Dr Teresa Bandeira
email: teresa.bandeira@hsm.min-saude.pt

Filipa Negreiro

Marisa Salgueiro

Luísa Lobo

Pedro Aguiar

Jc C Trindade

Differences between Obliterative Bronchiolitis and Problematic Severe Asthma in a pediatric population

Keywords: obliterative bronchiolitis, problematic asthma, children, overlap syndrome obliterative bronchiolitis, problematic asthma, children, computed tomography, lung function, atopy, overlap syndrome

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Introduction

The far most common form of bronchiolitis obliterans in children follows a severe lower respiratory-tract infection [START_REF] Del Pino | Growth in post-viral chronic lung disease[END_REF][START_REF] Teper | Respiratory sequelae of viral diseases: from diagnosis to treatment[END_REF]. Other causes include collagen vascular disease, toxic fume inhalation, aspiration syndromes, and Stevens-Johnson syndrome [START_REF] Moonnumakal | Bronchiolitis obliterans in children[END_REF]. The patient typically has wheezing, tachypnea, dyspnea, and persistent cough for weeks or months after the initial infection. The disease may persist for years after its onset and may worsen due to exacerbations caused by viral infections. There are no epidemiological data available, but, for unclear reasons, OB seems to occur more frequently in the southern hemisphere (southern Brazil, Uruguay, Argentina, Chile, New Zealand, and Australia) generating a demand for hospital and clinic services similar to that observed in patients with cystic fibrosis [START_REF] Teper | Respiratory sequelae of viral diseases: from diagnosis to treatment[END_REF][START_REF] Teper | Respiratory sequelae of viral diseases: from diagnosis to treatment[END_REF][START_REF] Moonnumakal | Bronchiolitis obliterans in children[END_REF] in other parts of the world.

OB is synonymous with the term "constrictive bronchiolitis" [START_REF] Chan | Bronchiolitis obliterans: an update[END_REF]. It has been shown that there are distinctive features of lung function (LF) and high-resolution CT (HRCT) that both characterizes the diagnosis and estimates the severity of attainment. LF results are usually characterized by fixed obstruction, minimum response to the administration of corticoids and normal total lung capacity [START_REF] Teper | Respiratory sequelae of viral diseases: from diagnosis to treatment[END_REF].. HRCT most characteristic findings are segmental or lobular areas of hypoattenuation that are associated with narrowing of the caliber of the pulmonary vessels (mosaic perfusion). These abnormalities are F o r P e e r R e v i e w 6 consistent with air trapping, and can be clearly demonstrated on CT scans of the chest performed during expiration or on inspiratory or lateral decubitus HRCT in the uncooperative pediatric patients [START_REF] Castro-Rodriguez | Adenovirus pneumonia in infants and factors for developing bronchiolitis obliterans: A 5-year follow-up[END_REF][START_REF] Colom | Clinical prediction rule to diagnose post-infectious bronchiolitis obliterans in children[END_REF][START_REF] Colom | Risk factors for the development of bronchiolitis obliterans in children with bronchiolitis[END_REF].

Bronchiectasis are also recognized and associated with OB, and tend to be peripheral and cylindrical in nature [START_REF] Chan | Bronchiolitis obliterans: an update[END_REF]. Most features presented by OB patients parallel those of COPD [START_REF]Global Initiative for Chronic Obstructive Lung Disease[END_REF].

In the last decade there is growing evidence for the overlap features between obstructive lung diseases (OLD) in the adult patients [START_REF] Gibson | The overlap syndrome of asthma and COPD: what are its features and how important is it?[END_REF] drawing a continuum from the variable airflow obstruction (asthma) to the incompletely reversible airflow limitation (COPD) with the concomitant diagnosis of asthma, chronic bronchitis, or emphysema being so common among OLD patients from the general population as to account for as much as half of the OLD population who are aged over 50 years [START_REF] Soriano | The proportional Venn diagram of obstructive lung disease: two approximations from the United States and the United Kingdom[END_REF].

In childhood the recognition of severe asthma is not an easy task [START_REF] Fleming | Difficult to Control Asthma in Children[END_REF]. In 2000 the ATS Proceedings classified refractory asthma mainly based on dose of corticosteroids prescribed [START_REF]Proceedings of the ATS Workshop on Refractory Asthma . Current Understanding, Recommendations, and Unanswered Questions[END_REF]. Recently Bush et al proposes the diagnosis of problematic severe asthma (PSA) for the poor control asthma patients that will afterwards be divided by the subspecialist in two subgroups either as difficult into difficult-to-treat asthma or as severe therapy-resistant asthma [START_REF] Bush | Severe childhood asthma: a common international approach?[END_REF][START_REF] Hedlin | Problematic severe asthma in children, not one problem but many: a GA2LEN initiative[END_REF]. Routine OB management at our centre included 3-6 monthly reviews, and the prescription of inhaled steroids and long-acting beta-2agonists and ipratropium or tiotropium. When bronchiectasis were present, chest physiotherapy was prescribed with advice to increase intensity when unwell, and antibiotics prescribed for exacerbations. recommended by the American Thoracic Society [START_REF] Miller | Standardisation of spirometry[END_REF]. The residual volume (RV), and total lung capacity (TLC) were determined in the body plethysmograph [START_REF] Wanger | Standardisation of the measurement of lung volumes[END_REF]. Using the reference equations from www.growinglungs.org.uk [START_REF] Stanojevic | Reference Ranges for Spirometry Across All Ages: A New Approach[END_REF], spirometric values were expressed as percent predicted and z-scores and lung volumes in % predicted in function to Zapletal or the ECCS according to age [START_REF] Pellegrino | Interpretative strategies for lung function tests[END_REF]. Weight and height z-scores were calculated after adjustment for age and sex. Zscore is calculated as (measured value -mean value)/Population SD.

It is a better way of expressing measurements than percent predicted, which now should be obsolete. For example, depending on the variance in the population, 80% predicted may be normal or abnormal, but a Z-score of -2.5 can be seen at once always to be

F

Atopy and inflammatory markers

Atopy was defined as either at least one positive specific immunoglobulin (Ig) E or one positive skin prick test to aeroallergens (cat, dog, house dust mite, grasses and fungi) [START_REF] Baldacci | Allergy markers in respiratory epidemiology[END_REF]. Furthermore the presence of increased number of eosinophils (>274cel.mm -3 ), and /or IgE higher that 2SD adjusted for the age was analyzed between groups [START_REF] Baldacci | Allergy markers in respiratory epidemiology[END_REF]. Inflammation pattern was considered based on a high Creactive protein (CRP) (mg/dl), sedimentation rate (SR) mm, fibrinogen (mg/dl), leukocytes (>13x10 9 /L) and alpha-1-antitrypsin (AAT) [START_REF] Cazzola | Outcomes for COPD pharmacological trials: from lung function to biomarkers[END_REF]. near-drowning in soapy water had occurred]Afterwards the majority of patients in both groups had persistent symptoms but fine crackles were heard more frequently in OB (60%) than in PSA patients (6.7%) (p< 0.001).. In both groups a high prevalence of exposure to tobacco smoke was found (p=0.542).

Statistical

Lung function

There

DISCUSSION

To our knowledge, this is the first study to establish clinical, radiological and physiological differences between Obliterative Bronchiolitis (OB) and Problematic Severe Asthma (PSA) in the transition through pediatric to young adult ages. Our main results

suggest that patients with OB start their symptoms early in life usually after a severe respiratory infection. Adenovirus is the most frequently identified etiological agent [START_REF] Colom | Postinfectious bronchiolitis obliterans[END_REF]although clinical studies frequently miss the identification of the causal agent as it occurred at this investigation [START_REF] Hodges | Causes and management of bronchiolitis with chronic obstructive features[END_REF][START_REF] Zhang | Clinical course of postinfectious bronchiolitis obliterans[END_REF]. In the second decade of life these patients still have persistent respiratory symptoms, poor nutrition and a significant airway obstruction and hyperinflation. Age and disease severity as shown by hospital admission and the need for ventilator support and persistent crackles distinguishes these from PSA patients. Moreover although persistent symptoms and an obstructive lung function pattern are found in PSA patients a significant less degree of compromise is evidenced. Advances in intensive therapy that have occurred over the last few years allowed many children to survive who, after being affected by serious respiratory infections, develop persistent and severe sequelae [START_REF] Teper | Respiratory sequelae of viral diseases: from diagnosis to treatment[END_REF].

Furthermore the different extension of lung compromise between OB and PSA patients is also revealed through the HRCT findings. Technical improvements have turned HRCT in the imaging modality of choice for the morphological assessment of pulmonary parenchyma, even in children [START_REF] Rossi | The radiology of chronic lung disease in children[END_REF].

We found that LFT results of obstructive ventilatory defect were common in both OB and PSA patients. Bronchial reactivity after beta-2-agonist inhalation was also commonly seen in both conditions especially if % variation from baseline was accounted for. No significant differences were found in FEV 1 /FVC ratio between OB and PSA. Severity of LF compromise expressed by the reduction of FEV 1 , FEF 25-75 and FVC must be taken in account as they tend to be more severe in OB than in PSA otherwise being difficult to differentiate OB and PSA on the basis of the LF defect alone. FVC was reduced in OB patients and close to normal in the PSA patients and OB patients showed higher air-trapping pattern. . Furthermore FVC and ∆FEV 1 post-BD (ml) emerged as the better LF variables capable of identifying third group of diagnosis with overlapping features. We have recently demonstrated that results of bronchodilation are dependent on previous lung volumes [START_REF] Bandeira | FEV 1 response to bronchodilation in Pediatric Patients With Non-Transplant Obliterative Bronchiolitis and Asthma: Similarities to Adult Patients in the Discriminative Value For Diagnosis[END_REF] in OB patients as has been

shown by others [START_REF] Casanova | Inspiratory-to-Total Lung Capacity Ratio Predicts Mortality in Patients with Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Verbanck | Small airways ventilation heterogeneity and hyperinflation in COPD: Response to tiotropium bromide[END_REF]. Casanova et al demonstrated that a 25% IC/TLC value had the best combined sensitivity, specificity, and positive and negative predictive values for mortality in COPD patients [START_REF] Casanova | Inspiratory-to-Total Lung Capacity Ratio Predicts Mortality in Patients with Chronic Obstructive Pulmonary Disease[END_REF] and another study showed the same for the ratio RV/TLC [START_REF] Nishimura | Dyspnea Is a Better Predictor of 5-Year Survival Than Airway Obstruction in Patients With COPD[END_REF].

Although we have not studied the IC because there are few reference values determined in pediatrics [START_REF] Tomalak | Reference values for forced inspiratory flows in children aged 7-15 years[END_REF] we found that FEV 1 and FVC are concomitantly decreased in OB patients determining that FEV 1 /FVC ratio is only slightly decreased and so not far different the mean value found in PSA patients. It has been stated before that this pattern reflects failure of the patient to inhale or exhale completely.

Another explanation, and that is our understanding, the patchy collapse of small airways early in exhalation may have a significant impact in the response to therapy [START_REF] Pellegrino | Interpretative strategies for lung function tests[END_REF][START_REF] Ferguson | Why Does the Lung Hyperinflate?[END_REF]suggesting that the on HRCT seems to be the key finding which may offer some confidence in suggesting a diagnosis of OB or overlap syndrome rather than asthma as has been shown before [START_REF] Jensen | High-resolution CT Features of Severe Asthma and Bronchiolitis Obliterans[END_REF]. Colom and Teper demonstrated that a mosaic pattern on HRCT was strongly associated with the diagnosis of post-infectious OB [START_REF] Colom | Clinical prediction rule to diagnose post-infectious bronchiolitis obliterans in children[END_REF]. Although there are different causes for the mosaic pattern of attenuation [START_REF] Stern | CT mosaic pattern of lung attenuation: distinguishing different causes[END_REF] we believe as Jensen [START_REF] Jensen | High-resolution CT Features of Severe Asthma and Bronchiolitis Obliterans[END_REF] that the mosaic pattern seen in OB results from a combination of air-trapping due to small airways disease and associated oligoemia secondary to reflex vasoconstriction to the areas of underventilated lung tissue.

peripheral
We found a great proportion of patients with atopic markers in both groups studied but variables from the atopic expression were more frequently is PSA than in OB patients. An interaction between atopy and induced viral wheezing is being increasingly described and a strong association between asthma and atopy comes from large epidemiologic studies [START_REF] Pinto-Mendes | Infecção na modulaçao da asma[END_REF][START_REF] Sly | Early identification of atopy in the prediction of persistent asthma in children[END_REF].

Although the importance of atopy as a cause of asthma in individuals may have been overemphasized [START_REF] Pearce | How much asthma is really attributable to atopy?[END_REF] and the available epidemiological evidence suggests that the population based proportion of asthma cases that are attributable to atopy is usually less than one half, another study on difficult asthma has shown increased proportion of atopy [START_REF] Bossley | Corticosteroid responsiveness and clinical characteristics in childhood difficult asthma[END_REF]. SPT in agreement with suggestion that quantitative measures of atopy especially cumulative titers of IgE specific for perennial inhalant allergens provide robust assessments of atopy-associated risk of current asthma and its severity [START_REF] Sly | Early identification of atopy in the prediction of persistent asthma in children[END_REF]. Atopy has also been related to other respiratory diseases but the association between atopy and the development of obstructive lung diseases remains to be clarified [START_REF] Baldacci | Allergy markers in respiratory epidemiology[END_REF].

The study does have a number of limitations. Patients were selected from a specialized clinic on a third care University Hospital and so the reduced number of patients over a large time span may turn difficult the generalization of findings. However both are rare diseases. This is the largest group of OB patients reported in Europe [START_REF] Cazzato | Airway inflammation and lung function decline in childhood post-infectious bronchiolitis obliterans[END_REF]. PSA constitutes a heterogeneous clinical diagnosis that only recently had their distinction clarified [START_REF] Hedlin | Problematic severe asthma in children, not one problem but many: a GA2LEN initiative[END_REF] so that the present study may add further discriminative features for severe obstructive diseases starting in pediatric ages and suggest a different pathophysiologic defect between them. We could have further studied peripheral airway measurements using different diagnostic tools [START_REF] Robinson | Inert Gas Washout: Theoretical Background and Clinical Utility in Respiratory Disease[END_REF][START_REF] Haruna | Relationship between peripheral airway function and patient-reported outcomes in COPD: a cross-sectional study[END_REF] HRCT HRCT images were acquired on inspiratory apnea from the lung apex to the diaphragm using 1 mm slice thickness at 10 mm intervals using a bone (high spatial detail) reconstruction algorithm, 50-80 mAs e 90-120 kVp (according to body volume) and 0,5 sec of rotation time. Images were photographed at lung window settings (1600/600) and mediastinal window settings (300/40) before March 2009. After March 2009 images were stored in PACS system. Most of the studies included inspiratory and expiratory images, typically one or two images at selected levels through the thorax with the patient in full expiration. In some cases volumetric acquisition ("combi") was performed: 3,2/1,6 mm, 80-100 mAs e 90-120 kVp, 0,5 ms rotation time, 1,75 pitch, followed by a pre-selected multilevel acquisition (3 a 4) of expiratory high resolution images.

Each HRCT was reviewed by a -trained pediatric radiologist. A score sheet modified from Jensen et al. was used to record the presence and extent of 24 HRCT findings. Each lung was divided into upper, middle and lower zones for a total of six lung zones for each patient.

Each of these zones was scored separately for the presence and/or extent of the 24 HRCT findings. Inspiratory decreased attenuation, expiratory air trapping and ground glass opacities were scored according to the cross-sectional area of lung involved in each zone 0no involvement; 1 -25% of the cross-sectional area of the lung affected; 2 -26-50% affected; 3 -51-75%; 4 -76-100% affected, bronchiectasis were classified according to [START_REF] Haruna | Relationship between peripheral airway function and patient-reported outcomes in COPD: a cross-sectional study[END_REF] into mild with broncho-arterial (BA) ratio lower than 2, moderate, from 2 to 3 and severe if broncho-arterial ratio was higher than 3. If a bronchiectasic collapse was found, a grade of 3 of the online supplement were scored as either present (score of 1) or absent (score of 0). Score sheets were than tabulated and composite scores for each HRCT feature wee than derived by adding the scores from the six lung zones for each patient.

Statistical analysis

The demographic data and outcomes between groups were compared using two-sided χ2 test for categorical variables and two-tailed Student's t-tests and oneway ANOVA for continuous variables. In small samples or in the absence of normality for continuous variables, non-parametric tests (Mann-Whitney and Kruskal-Wallis) were applied.

The ROC curve analysis only identified numerical parameters associated with diagnosis and cut-offs. The factors considered in the ROC analysis were: results from LFT (FVC%P; FEV 1 %P; FEV 1 /FVC; RV/TLC; change in FEV 1 after bronchodilator (ml and %); results from HRCT (inspiratory decreased attenuation, mosaic pattern, expiratory air trapping, bronchial wall thickening and bronchiectasies); for atopy [(skin prick test results, eosinophils (% of total cell)]; total IgE and specific IgE to D. Pteronyssinus); inflammation (leukocytes (total number of cells), sedimentation rate (SR), fibrinogen, C-reactive protein (CRP) and alpha-1antitrypsin (AAT) (Table 0 of the online supplement). Subsequently, in order to identify a "3rd diagnostic group" a cluster analysis was performed (k-means clustering method, SPSS®). In an initial phase two clusters were identified in order to validated this method and followed by another cluster analysis forcing the solution of three clusters. After achieved the solution of three clusters in each parameter, a cross between the final solution and diagnosis was performed according with the following scheme (see

7

 7 The purpose of our investigation was to describe the characteristic features and patterns distinctive for the diagnosis of OB and PSA through the analysis of the results of objective assessments consisting of conventional lung function tests, high resolution computed tomography (HRCT) and tests for atopy and inflammation. Our secondary goal was to determine the discriminative power of these variables for the diagnosis of OB or PSA or for the identification of an overlap syndrome. Studying overlap syndrome may shed light on the mechanisms of obstructive lung diseases development. observational study comparing two groups of patients with the diagnosis of OB and PSA was performed. The study was approved by the local research ethics committees and written informed parental or own consent was obtained prior to testing. Subjects OB patients were retrospectively identified in the computerized database of outpatients attending the pediatric respiratory clinic since 1975 at H. Santa Maria (HSM), using the following key diagnoses: OB, bronchiectasis, and post-adenoviral infection. The identified cases were checked to ascertain whether they met the following inclusion criteria[1-3]: initial episode of a severe respiratory infection or other relevant cause for OB in a previously healthy child followed by persistent cough, wheezing, retractions, crackles, abnormalities on chest radiograph for months or years, a high-resolution CT scan (HRCT) showing features consistent with the diagnosis: inspiratory decreased attenuation, expiratory air trapping and an attenuation mosaic pattern and lung function tests demonstrating persistent irreversible airflow limitation. Patients with the following diagnosis were excluded based on history or tests: cystic fibrosis, heart disease and other major polimalformative syndromes. The PSA group was selected from the Difficult Asthma Clinic at Pediatric Department of HSM based on the occurrence of persistent chronic symptoms of airways obstruction, frequent or severe exacerbations in last year despite multiple drug treatment associated with persistent airflow obstruction and variable obstructive pattern in LF tests sometimes with swings in lung function where the diagnosis of asthma was plausible and an appropriate work-up to exclude other diagnosis was done [14]. No attempt for discriminating between subgroups of PSA was done.

  airway involvement is an established component of OB and PSA patients. Early on in the evolution of the disease and in less severe patients a simple test of gas distribution such as the traditional VC N 2 single breath washout test would be a valuable tool of diagnosing OB and in the future should be considered in the workup for the diagnosis of these patients[START_REF] Robinson | Inert Gas Washout: Theoretical Background and Clinical Utility in Respiratory Disease[END_REF].Regarding high-resolution CT, our findings were in agreement with previous studies which documented that no isolated features could accurately diagnose OB as increased lung volume, inspiratory decreased attenuation, expiratory air trapping and mosaic pattern were frequent both in OB and in PSA patients. However OB patients showed higher scores for these HRCT findings and bronchiectasis were by far more frequent in OB patients. In our study, mosaic pattern provided the best power to predict the emergence of a third group of diagnosis. The mosaic pattern of attenuation of lung tissue

F o r P e e r R e v i e w 6 FEV 1

 61

2 2

 2 or higher was considered. The remaining HRCT features shown in table

  range of values from 18.2 to 70.6%P for FEV 1 (zscores -7.3 to -2.7), from 32.8 to 91% for FVC (z-scores -6.4 to -0.8), and from 46.8 to 92.6 for the FEV 1 /FVC ratio (z-scores -4.7 to -∆FEV 1 in ml, mosaic pattern, SPT and D.Pteronyssinus specific IgE were the best variables (online supplement and Table4).

	supplement), for HRCT findings (inspiratory decreased attenuation,
	mosaic pattern, expiratory air trapping and bronchiectasis) (Table 6
	of the online supplement) and for atopy (peripheral blood eosinophil
	1.0) compared to PSA patients (p<0.05; table 3). The RV/TLC ratio count, SPT, serum IgE level and D.Pteronyssinus specific IgE). No
	was significantly increased in patients with OB (range 27.83 to 71) (p inflammatory parameter showed enough discriminative power to be
	< 0.001), indicating higher hyperinflation compared with PSA patients included in further analysis. Cluster analysis for the identification of a
	(Table 3). ∆FEV 1 was greater than 12% in 12 (48.0%) OB patients Atopy and inflammation third group diagnosis showing overlap features determined that
	F o r and 8 (53.3%) of PSA patients, but only 8 (32%) OB patients had a 200 ml improvement after beta-2-agonist inhalation. Twelve (80%) F o r PSA patients had higher frequency of at least 1 positive allergen skin test (p=0.002), a greater mean number of positive skin tests per patient (p=0.001), significantly greater median serum IgE level F o r FVC%P, e F o r
	P P (p=0.007), and mean peripheral blood eosinophil count (p=0.004) P P
	e e e e
	e r e r e r e r
	R R R R
	e e e e
	v i e w v i e w v i e v i e Variables with power to discriminate between groups of patients ROC curve analysis demonstrated a good discriminative power (cut-w w
	off estimates higher than 75%) for LF variables (FVC%P, FEV 1 %P,
	FEV 1 /FVC%P, RV%TLC e ∆FEV 1 in ml) (Table 5 of the online
	16 17

was no statistical difference between the two groups in age and height at lung function test (LFT) but patients with OB showed a reduced z-score for weight and BMI compared with PSA patients (p< 0.05) (table 2 of the online supplement). Patients with OB showed a significant decrease in FEV 1 , FVC and FEV 1 /FVC ratio. LFT of OB 14 patients showed a PSA patients showed an improvement after bronchodilator greater than 200ml. The change of FEV 1 correlated significantly with baseline FVC and FEV 1 (p<0.01) in PSA patients but not in OB patients (data not shown).

CT findings

Among OB patients, the most frequent HRCT features were increased lung volume, inspiratory decreased attenuation and mosaic pattern in all examinations, and expiratory air trapping in all of the 24 (100%) studies that included expiratory images. Bronchial wall thickening were observed in 23 (92%) and bronchiectasis in 22 (88%). Among PSA patients, the most frequent HRCT features were increased lung volume in 13 out of 14 (92.9%) where this could be classified, inspiratory decreased attenuation in 13 (86.7%) and mosaic pattern in 11 out of 14 (78.6%). Expiratory air trapping was present in 11/14 15 (78.6%) studies that included expiratory images. Bronchial wall thickening was observed in 8/14 (57.1%) and bronchiectasis in 4 (26.7%). When comparing groups of diagnosis, OB patients showed significantly higher scores for inspiratory decreased attenuation, mosaic pattern, expiratory air trapping and bronchiectasis than PSA (table 3 of the online supplement). compared with patients with OB (table 4 of the online supplement).

Overall 14 (93.3%) patients with PSA had at least one allergy marker positive versus 11 (44%) of the OB patients.

Inflammatory variables studied were in the normal range for both groups and there was no significant difference between the two groups (see table E0 on online supplement).

  and IgE for D.Pteronyssinus

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	were identified as discriminative factors between OB and PSA. This is
	21

Table 1 -

 1 Demographic characteristics and tobacco smoke exposure by group of diagnosis (OB, PA)

	but mean

F o r P e e r R e v i e w 1 TABLES *Age at lung function tests; § only time of follow-up with the performance of LF studies is considered Page 28 of 44 John Wiley & Sons, Inc.

Table 2 -

 2 Clinical characteristics by group of diagnosis (OB, PSA)

	Obliterative bronchiolitis	Problematic Asthma
	n=25	%	n=15	%	p-value
			Past history

Table 3 -

 3 Lung function results at test by group of diagnosis (OB, PSA)

	Obliterative bronchiolitis	Problematic Asthma
		p-value
	n=25	n=15

Table 4 -

 4 Final cluster analysis presenting the 5 variables that proved the best adequability for group of diagnosis

	<0.001
	-0.8)

FVC: forced vital capacity; FEV 1 : forced expiratory volume in 1 second FEV 1 /FVC: forced vital capacity to forced expiratory volume in 1 second ratio; FEF 25-75 : forced expiratory flow at 25% to 75% vital capacity; RV: residual volume; TLC total lung capacity; sRtot:

table 1

 1

	of the online

Table 0 :

 0 Inflammation variables for diagnosis group

	OB	PSA
	(n=25)	(n=15)	p-value*

Table 1 :

 1 Three cluster analysis to identify the third diagnostic group (overlap syndrome) For individuals correctly classified as OB or PSA in this final cluster analysis, as well as those classified in the "3 rd diagnostic group," a comparative analysis of means and medians of the variables selected for the final cluster analysis of these 3 groups were performed. With this comparative analysis the values of central tendency that most distinguish these 3 groups can be observed. Values of p<0,050 were considered to be statistically significant.

	OB	PSA	Total

Table 2 -

 2 Anthropometric characteristics by group of diagnosis (OB, PSA)

						Page 40 of 44
				Obliterative bronchiolitis (n=25)	Problematic Asthma (n=15)	p-value
	Weight, z-score n	22	14
			mean (SD)	-0.54 (1.69)	0.66 (1.24)	0.028
			median (min ; max) -0.48 (-3.41 ; 2.00)	0.89 (-1.43 ; 3.13)
	Height, z-score n	22	14
			mean (SD)	-0.54 (0.92)	-0.39 (0.91)	0.633
			median (min ; max) -0.66 (-2.19 ; 1.14)	-0.74 (-1.39 ; 1.50)
	BMI (Kg/m	2 )	n	25	15
			mean (SD)	20.95 (6.10)	22.41 (5.35)	0.448
			median (min ; max) 20.55 (13.57 ; 40.14)	21.87 (15.84 ; 32.75)
	BMI, z-score	n	22	14
			mean (SD)	-0.34 (1.87)	0.93 (1.39)	0.036
			median (min ; max) -0.01 (-4.26 ; 2.87)	1.70 (-1.47 ; 2.96)
	BMI -body mass index

Table 3 -

 3 Comparison of OB and PSA scores for HRCT

	Obliterative	Problematic
	bronchiolitis n=25	Asthma n=15	p-value

Table 4 -

 4 Atopy results by group of diagnosis

		Obliterative bronchiolitis n=25	Problematic Asthma n=15	p-value
	Eosinophils (x10^9/L) median (min ; max)	130 (10 ; 1570)	410 (140 ; 710)	0.004
	Total IgE (kU/L) median (min ; max)	33.4 (6.9 ; 3034.0)	388.0 (8.9 ; 1338.0)	0.007
	D. pteronyssinus IgE (class) median (min ;max)	0 (0 ; 6)	4 (0 ; 6)	<0.001
	Skin prick tests (total of mm, all tests) median (min ; max)	0.00 (0 ; 22)	15.00 (0 ; 48)	0.001

Table 5 -

 5 Area under ROC curve (AUC) and concordance evaluation between the 2 clusters (cluster 1 as OB group patients and cluster 0 as PSA group patients) using Kappa Cohen coefficient for Lung Function results

				Kappa Cohen
		AUC	Cut-off		p-value
				coefficient
	FVC% predicted	0.899	77.79	0.605	<0.001
	FEV 1 % predicted	0.899	55.00	0.641	<0.001
	FEV 1 /FVC% predicted	0.747	79.82	0.392	0.011
	RV%TLC	0.872	36.88	0.395	0.004
	∆ ∆ ∆ ∆FEV 1 %t post-BD (ml)	0.800	235.00	0.333	0.012
	∆ ∆ ∆ ∆FEV 1 %t post-BD (%)	0.545	-----	0.000	>0.999
	AUC -area under ROC curve
	E-

Table 6 -

 6 Area under ROC curve (AUC) and concordance evaluation between the 2 clusters (cluster 1 as OB group patients and cluster 0 as PSA group patients) using Kappa Cohen coefficient for HR-CT findings

				Kappa Cohen
		AUC	Cut-off		p-value
				coefficient
	Inspiratory decrease attenuation	0,863	6,5	0,518	<0,001
	Expiratory air trapping	0,856	6,5	0,480	0,001
	Mosaic pattern	0,713	4,5	0,195	0,221
	Bronchial wall thickening	0,636	-	0,084	0,584
	Bronchietasis	0,861	1,5	0,125	0,102

Table 7 -

 7 Three clusters analysis. Adequability tax for LF results

		Adequability for the diagnosis	of	Adequability for the diagnosis
		OB and PSA		of the 3 rd group
	FVC% predicted	94%		87%
	FEV 1 % predicted	96%		66.6%
	FEV 1 /FVC% predicted	74.1		61.6
	RV%TLC	79.2%		37.6%
	∆FEV 1 %t post-BD (ml)	89.5%		85.8%
	∆FEV 1 %t post-BD (%)	60.7%		66.6%
	E-

Table 8 -

 8 Three clusters analysis. Adequability tax for HR-CT results

	Page 43 of 44	Pediatric Pulmonology
		Adequability for the diagnosis	Adequability for the diagnosis of
		of OB and PSA	the 3 rd group
	Inspiratory decreased
		75.9%	36.4%
	attenuation
	Expiratory air trapping	81.2%	76.2%
	Mosaic pattern	80.8%	92.4%
	Bronchial wall thickening	66.7%	40.0%
	Bronchiectasis	58.1%	0.0%
		John Wiley & Sons, Inc.

Table 9 -

 9 Three clusters analysis. Adequability tax for atopy results

				Page 44 of 44
		Adequability for the diagnosis	of	Adequability for the diagnosis of
		OB and PSA		the 3 rd group
	Skin prick tests (total of
		84%		80%
	mm, all tests)
	Eosinophils (x10^9/L)	77.3%		77.8%
	Total IgE (kU/L)	68.6%		0.0%
	D. pteronyssinus specific
		88.5%		83.4%
	IgE (class)

John Wiley & Sons, Inc.

Pediatric Pulmonology

John Wiley & Sons, Inc.Pediatric Pulmonology

John Wiley & Sons, Inc.


Noemi Fantini

Giancarlo Colombo

Antonella Riva

Paolo Morazzoni

Ezio Bombardelli

Mauro A M Carai
email: caraimam@tiscali.it

Andrea Giori

Evidence of glycemia-lowering effect by a Cynara scolymus L. extract in normal and obese rats

Keywords: Glycemia, Zucker rats, 18 Nutraceutical

Introduction 1 2 3 Artichokes (Cynara scolymus L., Asterceae family) are an ancient crop and medicinal plant, the 4 therapeutic potential of which was known to the ancient Egyptians, Greeks, and Romans 5 [START_REF] Lattanzio | Globe artichoke: A functional food 17 and source of nutraceutical ingredients[END_REF]. In the remote past artichoke preparations were used as a digestive aid. In 6 recent times, reports from folk medicine as well as preliminary data from scientifically designed 7 clinical studies have suggested that artichokes may have cholerectic, hypocholesterolemic, and 8 hypolipidemic properties (Gebhart, 1998;English et al., 2000;[START_REF] Bundy | Artichoke (Cynara 3 scolymus) reduces plasma cholesterol in otherwise healthy hypercholesterolemic adults: a 4 randomized, double blind placebo controlled trial[END_REF]Wider et al. 9 2009; [START_REF] Küskü-Kiraz | Artichoke leaf extract 13 reduces oxidative stress and lipoprotein dyshomeostasis in rats fed on high cholesterol diet[END_REF]. 10

Based on the notion that dietary fibers may be beneficial in carbohydrate metabolism a recent 11 study (Vinik et al., 1998) found that consumption of an artichoke food supplement for three 12 consecutive months reduced fasting and post-prandial glycemia in type-2 diabetic patients (Nazni 13 et al., 2006). Consistently, an independent study found that a meal of bread and wild artichokes 14 (Cynara cardunculus L.) attenuated post-prandial increase in glycemia in healthy subjects 15 (Nomikos et al., 2007). 16

The present study was designed to assess the ability of artichoke extract to reduce glycemia via a 17 mechanism other than the action of its fibers (Vinik et al., 1998). For this purpose, the present 18 study tested the capacity of a standardized, purified, fiber-free extract of Cynara scolymus L. to 19 lower post-prandial glycemia in laboratory rats. This extract was of particular interest with regard 20 to the study aims as it was completely fiber-free. Two different strains of rats were used: 21 unselected Wistar rats and genetically selected, obese Zucker rats; notably, Zucker rats display 22 fasting hyperglycemia and have been proposed as a possible animal model of pre-diabetes 23 (Romanovsky et al., 2008). The effect of the acutely administered artichoke extract on glycemia 24 was evaluated after the consumption of a large meal comprising a given amount of food. Rats were 25 5 corresponding to that of extracted plant material. Subsequently, the concentrate was purified on an 1 absorption resin through loading into a column packed with 900 mL polymeric resin 2 (Amberlite TM ); the resin was thoroughly washed with 1 L water, the purified extract eluted with 2 3 L ethanol, and was subsequently concentrated to dryness to yield 8.5 g of powder. Separation and 4 determination of caffeoylquinic acid derivatives and flavonoids in the extract was performed 5 simultaneously by a reversed phase HPLC method, using a stainless steel column (250 x 4.6 mm), 6 packed with octadecylsilyl silica gel for chromatography (5 µm), kept at 20°C, and with gradient 7 elution at a constant flowrate of 1.0 ml/min. Mobile phase A was composed by a mixture of 8 trifluoroacetic acid (0.01 % v/v, in water); mobile phase B was composed by a mixture of 9 trifluoroacetic acid (0.01 % v/v, in acetonitrile); the injection volume for all samples was 10 µl. 10 Detection was obtained with a UV spectophotometer at 263 nm. Samples and standards were 11 dissolved in methanol (40 % v/v, in water), filtered by a 0.45-µm polytetrafluoroethylene syringe 12 filter and then injected directly into the HPLC system. The extract obtained had a high 13 caffeoylquinic acid (≥50%), and flavonoid content, expressed as luteolin glycosides, of 14 approximately 2%. 15 16

Experimental procedure 17 18

In both experiments, rats were kept under a regimen of unlimited access to regular rodent chow 19 [Harlan, Global Diet 2018, Mucedola, Settimo Milanese, MI, Italy; main composition: 18.9% 20 crude protein, 5.7% crude oil, 3.8% crude fibre, 5.9% ash, 57.3% carbohydrate (42.2% starch, 21 4.9% sugar); digestible energy: 3.35 cal/g, metabolisable energy: 3.27 cal/g] and water for 24 22 hours/day. Rats were fasted 12 hours before the start of the experiment (this 12-hour period 23 coincided with the entire light phase of the light-dark cycle). In Experiment 1, on the test day 24 Wistar rats were divided into four groups of n=7-9, matched for body weight and glycemia at time 25 0, and treated with 0, 500, 1000, and 1500 mg/kg Cynara s. flowering head extract. In Experiment 26 flowering head extract was suspended in distilled water with a few drops of Tween 80 and 5 administered intragastrically by a metal gavage at an infusion volume of 4 ml/kg, 30 min before 6 lights off. At lights off, rats were given 8 g/kg food pellets; this food amount was chosen on the 7 basis of preliminary experiments demonstrating how fasted Wistar and Zucker rats consumed this 8 entire amount in less than 60 min (this laboratory, unpublished results). Glycemia was determined 9 0, 60, 120, and 360 min after food presentation. A small (0.05 ml) blood sample was collected 10 from the tip of the tail of each rat and analyzed enzymatically by means of GL5 Analox (Analox 11 Ltd, London, United Kingdom). 12 13

Statistical analysis 14 15

In both experiments, data on the effect of Cynara s. flowering head extract on time-course of 16 glycemia were analyzed by a 2-way (dose; time) ANOVA with repeated measures on the factor 17 "time". Data on the effect of Cynara s. flowering head extract on the area under the curve of the 18 time-course of glycemia [expressed in mg*min/100 ml; calculated using GraphPath Prism 3.03 19 (GraphPath Software, La Jolla, CA, USA)] were analyzed by a 1-way ANOVA. 20 The results of the present study indicate that the acute administration of a standardized extract of 9 Cynara s. flowering heads effectively reduced post-prandial increase in glycemia in two different 10 strains of rats, unselected Wistar and genetically obese Zucker rats. The experiment was designed 11 to oblige rats to consume large amounts of food (a regular rat chow made up of 57.3% 12 carbohydrates) in a single meal; to this end, rats were initially fasted and then given the maximum 13 amount of food that rats of both strains were able to consume entirely over a 1-hour period. In 14 Wistar rats the effect of Cynara s. flowering head extract produced a negligible dose-dependence 15 relationship. Additional studies should be undertaken to investigate whether alternative vehicles 16 may improve extract absorption resulting in a higher degree of separation between the effects of 17 the different doses. 18

To our knowledge, the results of the present study constitute the first experimental evidence of the 19 reducing effect of an artichoke preparation on glycemia in laboratory rodents. These results are 20 consonant with recent human studies reporting the capacity of dietary artichoke (both Cynara 21 scolymus L. and Cynara cardunculus L.) to lower post-prandial rise of glycemia in humans (Nazni 22 et al., 2006;Nomikos et al., 2007). 23

In the present study, potency and efficacy of Cynara s. flowering head extract tended to be higher 24 in Wistar than Zucker rats. The reason for this difference is unknown at present and may require 25 further investigation. 26 The present investigation did not specifically address the mechanism(s) of action and/or the active 1 principle(s) by which Cynara s. flowering head extract exerted its reducing effect on glycemia. At 2 present, we can however rule out the hypothesized contribution of fibers contained in dietary 3 artichokes [START_REF] Nazni | Hypoglycemic and 20 Hypolipidemic Effect of Cynara scolymus among Selected Type 2 Diabetic Individuals[END_REF]Nomikos et al., 2007), as the extract used in this investigation was 4 completely purified and devoid of any fiber content. Additional studies are needed to investigate 5 mechanism(s) of action, active principle(s), and changes in levels of hormones in specific tissues, 6 including pancreas, liver, gut, muscle, and adipose tissue.

  meal to ensure consumption of an equal amount of food by rats from each 1 experimental group (vehicle-or extract-treated rats) within the scheduled 1-hour access period. employed in the present study were in accordance with the European 7 Communities Council Directive (86/609/EEC) and the subsequent Italian Law on the Protection of 8 animals used for experimental and other scientific reasons. (Experiment 1) and Zucker (Experiment 2) rats (Charles River Laboratories, 13 Calco, LC, Italy) were used. In both experiments rats were individually housed in standard plastic 14 cages with wood chip bedding. The animal facility was under an inverted 12:12 hour light-dark 15 cycle (lights on at 11:00 p.m.), at a constant temperature of 22±2°C and relative humidity of 16 approximately 60%. Rats were extensively habituated to handling, intragastric infusion, and blood 17 drawing from the tail tip. 18 19 Extract preparation 20 21 Cynara scolymus flowering heads, Sardinian spiny variety, were chopped and frozen at the time of 22 harvesting. The biomass, maintained at -30°C until extraction, was cryoground and immediately 23 submitted to alcoholic extraction. 1 kg of Cynara scolymus flowering heads was extracted in a 24 percolator with ethanol/water (70:30), using a total volume of 7 L. The hydroethanolic extract was 25 concentrated under vacuum, to remove ethanol, obtaining 1 L of an aqueous concentrate, a volume 26

  the test day Zucker rats were divided into three groups of n=6-7, matched for body weight 1 and glycemia at time 0, and treated with 0, 500, and 1500 mg/kg Cynara s. flowering head extract. 2 Doses of Cynara s. flowering head extract were selected on the basis of the results of previous 3 experiments and literature data (Küskü-Kiraz et al., 2010) . In both experiments, Cynara s. 4

  Wistar rats averaged approximately 70 mg/dl in all rat groups (Figure1, top 1 panel). In vehicle-treated rats, meal consumption resulted in an increase of glycemia at both the 2 60-and 120-min recording times, when it averaged approximately 130 mg/dl (Figure1, top panel). 3At the 360-min recording time, glycemia in vehicle-treated rats approached basal levels (Figure1, 4 top panel). 5 ANOVA revealed a highly significant effect of treatment with Cynara s. flowering head extract on 6 post-prandial glycemia over the 6-hour recording period [F dose (3,30)=7.44, P<0.001; 7 F time (2,60)=129.31, P<0.0001; F interaction (6,60)=1.22, P>0.05]. At the 60-and 120-min recording 8 times, all three doses of Cynara s. flowering head extract produced a marked decrease in glycemia 9(Figure1, top panel). Glycemia increase (expressed as percentage of values recorded in fasted rats 10 at time 0) averaged approximately 85%, 65%, 60%, and 50% in 0, 500, 1000, and 1500 mg/kg 11 Cynara s. flowering head extract-treated rats, respectively, with negligible differences between the 12 60-and 120-min recording times (Figure1, top panel). 13 In close agreement with the above results, treatment with Cynara s. flowering head extract also 14 reduced the area under the curve of glycemia time-course [F(3,30)=7.23, P<0.001] (Figure 1Zucker rats averaged approximately 105 mg/dl in all rat groups (Figure 2, top 20 panel). In vehicle-treated rats, meal consumption resulted in an increase of glycemia at all three 21 recording times; it rose to 150 mg/dl at the 60-min recording time and declined slowly, being 22 approximately 135 mg/dl at the 360-min recording time (Figure 2, top panel). 23 ANOVA revealed a significant effect of treatment with Cynara s. flowering head extract on post-24 prandial glycemia over the 6-hour recording period [F dose (2,16)=5.22, P<0.05; F time (2,32)=1.09, 25 P>0.05; F interaction (4,32)=0.06, P>0.05]. At the 60-min recording time, both doses of Cynara s. 26 tended to reduce the glycemia (Figure 2, top panel). At the following time 1 intervals (120 and 360 min) a decrease in glycemia was observed only in the rat group treated with 2 1500 mg/kg Cynara s. flowering head extract (Figure 2, top panel). 3 Accordingly, treatment with Cynara s. flowering head extract reduced the area under the curve of 4 glycemia time-course [F(2,16)=4.60, P<0.05] (Figure 2, bottom panel). 5 6 Discussion 7

Figure 1 - 9 Figure 2 -

 192 Figure captions 1

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Acknowledgements 11


Shirley H Y Ho

Mohini ; Singh

Alison Christine Holloway

Denis James Crankshaw
email: cranksha@mcmaster.ca

Denis ; Crankshaw

Denis J Crankshaw

Dr D J Crankshaw

The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450

Keywords: Black cohosh, chaste tree berry, crampbark, false unicorn, cytochromes P450, fluorogenic substrates Black cohosh, chaste tree berry, crampbark, false unicorn, cytochrome P450, fluorogenic substrates

The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450

Introduction

Herbal products are gaining in popularity in large part because consumers believe that they are "natural", safer, more effective, have fewer side effects, and can be obtained at a lower cost than conventional therapies [START_REF]Should we be concerned about herbal remedies[END_REF].

Consequently, herbal products are widely used by women to alleviate discomfort associated with menstruation, pregnancy/labour, premenstrual syndrome and menopause [START_REF] Beal | Women's use of complementary and alternative therapies in reproductive health care[END_REF]. Herbals commonly used for these reproductive complaints include black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus), and false unicorn (Chamaelirium luteum) [START_REF] Beal | Women's use of complementary and alternative therapies in reproductive health care[END_REF][START_REF] Roemheld-Hamm | Chasteberry[END_REF][START_REF] Ross | Black cohosh (Actaea racemosa, syn. Cimicifuga racemosa): the queen of the female pharmacopeia[END_REF] Natural products are often thought to be safe, yet some herbal preparations contain constituents that inhibit cytochromes P450 (CYP450) [START_REF] Fugh-Berman | Herb-drug interactions: review and assessment of report reliability[END_REF]. Since many women take both herbals and prescription medications simultaneously [START_REF] Refuerzo | Use of over-the-counter medications and herbal remedies in pregnancy[END_REF] there is a potential for interactions to occur [START_REF] Fugh-Berman | Herb-drug interactions: review and assessment of report reliability[END_REF]. However, there is no predictive pharmacokinetic database that would assist users and prescribers in avoiding potential interactions. This is in stark contrast to the development of pharmaceuticals where predictive pharmacokinetics plays a major role in the progression from lead compound to candidate drug [START_REF] Cohen | In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates[END_REF]. Other important differences between pharmaceuticals and herbals are: i) that the former normally have only one active chemical entity whereas herbals -including those in this study -often have multiple components [START_REF] Gödecke | Variability in PXR-mediated induction of CYP3A4 by commercial preparations and dry extracts of St. John's wort[END_REF], and those components that are pharmacologically active are not necessarily the ones that are pharmacokinetically active; ii) that the relative amounts of various constituents of herbals can vary markedly from preparation to preparation [START_REF] Gödecke | Variability in PXR-mediated induction of CYP3A4 by commercial preparations and dry extracts of St. John's wort[END_REF].

The specific aim of this study was to establish predictive CYP450 interaction kinetics for specific commercial preparations of black cohosh root, chaste tree berry, crampbark and false unicorn that are readily available to consumers in Hamilton, Ontario. To achieve this we examined the ability of these herbal preparations to inhibit the biotransformation of fluorogenic substrates by the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 which account for 5%, 10%, 15%, 20-30%, and 40-45% of total drug biotransformations, respectively [START_REF] Ingelman-Sundberg | Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future[END_REF]. We also investigated their actions on human CYP1A1, which is implicated in both the activation and detoxification of a number of carcinogens [START_REF] Androutsopoulos | Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention[END_REF]. Since there is some controversy about the appropriateness of various methods for screening CYP activity [START_REF] Strandell | An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies[END_REF][START_REF] Unger | Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction[END_REF], a more general aim was to assess the usefulness of the rapid screening of herbals against human cytochromes P450 using fluorogenic substrates as an efficient way to acquire information on potential interactions.

Materials and Methods

Materials

All assay reagents were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Recombinant human cytochrome P450 enzymes (CYP450) were obtained from BD Biosciences (Mississauga, ON, Canada). All substrates and fluorescent product standards were obtained from BD Gentest (Woburn, MA, USA) except for the Vivid ® Red CYP2C9 (Vivid Red) substrate from Invitrogen (Burlington, ON, Canada).

Solvents were of analytic or high-performance liquid chromatography (HPLC) grade.

Commercial liquid (ethanolic) extracts of black cohosh and chaste tree berry (Gaia Herbs, Brevard, NC, USA) and crampbark and false unicorn (St Francis Farms, Cobermere, ON, Canada) were obtained from local retailers.

Preparation of Herbal Products

Commercial extracts of the herbal preparations were diluted in assay buffer so that the final concentration of ethanol in the assays was 1%.

Cytochrome P450 Assay

The velocity of biotransformation of 7-ethoxyresorufin (ERES) by CYP1A1 (0.2 pmol/well), 3-cyano-7-ethoxycoumarin (CEC) by CYP1A2 & CYP2C19 (0.5 and 2 pmol/well, respectively), Vivid Red by CYP2C9 (6.8 pmol/well), 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (AMMC) by CYP2D6 (5 pmol/well) and 7-benzyloxyquinoline (BQ) by CYP3A4 (1 pmol/well) were determined by fluorometric assays using the general principles as described [START_REF] Crespi | Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450[END_REF][START_REF] Marks | High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates[END_REF]. Reactions were carried out at 37°C in black 96-well potassium phosphate buffer pH 7.4 (0.15M), NADP (330 µM), glucose-6-phosphate (830 µΜ), MgCl 2 (850 µM), glucose-6-phosphate dehydrogenase (70-75 mU/well), substrate (variable), and recombinant human CYP450s. Assays were initiated by the addition of enzyme. Formation of product was monitored every 40s for 15 minutes using a SpectraMax Gemini XS reader (Molecular Devices) except in the case for CYP3A4 which was monitored for 7 minutes due to the high velocity of the reaction under the conditions used. Reaction velocities were calculated using the instrument's software (Softmax Pro). In all cases the fluorescence intensity was calibrated with authentic product and the duration of the incubation and the CYP450 concentration were in the linear range of metabolite formation. In experiments to examine the ability of standard compounds and herbal preparations to inhibit CYP450 activity, wells contained fixed concentrations of substrate (Table 1), the putative inhibitors, and the appropriate vehicle which was also present in control wells.

Detection of autofluorescence and fluorescence quenching

Increasing concentrations of herbal preparations were diluted in phosphate buffer or 1% ethanol and incubated with fixed concentrations of fluorescent products. The concentrations used were those that would be present in control wells of inhibition experiments at the end of the incubation period. They were determined from kinetic experiments and were as follows: resorufin (CYP1A1 and 2C9), 0.025 µM and 0.05

Data Analysis

Kinetic parameters for each of the enzymes were determined by fitting plots of the reaction velocity versus substrate concentration to different kinetic models by nonlinear least-squares regression. The simplest model was the Michaelis-Menten model given by the equation:

V = (V max * [S]) / (K m + [S]) (1)
where V is the reaction velocity at substrate concentration [S], V max is the maximum velocity of the reaction and K m is the Michaelis-Menten constant.

In some cases, the Hill model which can be described by equation 2 was used:

V = (V max * [S] n ) / (K m n + [S] n ) (2)
where n is the Hill coefficient.

In cases where a downturn in the enzyme kinetics at high substrate concentrations suggested the possibility of substrate inhibition, this was followed by fitting the data to equation (3), adopted from [START_REF] Kenworthy | Multisite kinetic models for CYP3A4: simultaneous activation and inhibition of diazepam and testosterone metabolism[END_REF] based on the principles defined by [START_REF] Segel | Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems New York[END_REF], which assumes that the substrate binds with equal affinity to two sites on the enzyme and allows for interaction between the two sites:

V = V max * (([S] / K m )+(β * [S]^2 / K m ^2) / (1 + (2[S] / K m )+([S]^2 / K m ^2))) (3)
where β reflects the degree to which the two occupied sites interact to alter the effective catalytic rate constant (when β = 1 there is no change in the rate of product ). The applicability of increasingly complex models over simple models was assessed using the F-test [START_REF] Motulsky | Fitting curves to data using nonlinear regression: a practical and nonmathematical review[END_REF] at P < 0.05.

The effects of putative inhibitors were expressed as a percentage of control according to the following equation:

E = ((V c -B) / (C-B))*100 (4)
where E represents the percentage change with respect to control, V c is the reaction velocity in the presence of putative inhibitor, B is the reaction velocity in the absence of enzyme and C is the reaction velocity in the absence of putative inhibitor.

Inhibitory effects were subsequently assessed using a nonlinear least-squares fitting to the simple competitive binding equation:

E = 100 -(100 / (1 + (10 (-pIC50 -log[X] ) ))) (5)
where [X] is the concentration of the putative inhibitor and pIC 50 is the negative log of the concentration that inhibits enzyme activity by 50%. For herbals the pIC 50 is in units of g/mL. For standard compounds, the molar pIC 50 values were converted to pK i (molar inhibition constant) values according to equation ( 6) adapted from [START_REF] Cheng | Relationship between the inhibition constant (K I ) and the concentration of inhibitor which causes 50 per cent inhibition (I 50 ) of an enzymatic reaction[END_REF] on the assumption that the nature of inhibition is independent of the kinetic model:

pK i = -log 10 ((10 -pIC50 ) / (1 + ([S]/K m )) (6)
When data were tabulated, the pIC 50 and pK i values were converted to IC 50 (µg/mL) and K i (µM), respectively, for ease of interpretation. IC 50 values were also compared to the concentrations (µg/mL) that would be attained from single recommended doses 1.

All herbal preparations produced some degree of fluorescence quenching or autofluoroescence at some wavelengths when used at high concentrations. This often reduced the maximum concentrations that could be used in the assays. By limiting the maximum concentrations used to those producing no more than a 10% change in the fluorescence intensity of the amount of product generated under standard conditions, we were still able to estimate the inhibitory potential of the herbal preparations. The maximum concentrations for each herbal used in each assay are listed in Table 3.

Concentration-dependent inhibition of CYP450 activity by chaste tree berry extract is shown in Fig. 2. The effects of all the herbal preparations on the human CYP450 enzymes are summarized in

Discussion

We chose to use fluorimetric, microplate-based assays because they are homogeneous, sensitive, easy to perform, and have a high throughput that would be ideal for initial of screening of large libraries of commercial herbal products for potential interactions with cytochromes P450 [START_REF] Crespi | Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450[END_REF][START_REF] Ghosal | Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes[END_REF][START_REF] Strandell | An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies[END_REF]. The kinetic parameters that we determined (Table 2) are in good agreement with previously published data [START_REF] Ghosal | Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes[END_REF][START_REF] Marks | High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates[END_REF][START_REF] Stresser | Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates[END_REF], some minor differences are likely due to different experimental conditions. We detected atypical kinetics for CYP1A2, CYP2C19, and CYP3A4

(Table 2). Complex allosteric interactions are fairly common among cytochromes P450 [START_REF] Houston | In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model[END_REF], but are not observed consistently [START_REF] Ghosal | Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes[END_REF][START_REF] Stresser | Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates[END_REF]. We validated the assays by showing that standard inhibitors of each respective CYP isoform acted with an appropriate potency (Table 1).

Again, there is good agreement between our data and those of others using quite diverse methods [START_REF] Ghosal | Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes[END_REF][START_REF] Marks | High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates[END_REF][START_REF] Shimada | Inhibition of human cytochrome P450 1A1-, 1A2-, and 1B1-mediated activation of procarcinogens to genotoxic metabolites by polycyclic aromatic hydrocarbons[END_REF][START_REF] Unger | Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction[END_REF].

We began this study with the assumption that the benefits of fluorimetric, microplate-based assays would outweigh those of alternate methods such as liquid chromatography/mass spectrometry [START_REF] Unger | Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction[END_REF]) which require complex instrumentation, dedicated personnel and additional sample extraction procedures.

One notable disadvantage of fluorogenic substrates is that results obtained do not always agree with those from conventional substrates. For instance there was a large discrepancy between the observed interaction of compounds with CYP3A4 when the fluorogenic probe Vivid Red was used as substrate as opposed to when the Vivid Blue and the conventional substrate S(+)-mephenytoin [START_REF] Cohen | In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates[END_REF].

This phenomenon puts some constraints upon the interpretation of the data [START_REF] Cohen | In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates[END_REF]. However, there is also lack of agreement among conventional substrates, and a general principle that the choice of the probe substrate has a significant impact on the drug interaction profile of a given agent has emerged [START_REF] Foti | CYP2C19 inhibition: the impact of substrate probe selection on in vitro inhibition profiles[END_REF]. Clearly, with a task as large as the initial screening of herbals for their interactions with CYP450s, the use of a library of substrates for each isoform is impractical. [START_REF] Cohen | In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates[END_REF] support the continued use of fluorogenic substrates for the initial identification of the potential of new chemical entities to interact with CYP450s, provided that follow-up studies are carried out on drug candidates with conventional probes. Similar principles could be adopted for herbals with follow-up studies being performed on the more potent interactions.

We compared the inhibitory potencies of the herbals to the concentrations that would be attained from single recommended doses of each preparation at three different volumes of distribution which corresponded to plasma volume, the volume of extracellular water and total body water by assuming 100% bioavailability of the active principle (Table 4). For black cohosh against CYPs 2D6 and 1A2

concentrations from a single dose distributed in total body water were approximately 7 and 35 times higher than the in vitro IC 50 values, respectively. Thus, the potencies of black cohosh against these two isoforms seem sufficiently high to merit concern, yet in vivo data argue against this [START_REF] Gurley | In vivo effects of goldenseal, kava kava, black cohosh, and valerian on human cytochrome P450 1A2, 2D6, 2E1, and 3A4/5 phenotypes[END_REF][START_REF] Gurley | Clinical assessment of CYP2D6-mediated herb-drug interactions in humans: effects of milk thistle, black cohosh, goldenseal, kava kava, St. John's wort, and Echinacea[END_REF]. Results of this study did identify some potent inhibition of CYP450 activity (Table 4) by commercially available herbal preparations. Black cohosh exhibited inhibitory activity against CYP 1A2, 2C19 and 2D6. Although some cycloartanoid triterpene glycoside components of black cohosh have weak inhibitory activity against CYP3A4 [START_REF] Tsukamoto | Isolation of CYP3A4 Inhibitors from the Black Cohosh (Cimicifuga racemosa)[END_REF], we found no effect of the commercial extract at the low concentrations we were able to test, and this is in agreement with in vivo data suggesting no clinically relevant interactions between black cohosh and CYP3A4 [START_REF] Gurley | Assessing the clinical significance of botanical supplementation on human cytochrome P450 3A activity: comparison of a milk thistle and black cohosh product to rifampin and clarithromycin[END_REF]. Similar in vivo experiments discount the clinical significance of the inhibition of CYP2D6 by black cohosh [START_REF] Gurley | Clinical assessment of CYP2D6-mediated herb-drug interactions in humans: effects of milk thistle, black cohosh, goldenseal, kava kava, St. John's wort, and Echinacea[END_REF] which was significant in our assays but was the lowest activity that we measured (Table 4). In vivo studies also do not support any clinically significant interactions between black cohosh and CYP1A2 [START_REF] Gurley | In vivo effects of goldenseal, kava kava, black cohosh, and valerian on human cytochrome P450 1A2, 2D6, 2E1, and 3A4/5 phenotypes[END_REF] whereas we found the inhibition to be quite potent (Table 4). However, the most potent inhibitory effect of black cohosh was against CYP2C19 (Table 4) for which no in vivo data are available at present. While the discrepancies might be explained by differences in the herbal preparations used in the in vivo studies compared to ours, a more likely explanation is that the active inhibitory components are poorly bioavailable. The majority of evidence (summarised by [START_REF] Unger | Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction[END_REF], suggests that polyphenolic constituents of herbals have a very low oral bioavailability, while other components are reported to have high bioavailability (see [START_REF] Strandell | An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies[END_REF].

Adverse events following the consumption of chaste tree berry are usually mild and reversible [START_REF] Daniele | Vitex agnus castus: a systematic review of adverse events[END_REF][START_REF] Dugoua | Safety and efficacy of chastetree (Vitex agnus-castus) during pregnancy and lactation[END_REF] with no drug interactions identified by systematic reviews of the literature. However, neither in vitro nor in vivo pharmacokinetic studies with this herb have been reported to date. We found that chaste tree berry extract inhibited CYPs 1A2, 2C19, 2D6 and 3A4 at concentrations in the µg/mL range or lower. In comparison to predicted concentrations in vivo, effects on CYPs 2C19 and 3A4 were particularly potent (Table 4).

There are no prior studies on the pharmacokinetics of crampbark and false unicorn. Both were potent inhibitors with IC 50 s close to 1 µg/mL; crampbark of CYPs 1A2 and 2C19 and false unicorn of CYPs 2D6 and 3A4 (Table 4).

Because of some necessary assumptions about pharmacokinetic properties of test substances and the nature of the assays themselves, CYP450 inhibition kinetics cannot be relied upon to accurately predict herb/drug interactions. This principle is aptly demonstrated in the case of black cohosh discussed above where its high in vitro potency against CYPs 2D6 and 1A2 does not lead to significant clinical effects.

However, the cost of performing in vivo experiments on all herbal preparations -let alone the enormity of the task -would be prohibitive. Therefore a more practical approach is to accept the lower predictive power of CYP450 inhibition kinetics alone, prioritise preparations according to their inhibitory potency and their frequency of use and then proceed directly to in vivo experiments. Although the methods we have described have some limitations brought about by inherent fluorescence properties of some herbals, they provide a relatively fast and efficient means of obtaining the necessary preliminary data on large numbers of preparations simultaneously.

In summary, the use of fluorogenic substrates to screen for the effects of herbal preparations on the activity of human CYP450s is limited by interference from

  ) with a final volume of 200 µL containing (final concentrations):

  µM; 3-cyano-7-hydroxycoumarin (CYP1A2 and 2C19) 0.2µM and 0.05µM; 3-[2-(N,N-Diethyl-N-methylammonium)ethyl]-7-hydroxy-4-methylcoumarin (CYP2D6), 0.1 µM; 7-hydroxyquinoline (CYP3A4), 2.25 µM. Incubations were carried out at 37°C in black 96-well plates (Costar) with a final volume of 200 µL. Single

  made using the SpectraMax Gemini XS reader (Molecular Devices) with the appropriate excitation and emission wavelengths.

  was used, on the other hand, there was good correlation for CYP2C19 between results obtained with the fluorogenic substrate

  Figure Legends Figure 1.

Figure 2 .

 2 Figure 2.

Table 4

 4 IC 50 values ranged from as low as 0.22 µg/mL to as high as 9.7 µg/mL, in all these cases, concentrations higher than those required to produce 50% inhibition of CYP activity would be attained from a single dose of herbal even if the active compound(s) were distributed in the total body water volume, assuming 100% bioavailability.

	. CYP1A2, CYP2C19, CYP2D6 and

Table 1 .

 1 components of the herbals themselves. Nevertheless, we have been able to demonstrate that several commercial preparations that are used by women are potent (IC 50 ~ 1 µg/mL) inhibitors of CYP450 activity. Caution in their concomitant use with pharmaceuticals that are biotransformed by these same enzymes is advised but in vivo research to investigate the clinical significance of the interactions is necessary. Assay conditions for the effects of putative inhibitors on the biotransformation of fluorogenic substrates by human cytochrome P450 enzymes and the effects of standard compoundsValues for pK i are means ± SEM from 3-4 experiments performed in duplicate. The K i values were derived from the pK i values to facilitate interpretation of the data.

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

Table 2 .

 2 Kinetic parameters for the biotransformation of fluorogenic substrates by human cytochrome P450 enzymes

	Enzyme	Substrate	Model	K m (µM)	V max	Coeff
					(pmol/min/pmol)
	CYP1A1	ERES	M-M	0.2 + 0.2	1.9 + 0.2	-
	CYP1A2	CEC	Hill	2.7 + 0.1	6.8 + 0.4	0.7 + 0.1
	CYP2C19	CEC	Hill	21 + 1	0.2 + 0.03	1.9 + 0.3
	CYP2C9	Vivid Red	M-M	1.3 + 0.1	0.07 ± 0.01	-
	CYP2D6	AMMC	M-M	1.4 + 0.01	0.8 + 0.1	-
	CYP3A4	BQ	SI	135	111	0.06
	Data are presented as means + SEM from 3-4 experiments, except for CYP3A4 where
	they are the means of two experiments. All experiments were performed in triplicate.
	MM, Michaelis-Menten; SI, substrate inhibition; Coeff, coefficient

Table 4 .

 4 Effects of herbal preparations on the biotransformation of fluorogenic substrates by human cytochromes P450The pIC 50 values are expressed as means ± SEM from 3-6 experiments performed in duplicate. IC 50 values and the concentrations attained from single recommended doses at different volumes of distribution were determined as described in Methods. Where no values are given, IC 50 values were greater than the maximum concentrations used in the assays (Table3). Neither CYP1A1 nor CYP2C6 were affected by any of the herbals tested at the concentrations shown in Table3.

						Single dose concentration
		CYP1A2	CYP2C19	CYP2D6	CYP3A4	(µg/mL) at different
						volumes of distribution
		IC 50 (µg/mL)	IC 50 (µg/mL)	IC 50 (µg/mL)	IC 50 (µg/mL)	3L
	Herbal	pIC 50 (g/mL)	pIC 50 (g/mL)	pIC 50 (g/mL)	pIC 50 (g/mL)	15L
						40L
	Black cohosh	1.8	0.37	9.7	-	167
		5.7 ± 0.1	6.44 ± 0.03	5.01 ± 0.06		33
						13
	Chaste tree berry	3.8	0.22	2.9	0.3	111
		5.4 ± 0.1	6.66 ± 0.05	5.5 ± 0.1	6.52 ± 0.08	22
						8
	Cramp bark	0.94	1.1	-	-	83
		6.03 ± 0.05	5.97 ± 0.05			17
						6
	False unicorn	-	-	0.8	0.5	67
				6.1 ± 0.1	6.30 ± 0.05	13
						5

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Acknowledgements

We thank Catherine Mielnik, Daniel Moldaver and Leila Nattagh for adapting the Vivid ® Red CYP2C9 assay for use in our laboratory. This work was supported by the SickKids Foundation and the Canadian Institutes for Health Research.