Esteban Keklikian¹

Manuel Sanchez-Solis²

Angel Jose Bonina³

Alejandra Meneguzzi²

Maria Dolores Pastor-Vivero

Pedro Mondejar-Lopez

; Luis

Garcia-Marcos Luis

Do risk factors for persistent asthma modify lung function in infants and young children with recurrent wheeze?

Keywords: Lung function, Infant, asthma, wheezing wheezing, infant, child, asthma, respiratory function test wheezing, infant, child, asthma, respiratory function test

Background : There is very limited information on how the risk of persistent asthma in recurrent wheezing (RW) infants modifies their lung function early in life. The aim of the present study is to compare lung function of RW infants and young children with a positive or negative asthma predictive index (API), an index previously used to anticipate asthma persistence into childhood and adolescence.

Methods: Two groups of RW infants and young children were recruited in two centres in Spain (Palma de Mallorca and Murcia). Lung function was measured according to the thoracho-abdominal compression technique (RCT), and values of the maximal flow at functional residual capacity (V' maxFRC ) were expressed as Z-scores. Other variables included in the study, as independents factors, were: gender, age, length, weight and parental smoking habits together with information regarding API.

Results: Expressed as mean±SD, API+ RW infants (n=50; age in months 11.9±4.9) had a lower V' maxFRC Z-score than API-RW ones (n=41; age in months 12.3±6.2) (-2.01±0.79 vs. -1.64±0.77, p=0.026, respectively). Centre and tobacco exposition did not have an effect on lung function.

Conclusion:

Among RW infants and young children, those having a positive API have a significant lower lung function as measured by V' maxFRC very early in life at an early age.

INTRODUCTION

Respiratory disorders associated to wheezing are very common in early ages.

Wheezing is the manifestation of different conditions which provoke narrowing of the airways, but most of the time it is triggered by a viral infection. Among those infants who suffer from several episodes of wheezing during their first months or years of life, different groups can be identified: those who repeat episodes during the first 3 years but not after that age (transient wheezers); those who do not outgrow their wheezing, have an elevated IgE and typically a family history of asthma or allergies (atopic persistent wheezers, or asthmatics); and those who suffer episodes beyond the age of three but usually not beyond the age of 13, which usually started with a severe bronchiolitis at early age and do not have a family history of asthma or allergies (non-atopic wheezers). [1] On the other hand, low lung function prior to any wheezing episode has been described in some infants (related to mother smoking during pregnancy, for example), leading to the hypothesis that the pre-existing impaired lung function may be the cause, rather than the effect, of the severe episodes of bronchiolitis which some infants suffer and which are associated with future recurrent episodes of wheezing. [2] However, the actual pathophysiology of frequent wheezing episodes in infants is not fully understood [3] and there is still much information lacking on lung mechanics in the early stages of the different phenotypes of wheeze. A recent study found that wheezing infants with high risk of suffering asthma have a lower lung function at early ages, as compared to frequent wheezers with low risk. [4] A different way of assessing the asthma risk is the so-called asthma predictive index (API), which includes major and minor criteria. [5] To the best of our knowledge, only the study by Borrego et al. [4] has compared lung The aim of the present study is to compare lung function of two groups of recurrent wheezing infants and young children: positive and negative to the asthma predictive index (API) described by Castro et al [5], hypothesizing that lung function might be -as a group-be lower in those with a positive API index who are more prone to suffer from future asthma.

MATERIAL AND METHODS

Population

Infants Children up to 24 months of age who had wheezed at least 3 times in their lives were selected from two different Paediatric Pulmonology clinics. Wheezing was always diagnosed by a paediatric pulmonologist. Infants Children with chronic respiratory disease or born preterm and/or with low birth-weight, were not included. A total of 79 91 steroid naïve wheezing Caucasian infants children (58 male) aged 2 to 24 months were included in the study. Forty were attended to the Paediatric Pulmonology unit at Palmaplanas Clinic, Palma de Mallorca, Spain; while 39 were studied at the Paediatric Pulmonology unit, at the Virgen de la Arrixaca University Children's Hospital, Murcia, Spain. Infants Children were recruited between 2004 and 2008.

Asthma predictive index was established as previously described; and the stringent index was applied [5]. Tobacco exposure was considered positive when either the father or the mother or both were smokers at the time of the lung function test.

Length and weight were measured at the time of the lung function measurement according to standard procedures. [6]. With the sedated infant breathing at tidal volume through a face mask connected to a pneumotachometer, a rapid thoracho-abdominal compression is made by means of a jacket which is instantly inflated at a given pressure. This pressure is increased in successive manoeuvers until the flow at functional residual capacity is maximal. This flow is the maximal flow at functional residual capacity (V' maxFRC ).

Lung function measurement

The best (highest flow from a minimum of three technically acceptable curves) value is reported, provided it is within 10% or 10 mL.s -1 (whichever is greater) of the next highest value [6]. Tests were performed with two different systems: a custom-built system following the guidelines of the ATS/ERS Working Group on Standardization of Infant Pulmonary Function Tests [6] in Palmaplanas Clinic and a MasterScreen BabyBody Plethysmograph from Jaeger (Germany) (Virgen de la Arrixaca University Children's Hospital). Lung function was performed after sedation with chloral hydrate (50-100 mg/Kg). Studies were performed in asymptomatic patients at least 3 weeks after the last wheezing episode. The same protocol was followed in both centers. Both systems used the same typemodel of face mask, and similar pneumotachometergraph types models (Jaeger's original in Murcia and Hans Rudolf, non heated 0-35 l/min [Shawnee, KS 66227, USA] in Palma de Mallorca); and they share the same dead space and resistance.

Investigators performing lung function tests were not aware to the API (positive or negative) of each infant. Continuous variables, including V' maxFRC (Z-scores calculated according to published regression formulas) [7] were compared by means of Student "t" test .

Statistical analysis

F

Proportions were compared with the Pearson Chi-square. A factorial analysis of variance analysis was performed in order to know the effect of API on V' maxFRC Zscores including age, sex, tobacco exposure and center as a covariate. The relative contribution of each studied variables (age, sex, length, weight, API, tobacco exposure and center) to the variablitlity of V' maxFRC was calculated by the sum of squares in the analysis of variance. All statistical calculations were carried out using the SPSS v15 software (Chicago, IL, USA).

Ethics

The study was approved by the two ethics committees of the two hospitals involved, Virgen de la Arrixaca University Children's Hospital and Palmaplanas Clinic, and informed consent was obtained before testing. Parents were asked for consent to perform a lung function study as part of the follow-up of their children.

RESULTS

Lung function studies were performed in a total 91 children. With this sample the estimated power of the study to ascertain a difference between the two wheezy groups was 60%. There were no differences in gender, age, weight, length, percentage of smokers parents, or z-score of V' maxFRC between the infants children recruited in both centers (Table 1 and Figure 1). Among the 91 wheezing infants children, 51 had a positive asthma predictive index (API+) while 40 had it negative (API-), and no statistical difference was found in proportion of API (+) between both centers (55% vs 54.9%; p=0.99). As is shown in Table 2 and Figure 2, no differences were found in age, length, weight and tobacco exposure between API+ and API-wheezing children. However, the API+ group had a V' maxFRC z-score significantly (p<0.026) lower than the API -one (mean±SD: -2.01±0.79 vs. -1.64±0.77respectively) (Figure 2).

In the factorial variance analysis, which included age, gender, center and smoke habits, as covariates and API as factor, the difference in z-score of V' maxFRC between the API+ and the API-groups persisted significant (F=5.153; p=0.026).

Finally, the main contributors for the variability of V' maxFRC was length (83.04%; p<0.001) and API (9.39%; p=0.038). The contribution of the others variables was not statistically significant (Figure 3).

DISCUSSION

The results of this study show that lung function is significantly lower, at a mean age of one year, in recurrent wheezing children with a positive asthma predictive index, than in those without these risk factoras compared to those with a negative one.

In the present study, wheezing infants and young children were recruited in two centres in Spain. In all instances, lung function tests were performed by two trained pulmonologists following the same protocol. There was no statistically significant difference in any of the variables between the two centers (Table 1), and the factorial variance analysis did not find that the effect of center modified the difference between V' maxFRC Z-score of API+ and API-groups. This suggests that the effect of risk of suffering from asthma is the more most important factor that explains the loss explaining the reduced in lung function. Although the use of the sum of squares is a method of establishing the relative contribution of several variables could be (83.04%), as expected, and API (9.39%), suggesting that very early in life the lung function may be influenced early in life, in some way, by the risk of suffering from asthma.

The impact of the "asthma predictive index" [5] on the airway obstruction has not been fully studied. The present studyresults shows that there is a difference in the size of the impairment between children with and without risk factors of suffering from asthma. Considering that children with risk of sufferinghaving future asthma are more likely to be persistent wheezers in late childhood, our findings emphasize the loss of lung function at very an early age.

With a very similar aim, a recent study by Borrego et al. [4] compared lung function (raised volume RTC technique, and tidal RTC technique) between wheezing

infants with high and low risk of suffering from asthma. High risk was defined as parental history of asthma or wheezing without a cold, plus eosinophilia>4%; while low risk was defined as the absence of both such features. In contrast, the present study defined risk factors according to a stringent index published previously [5] which include major (medical diagnosis of asthma in one parent or medical diagnosis of atopic dermatitis in the children) and minor (allergic rhinitis in the child, wheezing without colds, eosinophilia>4%) criteria. A positive asthma risk index requires at least one positive major criterion or two minor ones. Although very similar, the criteria used in the present study and that used in the one by Borrego et al. [4] are not exactly the same, as -for example-children diagnosed of atopic dermatitis would have been in different risk groups (API+ in the present study and low risk in that by Borrego et al. [4]). What is important to underline is that, the mean value of V' maxFRC Z-score among all founddid not find significant differences in V' maxFRC Z-score between high and low risk weezhing infants, perhapsprobably because the number of children in the wheezing group (n=50) was relatively too low to have a high statistical power, thus it is not striking that the difference was not statistically significant (although there was a trend for it: p=0.1). In the present study, the number of children in the wheezing group was relatively larger. This fact, andtogether with the somewhat different method to classify the asthma risk are probably enough to explainmay account for the apparent discordant results when comparing V' maxFRC Z-scores between high and low risk wheezing children in the two studies. The estimated powers of the study by Borrego et al. [4] and that of the present one are respectively 33% and 60% respectively. However, as compared with the aforementioned study, we acknowledge the main limitation of our own study isthat tidal RTC technique is less discriminative than the raised volume RTC to evaluate airway obstruction.

Lung function in transient wheezing children has been shown to be lower than in non-wheezing ones prior to the first wheezing episode. This lower lung function persists until the age of 16 years. In contrast, children with persistent wheezing have normal lung function before their first wheezing episode but this is reduced with time, and is lower at the age of 6 years [9]. Low lung function during the first months of life is associated with an increased prevalence of asthma in late childhood [10,11] and even with low lung function in adulthood [12,13]. In fact, low Z-scores of V' maxFRC at one month of life are associated with persistent wheezing at 11 years of age, and this loss of lung function has not been related to an increased airway reactivity or atopy in [10]. It has been hypothesized that small airways size could explain this [14].

However, and very interestingly, no difference was found in small-airway size between recurrent wheezing infants and normal controls when analyzed by high-resolution computed tomography images [15].

As shown here, with V' maxFRC , and previously by Borrego et al., [4] with forced expiratory lung volumes and flows (FVC and FEF 25-75 ), atopic risk is associated with lower lung function at an early age. Whether atopic children are born with structurally and/or functionally different airways, or respond to viral infections with greater tissue disorganisation and/or repair is not possible to say. What is known is that persistent wheezing infants respond differently than transtient wheezing ones to lower respiratory tract infections [16]. As atopy is the most important risk factor for persistent wheeze it could be hypothesized that -independently of the differences in lung tissue architecture at the time of birth-a distinct response to viral infections may affect lung tissue in a different way in API+ infants children as compared to API-ones.

The present study has two main potential limitations. Firstly, we do not have a prospective healthy control group; however, as our objective was to compare lung function between API+ and API-recurrent wheezing children, so a healthy control group does not seem strictly necessary, as device variability -if any-is most probably random and if having any effect it would have pushed the results towards the null hypothesis. Secondly, we used two different devices for measuring lung function.

However, face masks and dead spaces were the same, pneumotacometers were of the same type, values obtained from children were very similar between centers (Figure 1) and no centre effect was found in the regression analysis, thus making any bias derived from this limitation unlikely. -VmaxFRC (z-score) in Palma de Mallorca and Murcia. There were no differences statistically significant between both centers: p=0.36 (see Table 1). Results: Expressed as mean±SD, API+ RW infants (n=50; age in months 11.9±4.9) had a lower V' maxFRC Z-score than API-RW ones (n=41; age in months 12.3±6.2) (-2.01±0.79 vs. -1.64±0.77, p=0.026, respectively). Centre and tobacco exposition did not have an effect on lung function.

Conclusion:

Among RW infants and young children, those having a positive API have a significant lower lung function as measured by V' maxFRC at an early age. Wheezing is the manifestation of different conditions which provoke narrowing of the airways, but most of the time it is triggered by a viral infection. Among those infants who suffer from several episodes of wheezing during their first months or years of life, different groups can be identified: those who repeat episodes during the first 3 years but not after that age (transient wheezers); those who do not outgrow their wheezing, have an elevated IgE and typically a family history of asthma or allergies (atopic persistent wheezers, or asthmatics); and those who suffer episodes beyond the age of three but usually not beyond the age of 13, which usually started with a severe bronchiolitis at early age and do not have a family history of asthma or allergies (non-atopic wheezers). [1] On the other hand, low lung function prior to any wheezing episode has been described in some infants (related to mother smoking during pregnancy, for example), leading to the hypothesis that the pre-existing impaired lung function may be the cause, rather than the effect, of the severe episodes of bronchiolitis which some infants suffer and which are associated with future recurrent episodes of wheezing. [2] However, the actual pathophysiology of frequent wheezing episodes in infants is not fully understood [3] and there is still much information lacking on lung mechanics in the early stages of the different phenotypes of wheeze. A recent study found that wheezing infants with high risk of suffering asthma have a lower lung function at early ages, as compared to frequent wheezers with low risk. [4] A different way of assessing asthma risk is the socalled asthma predictive index (API), which includes major and minor criteria. [5] To the best of our knowledge, only the study by Borrego et al. [4] has compared lung The aim of the present study is to compare lung function of two groups of recurrent wheezing infants and young children: positive and negative to the asthma predictive index (API) described by Castro et al [5], hypothesizing that lung function might be -as a group-lower in those with a positive API index who are more prone to suffer from future asthma.

MATERIAL AND METHODS

Population

Children up to 24 months of age who had wheezed at least 3 times in their lives were selected from two different Paediatric Pulmonology clinics. Wheezing was always diagnosed by a paediatric pulmonologist. Children with chronic respiratory disease or born preterm and/or with low birth-weight, were not included. A total of 91 steroid naïve wheezing Caucasian children (58 male) aged 2 to 24 months were included in the study. Forty attended to the Paediatric Pulmonology unit at Palmaplanas Clinic, Palma de Mallorca, Spain; while 39 were studied at the Paediatric Pulmonology unit, at the Virgen de la Arrixaca University Children's Hospital, Murcia, Spain. Children were recruited between 2004 and 2008.

Asthma predictive index was established as previously described; and the stringent index was applied [5]. Tobacco exposure was considered positive when either the father or the mother or both were smokers at the time of the lung function test.

Length and weight were measured at the time of the lung function measurement according to standard procedures. [6]. With the sedated infant breathing at tidal volume through a face mask connected to a pneumotachometer, a rapid thoracho-abdominal compression is made by means of a jacket which is instantly inflated at a given pressure. This pressure is increased in successive manoeuvers until the flow at functional residual capacity is maximal. This flow is the maximal flow at functional residual capacity (V' maxFRC ).

Lung function measurement Lung function measurement Lung function measurement Lung function measurement

The best (highest flow from a minimum of three technically acceptable curves) value is reported, provided it is within 10% or 10 mL.s -1 (whichever is greater) of the next highest value [6]. Tests were performed with two different systems: a custom-built system following the guidelines of the ATS/ERS Working Group on Standardization of Infant Pulmonary Function Tests [6] in Palmaplanas Clinic and a MasterScreen BabyBody Plethysmograph from Jaeger (Germany) (Virgen de la Arrixaca University Children's Hospital). Lung function was performed after sedation with chloral hydrate (50-100 mg/Kg). Studies were performed in asymptomatic patients at least 3 weeks after the last wheezing episode. The same protocol was followed in both centers. Both systems used the same model of face mask, and similar pneumotachometer types (Jaeger's original in Murcia and Hans Rudolf, non heated 0-35 l/min [Shawnee, KS 66227, USA] in Palma de Mallorca); and they share the same dead space and resistance.

Investigators performing lung function tests were not aware to the API (positive or negative) of each infant. Continuous variables, including V' maxFRC (Z-scores calculated according to published regression formulas) [7] were compared by means of Student "t" test .

Statistical analysis Statistical analysis Statistical analysis Statistical analysis

Proportions were compared with the Pearson Chi-square. A factorial analysis of variance analysis was performed in order to know the effect of API on V' maxFRC Zscores including age, sex, tobacco exposure and center as a covariate. The relative contribution of each studied variable (age, sex, length, weight, API, tobacco exposure and center) to the variablitlity of V' maxFRC was calculated by the sum of squares in the analysis of variance. All statistical calculations were carried out using the SPSS v15 software (Chicago, IL, USA).

Ethics

The study was approved by the two ethic committees of the two hospitals involved, Virgen de la Arrixaca University Children's Hospital and Palmaplanas Clinic, and informed consent was obtained before testing. Parents were asked for consent to perform a lung function study as part of the follow-up of their children.

RESULTS

Lung function studies were performed in a total 91 children. With this sample the estimated power of the study to ascertain a difference between the two wheezy groups was 60%. There were no differences in gender, age, weight, length, percentage of smoker parents, or z-score of V' maxFRC between children recruited in both centers (Table 1 and Figure 1). Among the 91 wheezing children, 51 had a positive asthma predictive index (API+) while 40 had it negative (API-), and no statistical difference was found in proportion of API (+) between both centers (55% vs 54.9%; p=0.99). tobacco exposure between API+ and API-wheezing children. However, the API+ group had a V' maxFRC z-score significantly (p<0.026) lower than the API -one (mean±SD: -2.01±0.79 vs. -1.64±0.77respectively) (Figure 2).

As shown in

In the factorial variance analysis, which included age, gender, center and smoke habits, as covariates and API as factor, the difference in z-score of V' maxFRC between the API+ and the API-groups persisted significant (F=5.153; p=0.026).

Finally, the main contributors for the variability of V' maxFRC was length (83.04%; p<0.001) and API (9.39%; p=0.038). The contribution of the others variables was not statistically significant (Figure 3).

DISCUSSION

The results of this study show that lung function is significantly lower, at a mean age of one year, in recurrent wheezing children with a positive asthma predictive index, as compared to those with a negative one. Although the use of the sum of squares is a method of establishing the relative contribution of several variables could be controversial, only two variables contributed significantly to V' maxFRC : length (83.04%), as expected, and API (9.39%), suggesting that lung function may be influenced early in life, in some way, by the risk of suffering from asthma.

The impact of the "asthma predictive index" [5] on the airway obstruction has not been fully studied. The present results show that there is a difference in the size of the impairment between children with and without risk factors of suffering from asthma.

Considering that children with risk of having future asthma are more likely to be persistent wheezers in late childhood, our findings emphasize the loss of lung function at an early age. infants with high and low risk of suffering from asthma. High risk was defined as parental history of asthma or wheezing without a cold, plus eosinophilia>4%; while low risk was defined as the absence of both such features. In contrast, the present study defined risk factors according to a stringent index published previously [5] which include major (medical diagnosis of asthma in one parent or medical diagnosis of atopic dermatitis in the children) and minor (allergic rhinitis in the child, wheezing without colds, eosinophilia>4%) criteria. A positive asthma risk index requires at least one positive major criterion or two minor ones. Although very similar, the criteria used in the present study and that used in the one by Borrego et al. [4] are not exactly the same, as -for example-children diagnosed of atopic dermatitis would have been in different risk groups (API+ in the present study and low risk in that by Borrego et al. [4]). What is important to underline is that, the mean value of V' maxFRC Z-score among all wheezing children is fairly similar in the present study and in the aforementioned study by Borrego et al. [4] (-1.84±0.8 vs. -1.9±1.0, respectively). These authors did not find significant differences in V' maxFRC Z-score between high and low risk weezhing infants, probably because the number of children in the wheezing group (n=50) was too low to have a high statistical power, thus it is not striking that the difference was not statistically significant (although there was a trend for it: p=0.1). In the present study, the number of children in the wheezing group was larger. This fact, together with the somewhat different method to classify asthma risk may account for the apparent discordant results when comparing V' maxFRC Z-scores between high and low risk wheezing children in the two studies. The estimated powers of the study by Borrego et [4] and that of the present one are 33% and 60% respectively. However, as compared with the aforementioned study, we acknowledge that tidal RTC technique is less discriminative than raised volume RTC to evaluate airway obstruction.

Lung function in transient wheezing children has been shown to be lower than in non-wheezing ones prior to the first wheezing episode. This lower lung function persists until the age of 16 years. In contrast, children with persistent wheezing have normal lung function before their first wheezing episode but this is reduced with time, and is lower at the age of 6 years [9]. Low lung function during the first months of life is associated with an increased prevalence of asthma in late childhood [10,11] and even with low lung function in adulthood [12,13]. In fact, low Z-scores of V' maxFRC at one month of life are associated with persistent wheezing at 11 years of age, and this loss of lung function has not been related to an increased airway reactivity or atopy in childhood [10]. It has been hypothesized that small airways size could explain this [14].

However, and very interestingly, no difference was found in small-airway size between recurrent wheezing infants and normal controls when analyzed by high-resolution computed tomography images [15].

As shown here, with V' maxFRC , and previously by Borrego et al., [4] with forced expiratory lung volumes and flows (FVC and FEF 25-75 ), atopic risk is associated with lower lung function at an early age. Whether atopic children are born with structurally and/or functionally different airways, or respond to viral infections with greater tissue disorganisation and/or repair is not possible to say. What is known is that persistent wheezing infants respond differently than transtient wheezing ones to lower respiratory tract infections [16]. As atopy is the most important risk factor for persistent wheeze it could be hypothesized that -independently of the differences in lung tissue architecture The present study has two main potential limitations. Firstly, we do not have a prospective healthy control group; however, as our objective was to compare lung function between API+ and API-recurrent wheezing children, so a healthy control group does not seem strictly necessary, as device variability -if any-is most probably random and if having any effect it would have pushed the results towards the null hypothesis. Secondly, we used two different devices for measuring lung function.

However, face masks and dead spaces were the same, pneumotacometers were of the same type, values obtained from children were very similar between centers (Figure 1) and no centre effect was found in the regression analysis, thus making any bias derived from this limitation unlikely.

In summary, the present study shows that among recurrent wheezing infants and young children, those having a positive API have a significant lower lung function at an early age. -VmaxFRC (z-score) in Palma de Mallorca and Murcia. There were no differences statistically significant between both centers: p=0.36 (see Table 1). Pediatric Pulmonology

  with and without markers of future asthma, such as parental allergy.

  studied by means of the rapid thoraco-abdominal compression technique

  two variables contributed significantly to V' max FRC: length

  fairly similar in the present study and in the aforementioned study by Borrego et al.[4] (-1.84±0.8 vs. -1.9±1.0, respectively). These authors do not

  Figure legends:

Figure 2 .

 2 Figure 2.-VmaxFRC (z-score) in wheezing infants. Difference between API+ and

Figure 3 .

 3 Figure 3.-Percentage contribution of the different factors to the total explained

INTRODUCTION

  Respiratory disorders associated to wheezing are very common in early ages.

  with and without markers of future asthma, such as parental allergy.

  studied by means of the rapid thoraco-abdominal compression technique

  similar aim, a recent study by Borrego et al. [4] compared lung function (raised volume RTC technique, and tidal RTC technique) between wheezing

  of birth-a distinct response to viral infections may affect lung tissue in a different way in API+ children as compared to API-ones.

  Figure legends:

Figure 2 .

 2 Figure 2.-VmaxFRC (z-score) in wheezing infants. Difference between API+ and

Figure 3 .

 3 Figure 3.-Percentage contribution of the different factors to the total explained

Figure 2 .Figure 3 .

 23 Figure 2.-VmaxFRC (z-score) in wheezing infants. Difference between API [+] and API [-] is statistically significant (p=0.026). 147x180mm (600 x 600 DPI)

Table 2 ,

 2 no differences were found in age, length, weight and

Table 1 .

 1 -Demographic characteristics and lung function of wheezing infants from Palma de Mallorca and Murcia.

		All	Palma de		mean
		wheezing	Mallorca	Murcia	difference	p
		infants			(95% CI)
	Number of cases	91	40	51	--	--
	Male gender	72(79.1%)	30(75%)	42(82.4%)	--	0.39
	Smoker parents	34(37.4%) 15(37.5%) 19(37.3%)	--	0.98
	Asthma predictive index (+) 50(54.9%)	22(55%)	28(54.9%)	--	0.99
	Age at study (months)	12.05±5.5	11.1±4.6	12.8±6.1	1.6 (-0.7;3.9)	0.17
	Weight (Kg)	9.9±2.0	9.6±1.8	10.2±2.1	0.6 (-0.3;1.4)	0.19
	Length (cm)	75.6±6.8	74.5±5.7	76.5±7.4	2.01 (-0.8;4.8) 0.16
	V' maxFRC (z-score)	-1.84±0.80 -1.92±0.87 -1.77±0.74 0.15 (-1.8;0.5) 0.36
	Values are expressed as N(%) or as mean±SD

Table 2 .

 2 -Demographic characteristics and lung function of wheezing infants. All wheezing infants split in API (+) and API (-).

		API (-)	API (+)	Mean diference	p
		n=41	n=50	(95% CI)
	Male gender	33(80.5%)	39(78%)	--	0.77
	Smoker parents	14(34.1%)	20(40.0%)	--	0.57
	Age at study (months)	12.3±6.2	11.9±4.9	-0.4 (-2.8;1.9)	0.71
	Weight (Kg)	9.8±2.0	10.1±2.0	0.4 (-0.5;1.2)	0.39
	Length (cm)	75.2±7.5	76.0±6.2	0.7 (-2.1;3.6)	0.60
	V' maxFRC (z-score)	-1.64±0.77	-2.01±0.79	-0.4 (-0.7;-0.05)	0.026
	Values are expressed as N(%) or as mean±SD

Page 31 of 34 John Wiley & Sons, Inc.

  Figure 1.-VmaxFRC (z-score) in Palma de Mallorca and Murcia. There were no differences statistically significant between both centers: p=0.36 (see Table1). 146x148mm (600 x 600 DPI)

	Pediatric Pulmonology
	F o
	r
	P
	e e r
	R e v i e w

Page 32 of 34 John Wiley & Sons, Inc.

	Pediatric Pulmonology
	F o
	r
	P
	e e r
	R e v i e w

John Wiley & Sons, Inc.Pediatric Pulmonology

Acknowledgments:

The authors want to thank Mr. Anthony Carlson for his editorial assistance

Acknowledgments:

The authors want to thank Mr. Anthony Carlson for his editorial assistance


Alain Li

Peter Farndon
email: p.a.farndon@bham.ac.uk

Charles Craddock
email: charles.craddock@uhb.nhs.uk

Michael Griffiths
email: mike.griffiths@bwhct.nhs.uk

A Li-Wan-Po
email: a.liwanpo@talk21.com

Integrating pharmacogenetics and therapeutic drug monitoring: optimal dosing of imatinib as a case-example

Keywords: Chronic myeloid leukaemia, Imatinib resistance, Personalized medicine, Therapeutic drug monitoring

Integrating pharmacogenetics and therapeutic drug

monitoring: optimal dosing of imatinib as a case-example

Introduction

Imatinib is, even within the context of targeted therapies, a molecular therapeutic star [START_REF] Schwartz | A molecular star in the wars against cancer[END_REF][START_REF] Gdvmm | Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. A molecular star in the wars against cancer[END_REF]. Its major impact in the management of chronic myelogenous leukaemia (CML) and gastro-intestinal stromal tumours (GIST), is widely acknowledged and has arguably led to the growing and resurgent interest in translational research in medicine. Yet, despite its selectivity of action and relatively benign harmbenefit profile, imatinib does not cure. Using Paul Ehrlich's weaponry metaphor, the magic bullet stuns but does not cure, leaving behind resistant cancer cells to challenge the host on another occasion. The precise mechanisms for imatinib resistance and treatment failure are still unclear, but residual cancer stem cells and the emergence of new mutations made easier from the genomic instability characteristic of cancer cells [START_REF] Wardelmann | Acquired resistance to imatinib in gastrointestinal stromal tumours caused by multiple KIT mutations[END_REF] are plausible explanations for at least some of the cases.

An increasing body of evidence suggests that for a substantial number of cases, resistance may be apparent (pseudo-resistance) rather than true imatinib resistance, in that dose escalation leads to drug response [START_REF] Kantarjian | Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia[END_REF][START_REF] Kantarjian | Efficacy of imatinib dose escalation in patients with chronic myeloid leukemia in chronic phase[END_REF][START_REF] Jabbour | Imatinib mesylate dose escalation is associated with durable responses in patients with chronic myeloid leukemia after cytogenetic failure on standard-dose imatinib therapy[END_REF]. The reasons why the standard dose of imatinib fails to achieve the expected response in some patients include interindividual variability in the differential expression of the influx (hOCT1) and efflux (MDR1) transporters [START_REF] Thomas | Active transport of imatinib into and out of cells: implications for drug resistance[END_REF] and variable bioavailability [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF][START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF]. In an analysis of the clinical significance of imatinib pharmacokinetics [START_REF] Larson | Imatinib pharmacokinetics and its correlation with response and safety in chronic-phase chronic myeloid leukemia: a subanalysis of the IRIS study[END_REF], using data from the IRIS study [START_REF] O'brien | Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia[END_REF][START_REF] Druker | Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia[END_REF], subjects given 400 mg daily had trough levels ranging from 153 to 3910 ng/mL. It was also clear from this analysis that an adequate plasma concentration is important for a good drug response. Fixed dosing is therefore not the best approach to ensuring a good response. If blood level is important, however, the question then arises as to what the target level ought to be for therapeutic drug monitoring (TDM). The aim of our study was, therefore, to critically appraise the available evidence with a view to estimating this optimal blood level. In the Discussion section, we then suggest how TDM, with this target blood level, could interface with pharmacogenetic data to optimise therapy.

Method

We undertook a search for lead papers on dose-response with a keyword search on dose, monitoring, dose regimens and therapeutic monitoring, each with the qualifiers imatinib. We then retrieved the relevant papers based on the content of the abstracts and did historical searches through the references lists of those papers. We abstracted and subjected relevant data to statistical analysis, including receiver operating characteristic (ROC, signal detection theory) curve analysis [START_REF] Green | Signal detection theory and psychophysics[END_REF].

Results and discussion

In a recent report, Picard et al. [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF] studied the association between imatinib trough blood level and treatment response in CML patients who were given 400 or 600 mg imatinib daily, depending on whether they were in the chronic phase or accelerated phase of the disease. The treatment response was major molecular response, defined as a reduction in the BCR-ABL transcript level of at least 3 logs after 12 months of therapy from the standardised baseline. Despite the variable dosing and staging of the disease, using ROC curve analysis, the authors estimated, using major molec-ular response as outcome, that with the optimal sensitivity and specificity trade-off, the optimal threshold imatinib steady-state trough plasma level was 1002 ng/mL (1.70 µM; molecular weight 589.7).

In a second report, Singh et al. [START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF] studied 40 chronic phase CML patients treated with 400 mg imatinib daily and reported that mean steady-state trough plasma was significantly lower in non-responders than in responders (0.70 vs. 2.34 µM; p = 0.002). These researchers defined responders as those who achieved a complete haematological response within 3 months of initiating treatment or those who demonstrated a major cytogenetic response within 6 months of treatment initiation, both responses following the administration of imatinib mesylate at 400 mg/day. Although they did not mathematically estimate the optimum plasma level for a response, they did report the individual levels for both responders and non-responders. Using the approach adopted by Picard et al. [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF], we calculated the ROC curve for the data reported by Singh et al. [START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF] (Fig. 1) and obtained an area under the ROC curve of 0.78, which is the same as that reported by Picard et al. [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF]. We also estimated the target trough plasma level of imatinib, again using Singh et al.'s data [START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF] under conditions of a trade-off of sensitivity versus specificity (Fig. 2) and obtained a value of 0.95 µM;, which is lower than that reported by Picard et al. [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF]. Using the variance estimates of the blood levels, observed by Singh et al. [START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF] and Picard et al. [START_REF] Picard | Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia[END_REF] in their responder groups, we have calculated the 95% confidence intervals for optimal trough threshold to be 1.33-2.07 μM and 0.88-1.02 μM respectively. When we used the optimal cut-off mean blood level of 0.95 µM, obtained using Singh et al.'s data, the sensitivity was 75%, the specificity 85%, the positive predictive value 83%, the negative predictive value 77% and the percentage of subjects correctly classified 90%.

Dose escalation without TDM

If the target imatinib level is known for imatinib pseudoresistant CML, how effective would a simple dose adjustment be without measurement of the trough plasma level (i.e. without TDM)? In addition to an earlier preliminary report [START_REF] Kantarjian | Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia[END_REF], two very recent reports address this issue [START_REF] Kantarjian | Efficacy of imatinib dose escalation in patients with chronic myeloid leukemia in chronic phase[END_REF][START_REF] Jabbour | Imatinib mesylate dose escalation is associated with durable responses in patients with chronic myeloid leukemia after cytogenetic failure on standard-dose imatinib therapy[END_REF]. Kantarjian et al. [START_REF] Kantarjian | Efficacy of imatinib dose escalation in patients with chronic myeloid leukemia in chronic phase[END_REF], in their retrospective analysis of 106 patients enrolled in the IRIS trial [START_REF] O'brien | Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia[END_REF][START_REF] Druker | Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia[END_REF], reported that dose escalation from 400 to 600 or 800 mg daily, according to the predefined trial protocol or as recommended by European LeukemiaNet guidelines [START_REF] Baccarani | Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet[END_REF], led to about 40% of patients obtaining a cytogenetic response. Jabbour et al. assessed the longterm efficacy of imatinib dose escalation (400 to 800 mg, or 300 to 600 mg) in patients with chronic phase CML who had demonstrated a poor response or relapse on standard-dose imatinib [START_REF] Jabbour | Imatinib mesylate dose escalation is associated with durable responses in patients with chronic myeloid leukemia after cytogenetic failure on standard-dose imatinib therapy[END_REF]. After a median follow-up of 61 months from dose escalation, more than two-thirds of the patients were still alive: 40% achieved complete cytogenetic response, 88% of whom sustained their response beyond 2 years [START_REF] Jabbour | Imatinib mesylate dose escalation is associated with durable responses in patients with chronic myeloid leukemia after cytogenetic failure on standard-dose imatinib therapy[END_REF].

Those two studies [START_REF] Kantarjian | Efficacy of imatinib dose escalation in patients with chronic myeloid leukemia in chronic phase[END_REF][START_REF] Jabbour | Imatinib mesylate dose escalation is associated with durable responses in patients with chronic myeloid leukemia after cytogenetic failure on standard-dose imatinib therapy[END_REF], both of which were led by the Anderson Cancer Centre in Texas, were retrospective analyses of patients enrolled in other studies [START_REF] Kantarjian | Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia[END_REF][START_REF] Joensuu | Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor[END_REF][START_REF] Kantarjian | Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia[END_REF][START_REF] Kantarjian | Imatinib mesylate for Philadelphia chromosomepositive, chronic-phase myeloid leukemia after failure of interferon-alpha: follow-up results[END_REF], including those randomised to 400 mg imatinib in the IRIS randomised controlled study [START_REF] O'brien | Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia[END_REF]. To test the value of dose escalation further, Baccarani et al. [START_REF] Baccarani | Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphiapositive chronic myeloid leukemia: a European LeukemiaNet study[END_REF] compared response to 400 or 800 mg daily as the first-line treatment of patients with Philadelphia-positive chronic myeloid leukaemia, defined as high-risk on the basis of the Sokal prognostic classification, which focuses on percentage blasts, spleen size, platelet count and age. Their randomised controlled study surprisingly showed no significant difference between those two dosage regimens (64 vs. 58% with complete cytogenetic response in the 800 mg group vs. the 400 mg group). This led the authors to guard against extensive use of the higher dose [START_REF] Baccarani | Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphiapositive chronic myeloid leukemia: a European LeukemiaNet study[END_REF].

Value of TDM

Although the dose-plasma steady-state concentration relationship shows some dose-response linearity, at the higher dose levels, daily single-dose administration suggests some saturation (Fig. 3). Moreover, there is considerable interpatient variability, as shown by the wide inter-subject variability in steady-state plasma level reached in patients given the same dose (Fig. 3). Variability increases with increasing dose so that not all patients reach the target level, even at a dose of 600 mg daily. Figure 3 shows that at the 1200 mg daily dose, the 95% confidence interval extended from <3 µM to >9 µM. Therefore, patients given a higher dose may not achieve a given target concentration. Using Peng et al.'s data and assuming normality, on average, 26% of patients given a 400 mg dose would not achieve Picard et al.'s target trough level of 1002 ng/mL (1.70 µM) compared to less than 0.2% of those given 800 mg; using Singh et al.'s threshold of 0.95 µM , the corresponding values are 2.6 and 0.0001%. Therapeutic dose monitoring would therefore be useful for dose adjustment in order to minimise the number of cases of suboptimal dosing and to obviate the use of the higher dose for all patients. Moreover, the TDM data collected over time would also provide a valuable resource for estimating, through retrospective analyses, the optimum trough blood level for different subgroups of patients, such as those in the blast phase of the disease. These levels can then be efficiently validated through well-designed randomised controlled trials. Interfacing pharmacogenetics and TDM A recent UK House of Lords report [19] warned against genetic exceptionalism, which in the present context can be roughly defined as giving 'privilege to DNA-based information over other types of information', namely other clinical and background data 'that may be equally or more predictive' in the management of individual patients. The case presented here illustrates well the synergy to be derived from integrating pharmacogenetic information, namely the presence of the BCR-ABL1 gene, with TDM of the targeted agent. In CML confirmed by the presence of the appropriate genetic variant (cytogenetically as the Philadelphia chromosome or molecularly as the BRC-ABL1 gene, or the encoded constitutively activated oncoprotein), imatinib is now the first-line treatment in settings where the drug is affordable. This pharmacogenetictargeting benefits from conventional TDM, which ensures that the target level is reached without uncritical doseescalation, thereby reducing the risk of a severe unwanted adverse effect [20].

Although the dose-toxicity response for imatinib is not unequivocally positive, a number of published clinical reports suggest that high doses are less well tolerated. For example, in the LeukemiaNet study, at 12 months, the proportion of subjects discontinuing treatment because of adverse events were approximately twofold higher in those receiving 800 mg daily than in those on 400 mg daily (9/ 108 vs. 5/108) [START_REF] Baccarani | Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphiapositive chronic myeloid leukemia: a European LeukemiaNet study[END_REF]. In a recent study of dose escalation after the therapeutic failure of standard-dose imatinib, Jabbour et al. reported that dose reduction was necessary to resolve toxicity problems during long-term follow-up (median 61 months, range 7-89 months), most notably myelosuppression, which affected 13 of the 84 patients, and fluid retention, which affected seven patients. Moreover, in common with many of the newer targeted agents, imatinib is an expensive drug, costing an estimated US$42,000 per year at standard doses [START_REF] Pollack | As pills treat cancer, insurance lags behind[END_REF]. Optimum dosing and pharmaco-economic optimisation help in avoiding unnecessary waste of scarce resources [START_REF] Steinbrook | Saying no isn't NICE-the travails of Britain's National Institute for Health and Clinical Excellence[END_REF]. In some patients, disease control, even with the achievement of the target blood level, may still not be achieved. Current evidence suggests that this may be due to additional mutations conferring resistance [START_REF] Kim | Clinical relevance of a pharmacogenetic approach using multiple candidate genes to predict response and resistance to imatinib therapy in chronic myeloid leukemia[END_REF], and alternative strategies are then necessary, including the use of other targeted agents and combination therapy [START_REF] Quintas-Cardama | Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia[END_REF][START_REF] Volpe | Imatinib resistance in CML[END_REF]. However, neither nilotinib nor dasatinib appears to provide durable responses after failure of imatinib [START_REF] Garg | The use of nilotinib or dasatinib after failure to 2 prior tyrosine kinase inhibitors: long-term follow-up[END_REF]. Multikinase inhibitors, such as bosotunib, also appear to have only limited effects in such patients [START_REF] Quintas-Cardama | Chronic myeloid leukemia in the tyrosine kinase inhibitor era: what is the best therapy?[END_REF]. Allogenic stem-cell transplantation has also been suggested for eligible patients [START_REF] Jabbour | Treatment selection after imatinib resistance in chronic myeloid leukemia[END_REF].

While the targeting of BCR-ABL1 with imatinib is the first, pharmacogenetically guided, logical step in treating CML patients, the use of imatinib is complicated by other aspects of the pharmacogenetics of this drug. Imatinib is metabolised by several of the cytochrome P450 enzymes (CYP450). Of these, CYP3A4 and CYP3A5 appear to be the most important, although CYP1A2, CYP2D6, CYP2C9 and CYP2C19 may also participate [START_REF] Peng | Clinical pharmacokinetics of imatinib[END_REF][START_REF] Gardner | Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib[END_REF]. However, to date, none of the studied CYP450 metabolic enzyme genotypes have shown sufficiently strong predictive power to be useful for imatinib dose-blood level prediction in clinical practice [START_REF] Gardner | Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib[END_REF] when assessed using methodology we have described elsewhere for other types of genetic testing for both prognostic purposes [START_REF] Li-Wan-Po | When is a genetic test suitable for prime time? predicting the risk of prostate cancer as a case-example[END_REF] and critical evaluation of the impact of new variants [START_REF] Li-Wan-Po | Pharmacogenetics of CYP2C19: functional and clinical implications of a new variant CYP2C19*17[END_REF]. This may be due to the complex interactions in the expression of the CYP450 enzymes [START_REF] Meyer Zu Schwabedissen | Hepatic OATP1B transporters and nuclear receptors PXR and CAR: interplay, regulation of drug disposition genes, and single nucleotide polymorphisms[END_REF][START_REF] Willson | PXR, CAR and drug metabolism[END_REF][START_REF] Burk | Cytochrome P450 3A and their regulation[END_REF], including epigenetic effects, the participation of microRNAs [START_REF] Takagi | Posttranscriptional regulation of human pregnane X receptor by micro-RNA affects the expression of cytochrome P450 3A4[END_REF] and their polymorphisms [START_REF] Mishra | MicroRNA reexpression as differentiation therapy in cancer[END_REF] and the interplay between transporters and metabolic enzymes, which has yet to be fully unravelled [START_REF] Benet | The drug transporter-metabolism alliance: uncovering and defining the interplay[END_REF].

Polymorphism of genes coding for transporters are wellknown causes of drug resistance in cancer chemotherapy. The ABCB1 [START_REF] Gurney | Imatinib disposition and ABCB1 (MDR1, P-glycoprotein) genotype[END_REF] and ABCB2 [START_REF] Petain | Population pharmacokinetics and pharmacogenetics of imatinib in children and adults[END_REF] genotypes have been associated with imatinib clearance, although the strengths of association uncovered are, similar to those of the metabolic enzymes, relatively weak and unlikely to be clinically useful for predicting efficacy.

In addition to the gene products which might affect imatinib pharmacokinetics, a number of genes, possibly those involved in the signalling pathways, have also been evaluated with promising results [START_REF] Dressman | Correlation of major cytogenetic response with a pharmacogenetic marker in chronic myeloid leukemia patients treated with imatinib (STI571)[END_REF][START_REF] Mclean | Pharmacogenomic analysis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib[END_REF]. However, again the analyses had insufficient predictive power for routine clinical use.

Other considerations

Given the potential impact on the imatinib's clinical effects, several studies have considered the effect of potential drug interactions on its blood level. Thus, when the potent inducer of CYP3A4, rifampicin, was given concomitantly with imatinib, the area under the concentration-time curve [AUC (0-24 h) ] of the targeted agent decreased by 68% (95% confidence interval 64-70%). This magnitude of change is large enough to be most likely clinically significant, and such interactions should therefore be guarded against [START_REF] Bolton | Effect of rifampicin on the pharmacokinetics of imatinib mesylate (Gleevec, STI571) in healthy subjects[END_REF]. Imatinib, itself an inhibitor of CYP3A4, has been shown to significantly inhibit the metabolism of substrates of this enzyme [START_REF] O'brien | Effects of imatinib mesylate (STI571, Glivec) on the pharmacokinetics of simvastatin, a cytochrome p450 3A4 substrate, in patients with chronic myeloid leukaemia[END_REF]. Given the increased likelihood of combination therapy in cancer patients, constant vigilance is therefore necessary. As patients may inadvertently take non-prescribed herbal and over-the-counter medicines, or indeed dietary components, which may interact, TDM may be the only means of ensuring optimum therapy with critical drugs, such as imatinib. Therapeutic drug monitoring should also help in identifying the non-compliant patient or other unknown factors affecting the achievement of a target blood level despite adequate dosing.

Conclusion

Pharmacogenetic targeting of imatinib in patients with CML is done through the identification of the appropriate genetic variant. Our review and analysis shows that for those patients, a target threshold trough blood level of at least 1 µM and perhaps as high as 1.7 µM would be optimum initially. In the era of personalized medicine, this is best achieved through TDM given the large inter-patient variability in the pharmacokinetics of imatinib and the inadequate predictive power of genetic testing and profiling investigated to date, other than the presence of the BCR-ABL1 oncogene. Therapeutic drug monitoring should ensure that a highly effective drug is used even more effectively to minimise the incidence of pseudo-resistance.

Fig. 1

 1 Fig.1Calculation of the receiver operating characteristic (ROC) curve using the data of Singh et al.[START_REF] Singh | Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukaemia: comparing plasma levels of res-ponders and non-responders[END_REF]

Fig. 3 Fig. 2

 32 Fig. 3 Imatinib oral dose and steady-state trough plasma concentration relationship (closed circles: once-daily dosing; open circles: twice-daily dosing)

Eur J Clin Pharmacol (2010) 66:369-374