Dr Gloria Cereza

Antonia Agustí

Consuelo Pedrós

Antonio Vallano

Cristina Aguilera

Immaculada Danés

Xavier Vidal

Josep Maria Arnau

Arnau Jm

Effect of an intervention on the features of adverse drug reactions spontaneously reported in a hospital

Keywords: Pharmacovigilance, Adverse Drug Reactions Reporting Systems, Drug Toxicity, Intervention Studies, Hospital

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The spontaneous reporting system is currently the cornerstone of postmarketing signal detection in pharmacovigilance. [1][2] Detection of adverse drug reactions (ADRs) in hospitals is important because serious events may be identified, novel drugs are commonly used, new and unexpected ADRs may be detected early on and cases are usually well-documented with high quality information. Nevertheless, spontaneous reporting has been scarcely used as a method of ADRs detection in hospitals and underreporting is a major flaw, [3][4][5] as it is in the primary health care setting. [6] Several interventions (educational or others) aimed at solving this major drawback have been proposed and the effectiveness of some of them has been evaluated in a number of studies. [5,[7][8][START_REF] Scott | Physician reporting of adverse drug reactions. Results of the Rhode Island Adverse Drug Reaction Reporting Project[END_REF][START_REF] Fincham | A statewide program to stimulate reporting of adverse drug reactions[END_REF][START_REF] Mcgettigan | Reporting of adverse drug reactions by hospital doctors and the response to intervention[END_REF][START_REF] Kimelblatt | Improved reporting of adverse drug reactions[END_REF] In this sense we have reported that an intervention based on healthcare management agreements with economic incentives and educational activities was associated with a quantitative and qualitative improvement of spontaneous reporting of ADRs by hospital physicians. [START_REF] Pedrós | An intervention to improve spontaneous adverse drug reaction reporting by hospital physicians. A time series analysis in Spain[END_REF] Not only did the absolute number of spontaneous reports increase but so did the number of reports of serious or unexpected ADRs and ADRs associated with new drugs.

The pattern of adverse drug reactions notified by spontaneous reporting in hospitals has been described in a few studies. [START_REF] Maistrello | Adverse drug reactions in hospitalized patients: an operational procedure to improve reporting and investigate underreporting[END_REF][START_REF] Cox | Adverse drug reactions in patients admitted to hospital identified by discharge ICD-10 codes and by spontaneous reports[END_REF][START_REF] Ortega | Efficacy of an adverse drug reaction electronic reporting system 1491-6[END_REF][17][18][19] Furthermore, there are no studies assessing if the improvement in reporting in the hospital setting allows a more efficient detection of new drug safety issues or if it facilitates signal identification. The usefulness of an increase in spontaneous reporting depends on the increase in the number of meaningful reports that help in raising concerns. Therefore, the aim of this study was to analyze the effect of the spontaneous reporting increase after an intervention in the hospital on the features of the ADRs reported, the drugs involved and the signals identified. or clinical team, and c) physician. The financial incentive obtained was variable according to the objectives achieved, and was approximately 5-7% of the physician's salary. The size of the financial payment for ADR reporting accounted for less than 10% of the total of agreed incentives and was, on average, less than 1% of the physician's salary. In each clinical service, an initial meeting between physicians and the hospital pharmacovigilance team was held. In this first meeting, the hospital's pharmacovigilance activities, the aim of spontaneous ADR reporting and how to report ADRs, were presented. In addition to reinforcement, twice-yearly educational meetings were held in each clinical service, offering information about pharmacovigilance and emphasizing the priorities for spontaneous reporting ADRs (serious ADRs, unexpected ADRs, and those ADRs associated with new drugs). These meetings lasted 45-60 minutes, and consisted of a brief explanation of the number and main characteristics of ADRs detected in the whole hospital and in the specific clinical service. Signals identified by the PhVP and news about ADRs released by regulatory agencies were also commented on. Finally, reminder cards with the telephone number of the pharmacovigilance team and a list of the most important ADRs to be reported on (serious, unexpected and those associated with new drugs) were distributed to the hospital wards. Changes between the two periods of study (the 1 st period without intervention from 1998 to 2002 and the 2 nd period with intervention from 2003 to 2005) in the total number of spontaneous reports of ADRs, and in the number of serious reports, reports of unexpected ADRs and reports of ADRs related to recently marketed drugs were therein presented. [START_REF] Pedrós | An intervention to improve spontaneous adverse drug reaction reporting by hospital physicians. A time series analysis in Spain[END_REF] In this paper, changes between the two periods in the following variables of the longitudinal study have been analyzed: (a) The patients' characteristics, such as gender and age; (b) the reported ADRs, with their seriousness, outcome, medical assistance required, and previous knowledge; (c) the suspected drugs involved in the ADRs, including those recently marketed; and (d) the most frequent drug-ADR associations and the main signals identified.

The ADRs were classified in organ-system categories according to the World Health Organization terminology (WHOART -WHO Adverse Reaction Terminology). The suspected drugs were grouped in therapeutic classes according to the Anatomical Therapeutic Chemical classification system. The seriousness of the cases was classified according to the European Union's criteria and cases were considered serious (ADRs that result in death, are lifethreatening, require hospitalisation or prolongation of existing hospitalisation, result in persistent or significant disability or incapacity, are congenital anomaly or birth defect, or are important medical events) or non -serious (the remaining cases). [21] Its outcome was classified into recovery, permanent sequelae, death or unknown. According to the medical attention required, the cases were classified into those requiring hospital admission, in-hospital cases, cases requiring attention at the emergency room, and cases from the outpatient department. Previous knowledge of the ADRs was classified according to the Spanish Pharmacovigilance System's (SPhVS) causality algorithm in well-known ADRs, known from anecdotal reports, or unknown. [22] New-drugs were defined as those marketed for less than five years at the time when the ADRs began.

RESULTS

Number of spontaneous reports of adverse drug reactions

Of the total number of cases of ADRs identified by the PhVP, the proportion of spontaneous ADR reports increased from 29.5% (n=200) during the first period to 71.5% (n= 631) during the second period. More details about this increase were presented in the previous paper.

Patients' characteristics

Gender and age distribution of patients described in the spontaneous reports were no different between the two periods. The ratio female-to-male in the spontaneous reports from the first period was 1.08 (104 females and 96 males); it was 0.96 in the second one (310 females and 321 males; p=0.516).

Median age (minimum-maximum) was 64 (14-94) for patients described in both periods (p=0.150).

Medical attention required

Most cases reported during the first period either required medical attention at the emergency room without hospital admission (99; 49.5%) or were in- The number of cases requiring only medical attention at emergency room decreased during the second period (30; 4.8%). In-hospital cases slightly decreased in relative terms although the absolute number of reports increased (277; 43.9%). Conversely, cases requiring hospital admission increased both in relative and in absolute terms (236; 37.4%). On the other hand, cases from outpatient department not requiring attention at the emergency room or hospital admission began to be reported during the second period, accounting for 13.9% (88) of the overall reports received at the end of this period (p < 0.001).

Seriousness and outcome

In the second period, the proportion of serious cases almost doubled (63.1% vs 32.5%) and the number of serious cases increased by more than 6-fold (398 vs 65; p < 0.001).

The most frequently reported outcome in both periods was recovery (70% in the first one and 74.2% in the second one). The number of cases that recovered with sequelae was 2 in the first period and 16 in the second one. There were 9 fatal cases during the first period and 15 in the second one. The adverse reactions and drugs involved in these fatal cases are presented below in the drug-reaction associations section. Finally, the outcome was unknown in 49 cases in the first period and 132 in the second one (p=0.153).

Adverse reactions

Overall, 220 adverse reactions were described in the 200 first-period spontaneous reports. Cutaneous reactions were the most frequent, followed by cardiovascular and neurological reactions. In the 631 second-period spontaneous reports, 694 adverse reactions were described. Cutaneous reactions were the most frequent as well, followed in this case by haematological and gastrointestinal reactions (table 1). However, during the second period the frequency of endocrinological, urinary and hepatic reactions increased (3.7% vs. 0.5%, 4.6% vs. 1.8% and 9.2% vs. 5.5%, respectively).

The most commonly reported individual adverse reactions (with a frequency ≥ 3% of reports) during the first period were urticaria, angioedema, bronchospasm, hypotension, and haematoma. In the second period, individual reactions described in ≥ 3% of reports were rash and erythematous rash (table 2). During the second period the frequency of hyperthyroidism, thrombocytopenia and gastrointestinal haemorrhage increased by 4.3 and more than 2-fold, respectively.

Suspected drugs

Two hundred and eighty-five and 877 suspected pharmacological exposures were contained in the reports in the first and the second period respectively. In both periods, the most common therapeutic subgroups were systemic antibiotics, antithrombotics, and cardiac therapy. These three groups accounted for 38.2% and 30.3% of the total suspected pharmacological exposures in each period respectively (table 3). Regarding the remaining therapeutic subgroups, the greatest changes between periods were the increase in the proportion of immunostimulants, beta blocking agents, immunosuppressants and psychoanaleptics, and the decrease in the proportion of systemic antihistamines during the second period.

The drugs with the highest number of suspected exposures were amiodarone, amoxicillin-clavulanic acid, metamizole, and enoxaparin in both periods (table 4). Some drugs with no suspected exposures during the first period appeared during the second one (i.e., ceftriaxone, methotrexate, and tacrolimus). Additionally, a 9-fold increase was seen in the number of suspected exposures to ibuprofen, azithromycin and fluoxetine during the second period.

In both study periods, around 10% of suspected pharmacological exposures concerned recently marketed drugs. Nevertheless, both the overall number of suspected pharmacological exposures and the number of different drugs increased in the second period (97 vs. 28, and 50 vs. 19, respectively). The new drugs with the highest number of suspected exposures were clopidogrel, levofloxacin, and nevirapine (three exposures each) in the first period, and linezolid (eight exposures), tiotropium bromide, glatiramer acetate, oxcarbazepine, tenofovir, and voriconazole (five exposures each) in the second one.

Drug-reaction associations

In the first period, the most frequently reported drug-reaction associations were haemoperitoneum, retroperitoneal haemorrhage or muscular haematoma due to enoxaparin (11 cases, 5.5% of reports). In the second period, they were hepatotoxicity induced by amoxicillin-clavulanic acid and hyperthyroidism due to amiodarone (14 cases and 2.2% of reports each) (table 5).

Three out of nine fatal reports from the first period were cases of haemorrhagic reactions in patients treated with enoxaparin (two retroperitoneal haemorrhages and a gastrointestinal bleeding), three were cases of cardiovascular reactions (myocardial infarction after terbutaline treatment, salbutamol and levofloxacin-induced torsade de pointes, and cardiac arrest in a patient treated with levomepromazine and carbamazepine) and the remaining three reports described adverse reactions related to other systems. The most frequent reactions in the fifteen fatal cases reported during the second period were also haemorrhagic events (seven cases). There were four cases of intracranial haemorrhage (three of them with tenecteplase, enoxaparin and acetylsalicylic acid, and one with drotrecogin alpha and enoxaparin), one case

Previous knowledge/Signals identified

The absolute number of previously unknown or poorly known drug-reaction associations had a fourfold increase in the second period (54 vs. 13 in the first period). Most of them were serious cases (5 out of 13 in the first period and 40 out of 54 in the second one; p=0.047).

No signals were identified during the first period. In the second one, two serious and previously unknown or poorly known drug-reaction associations were considered signals that required the adoption of regulatory measures by the Spanish Agency for Medicines and Medical Devices (AEMPS). The first signal was pseudoproteinuria induced by gelatine succinate infusion. A warning regarding this problem was added in the summary of product characteristics [23] and a "Dear Dr letter" was sent to Spanish nephrologists and to the National Transplant Organization. The second signal was veralipride withdrawal syndrome; it required a risk-benefit assessment of the use of this orthopramide in the treatment of menopausal hot flushes and, as a result, the AEMPS withdrew this drug from the market. [24] Afterwards, the EMEA also recommended the withdrawal of this drug from the European market. [25] Additionally, the fatal case of cerebral haemorrhage due to drotrecogin alfa activated, which occurred in a patient who had recently undergone surgery, led to a review of safety data of this drug in the surgical population. As a result, the AEMPS proposed changes in the authorized conditions of use of drotrecogin alfa activated at the EMEA level. Those proposals contributed to updating the authorized conditions of use and contraindications of this drug. [26]

DISCUSSION

Our study has been the first one to assess the effect of a specific intervention on the features of ADRs spontaneously reported in a hospital setting, the drugs involved and the number of signals identified. It has shown that the improvement in spontaneous reporting of ADRs achieved following an intervention based on educational activities and economic incentives allowed us to detect some safety problems and to identify several signals. It is noteworthy that the reporting of all kind of adverse reactions and drugs increased.

The traditional methods and systems to detect ADRs in hospitals have been spontaneous reporting, intensive surveillance of hospital admissions and, more recently, computer-assisted approaches using routine data from hospital information systems. [3] Different methods identify different types of ADRs and drugs. Moreover, the type of suspect drugs, their rank order and the types of ADRs identified vary widely among studies. In studies that only or predominantly included ADR-related hospitalisations, nonsteroidal antiinflammatory drugs causing gastrointestinal bleeding play a major role, followed by cardiovascular drugs and drugs acting on the central nervous system. In contrast, antibacterials/antiinfectives very often cause ADRs in hospitalised patients (mainly allergic reactions), as do opioids, cardiovascular drugs and anticoagulants. [3] In our study the most frequently reported ADRs were similar in both periods, although some types of ADRs that were hardly reported on the first period increased in the second one. Cutaneous reactions were the most frequently reported ADRs in both periods: other ADRs commonly reported during both periods were cardiovascular, haematological, neurological and gastrointestinal reactions. Nevertheless, a high increase in endocrinological, urinary and hepatic reactions was observed in the second period. There are no other published studies comparing the effect of an intervention on the organ-system categories of reactions spontaneously reported in a hospital. A few studies have reported the pattern of spontaneously reported ADRs in hospitals. [16-19, 27,28] As in ours, in the majority of these studies dermatological reactions were the most frequently identified [17][18][19]27] and skin rash was the most commonly reported reaction. [17,18] Other ADRs such as neurological, gastrointestinal, and haematological reactions were also frequently reported. However, it is interesting to note that, in contrast with our study, endocrinological and urinary reactions were rarely reported in others. In our opinion, the intervention could be responsible for the increase in the reporting of these ADRs in the second period. Further studies should be carried out to analyze the effect of different interventions on the features of the spontaneously reported ADRs.

The majority of therapeutic subgroups of suspected drugs increased during the intervention. Systemic antibiotics, antithrombotics, and cardiac therapy drugs were the most commonly involved therapeutic subgroups in the spontaneous reports in both periods. These therapeutic subgroups were also frequently reported in other studies that assessed the drugs involved in spontaneously reported ADRs in hospitals. [18,19,27] In our study, the greatest changes between periods were the increase in the proportion of immunostimulants, immunosuppressants and psychoanaleptics. In addition, antineoplastics, a therapeutic subgroup highly reported in other studies, [17,19,27] also increased during the intervention. The therapeutic subgroups most commonly involved in the spontaneously reported ADRs were drugs frequently used in the hospital setting. Nevertheless, taking into account the differences in the pattern of suspect drugs in the two periods, it is likely that the intervention allowed us to identify during the second period some suspect drugs involved in ADRs infrequently reported in the first one. Furthermore, the absolute number of spontaneous reports from any origin increased during the second period, except for those from emergencies. It is worth noting that spontaneous reports from the outpatient hospital consultations began to be reported during the second period. The effect of the intervention was evident, since the physicians had been informed in the educational meetings that they could report ADRs from every assistance setting. Unexpectedly, spontaneous reporting of ADRs requiring only medical attention at emergency room did not increase in spite of the intervention. The reason for that is unknown, although high attendance-pressure, specially in this area, lack of time, and other clinical priorities have been argued as potential obstacles to the spontaneous reporting of ADRs by the doctors at our hospital. [5] Therefore, in the emergency area some other specific strategies aimed at increasing spontaneous reporting should be planned.

One of the main objectives of spontaneous reporting of ADRs is to detect drug safety concerns related to new drugs. Surprisingly, the novelty of suspect drugs has not been analyzed in published studies on spontaneous reporting in hospitals. In our study we observed that during the intervention the number of suspected pharmacological exposures to recently marketed drugs and the number of serious cases, including those with a fatal outcome, increased. It is noteworthy that one of the signals identified referred to a serious reaction to a recently marketed drug and arose through the reporting of a fatal case (i.e. intracranial haemorrhage associated to drotecogin alfa activated).

The majority of drug-reaction associations spontaneously reported in both periods of the study were well-known reactions related to old drugs (i.e., haemorrhage due to enoxaparin, hepatotoxicity due to amoxicillin-clavulanic acid, and hyperthyroidism due to amiodarone). However, the reporting of known ADRs can help to identify local problems related to the use of specific drugs. For example, in our hospital haemorrhages related to low-weight molecular heparins led to an analysis of the severe cases in order to identify possible risk factors. On the other hand, two previously unknown reactions associated with old drugs that were reported in the second period led to the withdrawal of the market of one drug and an update of the summary of product characteristics in the other one. This point is interesting because it shows that even old drugs must be kept under surveillance through their entire life-span to detect some possible previously unknown reactions.

Obviously our study has some limitations. First of all, it was carried out in only one hospital with specific characteristics (a teaching tertiary hospital; the biggest one in our geographical area). Additionally, the Catalan Centre of the Spanish Pharmacovigilance System is located in the hospital. Therefore, our findings could not be generalized to other hospitals. Secondly, since our study was a time series study without a control group, we can not rule out the influence of other factors in the observed results. Nevertheless, the number of the spontaneous ADRs reported by the other Catalan hospital to the Pharmacovigilance regional Centre during the two study's periods did not increase. Thirdly, the number of signals identified was low and we can not confirm the effect of intervention on its detection. Fourthly, we did not collect information on the category of physicians, medical speciality and other characteristics of the reporters that might have influenced the results. In spite of all these limitations, our study generates new information on the effect of a specific intervention on the features of the spontaneously reported ADRs, the drugs involved and the signals identified. On the one hand, that confirms the usefulness of the implementation of these types of strategies to improve reporting of ADRs in hospitals. On the other hand, these results can be and even should be used as an argument when stimulating the spontaneous reporting by health care professionals by means of supporting evidence of the usefulness of pharmacovigilance activities.

As a conclusion, an intervention based on educational activities and economic incentives led not only to a quantitative improvement in spontaneous reporting of ADRs by hospital physicians but also to a qualitative improvement in terms of seriousness of reactions identified affecting different organs or systems, and Only drugs with at least six suspect exposures in any period are displayed.

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

-25 - Results.-Gender and age distribution of patients described in the spontaneous reports were no different in the two periods. During the second period, spontaneously reported cases requiring hospital admission and those occurring in-hospital increased (236 from 2 in the first period and 277 from 99 in the first period, respectively) and cases from outpatient hospital consultations began to be reported (13.9% of reports). The spontaneous reporting on all kinds of ADRs and drugs increased during the second period. Cutaneous reactions were the most frequently spontaneous reported ADRs in both periods followed by cardiovascular and neurological reactions in the first period, and haematological and gastrointestinal reactions in the second one. However, during the second period the higher increase was for endocrinological, urinary and hepatic reactions. Systemic antibiotics, antithrombotics and cardiac therapy drugs were the most commonly therapeutic subgroups reported as suspected drugs in both periods, but in the second period the proportion of immunostimulants, beta blocking agents, immunosuppressants and psychoanaleptics increased. No

INTRODUCTION

The spontaneous reporting system is currently the cornerstone of postmarketing signal detection in pharmacovigilance. [1][2] Detection of adverse drug reactions (ADRs) in hospitals is important because serious events may be identified, novel drugs are commonly used, new and unexpected ADRs may be detected early on and cases are usually well-documented with high quality information. Nevertheless, spontaneous reporting has been scarcely used as a method of ADRs detection in hospitals and underreporting is a major flaw, [3][4][5] as it is in the primary health care setting. [6] Several interventions (educational or others) aimed at solving this major drawback have been proposed and the effectiveness of some of them has been evaluated in a number of studies. [5,[7][8][START_REF] Scott | Physician reporting of adverse drug reactions. Results of the Rhode Island Adverse Drug Reaction Reporting Project[END_REF][START_REF] Fincham | A statewide program to stimulate reporting of adverse drug reactions[END_REF][START_REF] Mcgettigan | Reporting of adverse drug reactions by hospital doctors and the response to intervention[END_REF][START_REF] Kimelblatt | Improved reporting of adverse drug reactions[END_REF] In this sense we have reported that an intervention based on healthcare management agreements with economic incentives and educational activities was associated with a quantitative and qualitative improvement of spontaneous reporting of ADRs by hospital physicians. [START_REF] Pedrós | An intervention to improve spontaneous adverse drug reaction reporting by hospital physicians. A time series analysis in Spain[END_REF] Not only did the absolute number of spontaneous reports increase but so did the number of reports of serious or unexpected ADRs and ADRs associated with new drugs.

The pattern of adverse drug reactions notified by spontaneous reporting in hospitals has been described in a few studies. [START_REF] Maistrello | Adverse drug reactions in hospitalized patients: an operational procedure to improve reporting and investigate underreporting[END_REF][START_REF] Cox | Adverse drug reactions in patients admitted to hospital identified by discharge ICD-10 codes and by spontaneous reports[END_REF][START_REF] Ortega | Efficacy of an adverse drug reaction electronic reporting system 1491-6[END_REF][17][18][19] Furthermore, there are no studies assessing if the improvement in reporting in the hospital setting allows a more efficient detection of new drug safety issues or if it facilitates signal identification. The usefulness of an increase in spontaneous reporting depends on the increase in the number of meaningful reports that help in raising concerns. Therefore, the aim of this study was to analyze the effect of the spontaneous reporting increase after an intervention in the hospital on the features of the ADRs reported, the drugs involved and the signals identified. marketed drugs were therein presented. [START_REF] Pedrós | An intervention to improve spontaneous adverse drug reaction reporting by hospital physicians. A time series analysis in Spain[END_REF] In this paper, changes between the two periods in the following variables of the ADRs, known from anecdotal reports, or unknown. [22] New-drugs were defined as those marketed for less than five years at the time when the ADRs began.

RESULTS

Number of spontaneous reports of adverse drug reactions

Of the total number of cases of ADRs identified by the PhVP, the proportion of spontaneous ADR reports increased from 29.5% (n=200) during the first period to 71.5% (n= 631) during the second period. More details about this increase were presented in the previous paper.

Patients' characteristics

Gender and age distribution of patients described in the spontaneous reports were no different between the two periods. The ratio female-to-male in the spontaneous reports from the first period was 1.08 (104 females and 96 males); it was 0.96 in the second one (310 females and 321 males; p=0.516).

Median age (minimum-maximum) was 64 (14-94) for patients described in both periods (p=0.150).

Medical attention required

Most cases reported during the first period either required medical attention at

Seriousness and outcome

In the second period, the proportion of serious cases almost doubled (63.1% vs 32.5%) and the number of serious cases increased by more than 6-fold (398 vs 65; p < 0.001).

The most frequently reported outcome in both periods was recovery (70% in the first one and 74.2% in the second one). The number of cases that recovered with sequelae was 2 in the first period and 16 in the second one.

There were 9 fatal cases during the first period and 15 in the second one. The adverse reactions and drugs involved in these fatal cases are presented below in the drug-reaction associations section. Finally, the outcome was unknown in 49 cases in the first period and 132 in the second one (p=0.153).

Adverse reactions

Overall, 220 adverse reactions were described in the 200 first-period spontaneous reports. Cutaneous reactions were the most frequent, followed by cardiovascular and neurological reactions. In the 631 second-period spontaneous reports, 694 adverse reactions were described. Cutaneous reactions were the most frequent as well, followed in this case by haematological and gastrointestinal reactions (table 1). However, during the second period the frequency of endocrinological, urinary and hepatic reactions increased (3.7% vs. 0.5%, 4.6% vs. 1.8% and 9.2% vs. 5.5%, respectively).

The most commonly reported individual adverse reactions (with a frequency ≥ 3% of reports) during the first period were urticaria, angioedema, bronchospasm, hypotension, and haematoma. In the second period, individual reactions described in ≥ 3% of reports were rash and erythematous rash (table 2). During the second period the frequency of hyperthyroidism, thrombocytopenia and gastrointestinal haemorrhage increased by 4.3 and more than 2-fold, respectively.

Suspected drugs

Two hundred and eighty-five and 877 suspected pharmacological exposures were contained in the reports in the first and the second period respectively. In both periods, the most common therapeutic subgroups were systemic antibiotics, antithrombotics, and cardiac therapy. These three groups accounted for 38.2% and 30.3% of the total suspected pharmacological exposures in each period respectively (table 3). Regarding the remaining therapeutic subgroups, the greatest changes between periods were the increase in the proportion of immunostimulants, beta blocking agents, immunosuppressants and psychoanaleptics, and the decrease in the proportion of systemic antihistamines during the second period.

The drugs with the highest number of suspected exposures were amiodarone, amoxicillin-clavulanic acid, metamizole, and enoxaparin in both periods (table 4). Some drugs with no suspected exposures during the first period appeared during the second one (i.e., ceftriaxone, methotrexate, and tacrolimus).

Drug-reaction associations

In the first period, the most frequently reported drug-reaction associations were haemoperitoneum, retroperitoneal haemorrhage or muscular haematoma due to enoxaparin (11 cases, 5.5% of reports). In the second period, they were hepatotoxicity induced by amoxicillin-clavulanic acid and hyperthyroidism due to amiodarone (14 cases and 2.2% of reports each) (table 5).

Three out of nine fatal reports from the first period were cases of haemorrhagic reactions in patients treated with enoxaparin (two retroperitoneal haemorrhages and a gastrointestinal bleeding), three were cases of cardiovascular reactions (myocardial infarction after terbutaline treatment, salbutamol and levofloxacin-induced torsade de pointes, and cardiac arrest in a patient treated with levomepromazine and carbamazepine) and the remaining three reports described adverse reactions related to other systems. The most frequent reactions in the fifteen fatal cases reported during the second period were also haemorrhagic events (seven cases). There were four cases of intracranial haemorrhage (three of them with tenecteplase, enoxaparin and acetylsalicylic acid, and one with drotrecogin alpha and enoxaparin), one case

Previous knowledge/Signals identified

The absolute number of previously unknown or poorly known drug-reaction associations had a fourfold increase in the second period (54 vs. 13 in the first period). Most of them were serious cases (5 out of 13 in the first period and 40 out of 54 in the second one; p=0.047).

No signals were identified during the first period. In the second one, two serious and previously unknown or poorly known drug-reaction associations were considered signals that required the adoption of regulatory measures by the Spanish Agency for Medicines and Medical Devices (AEMPS). The first signal was pseudoproteinuria induced by gelatine succinate infusion. A warning regarding this problem was added in the summary of product characteristics [23] and a "Dear Dr letter" was sent to Spanish nephrologists and to the National Transplant Organization. The second signal was veralipride withdrawal syndrome; it required a risk-benefit assessment of the use of this orthopramide in the treatment of menopausal hot flushes and, as a result, the AEMPS withdrew this drug from the market. [24] Afterwards, the EMEA also recommended the withdrawal of this drug from the European market. [25] Additionally, the fatal case of cerebral haemorrhage due to drotrecogin alfa activated, which occurred in a patient who had recently undergone surgery, led to a review of safety data of this drug in the surgical population. As a result, the AEMPS proposed changes in the authorized conditions of use of drotrecogin alfa activated at the EMEA level. Those proposals contributed to updating the authorized conditions of use and contraindications of this drug. [26] The traditional methods and systems to detect ADRs in hospitals have been spontaneous reporting, intensive surveillance of hospital admissions and, more recently, computer-assisted approaches using routine data from hospital information systems. [3] Different methods identify different types of ADRs and drugs. Moreover, the type of suspect drugs, their rank order and the types of ADRs identified vary widely among studies. In studies that only or predominantly included ADR-related hospitalisations, nonsteroidal antiinflammatory drugs causing gastrointestinal bleeding play a major role, followed by cardiovascular drugs and drugs acting on the central nervous system. In contrast, antibacterials/antiinfectives very often cause ADRs in hospitalised patients (mainly allergic reactions), as do opioids, cardiovascular drugs and anticoagulants. [3] In our study the most frequently reported ADRs were similar in both periods, although some types of ADRs that were hardly reported on the first period increased in the second one. Cutaneous reactions were the most frequently reported ADRs in both periods: other ADRs commonly reported during both periods were cardiovascular, haematological, neurological and gastrointestinal reactions. Nevertheless, a high increase in endocrinological, urinary and hepatic reactions was observed in the second period. There are no other published studies comparing the effect of an intervention on the organ-system categories of reactions spontaneously reported in a hospital. A few studies have reported the pattern of spontaneously reported ADRs in hospitals. [16-19, 27,28] As in ours, in the majority of these studies dermatological reactions were the most frequently identified [17][18][19]27] and skin rash was the most commonly reported reaction. [17,18] Other ADRs such as neurological, gastrointestinal, and haematological reactions were also frequently reported. However, it is interesting to note that, in contrast with our study, endocrinological and urinary reactions were rarely reported in others. In our opinion, the intervention could be responsible for the increase in the reporting of these ADRs in the second period. Further studies should be carried out to analyze the effect of different interventions on the features of the spontaneously reported ADRs.

The majority of therapeutic subgroups of suspected drugs increased during the intervention. Systemic antibiotics, antithrombotics, and cardiac therapy drugs were the most commonly involved therapeutic subgroups in the spontaneous reports in both periods. These therapeutic subgroups were also frequently reported in other studies that assessed the drugs involved in spontaneously reported ADRs in hospitals. [18,19,27] In our study, the greatest changes between periods were the increase in the proportion of immunostimulants, immunosuppressants and psychoanaleptics. In addition, antineoplastics, a therapeutic subgroup highly reported in other studies, [17,19,27] also increased during the intervention. The therapeutic subgroups most commonly involved in the spontaneously reported ADRs were drugs frequently used in the hospital setting. Nevertheless, taking into account the differences in the pattern of suspect drugs in the two periods, it is likely that the intervention allowed us to identify during the second period some suspect drugs involved in ADRs infrequently reported in the first one. Furthermore, the absolute number of spontaneous reports from any origin increased during the second period, except for those from emergencies. It is worth noting that spontaneous reports from the outpatient hospital consultations began to be reported during the second period. The effect of the intervention was evident, since the physicians had been informed in the educational meetings that they could report ADRs from every assistance setting. Unexpectedly, spontaneous reporting of ADRs requiring only medical attention at emergency room did not increase in spite of the intervention. The reason for that is unknown, although high attendance-pressure, specially in this area, lack of time, and other clinical priorities have been argued as potential obstacles to the spontaneous reporting of ADRs by the doctors at our hospital. [5] Therefore, in the emergency area some other specific strategies aimed at increasing spontaneous reporting should be planned.

One of the main objectives of spontaneous reporting of ADRs is to detect drug safety concerns related to new drugs. Surprisingly, the novelty of suspect drugs has not been analyzed in published studies on spontaneous reporting in hospitals. In our study we observed that during the intervention the number of suspected pharmacological exposures to recently marketed drugs and the number of serious cases, including those with a fatal outcome, increased. It is noteworthy that one of the signals identified referred to a serious reaction to a recently marketed drug and arose through the reporting of a fatal case (i.e.

intracranial haemorrhage associated to drotecogin alfa activated).

The majority of drug-reaction associations spontaneously reported in both periods of the study were well-known reactions related to old drugs (i.e., haemorrhage due to enoxaparin, hepatotoxicity due to amoxicillin-clavulanic acid, and hyperthyroidism due to amiodarone). However, the reporting of known ADRs can help to identify local problems related to the use of specific drugs. For example, in our hospital haemorrhages related to low-weight molecular heparins led to an analysis of the severe cases in order to identify possible risk factors. On the other hand, two previously unknown reactions associated with old drugs that were reported in the second period led to the withdrawal of the market of one drug and an update of the summary of product characteristics in the other one. This point is interesting because it shows that even old drugs must be kept under surveillance through their entire life-span to detect some possible previously unknown reactions.

Obviously our study has some limitations. First of all, it was carried out in only one hospital with specific characteristics (a teaching tertiary hospital; the biggest one in our geographical area). Additionally, the Catalan Centre of the Spanish Pharmacovigilance System is located in the hospital. Therefore, our findings could not be generalized to other hospitals. Secondly, since our study was a time series study without a control group, we can not rule out the influence of other factors in the observed results. Nevertheless, the number of the spontaneous ADRs reported by the other Catalan hospital to the Pharmacovigilance regional Centre during the two study's periods did not increase. Thirdly, the number of signals identified was low and we can not confirm the effect of intervention on its detection. Fourthly, we did not collect information on the category of physicians, medical speciality and other characteristics of the reporters that might have influenced the results. In spite of all these limitations, our study generates new information on the effect of a specific intervention on the features of the spontaneously reported ADRs, the drugs involved and the signals identified. On the one hand, that confirms the usefulness of the implementation of these types of strategies to improve reporting of ADRs in hospitals. On the other hand, these results can be and even should be used as an argument when stimulating the spontaneous reporting by health care professionals by means of supporting evidence of the usefulness of pharmacovigilance activities. Only drugs with at least six suspect exposures in any period are displayed.

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

-25 -

  analysis was carried out at Vall d'Hebron University Hospital (Barcelona, Spain) from 1998 to 2005 in order to assess the effectiveness of a continuous multifaceted intervention for improving spontaneous reporting of ADRs by hospital physicians to the Pharmacovigilance Programme (PhVP) of the hospital. The intervention was based on health care management agreements between hospital managers and clinical services, and was initiated in 2003. A framework agreement between hospital managers and physicians included different commitments linked to economic incentives. One of the objectives for physicians was to increase their participation in the PhVP increasing the number of spontaneous ADR reports. This objective was integrated into other clinical objectives at three levels: a) institution or whole hospital, b) clinical department

  analysis. The spontaneous reports of ADRs during the two studied periods were analyzed separately. Descriptive analysis of continuous variables was performed by means of median and range; and statistical differences were assessed by means of the Mann-Whitney test. Categorical variables were described using percentages; statistical differences were assessed by means of the χ 2 test. Significance was set at a level of 0.05, two tailed. The statistical analysis was performed using the SPSS version 15.0 statistical package (SPSS Inc. Chicago-Illinois).

  99; 49.5%). The two remaining cases (1%) required hospital admission.

  induced retroperitoneal haemorrhage, one case of intraabdominal haemorrhage due to enoxaparin, acenocoumarol and acetylsalicylic acid, and one case of acetylsalicylic acid-induced gastrointestinal haemorrhage. Of the eight remaining cases, three were toxic epidermal necrolysis and the other described reactions related to other systems.

  It would be interesting to investigate the effect of other interventions on the type of suspected drugs in spontaneously reported ADRs.

  of drugs involved, including those most recently marketed. Moreover, a number of signals were generated during the intervention and they contributed to the adoption of decisions by the regulatory authorities on either the withdrawal of drugs from the market or changes in the information contained in the summary of product characteristics of some drugs. Future studies should analyse the effect of other types of intervention on spontaneous reporting of ADRs in hospitals, the number of identified signals and quickness of their recognition. HD, Thacher-Renshaw A, Rosenbaum SE, Waters WJ Jr, Green M, Andrews LG, Faich GA. Physician reporting of adverse drug reactions. Results of the Rhode Island Adverse Drug Reaction Reporting Project. JAMA 1990; 263: 1785-8 10. Fincham J. A statewide program to stimulate reporting of adverse drug reactions. J Pharm Pract 1989; 2: 239-44 11. McGettigan P, Golden J, Conroy RM, Arthur N, Feely J. Reporting of adverse drug reactions by hospital doctors and the response to intervention. Br J Clin Pharmacol 1997; 44: 98-100 12. Kimelblatt BJ, Young SH, Heywood PM, Mandala AR, Gendelman S, Mehl B. Improved reporting of adverse drug reactions. Am J Hosp Pharm 1988; 45: 1086-9 13. Pedrós C, Vallano A, Cereza G, Mendoza-Aran G, Agustí A, Aguilera C, Danés I, Vidal X, Arnau JM. An intervention to improve spontaneous adverse drug reaction reporting by hospital physicians. A time series analysis in Spain. Drug Safety 2009; 32: 77-83 14. Maistrello I, Morgutti M, Maltempi M, Dantes M. Adverse drug reactions in hospitalized patients: an operational procedure to improve reporting and investigate underreporting. Pharmacoepidemiol Drug Saf 1995; 4: 101-6 15. Cox AR, Anton C, Goh CHF, Easter M, Langford NJ, Ferner RE. Adverse drug reactions in patients admitted to hospital identified by discharge ICD-10 codes and by spontaneous reports. Br J Clin Pharmacol 2001; 52: 337-9 16. Ortega A, Aguinagalde A, Lacasa C, Aquerreta I, Fernández-Benítez M, Fernández LM. Efficacy of an adverse drug reaction electronic reporting system

  A continuous intervention based on healthcare management agreements was associated in our hospital with an increase in the absolute number of spontaneous reporting of adverse drug reactions (ADRs), and also with an increase in the number of reports of serious or unexpected ADRs and ADRs associated with new drugs. Objective.-To analyze the effect of this intervention on the features of ADRs spontaneously reported in a hospital, the drugs involved and the number of signals identified. Methods.-A longitudinal study with two periods, the 1 st period without intervention from 1998 to 2002 and the 2 nd period with intervention from 2003 to 2005, was carried out in a teaching tertiary Hospital. Changes between the two periods in the following variables were analysed: (a) the patients' characteristics, such as gender and age; (b) the reported ADRs, and their medical assistance required; (c) the suspected drugs involved in the ADRs and (d) the main signals identified.

  during the first period, however two signals and one additional safety concern were identified during the second. Conclusion.-An intervention based on healthcare management agreements, was associated with an important increase in spontaneous reporting of ADRs by hospital physicians and also with a change in terms of type of ADRs identified affecting different organs or systems, and the therapeutic groups of drugs involved. Future studies should analyze the effect of different types of intervention on spontaneous reporting of ADRs in hospitals. Words account: 399 Keywords: Pharmacovigilance; Adverse Drug Reactions Reporting Systems; Drug Toxicity; Intervention Studies; Hospital

  analysis was carried out at Vall d'Hebron University Hospital (Barcelona, Spain) from 1998 to 2005 in order to assess the effectiveness of a continuous multifaceted intervention for improving spontaneous reporting of ADRs by hospital physicians to the Pharmacovigilance Programme (PhVP) of the hospital. The intervention was based on health care management agreements between hospital managers and clinical services, and was initiated in 2003. A framework agreement between hospital managers and physicians included different commitments linked to economic incentives. One of the objectives for physicians was to increase their participation in the PhVP increasing the number of spontaneous ADR reports. This objective was integrated into other clinical objectives at three levels: a) institution or whole hospital, b) clinical departmentor clinical team, and c) physician. The financial incentive obtained was variable according to the objectives achieved, and was approximately 5-7% of the physician's salary. The size of the financial payment for ADR reporting accounted for less than 10% of the total of agreed incentives and was, on average, less than 1% of the physician's salary. In each clinical service, an initial meeting between physicians and the hospital pharmacovigilance team was held. In this first meeting, the hospital's pharmacovigilance activities, the aim of spontaneous ADR reporting and how to report ADRs, were presented. In addition to reinforcement, twice-yearly educational meetings were held in each clinical service, offering information about pharmacovigilance and emphasizing the priorities for spontaneous reporting ADRs (serious ADRs, unexpected ADRs, and those ADRs associated with new drugs). These meetings lasted 45-60 minutes, and consisted of a brief explanation of the number and main characteristics of ADRs detected in the whole hospital and in the specific clinical service. Signals identified by the PhVP and news about ADRs released by regulatory agencies were also commented on. Finally, reminder cards with the telephone number of the pharmacovigilance team and a list of the most important ADRs to be reported on (serious, unexpected and those associated with new drugs) were distributed to the hospital wards.

  two periods of study (the 1 st period without intervention from 1998 to 2002 and the 2 nd period with intervention from 2003 to 2005) in the total number of spontaneous reports of ADRs, and in the number of serious reports, reports of unexpected ADRs and reports of ADRs related to recently

  longitudinal study have been analyzed: (a) The patients' characteristics, such as gender and age; (b) the reported ADRs, with their seriousness, outcome, medical assistance required, and previous knowledge; (c) the suspected drugs involved in the ADRs, including those recently marketed; and (d) the most frequent drug-ADR associations and the main signals identified.The ADRs were classified in organ-system categories according to the World Health Organization terminology (WHOART -WHO Adverse Reaction Terminology). The suspected drugs were grouped in therapeutic classes according to the Anatomical Therapeutic Chemical classification system. The seriousness of the cases was classified according to the European Union's criteria and cases were considered serious (ADRs that result in death, are lifethreatening, require hospitalisation or prolongation of existing hospitalisation, result in persistent or significant disability or incapacity, are congenital anomaly or birth defect, or are important medical events) or non -serious (the remaining cases).[21] Its outcome was classified into recovery, permanent sequelae, death or unknown. According to the medical attention required, the cases were classified into those requiring hospital admission, in-hospital cases, cases requiring attention at the emergency room, and cases from the outpatient department. Previous knowledge of the ADRs was classified according to the Spanish Pharmacovigilance System's (SPhVS) causality algorithm in well-known

7 -

 7 Statistical analysis. The spontaneous reports of ADRs during the two studied periods were analyzed separately. Descriptive analysis of continuous variables was performed by means of median and range; and statistical differences were assessed by means of the Mann-Whitney test. Categorical variables were described using percentages; statistical differences were assessed by means of the χ 2 test. Significance was set at a level of 0.05, two tailed. The statistical analysis was performed using the SPSS version 15.0 statistical package (SPSS Inc. Chicago-Illinois).

9 -

 9 fold increase was seen in the number of suspected exposures to ibuprofen, azithromycin and fluoxetine during the second period.In both study periods, around 10% of suspected pharmacological exposures concerned recently marketed drugs. Nevertheless, both the overall number of suspected pharmacological exposures and the number of different drugs increased in the second period (97 vs. 28, and 50 vs. 19, respectively). The new drugs with the highest number of suspected exposures were clopidogrel, levofloxacin, and nevirapine (three exposures each) in the first period, and linezolid (eight exposures), tiotropium bromide, glatiramer acetate, oxcarbazepine, tenofovir, and voriconazole (five exposures each) in the second one.

  induced retroperitoneal haemorrhage, one case of intraabdominal haemorrhage due to enoxaparin, acenocoumarol and acetylsalicylic acid, and one case of acetylsalicylic acid-induced gastrointestinal haemorrhage. Of the eight remaining cases, three were toxic epidermal necrolysis and the other described reactions related to other systems.

DISCUSSION

  Our study has been the first one to assess the effect of a specific intervention on the features of ADRs spontaneously reported in a hospital setting, the drugs involved and the number of signals identified. It has shown that the improvement in spontaneous reporting of ADRs achieved following an intervention based on educational activities and economic incentives allowed us to detect some safety problems and to identify several signals. It is noteworthy that the reporting of all kind of adverse reactions and drugs increased.

  It would be interesting to investigate the effect of other interventions on the type of suspected drugs in spontaneously reported ADRs.

Table 1 .

 1 Adverse drug reactions classified by organ or system affected

	Organ or system	First period N (%)	Second period N (%)
	Cutaneous reactions	44	(20.0)	128	(18.4)
	Haematological reactions	23	(10.5)	68	(9.8)
	Gastrointestinal reactions	19	(8.6)	68	(9.8)
	Cardiovascular reactions	27	(12.3)	67	(9.7)
	Hepatic reactions	12	(5.5)	64	(9.2)
	Neurological reactions	26	(11.8)	58	(8.4)
	Metabolic and nutritional reactions	10	(4.5)	43	(6.2)
	Respiratory reactions	19	(8.6)	39	(5.6)
	General disorders	9	(4.1)	37	(5.3)
	Urinary reactions	4	(1.8)	32	(4.6)
	Endocrinological reactions	1	(0.5)	26	(3.7)
	Psychiatric reactions	12	(5.5)	24	(3.5)
	Others	14	(6.3)	40	(5.8)
	Total	220	(100)	694	(100)

Table 2 .

 2 Most frequent adverse reactions

		First period	Second period
	Adverse reaction		(N=220)		(N=694)
		N	(%)	N	(%)
	Erythematous rash	5	(2.3)	31	(4.5)
	Rash	6	(2.7)	25	(3.6)
	Hepatitis	3	(1.4)	20	(2.9)
	Thrombocytopenia	2	(0.9)	19	(2.7)
	Hepatic enzymes increased	6	(2.7)	18	(2.6)
	Acute renal failure	4	(1.8)	18	(2.6)
	Hepatitis cholestatic	3	(1.4)	17	(2.4)
	Gastrointestinal haemorrhage	2	(0.9)	17	(2.4)
	Diarrhoea	3	(1.4)	16	(2.3)
	Anaphylactic shock	4	(1.8)	15	(2.2)
	Heart failure	2	(0.9)	14	(2.0)
	Hyperthyroidism	1	(0.5)	14	(2.0)
	Urticaria	10	(4.5)	12	(1.7)
	Angioedema	9	(4.1)	11	(1.6)
	Bronchospasm	9	(4.1)	10	(1.4)
	Haematoma	8	(3.6)	9	(1.3)
	Atrioventricular block	4	(1.8)	8	(1.2)
	Encephalopathy	4	(1.8)	8	(1.2)
	Extrapiramidal disorder	5	(2.3)	4	(0.6)
	Retroperitoneal haemorrhage	6	(2.7)	3	(0.4)
	Distonia	5	(2.3)	3	(0.4)
	Hypotension	9	(4.1)	2	(0.3)
	Only adverse reactions present in at least five reports of any period are displayed.

Table 3 .

 3 Suspect drugs by therapeutic subgroup

	Therapeutic subgroup	First period	Second period
		N	(%)	N	%
	Systemic antibiotics (J01)	62	(21.8)	149	(17.0)
	Antithrombotics (B01)	30	(10.5)	69	(7.9)
	Cardiac therapy (C01)	17	(6.0)	47	(5.4)
	Antiinflammatories and antirheumatics (M01)	12	(4.2)	46	(5.2)
	Psychoanaleptics (N06)	8	(2.8)	44	(5.0)
	Analgesics (N02)	17	(6.0)	35	(4.0)
	Drugs acting on the renin-angiotensin system (C09)	10	(3.5)	35	(4.0)
	Antiepileptics (N03)	9	(3.1)	35	(4.0)
	Antineoplastics (L01)	8	(2.8)	34	(3.9)
	Antimycobacterials (J04)	14	(4.9)	32	(3.6)
	Systemic antivirals (J05)	7	(2.5)	31	(3.5)
	Immunosuppressive drugs (L04)	4	(1.4)	29	(3.3)
	Diuretics (C03)	9	(3.1)	27	(3.1)
	Psycholeptics (N05)	9	(3.1)	24	(2.7)
	Drugs for obstructive airway diseases (R03)	6	(2.1)	22	(2.5)
	Beta blocking drugs (C07)	3	(1.1)	22	(2.5)
	Immunostimulants (L03)	0	(-)	18	(2.1)
	Systemic corticosteroids (H02)	4	(1.4)	16	(1.8)
	Lipid modifying drugs (C10)	6	(2.1)	15	(1.7)
	Systemic antihistamines (R06)	11	(3.9)	4	(0.5)
	Others	39	(13.7)	143	(16.3)
	Total	285	(100)	877	(100)

Table 4 .

 4 Most frequent suspect drugs

			First period			Second period
	Drug	Suspect exposures (N=285)	Reports (N=200)	Suspect exposures (N=877)	Reports (N=631)
		N	(%)	%	N	(%)	%
	Amiodarone	11	(3.9)	5.5	36	(4.1)	5.7
	Amoxicillin-clavulanic acid	16	(5.6)	8.0	35	(3.9)	5.5
	Metamizole	12	(4.2)	6.0	23	(2.6)	3.6
	Enoxaparin	16	(5.6)	8.0	21	(2.4)	3.3
	Enalapril	4	(1.4)	2.0	21	(2.4)	3.3
	Ibuprofen	2	(0.7)	1.0	18	(2.1)	2.9
	Acenocoumarol	2	(0.7)	1.0	15	(1.7)	2.4
	Acetylsalicylic acid	6	(2.1)	3.0	14	(1.6)	2.2
	Prednisone	2	(0.7)	1.0	13	(1.5)	2.1
	Phenytoin	3	(1.1)	1.5	10	(1.1)	1.6
	Levofloxacin	3	(1.1)	1.5	10	(1.1)	1.6
	Pyrazinamide	3	(1.1)	1.5	10	(1.1)	1.6
	Furosemide	3	(1.1)	1.5	9	(1.0)	1.4
	Azithromycin	1	(0.4)	0.5	9	(1.0)	1.4
	Fluoxetine	1	(0.4)	0.5	9	(1.0)	1.4
	Ciprofloxacin	6	(2.1)	3.0	8	(0.9)	1.3
	Spironolactone	1	(0.4)	0.5	8	(0.9)	1.3
	Linezolid	0	(-)	-	8	(0.9)	1.3
	Diclofenac	6	(2.1)	3.0	7	(0.8)	1.1
	Hydrochlorothiazide	3	(1.1)	1.5	7	(0.8)	1.1
	Cefepime	2	(0.7)	1.0	7	(0.8)	1.1
	Rifampicin-isoniazid	1	(0.4)	0.5	7	(0.8)	1.1
	Clarithromycin	5	(1.8)	2.5	6	(0.7)	1.0
	Carbamazepine	3	(1.1)	1.5	6	(0.7)	1.0
	Digoxin	3	(1.1)	1.5	6	(0.7)	1.0
	Moxifloxacin	2	(0.7)	1.0	6	(0.7)	1.0
	Azathioprine	1	(0.4)	0.5	6	(0.7)	1.0
	Vancomycin	1	(0.4)	0.5	6	(0.7)	1.0
	Ceftriaxone	0	(-)	-	6	(0.7)	1.0
	Methotrexate	0	(-)	-	6	(0.7)	1.0
	Tacrolimus	0	(-)	-	6	(0.7)	1.0
	Thiethylperazine	8	(2.8)	4.0	3	(0.3)	0.5

Table 5 .

 5 Most frequent drug-reaction associations

	First period	Second period
	(N=200 reports)	(N=631 reports)

a Concomitant administration of enoxaparin and acenocoumarol in two cases. b Enoxaparin concomitantly administered. c Drugs concomitantly administered: clopidogrel (in one case); clopidogrel, enoxaparin and abciximab (in one case); enoxaparin and streptokinase (in one case). d Drugs concomitantly administered: tenecteplase and acetylsalicylic acid (in four cases); drotrecogin alfa activated (in one case).

Table 1 .

 1 Adverse drug reactions classified by organ or system affected

	As a conclusion, an intervention based on educational activities and economic
	incentives led not only to a quantitative improvement in spontaneous reporting
	of ADRs by hospital physicians but also to a qualitative improvement in terms of
	seriousness of reactions identified affecting different organs or systems, and

Table 2 .

 2 Most frequent adverse reactionsOnly adverse reactions present in at least five reports of any period are displayed.

		First period	Second period
	Adverse reaction		(N=220)		(N=694)
		N	(%)	N	(%)
	Erythematous rash	5	(2.3)	31	(4.5)
	Rash	6	(2.7)	25	(3.6)
	Hepatitis	3	(1.4)	20	(2.9)
	Thrombocytopenia	2	(0.9)	19	(2.7)
	Hepatic enzymes increased	6	(2.7)	18	(2.6)
	Acute renal failure	4	(1.8)	18	(2.6)
	Hepatitis cholestatic	3	(1.4)	17	(2.4)
	Gastrointestinal haemorrhage	2	(0.9)	17	(2.4)
	Diarrhoea	3	(1.4)	16	(2.3)
	Anaphylactic shock	4	(1.8)	15	(2.2)
	Heart failure	2	(0.9)	14	(2.0)
	Hyperthyroidism	1	(0.5)	14	(2.0)
	Urticaria	10	(4.5)	12	(1.7)
	Angioedema	9	(4.1)	11	(1.6)
	Bronchospasm	9	(4.1)	10	(1.4)
	Haematoma	8	(3.6)	9	(1.3)
	Atrioventricular block	4	(1.8)	8	(1.2)
	Encephalopathy	4	(1.8)	8	(1.2)
	Extrapiramidal disorder	5	(2.3)	4	(0.6)
	Retroperitoneal haemorrhage	6	(2.7)	3	(0.4)
	Distonia	5	(2.3)	3	(0.4)
	Hypotension	9	(4.1)	2	(0.3)

Table 3 .

 3 Suspect drugs by therapeutic subgroup

	Therapeutic subgroup	First period	Second period
		N	(%)	N	%
	Systemic antibiotics (J01)	62	(21.8)	149	(17.0)
	Antithrombotics (B01)	30	(10.5)	69	(7.9)
	Cardiac therapy (C01)	17	(6.0)	47	(5.4)
	Antiinflammatories and antirheumatics (M01)	12	(4.2)	46	(5.2)
	Psychoanaleptics (N06)	8	(2.8)	44	(5.0)
	Analgesics (N02)	17	(6.0)	35	(4.0)
	Drugs acting on the renin-angiotensin system (C09)	10	(3.5)	35	(4.0)
	Antiepileptics (N03)	9	(3.1)	35	(4.0)
	Antineoplastics (L01)	8	(2.8)	34	(3.9)
	Antimycobacterials (J04)	14	(4.9)	32	(3.6)
	Systemic antivirals (J05)	7	(2.5)	31	(3.5)
	Immunosuppressive drugs (L04)	4	(1.4)	29	(3.3)
	Diuretics (C03)	9	(3.1)	27	(3.1)
	Psycholeptics (N05)	9	(3.1)	24	(2.7)
	Drugs for obstructive airway diseases (R03)	6	(2.1)	22	(2.5)
	Beta blocking drugs (C07)	3	(1.1)	22	(2.5)
	Immunostimulants (L03)	0	(-)	18	(2.1)
	Systemic corticosteroids (H02)	4	(1.4)	16	(1.8)
	Lipid modifying drugs (C10)	6	(2.1)	15	(1.7)
	Systemic antihistamines (R06)	11	(3.9)	4	(0.5)
	Others	39	(13.7)	143	(16.3)
	Total	285	(100)	877	(100)

Table 4 .

 4 Most frequent suspect drugs

			First period			Second period
	Drug	Suspect exposures (N=285)	Reports (N=200)	Suspect exposures (N=877)	Reports (N=631)
		N	(%)	%	N	(%)	%
	Amiodarone	11	(3.9)	5.5	36	(4.1)	5.7
	Amoxicillin-clavulanic acid	16	(5.6)	8.0	35	(3.9)	5.5
	Metamizole	12	(4.2)	6.0	23	(2.6)	3.6
	Enoxaparin	16	(5.6)	8.0	21	(2.4)	3.3
	Enalapril	4	(1.4)	2.0	21	(2.4)	3.3
	Ibuprofen	2	(0.7)	1.0	18	(2.1)	2.9
	Acenocoumarol	2	(0.7)	1.0	15	(1.7)	2.4
	Acetylsalicylic acid	6	(2.1)	3.0	14	(1.6)	2.2
	Prednisone	2	(0.7)	1.0	13	(1.5)	2.1
	Phenytoin	3	(1.1)	1.5	10	(1.1)	1.6
	Levofloxacin	3	(1.1)	1.5	10	(1.1)	1.6
	Pyrazinamide	3	(1.1)	1.5	10	(1.1)	1.6
	Furosemide	3	(1.1)	1.5	9	(1.0)	1.4
	Azithromycin	1	(0.4)	0.5	9	(1.0)	1.4
	Fluoxetine	1	(0.4)	0.5	9	(1.0)	1.4
	Ciprofloxacin	6	(2.1)	3.0	8	(0.9)	1.3
	Spironolactone	1	(0.4)	0.5	8	(0.9)	1.3
	Linezolid	0	(-)	-	8	(0.9)	1.3
	Diclofenac	6	(2.1)	3.0	7	(0.8)	1.1
	Hydrochlorothiazide	3	(1.1)	1.5	7	(0.8)	1.1
	Cefepime	2	(0.7)	1.0	7	(0.8)	1.1
	Rifampicin-isoniazid	1	(0.4)	0.5	7	(0.8)	1.1
	Clarithromycin	5	(1.8)	2.5	6	(0.7)	1.0
	Carbamazepine	3	(1.1)	1.5	6	(0.7)	1.0
	Digoxin	3	(1.1)	1.5	6	(0.7)	1.0
	Moxifloxacin	2	(0.7)	1.0	6	(0.7)	1.0
	Azathioprine	1	(0.4)	0.5	6	(0.7)	1.0
	Vancomycin	1	(0.4)	0.5	6	(0.7)	1.0
	Ceftriaxone	0	(-)	-	6	(0.7)	1.0
	Methotrexate	0	(-)	-	6	(0.7)	1.0
	Tacrolimus	0	(-)	-	6	(0.7)	1.0
	Thiethylperazine	8	(2.8)	4.0	3	(0.3)	0.5

Table 5 .

 5 Most frequent drug-reaction associationsOnly associations present in at least five reports of any period are displayed. Concomitant administration of enoxaparin and acenocoumarol in two cases. b Enoxaparin concomitantly administered. c Drugs concomitantly administered: clopidogrel (in one case); clopidogrel, enoxaparin and abciximab (in one case); enoxaparin and streptokinase (in one case).

	First period	Second period
	(N=200 reports)	(N=631 reports)

a d Drugs concomitantly administered: tenecteplase and acetylsalicylic acid (in four cases); drotrecogin alfa activated (in one case).

European Journal of Clinical Pharmacology

Acknowledgments

The authors would like to thank the hospital physicians who participated in this study for their collaboration. No sources of funding were used to assist in the development of this study. The authors have no conflicts of interest that are directly relevant to the content of this study.

Acknowledgments

The authors would like to thank the hospital physicians who participated in this study for their collaboration. No sources of funding were used to assist in the development of this study. The authors have no conflicts of interest that are directly relevant to the content of this study.


Ulrika ; Simonsson
email: ulrika.simonsson@farmbio.uu.se

Doaa Elsherbiny

Yuan Ren

Helen Mcilleron

Gary Maartens

Ulrika S H Simonsson

Dr Ulrika Simonsson

Population Pharmacokinetics of Lopinavir in Combination with Rifampicin-based Antitubercular Treatment in HIV-Infected South African Children

Keywords: lopinavir, ritonavir, pharmacokinetics, HIV, tuberculosis

Objective The population pharmacokinetics (PK) of lopinavir in tuberculosis (TB)/Human immunodeficiency virus (HIV) co-infected South African children taking super-boosted lopinavir (lopinavir/ritonavir ratio 1:1) as part of antiretroviral treatment in the presence of rifampicin, were compared to the population PK of lopinavir in HIV infected South African children taking standard doses of lopinavir/ritonavir (ratio 4:1).

Methods

Lopinavir concentrations were measured in 15 TB/HIV co-infected paediatric patients who were sampled during and after rifampicin-based TB treatment and in 15 HIV infected children without TB. During TB therapy, the dose of ritonavir was increased to lopinavir/ritonavir 1:1 in order to compensate the induction of rifampicin. The children received median (inter quartile range= IQR) doses of lopinavir 292 mg/m 2 (274, 309) and ritonavir 301 mg/m 2 (286, 309) twice daily. After TB treatment completion the children received standard doses of lopinavir/ritonavir 4:1 (median [IQR] lopinavir dose 289 mg/m 2 [286, 303] twice daily) as were those without TB (median [IQR] lopinavir dose 265 mg/m 2 [249, 289] twice daily).

Results Lopinavir oral clearance (CL/F) was about 30% lower in children without TB than in co-infected children treated with super-boosted lopinavir. However, the predicted lopinavir C min was above the recommended minimum therapeutic concentration during TB/HIV cotreatment in the 15 children. Lopinavir CL/F increased linearly during the dosing interval.

Conclusions Increasing the ritonavir dose to achieve a lopinavir/ritonavir ratio of 1:1 when given in combination with rifampicin-based TB treatment did not completely compensate the enhancement of lopinavir CL/F caused by rifampicin. The time-dependent lopinavir CL/F might be due to a time-dependent recovery from ritonavir inhibition of lopinavir metabolism during the dosing interval.

Introduction

Human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection is a major problem in sub-Saharan Africa. In South Africa, about 50% of children with TB aged between 0-12 years are HIV infected [START_REF] Jeena | Impact of HIV-1 coinfection on presentation and hospital-related mortality in children with culture proven pulmonary tuberculosis in Durban, South Africa[END_REF][START_REF] Lawn | Impact of HIV infection on the epidemiology of tuberculosis in a peri-urban community in South Africa: the need for age-specific interventions[END_REF]. Antiretroviral regimens including the lopinavir/ritonavir together with 2 nucleoside reverse transcriptase inhibitors (NRTIs) are commonly used in South African children. Lopinavir is a potent HIV protease inhibitor co-formulated with ritonavir in a ratio of 4:1. In this formulation, ritonavir acts as a booster of lopinavir concentrations due to its inhibitory effect on cytochrome P450 (CYP) enzyme 3A4 and Pglycoprotein which are involved in lopinavir metabolism and transport [START_REF]Lopinavir/Ritonavir (Kaletra)[END_REF][START_REF] Porche | Lopinavir/ritonavir[END_REF][START_REF] Hogeland | Lopinavir/ritonavir reduces bupropion plasma concentrations in healthy subjects[END_REF]. TB/HIV coinfected children should be treated with rifampicin for the whole TB treatment duration [START_REF] Who | Guidance for National Tuberculosis Programmes on the management of tuberculosis in children[END_REF]. However, by inducing CYP3A4 and P-glycoprotein expression [START_REF] Rae | Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays[END_REF][START_REF] Sun | Multidrug resistance P-glycoprotein: crucial significance in drug disposition and interaction[END_REF][START_REF] Niemi | Pharmacokinetic interactions with rifampicin: clinical relevance[END_REF], rifampicin reduces protease inhibitor concentrations to subtherapeutic concentrations [START_REF] Who | Guidance for National Tuberculosis Programmes on the management of tuberculosis in children[END_REF][START_REF] Who | ANTIRETROVIRAL THERAPY OF HIV INFECTION IN INFANTS AND CHILDREN: TOWARDS UNIVERSAL ACCESS[END_REF]. In adults, rifampicin reduces the area under the-concentration-time curve (AUC) and minimum concentration (C min ) of lopinavir by 75% and 99%, respectively [START_REF]Table 2. Recommendations for coadministering protease inhibitors and non-nucleoside reverse transcriptase inhibitors with RIFAMPIN[END_REF][START_REF] Porte | Pharmacokinetics of adjusted-dose lopinavirritonavir combined with rifampin in healthy volunteers[END_REF]. Such reduction can be overcome, in healthy adults, by administering extra ritonavir (super-boosted lopinavir with lopinavir/ritonavir in a 1:1 ratio) since it results in lopinavir C min similar to those obtained with a lopinavir/ritonavir (4:1) regimen without rifampicin [START_REF] Porte | Pharmacokinetics of adjusted-dose lopinavirritonavir combined with rifampin in healthy volunteers[END_REF]. This approach needs to be validated in children with TB/HIV co-infection since the expression of CYP enzymes and Pglycoprotein as well as protein binding may be altered by age or disease [START_REF] King | Antiretroviral pharmacokinetics in the paediatric population: a review[END_REF][START_REF] Sankatsing | Pglycoprotein in human immunodeficiency virus type 1 infection and therapy[END_REF]. The population pharmacokinetics of lopinavir in children has been published before by for example Jullien et al [START_REF] Jullien | Population analysis of weight-, age-, and sex-related differences in the pharmacokinetics of lopinavir in children from birth to 18 years[END_REF] and Rakhmanina et al [START_REF] Rakhmanina | Population pharmacokinetics of lopinavir predict suboptimal therapeutic concentrations in treatment-experienced human immunodeficiency virus-infected children[END_REF] . However, these models do not describe or take into account the pharmacokinetic interaction between lopinavir and rifampicin.

Neither is the population pharmacokinetics described when given together with the booster ritonavir. The latter has been described earlier [START_REF] Moltó | Simultaneous population pharmacokinetic model for lopinavir and ritonavir in HIV-infected adults[END_REF][START_REF] Crommentuyn | Population pharmacokinetics of lopinavir in combination with ritonavir in HIV-1-infected patients[END_REF] but not in children. As tuberculosis and HIV are very commonly co-existing in children in developing countries, it is a great need

Methods

Study design

The data were available from a study consisting of two parallel treatment groups (Table 1), TB/HIV co-infected children and children with HIV but not TB [START_REF] Ren | Effect of Rifampicin on Lopinavir Pharmacokinetics in HIV-infected Children with Tuberculosis[END_REF]. The TB/HIV co-infected children comprised 15 children (8 females) who aged from 7 to 34 months, weighed between 5-13 kg and had body surface area (BSA) measurements between 0.

Data analysis

Lopinavir population PK were described using nonlinear mixed effects modelling within NONMEM version VI (GloboMax, Austin, Texas, U.S.A) [START_REF] Beal | NONMEM user's guide, NONMEM Project Group[END_REF]. The typical population pharmacokinetic parameters, interindividual variability (IIV) and residual variability were estimated by the first-order conditional estimation method with interaction.

One-and two-compartment models with first order absorption were fitted to the data.

Elimination was assumed to take place from the central compartment.

IIV was described using an exponential error model to avoid negative individual parameter estimates:

) exp( . i P P i η = ( 1
)
where Pi is the individual parameter, P is the typical parameter value and i η is the IIV that describes the difference between the typical parameter value and the individual estimate. The variable i η is normally distributed with mean 0 and variance ω 2 . Investigated residual error models were additive, proportional and slope-intercept models.

Model selection was achieved by the use of the objective function value (OFV) which is minus twice the log likelihood of the data. A drop in the OFV of 3.84 between two nested models corresponding to p < 0.05 was regarded as statistically significant. Graphical analysis using Xpose version 3.104 and version 4 [START_REF] Jonsson | Xpose-an S-PLUS based population pharmacokinetic/pharmacodynamic model building aid for NONMEM[END_REF], parameter estimates standard error, and scientific plausibility were used for model selection as well. Covariate model building was done by forward inclusion and backwards deletion using an OFV change of > 10.83 (p < 0.001) as the cut-off for inclusion, followed by stepwise deletion using an OFV change of > 12.12 (p < 0.0001) to retain the effect on the selected parameters.

Results

Lopinavir showed time-dependent oral clearance (CL/F) in all children. Lopinavir CL/F increased linearly during the dosing interval. Ritonavir inhibits CYP3A4 and hence the elimination of lopinavir [START_REF]Lopinavir/Ritonavir (Kaletra)[END_REF]. Recovery from ritonavir inhibition during the dosing interval was described with a rate constant k r . The final model for lopinavir oral clearance (CL/F) was:

]) [ 1 ( ) / ( ) / ( . . 0 TAD k F CL F CL r + = (2)
where (CL/F) 0 is lopinavir CL/F at the beginning of the dosing interval and TAD is the time after dose in hours. The onset of recovery from enzyme inhibition was immediate since estimating a lag-time for the recovery did not lead to a drop in OFV. An exponential recovery from ritonavir inhibition did not offer any advantage over the linear recovery. Lopinavir (CL/F) 0 was 1.26, 1.17 and 0.90 L . h -1 in co-infected children during TB treatment, after TB treatment and in the group without TB, respectively. Recovery from ritonavir inhibition of lopinavir metabolism was linear with a k r of 0.0582 h -1 during TB treatment, and 0.0648 h -1 after TB treatment and in children without TB. This leads to a gradual increase in CL/F during the dosing interval reaching, at the end of the 12-hours dosing interval, 2.14, 2.08 and 1.59 Model predicted lopinavir concentrations, for all individuals, were above the recommended minimum therapeutic concentration (1 mg/L) [START_REF] Keulen | Recommendations for performing TDM of antiretroviral agents[END_REF].

L . h -1 in
The bootstrap validation results showing the medians and the 95% confidence intervals for 1000 bootstrap samples are represented in Table 2. Figure 1 shows visual predictive check plots obtained from simulation of 1000 datasets based on the final model.

Discussion

Increasing the dose of ritonavir to achieve a lopinavir/ritonavir ratio of 1:1 was not able to completely counteract the induction of lopinavir metabolism by rifampicin in this study as lopinavir CL/F was modestly higher during TB treatment than it was after TB treatment completion. Although giving larger doses of ritonavir may, in theory, fully compensate the effect of rifampicin on lopinavir CL/F, this needs to be evaluated in patients. When ritonavir was taken in therapeutic doses to act as HIV protease inhibitor, it induced CYP3A4 [START_REF] Gass | Neither dapsone hydroxylation nor cortisol 6beta-hydroxylation detects the inhibition of CYP3A4 by HIV-1 protease inhibitors[END_REF][START_REF] Ouellet | Effect of ritonavir on the pharmacokinetics of ethinyl oestradiol in healthy female volunteers[END_REF][START_REF] Geletko | Decreased methadone effect after ritonavir initiation[END_REF] and, in vitro, ritonavir activates the pregnane X receptor though it decreases microsomal CYP3A4 activity due to irreversible enzyme inhibition [START_REF] Luo | CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug metabolism and disposition: the biological fate of chemicals[END_REF].

Even after TB treatment, CL/F in the co-infected children was greater than in children without TB, indicating increased CL or reduced bioavailability of lopinavir, or both, in the TB/HIV co-infected group. This could be attributed to a disease effect, or sampling bias as the study was small. Lopinavir V/F was 2-fold larger in children with TB/HIV compared to children without TB. Lopinavir is 98-99% bound to plasma proteins, mainly alpha-1-acid glycoprotein and albumin [START_REF] Porche | Lopinavir/ritonavir[END_REF]. The larger V/F in the co-infected children might be attributed to lower albumin concentrations compared to children without TB [START_REF] Paton | Effects of tuberculosis and HIV infection on whole-body protein metabolism during feeding, measured by the [15N]glycine method[END_REF][START_REF] Paton | Body composition studies in patients with wasting associated with tuberculosis[END_REF], or to reduced bioavailability of lopinavir since the same trend was found in CL/F.

In healthy adults, administering extra ritonavir (lopinavir/ritonavir 1:1) resulted in similar lopinavir maximum concentration and AUC as obtained with the standard lopinavir/ritonavir (4:1) regimen without rifampicin [START_REF] Porte | Pharmacokinetics of adjusted-dose lopinavirritonavir combined with rifampin in healthy volunteers[END_REF]. The inability of the extra ritonavir dose to completely counteract rifampicin induction of lopinavir CL/F in this study may be due to the higher hepatic enzymatic activity in children between 1-4 years compared with adults [START_REF] King | Antiretroviral pharmacokinetics in the paediatric population: a review[END_REF]. However, the clinically relevant criterion for monitoring virological efficacy is the trough sample at 12 hours after dose for drugs given twice daily [START_REF] Keulen | Recommendations for performing TDM of antiretroviral agents[END_REF]. The model predicted C min suggest that administering extra ritonavir (lopinavir/ritonavir 1:1) is sufficient to maintain the C min above 1 mg/L in the studied children. However, this dosing regimen needs further evaluation in a larger population of children. It is important to note that the BSA-derived doses of lopinavir/ritonavir and ritonavir were rounded up to the nearest 0.5 mL of the oral solutions, such that the doses/BSA of lopinavir and ritonavir, respectively, were 27% and 31% greater than 230 mg/m 2 during TB treatment.

Lopinavir showed time-dependent PK where its CL/F increased linearly during the 12-hour dosing interval probably due to recovery from inhibition by ritonavir. The recovery rate constant, k r , was 0.0582 h -1 in the co-infected children during TB treatment, while after TB treatment and in children without TB, k r was and 0.0648 h -1 . The estimated k r values correspond to longer half-life values than those reported for ritonavir [START_REF] Kappelhoff | Development and validation of a population pharmacokinetic model for ritonavir used as a booster or as an antiviral agent in HIV-1infected patients[END_REF]30] indicating that enzymatic inhibition by ritonavir is mechanism-based and not only competitive. Assuming mechanism-based inhibition and that the half-life of the inhibitor is negligible in comparison to the turnover half-life of the enzyme, the rate constant for recovery from inhibition corresponds to the rate constant of enzyme turnover [START_REF] Venkatakrishnan | Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug-drug interaction risk assessment[END_REF]. The lower k r during TB treatment, in comparison to that after TB treatment and children without TB, may be due to the fact that patients with TB/HIV co-infection have lower protein anabolism than patients with HIV infection alone [START_REF] Paton | Effects of tuberculosis and HIV infection on whole-body protein metabolism during feeding, measured by the [15N]glycine method[END_REF]. Recovery from ritonavir inhibition of CYP3A4 in adults is nearly complete after 3 days of discontinuation [START_REF] Culm-Merdek | Effect of extended exposure to grapefruit juice on cytochrome P450 3A activity in humans: comparison with ritonavir[END_REF]. Our estimated recovery half-life was 11.9 h during TB treatment and 10.7 h after TB treatment and in the group without TB. This means that after 3 days of discontinuation of ritonavir, the recovery in the co-infected children during TB treatment would be 98% whilst after TB treatment and in the children without TB it would be 99%. A previous attempt to link lopinavir population PK to ritonavir exposure resulted in a time-independent inverse relationship between lopinavir CL/F and exposure to ritonavir [START_REF]Lopinavir/Ritonavir (Kaletra)[END_REF].

However, since ritonavir plasma concentrations were not available from our study, there was no relationship between lopinavir PK and ritonavir PK in the model.

Noncompartmental analysis of the data showed a non-significant trend for lopinavir half-life (t 1/2 ) to be larger in the co-infected children during TB treatment compared to the children without TB [START_REF] Ren | Effect of Rifampicin on Lopinavir Pharmacokinetics in HIV-infected Children with Tuberculosis[END_REF]. This is consistent with the estimated population pharmacokinetic parameters since lopinavir t 1/2 at the beginning of the dosing interval and 12 hours after the dose, respectively, were 11.5 h and 6.77 h for the co-infected children during TB treatment compared to 7.98 h and 4.49 h in children without TB.

Our study may have been too small to detect any effects of age or sex within the 6-month to 4-year age range of the study. Very young children (< 6-months) require increased doses/BSA of lopinavir/ritonavir in order to achieve the same lopinavir concentrations as those in older children and adults [START_REF] Verweel | Plasma concentrations of the HIV-protease inhibitor lopinavir are suboptimal in children aged 2 years and below[END_REF][START_REF] Chadwick | Pharmacokinetics, safety and efficacy of lopinavir/ritonavir in infants less than 6 months of age: 24 week results[END_REF][START_REF] Venkatakrishnan | Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug-drug interaction risk assessment[END_REF], and a 39% increase in CL/F has been reported for boys compared to girls in children older than 12 years [START_REF] Jullien | Population analysis[END_REF].

In conclusion, our study suggests that super-boosted lopinavir (lopinavir/ritonavir in the ratio

  pharmacokinetic models in order to simulate and investigate optimal doses and dosing recommendations.The aim of this analysis was to develop a model describing the dynamic PK interactions and comparing the population pharmacokinetics (PK) of lopinavir in TB/HIV co-infected South African children taking lopinavir/ritonavir in the ratio of 1:1 in the presence of rifampicin treatment, to the population PK of lopinavir in HIV infected South African children taking lopinavir/ritonavir in the ratio of 4:1.

  done by bootstrap resampling[START_REF] Ette | Stability and performance of a population pharmacokinetic model[END_REF] and visual predictive check. In the bootstrap resampling, parameter estimates where re-estimated for each of 1000 bootstrap samples. NONMEM estimates from the final model were compared with the bootstrap median and 95% confidence intervals. In the visual predictive check, the final model and parameter estimates were used to simulate 1000 datasets based on the original design. Observed concentrations were compared to the simulated median and 95% nonparametric prediction interval.

  Figure 2 shows plots of observed and individual predicted lopinavir concentration in children with poor, typical and best goodness-of-fit, categorized by the average of the absolute weighted individual residuals.

of 1 : 1 )

 11 in children treated concomitantly with rifampicin-based TB treatment results in therapeutic concentrations of lopinavir (C min > 1 mg/L). However, larger studies including younger and older children are needed to evaluate the pharmacokinetics, safety and efficacy of the combination of lopinavir, ritonavir and rifampicin in children. Lopinavir PK was timedependent probably due to recovery from ritonavir inhibition of lopinavir metabolism during the dosing interval.

F

Figure 1

 1 Figure 1

Figure 2 Observed

 2 Figure 2

  HIV= Human immunodeficiency virus, RSE= relative standard error which is the ratio of the estimated standard error of the parameter relative to its value, CI= confidence interval, (CL/F) 0 = lopinavir oral clearance at the beginning of the dosing interval, k r = rate constant for the dissipation of ritonavir enzyme inhibition, (CL/F)= oral clearance in F= volume of distribution, ka= absorption rate constant †Interindividual variability in V/F in TB/HIV group was only estimated during TB treatment.

Figure 1

 1 Figure 1 a

Figure 1

 1 Figure 1 b

Figure 1

 1 Figure 1 c

Figure 2

 2 Figure 2 a

Figure 2

 2 Figure 2 b

Table 2 .

 2 None of the PK parameters were related to age, weight or gender.

	The median sampling time (range) for the nominal 12-hour sample was 11.58 (10.50-12.07),
	11.50 (9.25-12.00) and 12.00 (11.55-12.42) hours after dose administration in the children

the children during TB treatment, after TB treatment and in the group without TB, respectively. The data did not support having different k r estimates for the co-infected group after TB treatment and the group without TB. Parameter estimates from the final pharmacokinetic lopinavir model are summarized in during and after TB treatment, and in those without TB, respectively. Due to the aforementioned wide ranges of the actual sampling time for the last sample, the nominal 12-

Table 1 Study design TB/HIV group HIV group

 1

	During TB treatment (n=15) *	n=15 *
	lopinavir:ritonavir (1:1)	lopinavir:ritonavir (4:1)
	230 mg/m 2 x 2: 230 mg/m 2 x 2	920 mg/m 2 x 2: 230 mg/m 2 x 2
	2 NRTIs	2 NRTIs
	Rifampicin 10 mg/kg/day
	After TB treatment (n=10) *
	lopinavir:ritonavir (4:1)
	920 mg/m 2 x 2: 230 mg/m 2 x 2
	* Blood sampling for PK determination was done after minimum 4 weeks of treatment

Table 2 Parameter estimates from the final pharmacokinetic lopinavir model

 2

	Parameter	Typical value	Interindividual variability
		Estimate	Bootstrap median	Estimate	Bootstrap median
		(RSE)	(95% CI)	(RSE)	(95% CI)
	(CL/F) 0 in TB/HIV
	group during TB	1.26 (0.11)	1.26 (1.06-1.5)	0.39 (0.24) 0.14 (0.081-0.21)
	treatment (L . h -1 )
	(CL/F) 0 in TB/HIV
	group after TB	1.17 (0.14)	1.16 (0.933-1.48)	0.5 (0.29)	0.23 (0.11-0.36)
	treatment (L . h -1 )
	(CL/F) 0 in HIV group (L . h -1 )	0.895 (0.14)	0.904 (0.703-1.13)	0.5 (0.29)	0.23 (0.11-0.36)
	k r in TB/HIV group
	during TB treatment	0.0582 (0.71)	0.06 (0.018-0.17)	1.9 (0.58)	3.1 (0.91-7.7)
	(h -1 )
	k r in TB/HIV group
	after TB treatment and	0.0648 (0.29) 0.064 (0.035-0.105) 0.7 (0.51)	0.48 (0.15-1.1)
	in HIV group (h -1 )
	V/F in TB/HIV group (L) †	20.9 (0.13)	20.5 (15.5-27.1)	0.86 (0.29)	0.74 (0.39-1.2)
	V/F in HIV group (L)	10.3 (0.17

European Journal of Clinical Pharmacology

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Acknowledgement

This study was supported by grants from the South African Department of Health (research programme for the comprehensive HIV and AIDS care, management and treatment plan for South Africa), the European and Developing Countries Clinical Trials Partnership (EDCTP), the Swedish International Development Cooperation Agency (SIDA) and the National Research Foundation South Africa (NRF).