent of the total man-days lost are due to asthma and other airway disorders9. The rising incidence of asthma over the past decades suggests that environmental and lifestyle factors are important8 . Biochemical pathways involved in the pathogenesis of asthma The biochemical pathways involving atopic asthma have been studied in great detail. Basically, two types of airway responses are initiated on allergen challenge of an appropriately sensitized asthmatic individual10 . The early phase is characterized with an acute bronchospasmatic event that begins 15-30 min after exposure and resolves over time. The process initiates with the recruitment of a subtype of CD4+ T cells, Th2, which produce predominantly interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13)), at the site of immune activation10,11 . IL-4 along with IL-13 induces B cells to produce immunoglobulin E (IgE)12,13. IL-13 also induces mucus secretion from the goblet cells 14,15 . IL-5 in association with interleukin-3 (IL-3) and granulocyte-monocyte colony stimulating factor (GMCSF) helps eosinophils to grow, mature and infiltrate into the lungs 16-18. Thus, asthma is mainly associated with an increase in Th2 cytokines both in the broncoalveolar lavage (BAL) and serum and with increased IgE levels in the sera19,20. Crosslinking of IgE receptor present on mast cells by fresh exposure of allergens initiates this acute phase. The late phase response begins 4-6 h after the initial insult and causes prolonged symptomology. The infiltration of leukocytes, particularly eosinophils, into the lungs and release of vasoactive mediators from mast cells set the stage for asthmatic inflammation20. Along with cytokines, chemokines play a major role in asthma pathogenesis as they are potent leukocyte chemoattractants, cell activating factors, and histamine-releasing factors. In particular, the eotaxin subfamily of chemokines and their receptor CC chemokine receptor 3 (CCR3) have emerged as central regulators of the asthmatic response21,22. Recent studies have provided an integrated mechanism for understanding the coordinate interaction between IL-13 and chemokines in the pathogenesis of asthma 23 . Finally, structural alterations, including airway wall thickening, lung fibrosis, mucus metaplasia, hyperplasia and hypertrophy of the myocyte are certain features which are generally observed in the airway of asthmatics2 , 3. Contribution of genes in the pathogenesis of asthma Asthma is a complex disorder of multi-factorial origin. Atopic asthma in children is found to be strongly familial and a genetic basis is indicated by familial aggregation and the identification of candidate genes and chromosomal regions linked to asthma risk24. The risk of a first-degree relative of an asthmatic individual being asthmatic is two to almost six times higher than the risk for an individual from the general population to develop the disease 24-26. Both shared genes and shared environment account for such a huge risk. Twin studies have shown that the incidence of asthma is significantly higher in monozygotic twins than dizygotic twins 27-29. It has earlier been shown that atopic asthma was influenced by a few genes with moderate effects30 . Similarly few other studies have implicated the maternal inheritance of atopy31. A previous study has suggested that early breastfeeding may increase the risk of allergic disease in genetically susceptible children32 . Although asthma has a significant heritable component, the mode of inheritance is controversial GHOSH et al : GENETICS OF ASTHMA due to the complex nature of the disorder. In a study conducted in Taiwan, it was concluded that a history of asthma in parents is a strong risk factor for asthma in the offspring33 . Under the assumption of applied segregation, it was reported that at least one major gene exists that could be involved in the development of allergy. In addition, a polygenic/ multifactorial (genetic and environmental factors) influence with a recessive component inheritance may be involved in the pathogenesis of asthma 33 . Further, there are gene-gene interactions that may lead to increased risk of developing asthma 8,34,35. Polymorphisms in several candidate genes have been found to be associated with asthma and allergic disorders (Table I). Atopy was linked to a genetic marker on chromosome 11q13 36,37. In different independent studies, polymorphism in the beta chain of high-affinity receptor for IgE (FcεRI-β) in the same chromosomal location was found to be associated with asthma, atopy, bronchial hyperresponsiveness and severe atopic dermatitis 37,38. A significant association of total serum IgE concentrations and asthma with genetic markers within the IL4 gene cluster (5q31.1) has been established39,40. Interestingly, this region, contains a large number of important candidate genes that encode IL4, IL13, IRF1, IL9, CD14, IL-12β and β2-adrenergic receptor39. Recently, polymorphisms have been recognised in several of these genes which may contribute to the pathophysiology of allergic diseases41,42. It has been proposed that these genes are co-ordinately expressed due to the presence of some common regulatory motifs, therefore, polymorphisms within this cluster could be due to linkage disequilibrium with other known or unknown genes39. In a preliminary study conducted in the Indian population, it has been observed that polymorphisms in the proximal promoter and a CA repeat in intron 2 of IL4 are less likely to be associated with asthma (Nagarkatti and Ghosh, unpublished data). Chromosome 12q is another interesting region for both asthma and atopy because of the presence of several candidate genes encoding IFN-γ43,44, signal transducer and activator of transcription (STAT6) 45-49, a mast cell growth factor and a 187 β-subunit of nuclear factor-Y. Studies with AfroCaribbean and Caucasian populations found an association of serum IgE and asthma to markers on chromosome 12q 50 . Earlier studies in several populations have observed that IFN-γ gene was linked to atopy and asthma 43,44. Recent studies carried out in the Indian population have shown a significant positive association of (CA)n repeat in IFN-γ with asthma phenotype and serum IgE levels 43. STAT-6 plays a major role in the initiation of signals from activated Th2 cells, specifically through IL-4 and IL-13 receptors48. In a study conducted in the Indian population, novel polymorphisms in the STAT6 gene had been identified51. Using a novel CA repeat region in the proximal promoter region [denoted as R1] and a previously identified CA repeat in the 5'-UTR [denoted as R3], it has been demonstrated that a haplotype, containing 17 CA repeats at the R1 locus and 15 CA repeats at the R3 locus was significantly associated with asthma in the Indian population (Nagarkatti and Ghosh, unpublished data). A polymorphism in the IL4Rα coding region has been associated with asthma 52. Also, polymorphism in TNF-α has been found to be associated with asthma 53 . An increased risk of aspirin-induced asthma is found to be associated with polymorphism in the leukotrine C4 synthase (LTC4S) promoter54 . There is a significant difference in the linkage in candidate genes among various ethnic populations. Studies of asthma conducted in Japan, UK, and USA have implicated chromosome 5q as the region containing one or more susceptibility genes for asthma 55-58 . However, in studies conducted in Australian, Finnish, British, Scottish and German populations, chromosome 5q did not be appeared to be linked with asthma or atopy59-63. These studies on candidate genes have been mostly done on limited sample sizes. For the utility of these studies a largescale epidemiological study is required to classify various classes of allergies and asthma. In addition to studies on candidate genes, several genome-wide searches have been carried out. In this approach, genetic markers throughout the genome are mapped in family members and are used to identify chromosomal regions that are co-inherited 188 INDIAN J MED RES, MAY 2003 Table I. Major chromosomal locations with prime candidate genes Chromosomal location Candidate gene Function Association obtained 5q31.1-33.3 IL3, IL4, IL5, IL13, IL9, CSF2, CD14 IgE class switching, eosinophil, basophil and mast cell maturation BHR, asthma, atopy ADRB2 G-protein receptor Total IgE, BHR, Asthma, atopy GRL Modulates inflammation Asthma, atopy HLAD Antigen presentation Specific IgE, IgE TNF-α Mediates inflammation Asthma FcεRIβ Signal transduction BHR, asthma, IgE, high eosinophils counts, allergic dermatitis, atopy FGF3 Cellular proliferation IFN-γ Inhibits IL4 production Produces IL4 Upregulates IL4 transcription Cytokine transcription factor Total IgE, BHR, asthma, atopy Interacts with MHC complex Activates immunoregulator y genes BHR, asthma, IgE 6p2l.3 11q13 12q14.3-24.1 SCF NFYβ STAT6 14q11.2-13 TCR-α, TCR-δ NFKβ-1 16pl2.1-11.2 IL4RA Signal transduction and activation Atopy, IgE 2q33 CD28, CTLA4 Atopy, asthma 20p13 ADAM33 Antigen presentation Membrane anchored metalloprotease BHR, bronchial hyper responsiveness Asthma GHOSH et al : GENETICS OF ASTHMA with a particular phenotype such as asthma, bronchial hyperresponsiveness (BHR), or a positive SPT. The data gathered from these studies where the linkage has been verified in at least two populations, have been summarised (Table II). Attempts are underway to locate the genes in these regions by fine mapping. ADAM33 is an important gene located on 20p13 identified as a result of such fine mapping63 . Contribution of environment to the pathogenesis of asthma In addition to genes, environmental factors, such as allergens, food, childhood viral infection etc., also play significant roles in causing asthma. The incidence of asthma is rising with an alarming rate in developed as well as in the developing countries. It has been postulated that the immune deviation resulting in asthma takes place much earlier in utero 74 . Depending on the genetic status of the mother during pregnancy and exposure to various allergens, it is possible that the child may be born with an intrinsic propensity to be atopic. Genetically predisposed children when exposed to environmental allergens develop asthma even in very early phase of life75 . Evidence of polymorphism in the CD14 (LPS receptor) gene supports this hypothesis 41. In a recent study conducted in Canada, it has been shown that daily visits to a local hospital due to asthma increased significantly with increases in level of pollens and pollution in the air 76 . Similarly, in a study carried out in US, it has been shown that with increase in air pollution levels in Cincinatti, Cleveland and Columbus, the visits to the asthma clinic increased significantly 77 . In a study carried out in Palestinian children it has been shown that familial atopic diseases are predictors of asthma in children, however the indoor environment, such as the presence of cats, dogs, etc., also play a major role 78 . In contrast, it has also been shown that the prevalence of asthma in the western countries is increasing even though the environment is cleaner than earlier79,80. For example, the incidence of atopic disorders including asthma in East Berlin increased 189 after the unification of Germany 81-84. Similarly, many surveys have identified an inverse relationship between prior microbial exposure and the development of atopy79. Further, it has been seen that respiratory allergy appears less frequently in people exposed to orofaecal and food-borne microbes. Thus, improved hygiene, early infection and antibiotic use, and semi-sterilized diet may facilitate atopy by influencing exposure to commensals and pathogens that stimulate cell populations such as gut associated lymphoid tissue 85,86 . It is, therefore, proposed (hygiene hypothesis) that the cleaner environment in the western countries is not favourable for providing signals for Thl development, especially in children born of atopic parents79 . The underlying reason of these apparently contradictory observations is not understood as yet. Nevertheless, it seems very likely that environment is only a triggering factor. A genetically predisposed individual will develop the disorder anyway once the ‘proper’ environmental exposure is provided irrespective of the specific nature of the trigger. Therefore, the identification of the environmental factors that trigger asthma offers the possibility of prevention of disease. Current mode of asthma therapy A large number of drugs are now available (Table III), which help to control the signs and symptoms of asthma 87,88. The anti-leukotrines are the newest class of anti-asthmatic drugs available. Although, they do not provide any quick relief, they help to control the symptoms of asthma in the longterm. Despite the introduction of such new agents, corticosteriods are the anti-inflammatory drugs of choice for the majority in the treatment of asthma 89 . Both intravenous and oral forms are available and are equally effective in the treatment of mild to severe asthma 89,90. However, when inhaled, the dose is not sufficient to cause complete relief. Moreover, the therapy is associated with side effects like kidney, liver failure, increased hunger, compromised immune system, high blood pressure, etc . 190 INDIAN J MED RES, MAY 2003 Table II. Major chromosomal locations identified in various genome-wide scans in various populations Chromosome Location Study population Sample size Phenotypes Statistical method/ Programme used LOD score/ P value 1p D1S468 Hutterites 64 693 Inbred Strict asthma LR (χ 2)/TDT P=0.0002 1p36.2 Japanese4 67 ASP Severe allergic rhinitis, Total IgE GENEHUNTER P<0.002 2p D2S1780 Chinese6 2551 individuals Slope BHR Unified Haseman Elston method P=0.00002 2q D2S2944 Hutterites 64 693 Inbred SPT cockroach LR(χ2)/TDT P=0.00004 D2S116 German65 156 ASP Total IgE GENEHUNTER P=0.0016 173-210 cM from pter Dutch11,66 1174 individuals Total IgE Eosinophils Linkage LOD=1.96 LOD=1.49 D3S3564 Hutterites 64 693 Inbred Loose asthma LR (χ 2)/TDT P=0.00004 3p24.1 Japanese4 67 ASP Total IgE GENEHUNTER P<0.001 D4S1467 Chinese6 2551 individuals SPT Unified Haseman P=0.0003 4q24-27 Danish67 33 ASP Allergic rhinitis MAPMAKER/SIBS LOD=2.83 D4S2417 -D4S408 Japanese68 65 ASP Mite sensitive asthma MAPMAKER/SIBS MLS=2.7 D4S426 Busselton69 172 ASP Slope BHR HasemanElston sib pair Technique P<0.0005 D5S268 French70 297 ASP Slope BHR GENEHUNTER P=0.001 D5S1470 Hutterites 64 693 Inbred BHR LR (χ 2)/TDT P=0.001 D5S820 Japanese68 65 ASP Mite-sensitive asthma MAPMAKER/SIBS MLS=4.8 D5S2014 Hutterites 64 693 Inbred Asthma symptoms LR (χ 2)/TDT P=0.0009 130-172 cM from pter Dutch11,66 1174 individuals Total IgE Linkage LOD=2.73 5q33.1 Japanese4 67 ASP Total IgE GENEHUNTER P<0.001 D6S276 Busselton69 172 ASP Eosinophils, Atopy, Total IgE HasemanElston sib pair Tehnique P<0.0001 P<0.005 P<0.05 30-40 cM from pter Caucasians71,72 CSGA (266 Families) Asthma Multi-point analysis LOD=1.91 D6S276 D6S291 D6S426 D6S291 German65 156 ASP Total IgE, RAST Eosinophils, Asthma GENEHUNTER P=0.0012 P=0.0011 P=0.0005 P=0.0081 D6S1959D6S2439 Japanese68 65 ASP Mite-sensitive asthma MAPMAKER/SIB MLS=2.1 D7S484 D7S2250 D7S484/ D7S2250 Busselton69 172 ASP BHR, Total IgE, Eosinophils HasemanElston sib pair Technique P<0.0005 P<0.005 P<0.05 3p 4q 5p 5q 6p 7p P<0.002 Contd... GHOSH et al : GENETICS OF ASTHMA 191 7p14-15 Finnish73 220 affected IgE, Asthma Non-parameteric linkage P<0.0001 D7S484 French70 297 ASP Eosinophils GENEHUNTER P=0.002 7q 98-109 cM from pter Dutch11,66 1174 individuals Total IgE, SPT aeroallergens Linkage LOD=3.36 LOD=1.04 11q FCER1B Busselton69 172 ASP Skin test index, Total IgE HasemanElston sib pair Technique P<0.00005 P<0.005 D11S2002 AfricanAmerican71,72 CSGA (266 families) Asthma ASP two-locus analysis, Conditional analysis LOD=2 D12S366 D12S78D12S79 French70 Japanese68 297 ASP 65 ASP Eosinophils Mite-sensitive asthma GENEHUNTER MAPMAKER/SIBS P=0.0003 MLS=1.9 111-134 cM from pter Dutch11,66 1174 individuals Total IgE Linkage LOD=2.46 12q24.2 Japanese4 67 ASP Total IgE GENEHUNTER P<0.001 D13S787 Hutterites 64 693 Inbred Asthma symptoms LR (χ 2)/TDT P=0.0006 D13S175D13S217/ D13S153 Japanese68 65 ASP Mite-sensitive asthma MAPMAKER/SIBS MLS=2.4/2.0 6-45 cM from pter Dutch11,66 1174 individuals Total IgE SPT Linkage LOD=2.28 LOD=1.27 D13S153 Busselton69 172 ASP Atopy HasemanElston sib pair Technique P<0.001 D13S170 French70 297 ASP Eosinophils GENEHUNTER P=0.002 D16S412 Chinese6 2551 individuals Forced vital capacity Unified Haseman Elston method P=0.0006 16p12.3 Japanese4 67 ASP RAST (orchard grass) GENEHUNTER P<0.001 D16S289 Busselton69 172 ASP Total IgE, Slope BHR HasemanElston sib pair Technique P<0.0005 P<0.05 D16S539 Hutterites 64 693 Inbred SPT (molds) LR (χ 2)/TDT P=0.0008 D17S250 French70 297 ASP SPT, Asthma GENEHUNTER P=0.001 P=0.003 62-100 cM from pter Dutch11,66 1174 individuals Eosinophils, SPT (Mite) Linkage LOD=1.97 LOD=1.21 D19S900 Hutterites 64 693 Inbred BHR LR (χ 2)/TDT P<0.001 D19S433 Chinese6 2551 individuals BHR Unified HasemanElston method P=0.002 12q 13q 16p 16q 17q 19q The numbers in superscript denote references IgE, Immunoglobulin E; BHR, Bronchial hyperresponsiveness; SPT, Skin Prick Test; RAST, Radio allergo sorbent test; LR, Likelihood ratio; TDT, Transmission disequilibrium test; ASP, Affected sib pair; CSGA, Collaborative Study on Genetics of Asthma; LOD, Log of odds; pter, Genetic distance (cM) based on Marshfield map 192 INDIAN J MED RES, MAY 2003 Table III. Major classification for types of drugs used in asthma therapy Drug type Mechanism of action Route of administration Example Bronchodialators Relax smooth muscles in the airways Beta-adrenergics Inhaled, subcutaneous, oral Epinephrine, Isoproteronol Methyl-xanthines Oral/iv Theophylline/Aminophlline Anti-cholinergics Inhaled only Atropine, Atrovent Oral; Intramuscular; intravenous Beclomethasone, Dexamethasone Mediator-release inhibitors Inhaled only Nedocromil sodium Anti-leukotrine drugs Oral only Anti-Inflammatory drugs Decrease cellular response of inflammation Corticosteroids Additionally, in 25 per cent of the cases there may be resistance to treatment with the intensity of sideeffects increasing. Response to asthma therapy varies with individual’s genetic make-up Various clinical trials have shown that there is considerable variation in the treatment response from individual to individual. These differences may be due to genetic variations between individuals along with variable expression of metabolic enzymes and receptors for drugs 91 . These factors contribute in the varying efficacy of the treatment regime. For example, patients with polymorphisms in the core promoter of ALOX5 leading to decreased promoter activity in vitro, have failed to respond to treatment with ALOX5 inhibitors like ABT-76192 . It has been noted that the promoter of ALOX5 contains 3-6 copies of Sp-1 binding sites. Only individuals with wild type ALOX5 promoter (5 Sp-1 binding sites in both chromosomes) responded to the therapy, whereas individual with mutant alleles (any other combination other than 5) failed to show any improvement of lung function when treated with ABT-761. Thus scanning of the ALOX5 promoter for Sp-1 binding sites will provide the opportunity to administer the drug according to the genetic make-up of the individual. Sanak and Szczeklik 54 have described a polymorphism in the leukotriene C4 synthase (LTC4S) promoter that resulted in higher risk of asprin-induced asthma. This genetic variant may also alter the response to treatment with drugs directed against leukotrines. Similarly, variations in the β2-adrenergic receptor (ADRB2) does not lead to the loss of functionality of the receptor, however, the response of patients to treatment with drugs varies from individual to individual93. Drysdale et al84 have demonstrated that only a limited number of β2AR haplotypes can be found in several ethnic groups 94. Also, transfection studies have shown that certain haplotypes were associated with a better response to β2-agonist drugs. GHOSH et al : GENETICS OF ASTHMA Future perspective The goal of current therapy for asthma is to render the patient as symptom-free as possible and to reduce or eliminate the need for rescue therapy and hospitalisation. Even with the availability of a large range of drugs, most patients show considerable Table IV. Novel strategies for the inhibition and prevention of asthma Target Agent Prenvention of T-cell activation Anti-CD4 CTLA4 Prevention of reversal of Th2 expression Inhibition of Th2 cytokines Anti-IL4 STAT6 inhibition Anti-IL5 GATA inhibition Anti-IL9 Soluble ILI3Rα Promotion of Th1 cytokines IFN-γ IL12 IL18 Immunotherapy Specific Immunotherapy Peptide immunotherapy Mycobacterium vaccae vaccination CpG Inhibition of downstream mediators Anti-inflammatory cytokines IL10 IL1Rα Inhibition of eosinophil migration and activation CCR3 Antagaonist CCR3 Antisense Met-RANTES Blocking cell adhesion molecules VLA4 inhibitor ICAM-1 inhibitor IgE inhibition Monoclonal anti-IgE (E25) STAT, signal tranducer and activator of transcription; IFN, Interferon; IL, Interleukin; CCR, chemokine receptor; MetRANTES, Methionine-regulated on activation, normal T cell expressed and secrefed; VLA, very late antigen; ICAM, Intercellular cell adhesion molecule; CTLA, cytotoxic T lymphocyte antigen 193 heterogeneity in terms of the type and extent of inflammatory response, response to environmental triggers and degree of atopy95,96. A major challenge in asthma therapy has therefore been the identification of novel therapeutic targets, which are safer and more specific in their action. The major abnormality in asthma is the presence of activated CD4+-Th2 cells, eosinophils and increased levels of certain Th2 cytokines. These findings, therefore, suggest that most asthmatics may benefit from an approach that targets the mechanism of allergic sensitisation and inflammation97,98. A few of these novel strategies are listed in Table IV. Recent advances in the techniques for the synthesis and manufacture of monoclonal antibodies, synthetic peptides and peptidomimetic small molecules have increased the potential for the creation of specific inhibitors of immune processes in allergic inflammation97. While preliminary data from studies on these agents appear promising, these agents will have to endure rigorous evaluation of efficacy, long-term safety and minimal side effects along with cost effectiveness. 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