Conditions: Systemic Lupus Erythematosus (SLE) Juvenile SLE Cutaneous Lupus
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 93
Healthy volunteers: no

Brief summary:
No drug treatment is completely free of risk and lack of response, adverse events and poor adherence may affect its effectiveness. Within this context, this project aims to evaluate the importance of monitoring blood levels and salivary drug used in rheumatic autoimmune diseases in the monitoring of adherence to therapy. In addition, this project intends to use the monitoring of drug levels, based on pharmacokinetic studies and pharmacokinetics/pharmacodynamics modeling, to broaden the understanding of the possible cellular, tissue and immunological mechanisms involved in efficacy and adverse effects of these drugs with the prospect of reducing the damage and maintain therapeutic efficacy. The high-performance liquid chromatography (HPLC) coupled to mass spectrometry, which will be used to evaluate hydroxychloroquine, thalidomide, glucocorticoids, is considered the gold standard technology to qualitative and quantitative analysis of drugs in blood and its comparison with the dosage in the saliva is an improvement in simplification of the process. For biological agents the focus will be on the understanding the loss of efficacy and the possible role of anti-TNF antibodies using ELISA capture methodology.This project will be divided into four sections with their respective sub-projects according to the medications that will be studied: hydroxychloroquine, thalidomide, biologic agents and glucocorticoids.

Detailed description:
No drug treatment is completely free of risk and lack of response, adverse events and poor adherence may affect its effectiveness. There is also a large inter-individual variability in response to treatments with regard to efficacy and toxicity, and for many drugs, there is also a period of weeks to months to establish its efficacy. Within this context, this project aims to evaluate the importance of monitoring blood levels and salivary drug used in rheumatic autoimmune diseases in the monitoring of adherence to therapy. In addition, this project intends to use the monitoring of drug levels, based on pharmacokinetic studies and pharmacokinetics/pharmacodynamics modeling, to broaden the understanding of the possible cellular, tissue and immunological mechanisms involved in efficacy and adverse effects of these drugs with the prospect of reducing the damage and maintain therapeutic efficacy. The high-performance liquid chromatography (HPLC) coupled to mass spectrometry, which will be used to evaluate hydroxychloroquine, thalidomide, glucocorticoids, is considered the gold standard technology to qualitative and quantitative analysis of drugs in blood and its comparison with the dosage in the saliva is an improvement in simplification of the process. The implementation of this methodology dedicated to research in our center, with the necessary training of human resources, will enable the standardization and availability of this advanced technology to other muldisciplinary projects in various areas of science. For biological agents the focus will be on the understanding the loss of efficacy and the possible role of anti-TNF antibodies using ELISA capture methodology.This thematic project will be divided into four sections with their respective sub-projects according to the medications that will be studied: hydroxychloroquine, thalidomide, biologic agents and glucocorticoids.

Arms:
arm 1: This subproject includes only one arm of lupus patients with active and refractory cutaneous disease and eligible for Thalidomide 100mg/day for 12 months. arm 2: This subproject includes one arm of lupus patients with inactive disease, in which will be maintained on standard dose of Hydroxychloroquine (400mg/day). arm 3: This subproject includes one arm of lupus patients with inactive disease: in which the dose will be reduced to 400mg 3 times a week (Hydroxychloroquine reduced).

Interventions: intervention 1: Thalidomide intervention 2: Hydroxychloroquine reduced intervention 3: standard dose of HCQ
Intervention descriptions: intervention 1: Thalidomide 100 mg/day intervention 2: Hydroxychloroquine 2.5 mg/kg/day intervention 3: Hydroxychloroquine 5.0 mg/kg/day

Protocol document:
Prot_SAP_000.pdf: 
COVER PAGE  
 
Official Title: Relevance of Monitoring Blood and Salivary Levels of Drugs Used in Rheumatic 
Autoimmune Diseases  
NCT number: NCT03122431  
Date of the document: April 1, 2017 
 
 


 
THEMATIC PROJECT 
ASSESSMENT OF RELEVANCE OF BLOOD LEVELS OF DRUGS IN THE 
MONITORING RHEUMATIC AUTOIMMUNE DISEASES: SAFETY/ EFFECTIVENESS OF 
THERAPY, DISEASE ACTIVITY AND ADHERENCE TO THERAPY 
Responsible Researcher: 
Prof. Dr. Eloisa S. D. O. Bonfá (Rheumatology) 
Principal Researchers: 
Prof. Dr. Clovis A. A. Silva (ICR, HCFMUSP) 
Dr. Sandra Gofinet Pasoto (Rheumatology, HCFMUSP) 
Dr. Nadia E. Aikawa (CEDMAC**- Rheumatology, USP) 
Associated Researchers: 
Prof. Dr. Alberto J. S. Duarte (DLC*, HCFMUSP) 
Dr. Ana C. R. Medeiros (CEDMAC**, Rheumatology, USP) 
Dr. Carla G. S. Saad (CEDMAC**, Rheumatology, USP) 
Prof. Dr. Eduardo F. Borba Neto (Rheumatology, USP) 
Dr. Ésper Georges Kallás (Clinical Immunology and Allergy, USP) 
Prof. Dr. Gilberto De Nucci (Department of Pharmacology, USP and UNICAMP) 
Dr. Pedro Carlos Carricondo (Ophthalmology, HCFMUSP) 
Prof.Dr. Rosa M. R. Pereira (Rheumatology) 
Dr. Valdemir Melechco Carvalho (FCF***, USP) 
Dr. Vilma S. T. Viana (LIM****-17, HCFMUSP) 
Establishment: Hospital das Clínicas da Faculdade de Medicina da 
Universidade de São Paulo (HC-FMUSP) 
*DLC = Central Laboratory Division 
**CEDMAC = High Cost Medication Dispensing Center of HC-FMUSP 
FCF = Faculty of Pharmaceutical Sciences, USP 
LIM = Medical Research Laboratory 
*UNICAMP/ USP = Pharmacology Discipline of UNICAMP/USP 
Clinical Support Team: 
● Adult Rheumatology (Lupus Group): 
Danieli C. O. Andrade, Michelle Ugolini 
● Pediatric Rheumatology: 
Cláudia G. Schainberg, Adriana M. E. Sallum, Katia T. Kozu, Lucia M. A. Campos, 
Natali W. S. Gormezano 
● High Cost Drug Dispensing Center (CEDMAC): 


Julio Cesar Bertacini de Moraes, Luciana P.C. Seguro, Karina Bonfiglioli, Mariana Gioielli 
Waisberg 
● Department of Ophthalmology: 
Marcelo Hatanaka, Sergio L. G. Pimentel 
● Department of Pathology: 
Prof. Dr. Mirian N. Sotto, Valeria Aoki 
● Department of Dermatology: 
Ricardo Romiti 
● Department of Neurology: 
Carlos O. Heise 
FMUSP Technical Support Team: 
● Humoral Immunology Laboratory of the Discipline of Rheumatology: 
Elaine P. Leon, Margarete Vendramini, Cleonice Bueno 
● Laboratory of Bone Metabolism of the Discipline of Rheumatology: 
Lilian Takayama, Valeria F. Caparbo 
● Statistical Support: 
Ana C. Medeiros (CEDMAC- Rheumatology USP) 
Team of Researchers and Technical Support Personnel of the Central Laboratory Division 
(DLC) of HC-FMUSP: 
Nilo Jose Duarte Coelho 
Nairo Sumita 
Paschoalina Romano 
Persio de Almeida Rezende Ebner 
 
Summary 
No drug treatment is completely free of risk and lack of response, adverse events and poor 
adherence may affect its effectiveness. There is also a large inter-individual variability in response to 
treatments with regard to efficacy and toxicity, and for many drugs, there is also a period of weeks to 
months to establish its efficacy. Within this context, this project aims to evaluate the importance of 
monitoring blood levels and salivary drug used in rheumatic autoimmune diseases in the monitoring of 
adherence to therapy. In addition, this project intends to use the monitoring of drug levels, based on 
pharmacokinetic studies and pharmacokinetics/pharmacodynamics 
modeling, to broaden the 
understanding of the possible cellular, tissue and immunological mechanisms involved in efficacy and 
adverse effects of these drugs with the prospect of reducing the damage and maintain therapeutic efficacy. 
The high-performance liquid chromatography (HPLC) coupled to mass spectrometry, which will be used 
to measure hydroxychloroquine, thalidomide, glucocorticoids levels, is considered the gold standard 
technology to qualitative and quantitative analysis of drugs in blood and its comparison with the dosage 
in the saliva is an improvement in terms of simplification of the process. The implementation of this 
methodology dedicated to research in our center, with the necessary training of human resources, will 
enable the standardization and availability of this advanced technology to other muldisciplinary projects 


in various areas of science. For biological agents the focus will be on the understanding the loss of 
efficacy and the possible role of anti-TNF antibodies using ELISA capture methodology. This thematic 
project will be divided into four sections with their respective sub-projects according to the medications 
that will be studied: hydroxychloroquine, thalidomide, biologic agents and glucocorticoids. 
Keywords: hydroxychloroquine, thalidomide, anti-TNF, glucocorticoids. 
 
Introduction 
No drug therapy is completely risk-free, and the values associated with non-response or adverse 
effects may exceed the cost of therapy. There is also great interindividual variability in the response to 
treatments with regard to efficacy and toxicity and, for many drugs, there is also a period of weeks to 
months for the establishment of this efficacy (Clark et al., 1993). On the other hand, in a recent study a 
gap was reported between the success rates of current treatments and those believed to be viable (Agency 
for Healthcare Research and Quality,2003). This difference has been attributed in part to the patient's 
poor adherence to the recommended treatment (Agency for Healthcare Research and Quality, 2003); 
Pathman et al., 1996). Poor adherence to medication is common (Osterberg & Blaschke, 2005; World 
Health Organization, 2003) and studies consistently showed that 20% to 30% of drug prescriptions are 
not adequately filled and that approximately 50% of chronic disease medications are not used as 
prescribed (Peterson et al., 2003; Haynes et al., 2008). This poor adherence has dramatic effects on 
health. In the United States, it is estimated that this factor was a direct cause of about 125,000 deaths as 
well as at least 10% of hospitalizations poor adherence is also related to a substantial increase in 
morbidity and mortality. The cost of non-adherence was estimated for the U.S. health system between 
$100 billion and $289 billion annually (Viswanathan et al., 2012). 
Adherence is a parameter generally defined as the percentage of prescribed doses of medications 
that are actually taken by patients over a specified period (Haynes et al., 2002; Shishov et al., 2005). 
Although poor adherence has negative effects on the prognosis of the patient (Uribe et al., 2004), there is 
no universally accepted criterion for measuring medication adherence and, its application in daily clinical 
practice is generally very difficult. Assessment of adherence by tablet counting is a complex task, while 
electronic dosing systems are expensive. The data of the number of patients, although readily available in 
the clinic, as already mentioned, are prone to inaccuracy (Finney et al., 1993; Wagner et al., 2001). 
Several questionnaires of self-reports of adherence to medication were evaluated, among them, the 
CQR(Compliance - Questionnaire - Rheumatology) (Klerk et al., 2003) and the Medication Adherence 
Self-report Inventory (MASRI) (Walsh et al., 2002; Koneru et al., 2007). However, its use in clinical 
practice has been quite limited. Pharmacological drug exposure tests seem a promising solution not only 
to the issue of adherence but would also meet dose adjustmentand to a single specific patient, which is a 
growing clinical and financial concern. Alternatively, recent studies with a very limited number of drugs 
suggest that monitoring drug concentrations can also be performed in saliva with the advantage of 
determining the concentration of its free form and because due to easier and less invasive sample 
collection (Dwivedi et al., 2015a). With respect to this, monitoring in blood and saliva as evidenced in 
antiepileptic therapy proved to be of great value in order to individualize and optimize the management of 
drugs in patients with epilepsy (Johannessen and Landmark, 2008; Dwivedi et al., 2015b). Drugs are 
associated with plasma proteins such as albumin and alpha-glycoprotein in different proportions. Since 
the 1970s, studies have shown that the free fraction of the drug corresponds to the active form responsible 
for its therapeutic and toxic effects. Thus, the determination of the concentration of the drug in oral fluid 
may have a higher predictive value of clinical prognosis and toxicity (Ghareeb and Akhlaghi, 2015). 
Several conditions affecting the liver and kidneys alter the proportions between free and bound fractions, 
altering the therapeutic efficiency of the drugs. However, few studies have explored the potential to 
simultaneously monitor the free and total fraction and correlate with the clinical data of patients. The 
equivalence of free plasma levels of some drugs has already been demonstrated with those found in saliva 


and tears matrices. The relationship between the concentration of the drug's free form in saliva and 
plasma presents minimal intra/inter individual variability and is independent of time and dose. 
Nevertheless, these matrices, other than plasma, were little explored due to the lack of sensitivity of the 
available methodologies. The recent development of high-performance tandem mass spectrometers has 
made possible to determine concentrations of the order of pg/mL, enabling the monitoring of reduced 
values of free drugs in reduced volumes of alternative matrices. In the area of Rheumatology, there are 
few studies and are restricted to blood dosage and there is only one study that used saliva for 
glucocorticoids dosage. Saliva dosage offers a good alternative for serum dosage, because it is less 
invasive and because it has, in general, a good correlation with the free drug in serum (Ruiter et al., 
2012). 
In systemic lupus erythematosus (SLE), tests are restricted to blood measurement and there is 
only one open study with 50 patients demonstrating that clinical responses with the azathioprine (AZA) 
seem to occur at 6-TGN levels below the target established for IDI (Cuffari et al., 2001; Goldenberg et 
al., 2004) and it has been reported that the dosages of metabolites of azathioprine may provide a rational 
approach to safety (Askanase et al., 2009). For rheumatoid arthritis (AR), it has been demonstrated that 
levels of the active metabolite of MTX, PG-MTX, can be measured in patients and are correlated with 
disease activity (DAS28),but there are no data on adherence or correlation of PG-MTX levels with 
adverse events (Angelis-Stoforidis et al., 1999; Dervieux et al., 2004; Dervieux et al., 2006; Stamp et al., 
2009; Stamp et al., 2010). 
For antimalarials, there is only one study in adults with SLE (Costedoat-Chalumeau et al., 2006) 
and there are no blood control studies in juvenile lupus. These drugs have an exceptional 
immunomodulatory role in SLE and their withdrawal is associated with disease outbreaks (Tsakonas et 
al., 1998). Today, there are recommendations for its continuous use in all patients with SLE, except for 
those who have formal contraindications, including during pregnancy. In fact, in recent years, additional 
beneficial effects of hydroxychloroquine on cardiovascular complications, glycemic control, 
dyslipidemia, thrombotic events and mortality has been demonstrated (Costedoat-Chalumeau et al., 
2014). 
The issue of non-adherence for drugs with continuous use in SLE is a difficult problem to 
measure and can reach 76% of cases, bringing serious consequences for the patient related to changes in 
prescriptions (Costedoat-Chalumeau et al., 2013). In this context, it is relevant that some studies have 
shown that plasma dosage of hydroxychloroquine is a reliable parameter for adherence because the drug 
has a long half-life and can be detected in blood tests. 
In adult SLE, it has been reported that HCQ blood concentrations below 1000 ng/mL are 
associated with disease activity, it is a strong predictor of exacerbation in the following six months 
(Costedoat-Chalumeau et al., 2006). It is emphasized, however, that the concentrations of HCQ in the 
total blood varied greatly among the patients in this study, despite the prescription of the same daily dose. 
In this study, however, the evaluation of lupus activity was global and did not focus on renal involvement, 
and it was only mentioned that 10 patients had active nephritis. Other studies are therefore necessary to 
define blood levels of HCQ that will be predictors of outbreaks of renal activity, whose involvement is 
present in more than 50% of patients and which is the greatest therapeutic challenge of the disease. In this 
context, it is interesting that the use of this drug is associated with higher renal survival (Fessler et al., 
2005) together with a better response to standard treatment with mycophenolatote mofetil in patients with 
SLE (Kasitanon et al., 2006), reinforcing the importance of an adequate control of its use in this 
condition. It is also relevant in the context of treatment that antimalarials have a long half-life of 
approximately 40 days and there is a delay to reach plasma concentration with a therapeutic effect 
expected only after 3 months or more of treatment (Rodriguez-Caruncho et al., 2014). Monitoring the 
level of this medication, which was established as at least 1000 ng/mL, would certainly be very useful in 
determining whether a higher attack dose would allow this therapeutic effect to be achieved more quickly. 
In addition, monitoring HCQ levels would allow assessing whether a reduction in the weekly HCQ dose 


after a prolonged period of treatment with tissue depot of the drug would be possible to avoid the most 
feared side effect that is retinopathy with loss of vision. This side effect is associated with the cumulative 
dose of an (Wolfe et al., 2010; Jallouli et al., 2015). 
Ophthalmological monitoring of the early state of the retinian toxicity of chloroquine and 
hydroxychloroquine is critical for the prevention of vision loss, since the progression of structural and 
functional deficits may occur even after cessation of therapy with these drugs (Michaelides et al., 2011). 
The new recommendation of the American Ophthalmology Association (2011) determines that 
ophthalmological control of the use of antimalarial should necessarily include complete ophthalmologic 
examination under mydriasis and automated visual field testing Humphrey in protocol 10-2 (campimetry) 
and at least one more objective examination such as multifocal electroretinogram, spectral domain optical 
coherence tomography (SD-OCT), autofluorescence or multifocal electroretinography (mfERG) (Marmor 
et al, 2011). There is some evidence showing that the detection of early functional loss associated with the 
use of these drugs may precede structural changes (Marmor et al., 2014; Greenstein et al., 2015). 
However, it cannot be excluded that these observations are based on tests with false-positive results, or 
that any signs of retinal toxicity may not be detected, since the visual deficits in these cases result from 
retinal pigment epithelium damage caused by the deposit of antimalarials (Bernstein et al., 1991). New 
technologies, SD-OCT, with the realization of a quantitative analysis of the retinian external layers, 
contribute to better diagnostic accuracy (Greenstein et al., 2015). Our objectiveis to determine the 
mechanism of HCQ-induced injury through an evaluation of the correlation of functional and structural 
damage of the retina, evaluated by central visual field examination (campimetry) and SD-OCT 
measurements, with segmentation of the outer layer of the retina (pigment epithelium). 
Another interesting aspect related to antimalarial is that the mechanism of its beneficial effect as 
an immunomodulator has not yet been fully clarified, but it is believed to be correlated with the blockade 
of toll-like receptors (TLR-7 and -9) in dendritic cells and inhibition of alpha interferon production, 
which plays a crucial role in the pathogenesis of SLE. In fact, an in vitro study observed that the use of 
hydroxychloroquine inhibits the production of interferon-alpha and TNF by plasmocytoid dendritic cells 
of SLE patients in european population and, apparently, this inhibition is more effective for TLR-9 than 
for TLR-7 (Sacre et al., 2012). However, there is still a need to determine the possible correlation 
between the level of antimalarial and the degree of inhibition of the two different receptors. 
Thalidomide is another immunomodulatory agent used to treat the cutaneous activity of SLE and 
LEC, even in patients refractory to other medications (Knop et al., 1983; Kyriakis et al., 2000; Coelho et 
al., 2005; Cuadrado et al., 2005). Its high efficacy associated with a very rapid response of the cutaneous 
lupus, made thalidomide a first choice drug in patients without formal contraindication related to 
teratogenesis. The most common adverse effects include peripheral polyneuropathy. In a study of lupus 
patients treated with thalidomide (25 to 100 mg/day), peripheral polyneuropathy occurred in 27% of 
patients (Cuadrado et al., 2005). 
Thalidomide acts mainly by inhibiting the synthesis of TNF-α and modifying the expression of 
the adhering molecules on the surface of endothelial cells (Nakamura et al., 2007; Kuhn et al., 2011). 
Reinforcing this possibility, it has been demonstrated that TNF-α and IFN-1 are increased in the skin 
lesion of lupus (Yu et al., 2013). In addition, thalidomide seems to inhibit keratinocyte apoptosis induced 
by ultraviolet B radiation (Lu et al., 2003), another relevant aspect in the pathogenesis of cutaneous lupus 
(Reefman et al., 2006). In order to better understand the mechanisms involved in this therapeutic 
response, we intend to evaluate whether plasma levels of TNF-α and tissue expression of this cytokine 
would be relevant parameters for the action of thalidomide in the cutaneous manifestation of SLE. 
The neurotoxicity mechanism of this drug is also unclear and there is work suggesting that 
thalidomide could have a direct toxic effect on neurons of the posterior root ganglion (Giannini et al., 
2003). In this respect, the recent description that thin fiber neuropathy is associated with anti-TNF-
therapy, with clinical findings and skin biopsy suggestive of posterior dorsal root involvement in patients 


with AR (Birnbaum & Bingham, 2014), reinforces this possibility. We speculate that this same 
mechanism would be involved in the neurotoxicity of thalidomide in SLE. This finding would have great 
clinical relevance, because the diagnosis of this complication still depends on electroneuromyography, 
which is an expensive, time-consuming, painful and operator-dependent. Skin biopsy is an interesting 
alternative because it is a low-cost, fast, low-risk and easily accessible procedure that would open the 
prospect of performing a more accurate and early diagnosis of neuropathy, besides providing 
histopathological characteristics of the process of injury of these fibers. 
Within this same line, one of the greatest advances in the treatment of rheumatic diseases was the 
emergence of biological therapy using immunological TNF blockers (anti-TNF antibodies). However, 
throughout treatment with these biological drugs, some patients develop anti-drug antibodies (ADA), 
which compete with their anti-TNF action via blocking of their binding site, reducing the therapeutic 
effect of these biologics. This immunogenicity seems to be related to numerous clinical impacts and 
adverse events, including reactions related to infusion, autoantibody production and even drug-induced 
lupus. The clinical usefulness of detecting the presence of these antidrug antibodies concomitantly with 
the determination of serum levels of the biological agent in the first months of therapy was recently 
published in the Lancet journal in 2015 only for rheumatoid arthritis, where it was shown that the dosage 
at three months is predictive of therapeutic response at 12 months (Jani et al., 2015). However, the biggest 
challenge in the treatment of these patients is long-term management, where a significant number of 
patients require the exchange of biological agents after an effective initial response. In such cases, there 
are several dilemmas that include exchange for another biological of the same class, drug class change, 
reduction of the range or increase of dose. For this group of patients, which we call ‘switchers’, the 
longitudinally evaluation of antidrug antibodies seems to be even more relevant and the only study in 
spondyloarthritis evaluated only the first anti-TNF and did not analyze the subgroups of disease that is 
known to have a distinct therapeutic response characteristic, not allowing a precise conclusion about the 
findings. There are no data on JIS yet. On the other hand, the evaluation of antidrug antibodies (ADA) 
and serum levels of the biological agent anti-TNF in patients with good clinical response could also 
enable the reduction of the dose or increase of the interval between applications, reducing costs and the 
risk of toxicity. 
Our Discipline has a biorepository of plasma of patients monitored since the creation of our 
Biological Center in 2007 with the support of a thematic project granted by FAPESP. In this Center, we 
have 110 patients with spondyloarthritis (AS) who presented therapeutic failure with the need for dose 
change or exchange of biological agent (switchers). All have biological samples obtained in the long-term 
follow-up within a collection protocol accompanied with clinical, laboratory and imaging data. This 
biorepository provides us with an opportunity to define in spondyloarthritis the relevance of antidrug 
antibodies in the switchers group, compared to non-switchers. It will also be possible to perform for the 
first time the concomitant longitudinal analysis of the production of antidrug antibodies and 
autoantibodies associated with rheumatologic autoimmune diseases. This would make it possible to 
understand whether the mechanism involved in the production of antidrug antibodies (ADA) is an 
immune response directed only to anti-TNF or is the result of broader polyclonal activation involving the 
production of autoantibodies. In addition, it was observed the concomitant use of Disease-modifying anti-
rheumatic drugs (DMARDs) in RA can reduce the immunogenicity directed against anti-TNF (Krieckaert 
et al., 2010) and this aspect was not determined in AS. 
Prednisolone and prednisone are drugs widely used in the induction and maintenance of 
immunosuppressive regimens in rheumatologic diseases. Prednisolone is the active part of the drug, while 
prednisone is a prodrug and an inactive metabolite of prednisolone. There is only one open study that 
evaluated the blood level of this drug with a single dose in juvenile SLE. This study with only eight 
patients demonstrated that there is a great individual variability and a possible association of the dose 
with disease activity (Sagcal-Gironella et al., 2011). Patient adherence for these drugs is particularly 
difficult due to the aesthetic changes (acne, stretch marks and obesity) related to their use. However, the 


efficacy and rapid onset of action related to these drugs allow the treatment of severe activity and acute 
exacerbations (flares), being used either for induction of remission, or as maintenance therapy in SLE and 
JSLE (Mukhtyar et al., 2009; Mukhtyar et al., 2009a). 
Despite the established role of glucocorticoids (GCs) in controlling inflammation (Karan et al., 
1995), its potential adverse effects (AEs) include cardiovascular, infectious, gastrointestinal, 
psychological, behavioral, endocrine, metabolic, dermatological, musculoskeletal and ophthalmological 
manifestations (Hoes et al., 2007; Wang et al., 2009; Carla et al., 2013; Han et al., 2013). GC is today the 
leading cause of damage in SLE and is responsible for more than 60% of permanent lesions in these 
patients (Glad man et al., 2003). In bone, it is known that, in patients using GC, fracture can occur even 
with normal bone mineral density assessed by densitometry, and the mechanisms of this condition have 
not yet been clarified. In this sense, we intend to evaluate the bone structure and volumetric density, 
currently possible with the use of HRpQCT, a device acquired by our group with multi-user FAPESP 
project. This equipment provides important additional information on bone quality (Tang et al., 2012; 
Tang et al., 2013). We believe that this first prospective study accompanying the introduction of 
glucocorticoid in these patients may provide relevant data on the mechanisms involved in bone loss, in 
addition to understanding the dynamics of corticoid-induced bone alteration, as well as determining the 
predictive factors of fractures in this population. This evaluation will be accompanied by the 
determination of markers of bone metabolism. 
On the other hand, ocular complications are also very frequent in patients who use GC and, 
among these, is the carotidal thickness increase (Han et al., 2013). As this condition has been implicated 
in many retinal diseases, such as central serous chorioretinopathy and pachychoroid neovasculopathy 
(Han et al., 2013), patients undergoing GC therapy may have an increased risk of developing these 
conditions. Thus, one of the objectives of this study is to understand this process of injury through the 
characterization of the effects of CG on the chroidal thickness, which would contribute not only to the 
understanding of the mechanism of choroidal injury but also to the identification of patients with retinal 
diseases associated with chronic use of GC. 
For GC-induced cataract, the mechanism involved s not yet known, but it may be associated with 
gene activation, non-enzymatic binding of corticosteroids to the ocular lens, altered osmotic balance, or 
oxidative damage (Manabeet al., 1984; McLean et al., 1995; Zhou et al., 2015). Regarding oxidative 
damage, serum levels of oxidative stress products have been shown to be increased in cataract associated 
with age (Chang et al., 2013), but there are no data for corticosteroid-induced cataract. It is interesting 
that, in SLE, oxidative stress is present and seems to contribute to the immunomodulation of the disease 
(Shahet al., 2014). In this respect, we speculate whether the detection and quantification of oxidative 
stress products in the blood could eventually help elucidate the pathophysiological mechanisms involved 
in the development of corticosteroid-induced cataract or be a predictive factor of their onset in patients 
with SLE on chronic corticosteroid therapy. 
Therefore, this project aims to evaluate the relevance of monitoring blood and salivary levels of 
drugs used in rheumatologic autoimmune diseases by monitoring efficacy and adherence to therapy. In 
addition, this project aims to broaden the understanding of the possible mechanisms involved in the 
adverse effects of these drugs, aiming damage reduction, and maintaining therapeutic efficacy. High-
efficiency chromatography (HPLC) coupled with tandem mass spectrometry (HPLC/MS-MS) is 
considered the gold standard in qualitative and quantitative analysis of drugs in biological matrices. The 
advantages of this methodology could be grouped into two categories: bioanalytical quality and logistics. 
At present, there is no other methodology that equals HPLC-MS/MS for the analytical quality of the data 
obtained. This technique allows the measurement of multiple drugs in a single analysis run, a fact that 
helps to reduce the cost and the time required to perform the analyses. The high sensitivity related to 
HPLC/MS-MS allows to detect compounds in very low concentrations, especially for free compounds 
that will be measured in saliva. The specificity obtained allows precise determinations and elimination of 
interference generated by metabolites of drugs and other endogenous and xenobiotic interfering factors. 


In the proposed project, there is a special need to obtain high accuracy due to the toxicity of the drugs 
studied and the need for precise adjustment of therapeutic levels. In the logistics aspect, the methodology 
allows simplification in the procedures of extraction of the analytes and reduction in the acquisition time 
of bioanalytical data. Consequently, with smaller turn-around times there is an increase in processing 
capacity in the analytical stage, enabling the analysis of hundreds/thousands of samples in the time frame 
compatible with the proposed study. The multiplexing capacity of the determinations is an unattainable 
aspect regarding other quantification methods, however using HPLC/MS-MS in this project will allow the 
determination of the intact drug and its metabolites in a single acquisition, generating more 
comprehensive results that could reveal in more depth the therapeutic dynamics. 
The implementation of this methodology for research and routine in our center, with the 
necessary training of human resources, will allow the standardization and availability of this advanced 
technology for other multidisciplinary projects in various areas of science. In addition, drug 
concentrations obtain from patients in this study will be used to establish pharmacokinetic-
pharmacodynamic models for GC, thalidomide and hydroxycloroquine use in patients with SLE. 
Pharmacokinetic-pharmacodynamic modeling is a technique that combines two important aspects in 
medical therapy: pharmacokinetics and pharmacodynamics. This integration is done in a series of 
mathematical expressions that allows the description of the duration of the therapeutic effect in relation to 
the dose administered (Poggi JC et al., 2006). This thematic project will be divided into four sections with 
their respective subprojects, according to the medications that will be studied: 
1. hydroxychloroquine; 
2. thalidomide. 
 
Project 1: HYDROXYCHLOROQUINE (1A, 1B and 1C) 
Evaluation of the relevance of determining blood levels of hydroxychloroquine and its 
metabolites in rheumatologic autoimmune diseases: safety/efficacy of therapy, disease activity, 
adherence to therapy and cost-effectiveness 
TITLE OF THE SUBPROJECT 
Correlation of blood levels of hydroxychloroquine (HCQ) with the indices of disease 
activity and adherence to therapy in a cross-sectional study diseases Systemic lupus erythematosus 
(SLE), and Juvenile systemic lupus erythematosus (JSLE). 
 
Background 
HCQ has been used in the treatment of autoimmune rheumatologic diseases, including SLE 
(Ruiz-Irastorza et al., 2010). However, only one study that evaluated blood levels of HCQ in adult SLE 
(Costedoat-Chalumeau et al., 2006), without data on juvenile SLE. The issue of non-adherence in SLE for 
drugs of continuous use is difficult to measure and can reach 76% of cases, bringing serious consequences 
for the patient related to changes in prescriptions (Costedoat-Chalumeau et al., 2013). In this context, it is 
relevant that some studies have shown that plasma dosage of hydroxychloroquine is a reliable parameter 
of adherence, as the drug has a long half-life and is detected in blood samples. 
In adult patients with SLE, it has been reported that concentrations below 1000 ng/mL of HCQ 
in the blood are associated with disease activity, and a strong predictor of disease exacerbation in the 
following six months (Costedoat-Chalumeau et al., 2006). It is emphasized, however, that the 
concentrations of HCQ in the total blood varied greatly among the patients in this study, despite the 
prescription of the same daily dosage. In this study, however, the evaluation of lupus activity was global 
and did not focus on renal involvement, and it was only mentioned that 10 patients had active nephritis. 


Other studies are therefore necessary to define blood levels of HCQ that will be predictors of outbreaks of 
renal activity, whose involvement is present in more than 50% of patients and is the greatest therapeutic 
challenge of the disease. In this context, it is interesting that the use of this drug is associated with higher 
renal survival (Fessler et al., 2005) and a better response to standard treatment with mycophenolatote 
mofetil in patients with SLE (Kasitanon et al., 2006), reinforcing the importance of an adequate control of 
its use in this condition. 
Alternatively, to blood measurement, recent studies with a very limited number of drugs suggest 
that monitoring drug concentrations can be performed in saliva, with the advantage of detecting its free 
form (Dwivedi et al., 2015a). In the area of Rheumatology, there are few studies and are restricted to 
blood measurement and there is only one study that used saliva for glucocorticoid dosage. Saliva dosage 
offers a good alternative to serum level, because it is less invasive and because it generally has a good 
correlation with the free drug in serum (Ruiter et al., 2012). 
Another interesting aspect in relation to antimalarial is that the mechanism of its beneficial effect 
has not yet been fully clarified, but it is believed to be correlated with the blockade of toll-like receptors 
(TLR7-9) and inhibition of alpha interferon production, which plays a crucial role in the pathogenesis of 
SLE. In fact, an in vitro study observed that the use of hydroxychloroquine inhibits the production of 
interferon-alpha and TNF by plasmocytoid dendritic cells of patients with SLE and, apparently, this 
inhibition is more effective for TLR-9 than for TLR-7 (Sacre et al., 2012). However, there is still a need 
to determine the possible correlation between the level of antimalarial and the degree of inhibition of the 
two different receptors. 
Objectives primary: 
- Monitoring of HCQ blood levels: possible relevance on disease activity in JSLE and renal adult 
SLE to inclusion and 6 months following inclusion.  
Secondary: 
- Evaluate the predictive value of HCQ levels for JSLE disease activity and renal disease activity 
in adult SLE within 6 months of inclusion. 
- To verify the possible association between blood levels of HCQ with adherence to treatment 
assessed by a specific questionnaire. 
- Evaluate the correlation between blood and salivary levels of HCQ in a subgroup of patients 
with SLE and JSLE and their possible association with therapeutic response and safety. 
- Check whether the production of interferon-alpha and TNF by plasmocytoid dendritic cells 
correlates with blood HCQ levels. 
NUMBER OF INDIVIDUALS 
Total patients required for the study = 309, being: SLE (n= 206), SLEJ (n= 103). 
STUDY DESIGN 
Initially, a cross-sectional study will be conducted with patients using HCQ at a dose of 5-6.5 
mg/kg/day (maximum of 400 mg/day) for at least 6 months (Clark et al., 1993). At the entrance, 
peripheral blood samples will be taken to determine HCQ levels and patients will be evaluated for the 
activity of the underlying disease in a "blind" way to HCQ concentrations. 
The following indices will be used to assess disease activity every 3 months: 
- SLE and SLEJ: Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) (Glad 
man et al., 2002) and active disease randomly defined asSLEDAI-2K≥ 6. 


- Renal activity: blood pressure (BP), urine type I, protein and creatinine in an isolated urine 
sample, serum keratinize, C3, C4, anti-dsDNA, according to the recommendations of the American 
College of Rheumatology (ACR) (Hahnet al., 2012). 
Adherence will be evaluated by the Medication Adherence Self-report Inventory (MASRI) 
applied by a pharmacist not directly involved in patient care (Koneru et al., 2007). 
SLE and JSLE: Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) (Gladman 
et al., 2002) and active disease randomly defined as SLEDAI-2K≥ 6. 
- Renal activity: blood pressure (BP), urine type I, protein and creatinine in an isolated urine 
sample, serum creatinine, C3, C4, anti-dsDNA, according to the recommendations of the American 
College of Rheumatology (ACR) (Hahnet al., 2012). 
Adherence will be evaluated by the Medication Adherence Self-report Inventory (MASRI) 
applied by a pharmacist not directly involved in patient care (Koneru et al., 2007). 
The production of interferon-alpha and TNF-alpha by plasmocytoid dendritic cells will be 
evaluated in vitro using mononuclear cell preparations isolated from peripheral blood from a random 
sample of the same number of consecutive patients with active and inactive SLE. The cells will be 
stimulated with the TLR-9 agonist, Oligodeoxynucleotides CpG (ODN CpG-A) -2216, and with the TLR-
7 agonist, imiquimod. The percentage of monocytes, B cells, dendritic cells and natural killers (NK) in 
the PBMC fraction will be determined by the phenotypic characterization of these cell subpopulations by 
flow cytometry using monoclonal fluorochrome-conjugated antibodies. Intracellular detection of IFN-
alpha and TNF-alpha in plasmocytoid dendritic cells will also be performed by flow cytometry and 
specific monoclonal antibodies (Sacre et al., 2012). 
TARGET POPULATION 
Inclusion criteria: 
- LES and LESJ: Criteria of the American College of Rheumatology (ACR) (Hochberg, 1997); 
- use of HCQ at a dose of 5-6.5 mg/kg/day (maximum 400 mg/day) for at least 6 months. 
Exclusion criteria: 
- alcoholism; 
- renal dialysis; 
- concomitant infectious process; 
- acute and chronic liver diseases and 
- concomitant use of some drugs that interact with HCQ (cimetidine, antacids, digoxin, 
aminoglycosides, penicillin, neostigmine, pyridostigmine). 
RELEVANT METHODS 
HPLC MS/MS: The determination of blood and salivary levels of hydroxychloroquine and its 
metabolites bisdesetylchloroquine (BDCQ), desetilhydroxychloroquine (DHCQ) and desetylchloroquine 
(DCQ) will be performed by HPLC and tandem mass spectrometry. The process of extracting the analytes 
will be as described by Munster et al. (2002): 10 mL of blood will be collected in silanized tubes 
containing heparin, divided in aliquots and stored at -80ºC until analysis. After thawing, the sample will 
be alkalized with NaOH 10 N and supplemented with internal control (chloroquine or HCQ-D4 sulfate). 
The sample will then be subjected to extraction with acetonitrile and the residue obtained after 
evaporation will be reconstituted with the mobile phase (methanol:acidified water with formic acid) and 
injected into the LC-MS/MS system according to the Soichot protocol, 2014. Chromatographic separation 
will be obtained using hypersilgold aQ analytical column and, as mobile phase, water gradient and 


acidified methanol with 0.1% formic acid. The detection of analytes will be by tandem mass spectrometry 
with mass spectrometer equipped with an electrospray ionization source operating in ion-positive mode. 
HCQ sulfate and its metabolites DHCQ, DCQ, BDCQ will be added in increasing concentrations to a 
blood sample of healthy individuals as standards in the elaboration of the calibration curve to be used in 
the quantitative calculation of the drug and its derivatives and as quality control of the procedure. 
Phenotypic characterization of PBMC subpopulations: mononuclear cells will be isolated from 
peripheral blood in physio-Hypaque gradient, placed on plates containing RPMI 1640 culture medium 
supplemented with fetal serum and antibiotics. The percentage of monocytes, B cells, dendritic cells, and 
natural killers (NK) in this preparation will be determined by flow cytometry, using monoclonal 
antibodies conjugated to fluorochromes (CD123, CD20, Cd11b, CD16, CD14, HLA-DR). 
Intracellular detection of IFN-alpha and TNF-alpha cytokines in plasmocytoid dendritic cells: 
PBMC cells will be stimulated with TLR-7 and TLR-9 agonists, imiquimod (5 μg/mL) and CpG-A ODN 
2216 (5 μM) for 5 hours at 37ºC. After 3 hours of stimulation, Brefeldin A will be added to induce the 
accumulation of cytokines in the endoplasmic reticulum by its inibiotic action on intracellular protein 
transport to the Golgi complex. Phenotypic cell characterization (PBMC) and intracellular detection of 
cytokines will be performed by sequential membrane marking and intracellularly using specific 
monoclonal antibodies labeled with fuorochromes followed by evaluation by flow cytometry (Sacre et al., 
2012). 
WITHDRAWALS 
It doesn't apply. 
ORIGINALITY WITH IMPACT 
Healthcare 
To date, there is no study that has evaluated blood and salivary levels of hydroxychloroquine 
(HCQ) and its predictive value for disease activity in JSLE and adult lupus nephritis. 
The expected impact with the availability of this technique in clinical practice is an improvement 
in therapeutic adherence and an earlier intervention with reduction of outbreaks of disease activity. The 
use of saliva as an alternative to this monitoring would provide a less invasive technique for this process. 
ADVANCES IN MECHANISMS 
The expected progress is a better understanding of the mechanism of action of HCQ on TLR-7-9, 
previously suggested as possible therapeutic targets for the disease. 
STATISTICAL PLAN 
SAMPLE SIZE 
The sample size was calculated based on the HCQ levels observed in the study by Costedoat-
Chalumeau et al., 2006, in patients with SLE and assuming: 95% confidence interval, α error of 5%; 
power of 80%; mean HCQ concentration in active patients: 694 ± 448 ng/mL; mean HCQ concentration 
in inactive patients: 1079 ± 526 ng/mL; aggregate standard deviation: 532 ng/mL and loss: 10%. 
For the SLE group with nephritis, 16% of SLE patients in our outpatient clinic present active 
renal disease, then we expect 33 patients with renal activity out of the 206 patients which will be 
included. 
For the JSLE group, 34% of our outpatient clinic patients present active, then 35 with disease 
activity are expected out of a total number of 103 patients to be included statistical analysis The groups of 
patients with and without disease activity will be compared for categorical variables by fisher's exact test 
and, for quantitative variables, by student and mann-whitney t tests, when applicable. 


TITLE OF THE 
SUBPROJECT 1B 
Prospective, randomized and controlled study comparing the frequency of SLE and jSLE activity 
of patients with a stable dose of HCQ and patients with dose reduction of this drug and its correlation 
with blood levels of its metabolites diseases.  
SLE AND jSLE 
Background 
It takes at least 6 months of HCQ use to achieve its best efficacy (Clark et al., 1993). In SLE, 
regular use of HCQ promotes a decrease in inflammatory outbreaks of the disease (Ruiz-Irastorza et al., 
2010) and its withdrawal is associated with worsening activity remission (Tsakonas et al., 1998 - The 
Canadian Hydroxychloroquine Study Group). Its action is due to a modulating effect on the immune 
response, influencing the presentation of antigens and production of pro-inflammatory cytokines, which 
are probably mediated mainly by inhibition of Toll-like receptors-7 and -9 (Wallace et al., 2012). 
Adequate serum levels of HCQ metabolites are related to a better efficacy in the treatment of the 
cutaneous form of lupus (Francès et al., 2012) and HCQ concentrations in the blood lower than 1,000 
ng/mL determined by HPLC have been associated with higher inflammatory activity of SLE (Costedoat-
Chalumeau et al., 2006). Monitoring of HCQ in the blood would allow assessing whether, after a 
prolonged period of treatment, which causes a tissue deposit of the drug in the skin, a reduction in the 
weekly dose would be possible. In addition, the measurement of HCQ in saliva could offer a good 
alternative for blood levels, because it is less invasive, as previously demonstrated with other drugs 
(Ruiter et al., 2012). The goal for reduced HCQ dose is to avoid retinopathy with vision loss, without risk 
of increased outbreaks of disease activity. Retinopathy is the most feared side effect of HCQ use and is 
associated with the cumulative dose of antimalarial (Wolfe et al., 2010). The new recommendation of the 
American Ophthalmology Association of 2011 determines that ophthalmological control of the use of 
antimalarials must necessarily include campimetry and at least one more objective examination such as 
multifocal electroretinogram, optical coherence tomography (SD-OCT) (Marmor et al., 2011). Our 
objective is to determine the mechanism of HCQ-induced injury through an evaluation of the correlation 
of functional and structural retinal damage, evaluated by central visual field examination (campimetry) 
and SD-OCT measurements, with segmentation of the outer layer of the retina (pigment epithelium). The 
proposed acquisition of a campimeter dedicated to the research intends to validate the isolated use of 
campimetry, limited to the 10-2 Humphrey field, as the gold standard of this evaluation. 
In addition, it is extremely important to evaluate whether the reduction of the weekly dose of 
HCQ after a long period of use of this therapy would be able to maintain inhibition of TLR-7 and -9. 
Objectives primary: 
- Compare the frequency of outbreaks of disease activity (SLE and jSLE) in patients using 
standard dose (HCQ 400 mg/day) vs. patients with reduced dose (HCQ 400 mg/3X week) and its 
correlation with blood and saliva levels of the drug. Secondary: - Compare frequency of retinopathy in 
patients using standard dose (HCQ 400 mg/day) vs. patients with reduced dose (HCQ 400 mg/3X week) 
and its correlation with HCQ blood levels. 
- Verify a possible longitudinal correlation of HCQ concentrations with the production of 
interferon-alpha and TNF by plasmocytoid dendritic cells. 
- Correlate blood and salivary levels of HCQ and its metabolites in patients with SLE and JSLE 
with adherence to this treatment. 
- Correlate functional and structural damage of the retina, evaluated by central visual field 
examination (campimetry) and SD-OCT measurements, with segmentation of the outer layer of the retina 
(pigmental epithelium). 


- Establishment of a pharmacokinetic-pharmacodynamic model of hydroxychloroquine dose 
reduction in patients with SLE. 
NUMBER OF INDIVIDUALS 
Total number of patients required for the study= 256 (SLE and JSLE). 
STUDY DESIGN 
Randomized controlled prospective study of non-inferiority of one year of follow-up of patients 
with SLE and JSLE in remission with HCQ use (stable dose of 400 mg/day for at least five years). 
Patients will be randomized into two groups: no daily dose reduction group (HCQ 400 mg/day) 
and reduction group (HCQ 400 mg/day - 3X week). Patients will be evaluated for the activity of the 
underlying disease in a "blind" way. HCQ concentrations will be measured at the entrance, 6 months and 
12 months after. 
The groups will be compared for episodes of disease activity (clinical and laboratory 
parameters). The production of interferon-alpha and TNF by plasmocytoid dendritic cells will be 
evaluated in the same period in a subgroup of patients. 
The following indexes will be used to assess disease activity: 
- SLE and jSLE: SLEDAI-2K (Gladman et al., 2002) and active disease randomly defined as 
SLEDAI-2K≥ 6. 
- Renal activity: BP, urine type I, protein and creatinine in an isolated urine sample, serum 
creatinine, C3, C4, anti-dsDNA, according to the recommendations of the ACR (Hahn et al., 2012). 
Adherence will be assessed by the MASRI questionnaire applied by a pharmacist not directly 
involved in patient care (Koneru et al., 2007). 
The following clinical, laboratory and treatment data will be evaluated in the groups (active 
disease and inactive disease): age, sex, race, weight, height, BMI, ideal weight; disease time, time of 
HCQ use, adherence to treatment; smoking; use and dose of prednisone, methotrexate, azathioprine, 
mycophenolate 
mofetil, 
cyclophosphamide, 
non-hormonal 
anti-inflammatory 
drugs; 
urinary 
protein/creatinine ratio; serum creatinine; blood concentrations of HCQ by HPLC; specific adverse 
events. 
- Complete ophthalmologic assessment (at the entrance and at the end of the study), including 
campimetry and SD-OCT measurements, with segmentation of the outer layer of the retina (pigmentary 
epithelium). 
- The production of interferon-alpha and TNF-alpha by plasmocytoid dendritic cells will be 
evaluated in vitro using mononuclear cell preparations isolated from peripheral blood from a random 
sample of the same number of consecutive patients with active and inactive SLE. The cells will be 
stimulated with the TLR-9 agonist, Oligodeoxynucleotide S CpG (ODN CpG-A)-2216 and with the TLR-
7 agonist, imiquimod. The percentage of monocytes, B cells, dendritic cells and natural killers (NK) in 
the PBMC fraction will be determined by the phenotypic characterization of these cell subpopulations by 
flow cytometry using monoclonal fluorochrome-conjugated antibodies. Intracellular detection of IFN-
alpha and TNF-alpha in plasmocytoid dendritic cells will also be performed by flow cytometry and 
specific monoclonal antibodies (Sacre et al., 2012). 
- Establishment of a pharmacokinetic-pharmacodynamic model of hydroxychloroquine use with 
two groups of 5 patients: one with 5mg/kg/day daily and another 5mg/Kg/day 3 times a week. On 
inclusion both will be on hydroxychloroquine use for at least 6 months and should have the stable level of 
the drug (> 1000ng/mL). 
Outcomes: assessment on days 0 and 120 days. 


a) Worsens no SLEDAI -2K. Change in SLEDAI-2K evaluating activity criteria from 0-105 in 9 
organs and 24 items (clinical and laboratorial). The increase represents clinical/laboratory worsening of 
the disease. 
b) Cytokine measurement. Plasma levels of TNF-α and IFN-1 will be determined by Luminex 
MAP (EMD Millipore Corporation®). Plasma samples will be stored at -80oC until use. 
TARGET POPULATION 
Inclusion criteria: 
- LES and jSLE (ACR criteria) (Hochberg, 1997); 
- use of HCQ at a dose of 400 mg/day for at least 3 years. 
Exclusion criteria: 
- alcoholism; 
- renal dialysis; 
- concomitant infectious process; 
- acute and chronic liver diseases; 
- concomitant use of some drugs that interact with HCQ (cimetidine, antacids, digoxin, 
aminoglycosides, penicillin, neostigmine, pyridostigmine). 
RELEVANT METHODS - Ophthalmological assessment: central visual field examination 
(campimetry) and SD-OCT measurements, with segmentation of the outer layer of the retina (pigmentary 
epithelium). 
- Blood levels of HCQ will be determined by HPLC MS/MS (Munster et al., 2002). 
- Pharmacokinetic analysis: The pharmacokinetic parameters of hydroxychloroquine will be 
calculated in the 24 h dose range based on the total plasma concentration versus time curves. The 
Winnonlin program, version 4.0 (PharsightCorp., Mountain View, California, USA) and theNon-linear 
mixed effect level(NONMEN) program will be employed. The area under the plasma concentration curve 
versus time will be calculated in the 24-h dose interval by the trapezoid method on days 0 and 120. The 
apparent total clearance (ClT/F) will be obtained through the equation: ClT/F= Dose/AUC0-24. The 
apparent distribution volume (Vd/F) will be calculated by the standard software equation. The 
hydroxychloroquine-free concentration will be determined by the dosage in saliva. The PK-PD analysis 
will be performed using as pharmacokinetic parameters the total and free plasma concentrations of 
hydroxychloroquine and, as a pharmacodynamic parameter, we will use the clinical/laboratory indices 
obtained by the evaluation of SLEDAI-2K (American College of Rheumathology). The PK-PD model 
will be developed using the non-linear mixed effect level (NONMEM). 
- Others, the same as described in subproject 1A. 
Safety 
Monitoring of outbreaks of inflammatory activity(flare) and identification of retinopathy. 
WITHDRAWALS 
Signs of retinopathy. 
ORIGINALITY WITH IMPACT on 
Healthcare 


To date, there is no study demonstrating that the reduction of HCQ dosage after tissue depot 
caused by a prolonged time of use of the drug can be safely performed in SLEJ and SLE, without 
increasing the number of outbreaks of inflammatory activity. 
The expected care impact is a reduction of severe ophthalmologic event with the reduction of the 
HCQ dose and the expansion of the time of use of the drug that has a direct impact on the control of 
disease activity. 
ADVANCES IN MECHANISMS Advances in understanding the role of cutaneous HCQ depose 
acquired after prolonged previous treatment in maintaining drug-induced TLRs-7 and -9 inhibition would 
justify reducing the therapeutic dose based on the drug's immunomodulatory mechanisms of action. 
SAMPLE SIZE 
The sample size was calculated based on the difference in Reactivation of SLE/SJS in one year 
after the removal or maintenance of hydroxychloroquine, as observed in the study by Tsakonas et al., 
1998 and assuming: 95% confidence interval, α error of 5%; power of 80%; expected success rate after 1 
year in the group that kept HCQ at the standard dose (Tsakonas et al., 1998)- 84%; expected success rate 
after 1 year in the group that fully removes HCQ (Tsakonas et al., 1998)- 59%; no-inferiority margin- 
50% of the difference between maintaining chloroquine or removing it completely, i.e., delta of 12%; 
losses- 10%; n in each group- 128; total number of patients to be recruited: 256 (for SLE/JSLE). 
statistical analysis The comparison of the risk of inflammatory activity of the disease in patients 
using standard dose (HCQ 400 mg/day) vs. patients with reduced dose (HCQ 400mg/day - 3X week) will 
be made through a survival curve by the log-rank test. The categorical variables will be evaluated by 
fisher's exact or chi-square test and quantitative variables by student and mann-whitney t tests, when 
applicable. 
 
TITLE OF THE SUBPROJECT 1C 
Prospective study of the pharmacokinetics of hydroxychloroquine in patients with Systemic 
Lupus Erythematosus using attack dose diseases SLE and jSLE. 
Justification 
 
Currently, it is recommended that all patients with SLE should be treated with antimalarials 
unless specific contraindications (Wallace et al., 2012; van Vollenhoven et al., 2014). Its use is justified 
because of the demonstrated effectiveness in the treatment of the disease (Ruiz-Irastorza et al., 2010), 
with a decrease in inflammatory episodes (flares) (Clark et al., 1993). Furthermore, it was demonstrated 
that HCQ withdrawal increases the risk of disease reactivation (The Canadian Hydroxychloroquine Study 
Group,1991; Tsakonas et al., 1998). There is a modulating effect on the immune response with its use, 
promoting a change in the production of pro-inflammatory cytokines, and these effects are probably 
mediated mainly by inhibition of receptor-7 and -9 Toll-like (Wallace et al., 2012). The most commonly 
used dosage is 400 mg/day of HCQ. It is also relevant in the context of treatment that antimalarials have a 
long half-life of approximately 40 days and there is a delay to reach plasma concentration with a 
therapeutic effect expected only after 3 months or more of treatment (Rodriguez-Caruncho et al., 2014). 
Monitoring the level of this medication, which was established as at least 1000 ng/mL, would certainly be 
very useful for determining whether a higher attack dose would allow this therapeutic effect to be 
achieved more quickly. Lower blood concentrations of HCQ (< 1,000 ng/mL) determined by HPLC 
MS/MS are associated with higher inflammatory activity of SLE (Costedoat-Chalumeau et al., 2006). 
Adequate serum levels of the metabolites of this drug are related to a better efficacy in the treatment of 
the cutaneous form of lupus (Francès et al., 2012), but even using the same daily dose of the drug, there is 
a great individual variability among patients (Munster et al., 2002). Therefore, in rheumatoid arthritis, it 


has been suggested that an attack dose in the first weeks of treatment is related to an increase in response 
in this period (Emery, 1999; Furst et al., 1999). There are no studies in SLE that assess how long after the 
start of treatment is necessary to achieve adequate HCQ blood level, nor whether a higher initial dose 
(attack) could anticipate these levels in order to provide an earlier clinical/laboratory response. 
Objectives Primary objective: 
- Compare the time required to reach the level of 1000 ng/mL of HCQ in patients with SLE, 
using standard dose vs. attack dose. 
Secondary objective: 
- Evaluate the clinical/laboratory response and adverse events in both groups. 
- Verify a possible correlation of HCQ concentrations in blood and saliva with interferon-alpha 
and TNF production levels by plasmocytoid dendritic cells and disease activity parameters. 
- Correlate blood and salivary levels of HCQ and its metabolites in Patients with SLE with 
adherence to this treatment. 
NUMBER OF INDIVIDUALS 
Total patients needed for the study = 96 (SLE). 
STUDY DESIGN 
Prospective randomized study controlled for a period of six months of patients with SLE who 
will start the use of HCQ. 
Patients will be randomized randomly into two groups: traditional group - without increasing the 
daily dose (HCQ 400 mg/day); attack dose group - with increased daily dose (HCQ 800 mg/day for 6 
months). Patients will be evaluated for the activity of the underlying disease in a "blind" way. HCQ 
concentrations will be assessed at the entrance and at the end of the study. 
The groups will be compared for episodes of disease activity (clinical and laboratory 
parameters), inflammatory cytokines, and expression of TLR-7 and TLR-9 receptors. 
Blood concentrations of HCQ and metabolites will be determined in both groups by HPLC 
(Munster et al., 2002). 
The following indexes will be used to assess disease activity: 
- SLE: SLEDAI-2K (Gladman et al., 2002) and active disease randomly defined as SLEDAI-
2K≥ 6. 
- Renal activity: BP, urine type I, protein and creatinine in an isolated urine sample, serum 
creatinine, C3, C4, anti-dsDNA, according to the ACR Guidelines (Hahnet al., 2012). 
Adherence will be assessed by the MASRI questionnaire applied by a pharmacist not directly 
involved in patient care (Koneru et al., 2007). 
The following clinical, laboratory and treatment data will be evaluated in the groups (traditional 
dose and attack dose): age, sex, race, weight, height, BMI, ideal weight; disease time, time of HCQ use, 
adherence to treatment; smoking; use and dose of prednisone, methotrexate, azathioprine, mycophenolate 
mofetil, cyclophosphamide, non-hormonal anti-inflammatory drugs; urinary protein/creatinine ratio; 
serum creatinine; HCQ blood concentrations by HPLC MS/MS; general- gastrointestinal and cutaneous 
adverse events. 
- The production of interferon-alpha and TNF-alpha by plasmocytoid dendritic cells will be 
evaluated in vitro using mononuclear cell preparations isolated from peripheral blood from a random 
sample of the same number of consecutive patients with active and inactive SLE. The cells will be 


stimulated with the TLR-9 agonist, Oligodeoxynucleotides CpG (ODN CpG-A)-2216 and the TLR-7 
agonist, imiquimod. The percentage of monocytes, B cells, dendritic cells and natural killers (NK) in the 
PBMC fraction will be determined by the phenotypic characterization of these cell subpopulations by 
flow cytometry using monoclonal fluorochrome-conjugated antibodies. Intracellular detection of IFN-
alpha and TNF-alpha in plasmocytoid dendritic cells will also be performed by flow cytometry and 
specific monoclonal antibodies (Sacre et al., 2012). 
TARGET POPULATION 
Inclusion criteria: 
- SLE (ACR criteria) (Hochberg, 1997); 
- without using HCQ at a dose of 400 mg/day for at least 6 months. 
Exclusion criteria: 
- alcoholism; 
- renal dialysis; 
- concomitant infectious process; 
- acute and chronic liver diseases; 
- concomitant use of drugs that interact with HCQ (cimetidine, antacids, digoxin, 
aminoglycosides, penicillin, neostigmine, pyridostigmine). 
RELEVANT METHODS 
See subproject 1A. 
Safety. Monitoring of adverse events and outbreaks of inflammatory activity (flare). 
ORIGINALITY WITH IMPACT ON ASSISTANCE 
There are no studies in SLE that assess how long after the start of treatment is necessary to 
achieve adequate HCQ blood and salivary level, nor whether a higher initial dose (attack) could anticipate 
these levels to provide an earlier clinical/laboratory response without increasing adverse effects. 
The expected impact would be that the dosage of HCQ levels would allow to perform a control 
of the inflammatory activity of the disease earlier and safely. 
ADVANCES IN MECHANISMS To compare the inhibitory action of different doses of HCQ in 
TLRs -7 and -9 and its relevance to the immunomodulatory action of the drug. 
SAMPLE SIZE 
The sample size was calculated considering that the faster the 1000 ng/mL blood level is 
reached, the greater the improvement in lupus activity and the lower the recurrence rate in the groups is 
observed, 6 months after the onset of HCQ treatment. In the study by Tett et al., 2000, in patients with 
AR, in the group that started HCQ at a dose of 400 mg/d, the mean seric level was 870.3 ± 329.3 ng/mL, 
that is, about 34% of patients reached > 1000 ng/mL blood level after 6 months. It is estimated that more 
than 20% of patients will reach this value with twice the attack dose, we calculate (based on: 95% 
confidence interval, error α 5%; power of 80%) expected frequency of patients with 1000 ng/mL > level 
after 6 months in the 400 mg/d group: 34%; expected frequency of patients with 1000 ng/mL > level after 
6 months in the 800 mg/d group: 55 %; losses: 10%; n = 96. 
STATISTICAL PLAN 


The comparison of the time required to reach the level of 1000 ng/mL of HCQ in patients with 
SLE and JSLE, using survival curve by log-rank test. Categorical variables will be evaluated by fisher's 
exact or chi-square test and quantitative variables by Student and Mann-Whitney t tests, when applicable. 
 
Project 2: THALIDOMIDE 
EVALUATION OF THE TREATMENT OF CUTANEOUS LUPUS ERYTHEMATOSUS 
WITH THALIDOMIDE: CLINICAL, LABORATORY AND HISTOLOGICAL FACTORS 
ASSOCIATED WITH CLINIC RESPONSE AND ADVERSE EFFECTS 
Diseases 
Systemic lupus erythematosus with cutaneous involvement (LEC). 
Justification 
Thalidomide is another immunomodulatory agent used to treat the cutaneous activity of SLE and 
LEC, even in patients refractory to other medications (Knop et al., 1983; Kyriakis et al., 2000; Coelho et 
al., 2005; Cuadrado et al., 2005). Its high efficacy associated with a very rapid response of the cutaneous 
picture, made this drug one of the first choices in patients without contraindication. Cuadrado et al. 
demonstrated in a retrospective study with 48 patients with SLE and skin lesions refractory to treatment 
with antimalarials, prednisone, azathioprine, cyclosporine and cyclophosphamide a high partial or 
complete clinical response rate (81%) with doses of 25, 50 or 100 mg/day of thalidomide (Cuadrado et 
al., 2005). Thus, thalidomide is very useful as an agent inducing remission of skin activity of SLE 
(Cuadrado et al., 2005). In this study, the treatment time was around one year. Thrombotic events were 
not observed, although some patients had positive antiphospholipid antibodies. The most common 
adverse effects were dizziness, abdominal pain, and peripheral polyneuropathy. Peripheral 
polyneuropathy occurred in 27% of patients, including 18.8% with neurological symptoms and alterations 
in ENMG, 4.2% only with findings at ENMG and 4.2% with neurological symptoms despite normal 
MGS. Regarding the time of disease, this effect seems to occur around 14 months of treatment (Briani et 
al., 2004). 
In Brazil, thalidomide is indicated for use in SLE, subacute cutaneous LE and discoid LE 
(Ministry of Health, 2013). However, due to its known teratogenicity when used during pregnancy, it is 
recommended for male patients (who agree to use condoms throughout the treatment period and up to 30 
days after the end of the same, even if they have already undergone vasectomy, as its possible excretion 
in the seminal fluid is not known) and to women without fertility potential (i.e., who have been in the 
postmenopausal phase for at least one year, or who have undergone tubal ligation or hysterectomy) and 
who agree to use it according to the Term of Responsibility/Clarification (Ministry of Health, 2013; 
System for Thalidomide Education and Prescribing Safety, Zeldis et al., 1999). 
Thalidomide acts mainly by inhibiting the synthesis of TNF-α (Nakamura et al., 2007; Kuhn et 
al., 2011). In order to better understand the mechanisms involved in this therapeutic response, we intend 
to evaluate whether plasma levels and tissue expression of TNF-α at the beginning of treatment would be 
relevant parameters as predictors of response. 
In addition to blood monitoring, salivary drug dosage has been suggested as a practical and low-
invasive method to individualize and optimize drug management in patients with chronic diseases such as 
epilepsy (Johannessen and Landmark, 2008; Dwivedi et al., 2015b). There are no studies in the literature 
to monitor levels of thalidomide in saliva in patients with SLE. 
In addition, it would be important to determine whether the longitudinal evaluation of these 
inflammatory markers and thalidomide concentration would play a role in monitoring the efficacy of this 
therapy in SLE. Reinforcing this possibility, it has been demonstrated that TNF-α and IFN-1 are increased 
in the skin lesion of lupus (Yu et al., 2013). In addition, thalidomide seems to inhibit keratinocyte 


apoptosis induced by ultraviolet B radiation (Lu et al., 2003), another relevant aspect in the pathogenesis 
of cutaneous LE (Reefman et al., 2006). 
The mechanism of neurotoxicity of thalidomide is also unclear and there is work suggesting that 
this drug could have a direct toxic effect on neurons of the posterior root ganglion (Giannini et al., 2003). 
In this respect, the recent description that thin fiber neuropathy is associated with anti-TNF-therapy, with 
clinical findings and skin biopsy suggestive of posterior dorsal root involvement in patients with AR 
(Birnbaum & Bingham, 2014), reinforces this possibility. We speculate that this same mechanism would 
be involved in the neurotoxicity of thalidomide in SLE. This finding would have great clinical relevance, 
because the diagnosis of this complication today depends on electroneuromyography, which is an 
expensive, time-consuming, painful and operator-dependent examination. Skin biopsy is an interesting 
alternative because it is today a low-cost, fast, not painful and easily accessible procedure that would 
open the prospect of performing a more accurate and early diagnosis of neuropathy, besides providing 
histopathological characteristics of the process of injury of these fibers. 
Objectives primary: 
Evaluate in SLE patients with cutaneous involvement (SLE): 
- The possible combination of plasma levels of TNF-, αIFN-1, BAFF (B-cell activator factor) 
and thalidomide levels in the blood and saliva with the therapeutic response and the adverse effects. 
Secondary: 
- Evaluate the possible association between TNF-levels, adjoin molecules, apoptosis markers and 
growth factors in skin biopsy samples with efficacy and adverse effects. 
- Evaluate the presence of fine fiber neuropathy in skin biopsy specimens. 
- Establishment of a pharmacokinetic-pharmacodynamic model of thalidomide dose reduction in 
patients with SLE. 
NUMBER OF INDIVIDUALS 
40 patients with lupus (SLE) and active cutaneous involvement will be included. 
STUDY DESIGN 
A prospective study for a period of 12 months with adult patients with SLE (according to the 
classification criteria of the American College of Rheumatology- ACR) (Hochberg, 1997) with active 
skin lesions at the entrance regardless the use of hydroxychloroquine and/or dapsone and/or prednisone 
and/or immunosuppressants, and who are eligible for the use of thalidomide (see inclusion criteria) 
(Ministry of Health, 2013; System for Thalidomide Education and Prescribing Safety, Zeldis et al., 1999). 
The initial dose of thalidomide will be 100 mg/day for 3 months and reduced to 50 mg/day and 
25 mg/day, at any time depending on the therapeutic response until complete 12 months of treatment. 
All patients will be treated simultaneously with hydroxychloroquine, unless there is 
contraindication for use of this medication. 
The other drugs used for SLE control will be maintained during the study, including prednisone 
(with subsequent gradual dose decrease in case of improvement of skin lesions). 
Patients will be clinically evaluated at baseline, then 1 month, 6 months and 12 months after 
inclusion for: 
- the global activity of the disease through the SLEDAI-2K (Systemic Lupus Erythematosus 
Disease Activity Index 2000) (Gladman et al., 2002; 
- the index of activity and skin severity (CLASI) (Albrecht et al., 2005); 


- possible adverse effects and adherence by the Medication Adherence Self-report Inventory 
(MASRI) applied by a pharmacist not directly involved in patient care (Koneru et al., 2007); 
- quality of life by the visual analog scale and the SF36 questionnaire (Mosca et al., 2010). 
Plasma levels of TNF-, IFN-1, BAFF, transforming growth factor-beta (TGF-β) and thalidomide 
levels in the blood and saliva will be performed at zero time, at 30 days, 3 months, 6 months, 9 months 
and 12 months. 
The clinical evaluation of peripheral neuropathy will be performed at each consultation by 
anamnesis and through THE ENMG at baseline, at 6 months and at 12 months. 
Skin biopsy of an active lesion will be performed at baseline for histological evaluation, 
including the study of immunohistochemistry expression of TNF-, TGFαβ (Kuhn et al., 2010; Yu et al., 
2013) and plasmacytoid and pro-apoptotic dendritic cell markers such as caspase 3, CD25, CD35, CD21, 
CD36, CD68, CD31, detection of IgG, IgM, C3 and T and B cell markers (Bălănescu et al., 2010). 
Skin biopsies of uninjured skin will be obtained from the thigh area, at baseline and at 6 months 
for the fine fiber neuropathy assessment through immunostaining with PGP 9.5 (Birnbaum & Bingham, 
2014). 
- Pharmacokinetic-pharmacodynamic model of use of thalidomide will be performed in two 
groups of 5 patients with different doses (50 mg/day and 100 mg/day). 
Outcomes: evaluations on days 0 and 30. 
a) Improvement in skin symptoms and severity scores [(Cutaneous Lupus Area and Severity 
Activity Index score (CLASI)]. Change in CLASI score assessing the activity/severity criteria from 0 to 
70 will be performed. The reduction represents an improvement in skin injury related to lupus in terms of 
activity and/or severity. CLASI evaluates erythema and scar/hypertrophy/atrophy in 13 anatomical areas. 
Membrane lesions and alopecia are also part of this index. 
b) Cytokine dosage. Plasma levels of TNF-α and IFN-1 will be determined by Luminex MAP 
(EMD Millipore Corporation®). Plasma samples will be stored at -80oC until use. 
TARGET POPULATION 
Inclusion criteria: 
- Adult patients with SLE (according to the classification criteria of the American College of 
Rheumatology) (Hochberg, 1997) with active skin lesions at the entrance to the research despite the use 
of hydroxychloroquine and/or prednisone and/or immunosuppressants who are eligible for thalidomide 
use. 
- Male patients (who agree to use condoms during the entire period of treatment with 
thalidomide and up to 30 days after the end of it, even if they have already undergone vasectomy*) and 
women without fertility potential [i.e., who are in the postmenopausal phase (with amenorrhea > 1 year 
and confirmed by estradiol, FSH and LH levels), or who have undergone tubal ligation or hysterectomy] 
and who agree to the use of this drug according to the Term of Responsibility/Clarification to the 
introduction of thalidomide and each renewal of the prescription (Ministry of Health, 2013). 
- Wish to participate in the study according to the signed TCLE. 
- NORMAL ENMG at study entry. 
Exclusion criteria: 
Alcoholism. 
- The patient's desire not to participate or leave the study at any time (without any harm to their 
care). 


- History of peripheral neuropathy. 
- Previous history of thrombophilia or presence of positive antiphospholipid antibodies. 
- Renal and/or central nervous system and/or hematological activity (hemolytic anemia and/or 
platelet to < 50,000 platelets) concomitantly. 
*Condoms will be provided by researchers (Ministry of Health, 2013). 
 
RELEVANT METHODS 
Laboratory determinations 
HPLC MS/MS: The determination of blood and salivary levels of thalidomide will be performed 
by HPLC and tandem mass spectrometry (Teo et al., 2004). 
- Pharmacokinetic analysis: The pharmacokinetic parameters of thalidomide will be calculated in 
the 24 h dose range based on the total plasma concentration versus time curves. The Winnonlin program, 
version 4.0 (PharsightCorp., Mountain View, California, USA) and the Non-linear mixed effect 
(NONMEN) program will be employed. The area under the plasma concentration versus time curve will 
be calculated in the 24-h dose interval by the trapezoid method on days 0 and 30. The apparent total 
clearance (ClT/F) will be obtained through the equation: ClT/F= Dose/AUC0-24. The apparent volume of 
distribution (Vd/F) will be calculated by the standard software equation. The free concentration of 
thalidomide will be determined by the dosage in saliva. The PK-PD analysis will be performed using as 
pharmacokinetic parameters the total and free plasma concentrations of thalidomide and, as a 
pharmacodynamic parameter, we will use the clinical/laboratory indices obtained by the evaluation of 
SLEDAI-2K (American College of Rheumathology). The PK-PD model will be developed using the non-
linear mixed effect (NONMEM). 
Cytokines. Plasma levels of TNF-α,IFN-1, BAFF and TGF-β will be determined by Luminex 
MAP (EMD Millipore Corporation®) technology. Plasma samples will be stored at -80oC until use. 
thalidomide. Heparinized plasma samples will be immediately transferred to ice bath and 
acidified with citric acid 0.2 M pH 1.5 (v/v), to prevent spontaneous hydrolysis of thalidomide and stored 
at -80ºC until use (Eriksson, 1992). Plasma thalidomide levels will be determined by LC-MS (Bai,2013). 
For this, internal reference standard, deutered thalidomide (d4-thalidomide), will be added to the samples, 
prior to the addition of the mixture of methanol:ammonium acetate (50:50%, v/v) containing 0.2% formic 
acid. The extraction stage will be made by adding a mixture of ether-dichloromethane (3:2) and, after 
centrifugation, the organic phase will be collected and subjected to evaporation. The residue will be 
reconstituted in a mobile phase and injected into the LC-MS/MS system for analysis, using analytical 
column C18 (with particles of 5 μm) and under isocratic elution with mobile phase consisting of 
methanol-ammonium acetate-formic acid (60:40:0.04, v/v/v). Mass spectrometry with electrospray-type 
ionization source will be operated in positive mode. Plasma blank samples will be used for the calibration 
step of the HPLC MS/MS procedure by adding the calibrator (thalidomide) at various concentrations and 
processed similarly. 
Histological analyses 
Histological preparations will be obtained by skin biopsy using a punch of up to 6 mm or spindle 
excision. The skin specimens will then be fragmented. One fragment will be immersed in Michel's 
medium for direct immunofluorescence examination and the other fixed in pH buffered formalin 7.4 for 
histological and immunohistochemistry techniques. 
Skin specimens fixed in formalin will be submitted to routine histological techniques, 
hematoxylin-eosin and periodic Schiff acid. All specimens will be evaluated for confirmation of the 
diagnosis of lupus erythematosus (Ackerman, 1978). 


For immunohistochemistry techniques, the primary antibodies and their origins will be as 
follows: CD3, CD25, CD21, CD35, CD36, CD31, CD68, CD123, TNF-α, TGF-β1, caspase 3 cleaved and 
PGP 9.5. 
Histological sections submitted to immunohistochemistry techniques to demonstrate cellular and 
cytokine elements will be digitized by the Pannoramic Scan (3D Histech, Hungary) scanner to obtain 
virtual histological slides. The reading of the material and capture of the images to be analyzed will be 
carried out with the program Pannoramic Viewer version 1.15.2.21080 (3D Histech, Budapest, Hungary). 
The quantification of immune tagged cellular elements will be performed with the aid of image analysis 
program Image Pro Plus version 4.5.0.29 (MediaCybernetics Inc. , Rockville, MD, USA). 
The quantification of fine nerve fibers will be performed by counting intraepidermal nerve fibers 
per millimeter of dermal epidermal junction and the relationship between area of marked subepidermal 
nerve fibers and total area of papillary dermis examined (Rodrigues Júnior, 2011). 
Safety 
Patients who fit the safety recommendations of the Ministry of Health will be included. 
Clinical (with special attention to symptoms of peripheral polyneuropathy) and laboratory of 
adverse effects (including leukocyte count) will be performed at the entrance into the study and each 
medical consultation. 
The ENMG will be performed to include in the study, at 6 months and at the end of the study. 
If there is no clinical response within 2 months of the use of thalidomide, or in the clinical 
suspicion or enmg of peripheral neuropathy at any time, thalidomide will be suspended (out of the study) 
and follow-up of the patient will be continued in the outpatient clinics of the Rheumatology and 
Dermatology Services of HC-FMUSP. 
WITHDRAWALS 
See item Security. 
ORIGINALITY WITH CARE IMPACT 
There are no studies correlating tissue expression of cytokines with blood and salivary levels of 
thalidomide and its possible role as a predictor of response and adverse effects in patients in skin lesions 
of SLE. In addition, there are no studies on the value of skin biopsy in the diagnosis of thalidomide-
induced neuropathy. 
The expected impact with the availability of blood and salivary dosage of thalidomide in clinical 
practice is that this parameter may be a predictor of clinical response and neuropathy. 
ADVANCES IN MECHANISM 
The expected advance is a better understanding of the role of thalidomide in the expression of 
cytokines in the cutaneous lesion of SLE. 
In addition, we hope to determine whether the mechanism of thalidomide-induced neuropathy 
would target thin nerve fiber injury to histological skin analysis of patients treated with this medication. 
SAMPLE SIZE 
This is a convenience sample. 
STATISTICAL PLAN 
Patients with vs. without therapeutic response to thalidomide and vs. without signs of peripheral 
polyneuropathy will be compared for categorical variables by fisher's exact or chi-square test and, as for 


quantitative variables, by t-Student or Mann-Whitney tests, when applicable. The influence of the 
different variables on disease activity and polyneuropathy will be evaluated by multivariate analysis. 
 
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ICF_001.pdf: 
COVER PAGE  
 
Official Title: Relevance of Monitoring Blood and Salivary Levels of Drugs Used in Rheumatic Autoimmune 
Diseases  
NCT number: NCT03122431  
Date of the document: April 1, 2017 
 
 


HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO 
PAULO-HCFMUSP FREE AND INFORMED CONSENT TERM 
__________________________________________________________________________________________ 
IDENTIFICATION DATA OF THE RESEARCH SUBJECT OR LEGAL GUARDIAN 
1. NAME:.................................................................................................................................................................... 
ID No:........................................................... SEX: M □ F □ 
DATE OF BIRTH: ......../......../................ 
Address:............................................................................................................ Nº: .................. Fit:........................... 
Neighborhood:........................................................................ City:............................................................................ 
Zip code:......................................PHONE:DDD (............) ........................................................................................ 
2. LEGAL GUARDIAN.......................................................................................................................................... 
NATURE (degree of kinship, tutor, healer, etc.):...................................................................................................... 
ID No: ....................................................... SEX: M □ F □ 
DATE OF BIRTH: ......../......../................ 
Address:............................................................................................................. Nº: ................. Fit:........................... 
Neighborhood:........................................................................ City:................................................ ........................... 
Zip code:......................................... PHONE: DDD (............) ................................................................................... 
__________________________________________________________________________________________ 
RESEARCH DATA 
1. TITLE OF THE RESEARCH PROTOCOL: 
THEMATIC PROJECT: EVALUATION OF THE RELEVANCE OF BLOOD LEVELS OF DRUGS USED 
IN RHEUMATIC AUTOIMMUNE DISEASES IN THE MONITORING OF THE SAFETY/EFFICACY OF 
THERAPY, DISEASE ACTIVITY AND ADHERENCE TO THERAPY. 
SUBPROJECT: Prospective, randomized and controlled study comparing the frequency of SLE and 
JSLE activity of patients with a stable dose of HCQ and patients with dose reduction of this drug and its 
correlation with the blood levels of its metabolites. 
2. PRINCIPAL INVESTIGATOR: 
ELOISA BONFA 
Position/ Role: Full Professor of the Discipline of Rheumatology, Faculty of Medicine, HC-FMUSP. 
Registration at Regional Council No. 42,708 
HC-FMUSP Unit: Discipline of Rheumatology of HCFMUSP 
3. RESEARCH RISK ASSESSMENT: 
MINIMUM RISK □ MEDIUM RISK X LOW RISK □ INCREASED risk □ 
4. SEARCH DURATION: 12 months 
 
 


INTRODUCTION: You are being invited to participate in this research because you have a rheumatologic 
disease (systemic lupus erythematosus) and are using hydroxychloroquine as a treatment for your disease. The 
information about the study is detailed in this document and will be explained by the researchers, who will 
answer any questions. We ask you to read this term carefully and feel free to ask any questions that arise to you 
about it. 
STUDY PURPOSE: Hydroxychloroquine is used to treat various autoimmune rheumatologic diseases including 
systemic lupus erythematosus (SLE). Despite regular daily use of 1 tablet (400 mg) to control the inflammatory 
activity of the disease, each patient has a different level in the blood. It is important to know that the 
effectiveness of this drug depends on the drug blood levels. The aim of this research is to measure the level of 
hydroxychloroquine in your blood and saliva when you are taking this drug. What we want to know is whether 
this test that measure the blood level can identify patients who have a good response to the drug or not. 
INCLUSION CRITERIA: Patients with systemic lupus erythematosus (SLE) who will experiences 
withdrawal of hydroxychloroquine may participate in this study. Your participation is voluntary and if you do 
not wish to participate, or want to leave the study, this will not change your follow-up in the Hospital at all. Only 
patients who agree to sign this document will be accepted in the study. 
EXCLUSION CRITERIA: Patients with infection, liver disease, alcohol disease, dialysis, or using some 
drugs that interact with hydroxychloroquine (cimetidine, antiacids, digoxin, aminoglycosides, penicillin, 
neostigmine, pyridostigmine) may not participate in the study. 
PROCEDURES: Upon entering the study, you will make a medical consultation and along with the blood tests 
that are normally done, an additional blood sample will be taken for the hydroxychloroquine dosage. You will be 
selected to use the normal dose of this medication (1 tablet) or a smaller dose (half) for 1 year. During the study, 
all participating patients will be evaluated at the entrance and after 3, 6 and 12 months to determine the effects of 
the medication. In each of these assessments, the patient will undergo medical consultation and blood test 
collection. You will also be asked to spit a little in a tube for saliva collection. 
RISKS AND DISCOMFORTS: The blood test will be collected together with the other tests and a little pain or 
swelling may occur at the site of blood collection. 
VOLUNTARY PARTICIPATION/ EXIT FROM THE STUDY: Your participation is completely voluntary, 
that is, you may decide whether or not to participate in this study and you are also free to leave the study at any 
time, without any prejudice or loss of your rights to follow-up and treatment in the Hospital. If you decide to 
participate, you must sign this informed consent (last sheet) and initial (make a small signature) on all other 
sheets. One way will be provided to you and the other will be saved by the researcher. If you decide not to 
participate in the study, or leave it, this decision will have no influence on your future medical care. 
BENEFITS: The main benefit is whether the drug is doing a good control of the inflammation of your disease. 
There are no financial benefits for you or researchers. You will be informed of all test results. 
CONFIDENTIALITY: This search is confidential and your personal data will not be disclosed in any event to 
others. The secrecy of the study will only be broken in cases where there is legal authorization to do so. The 
results of this study may be presented in congresses or in scientific publications, but their identity will always be 
preserved. The data collected in this study will be used only for this research. 
COMMERCIAL INTERESTS: The study will be free of charge. Since there are no economic or financial 
interests on the part of participating institutions, you, your family or any of the researchers are not expected to 
receive any payment for participating in it. 
 
 


MORE INFORMATION: This consent was approved by the Ethics, Teaching and Research Committee 
(institutional review board) of this institution. If you have additional questions regarding the survey or any other 
questions or symptoms, please contact: 
Prof. Eloisa Bonfa, MD, Dr. Eduardo Ferreira Borba Neto, at the addresses and phone numbers below: 
- Address: Faculdade de Medicina da USP Av. Dr. Arnaldo, No. 455 - 3rd floor, room 3150. Cerqueira César, 
São Paulo - SP. Zip code: 01246-903 
- Phones: (+5511) 3061-7492 / (+5511) 3061-7490 (Secretary of the Rheumatology Division, Faculdade de 
Medicina da USP), (+5511) 2661-6105 (Outpatient Clinic of the Rheumatology Division, Hospital das Clinicas, 
Faculdade de Medicina da USP) 
- Email: reumato@usp.br 
If you have any considerations or doubts about the ethics of this research, please contact the Research Ethics 
Committee (REC) at the following address: Ovídio Pires de Campos Street, 225 - 5th floor. Phones: (+5511) 
2661-7585, 2661-1548, and 2661-1549. E-mail: cappesq.adm@hc.fm.usp.br. 
 
I believe I have been sufficiently informed about the information I have read or read to me, describing the study 
"Prospective, randomized and controlled study comparing the frequency of SLE and JSLE activity of 
patients with a stable dose of HCQ and patients with dose reduction of this drug and its correlation with 
the blood levels of its metabolites". I argued with Prof. Dr. Eloisa Bonfa on my decision to participate in this 
study. They became clear to me what are the purposes of the study, the procedures to be performed, their 
discomforts and risks, the guarantees of confidentiality and permanent clarification. It was also clear that my 
participation is free of expenses and that I have guaranteed access to hospital treatment when necessary. I 
voluntarily agree to participate in this study and may withdraw my consent at any time, before or during it, 
without penalty or loss or loss of any benefit I may have acquired, or in my service on that Service. 
__________________________________________________________________________________________ 
Patient/ legal representative signature Date: ___/____/_____ 
__________________________________________________________________________________________ 
Witness signature Date: ___/____/_____ 
For cases of patients under 18 years old, illiterate, semi-illiterate or with hearing or visual impairment. 
(Only for the researcher) 
I declare that I have appropriately and voluntarily obtained the Informed Consent of this patient or legal 
representative to participate in this study. 
__________________________________________________________________________________________ 
Researcher in charge signature Date: ___/____/_____ 
 
 


 
HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO 
PAULO-HCFMUSP FREE AND INFORMED CONSENT TERM 
__________________________________________________________________________________________ 
IDENTIFICATION DATA OF THE RESEARCH SUBJECT OR LEGAL GUARDIAN 
1. NAME:.................................................................................................................................................................... 
ID No:........................................................... SEX: M □ F □ 
DATE OF BIRTH: ......../......../................ 
Address:............................................................................................................ Nº: .................. Fit:........................... 
Neighborhood:........................................................................ City:............................................................................ 
Zip code:......................................PHONE:DDD (............) ........................................................................................ 
2. LEGAL GUARDIAN.......................................................................................................................................... 
NATURE (degree of kinship, tutor, healer, etc.):...................................................................................................... 
ID No: ....................................................... SEX: M □ F □ 
DATE OF BIRTH: ......../......../................ 
Address:............................................................................................................. Nº: ................. Fit:........................... 
Neighborhood:........................................................................ City:................................................ ........................... 
Zip code:......................................... PHONE: DDD (............) ................................................................................... 
__________________________________________________________________________________________ 
RESEARCH DATA 
1. TITLE OF THE RESEARCH PROTOCOL: 
PROJECT THEME: EVALUATION OF THE RELEVANCE OF BLOOD DRUG LEVELS USED IN 
AUTOIMMUNE 
RHEUMATOLOGICAL 
DISEASES 
IN 
THE 
MONITORING 
OF 
SAFETY 
/ 
EFFECTIVENESS OF THERAPY, DISEASE ACTIVITY AND ADHERENCE TO TREATMENT. 
SUB-PROJECT: TREATMENT EVALUATION OF CUTANEOUS LUPUS ERYTHEMATOSUS WITH 
THALIDOMIDE: CLINICAL, LABORATORY AND HISTOLOGICAL FACTORS ASSOCIATED WITH 
CLINICAL RESPONSE AND ADVERSE EVENTS. 
2. MAIN RESEARCHER: 
ELOISA BONFA 
Position / Function: Full Professor of the Department of Rheumatology, 
Faculdade de Medicina, HCFMUSP. 
Registration with the Regional Council Nº: 42,708 
HC-FMUSP Division: Discipline of Rheumatology, HCFMUSP 
3. RESEARCH RISK ASSESSMENT: 
MINIMUM RISK LOW RISK MEDIUM RISK HIGH RISK 
4. RESEARCH DURATION: 12 months 
 
 


INTRODUCTION: You are being invited to participate in this research because you have lupus erythematosus, 
which, at the moment, is causing skin inflammation, even though you are already receiving medication for lupus, 
such as prednisone and / or hydroxychloroquine and / or dapsone and / or immunosuppressive drugs (which 
lower immunity). The information about the study is detailed in this document and will be explained to you by 
the researchers, who will answer any questions. We ask you to read this term carefully and feel free to ask any 
questions that may arise from it. 
INCLUSION CRITERIA: Adult patients with lupus erythematosus who, at the time of entry into the survey, 
present inflamed lesions on their skin and need treatment can participate in this study. Your participation is 
voluntary and, if you do not wish to participate, or want to leave the study, this will not change your follow-up at 
the Hospital in any way. Only patients who agree to sign this document will be accepted into the study. In 
addition, as Thalidomide is a teratogenic medication (i.e., it can cause fetal malformations), only the following 
patients will be included: 1. Adult men, who agree to use condoms in all sexual relations during the study period 
and up to one month after stopping medication (13 months in total), even if they have already had a vasectomy. 
Note: condoms will be provided free of charge by the researchers. 2. Women who have been �in menopause� 
for more than a year (and we will order blood tests to confirm �menopause�), or who are sure they have had 
their tubes sterilized. 
EXCLUSION CRITERIA: Will not be able to participate in the study: 1. Any woman who is at risk of getting 
pregnant, even if she is taking contraceptive medication or using an IUD. 2. Male patients who do not agree to 
use condoms as explained above. 3. Patients with a history of peripheral neuropathy (disease that affects the 
nerves of the legs and arms), thrombosis, pulmonary embolism or alcoholism. 
PROCEDURES TO BE FOLLOWED: Upon entering the study, each patient must collect blood tests and 
undergo a medical consultation. Next, patients will be subjected to two skin biopsies, one in the area affected 
(inflamed) by the disease and the other in the skin of the thigh (not inflamed). Finally, an exam called 
Electroneuromyography will be performed, which serves to study the functioning of the nerves in the legs and 
arms (in this exam, small electrodes are placed in the arms and legs, to see if the nerves are working properly, 
lasting about half an hour). Skin biopsies are performed under local anesthesia, and a small piece (about 6 
millimeters) is removed for examination. If there is no contraindication (alteration of nerves) in these 
evaluations, we will start treatment with Thalidomide orally at the dose of 1 tablet of 100 mg once daily. Note: 
You will keep taking your current medications for lupus. The dose of thalidomide and prednisone will be 
progressively decreased if the skin lesions improve. During the study, all participating patients will be evaluated 
periodically to determine the effects of the medication and possible side reactions (which are explained below). 
The first reevaluation should take place within two weeks, the second in 1 month and, thereafter, every 3 
months, until completing 1 year of follow-up. In each of these assessments, the patient will undergo medical 
consultation and blood test collection. You will also be asked to spit in a test tube to collect a saliva sample. 
After 6 months of follow-up, patients should undergo a new skin biopsy of the leg and a second 
Electroneuromyography. Finally, after completing the 12 months of the study, a third Electroneuromyography 
will be performed. Note: If there is no clinical response within 2 months of using thalidomide, this medication 
will be suspended (you will no longer participate in this research) 
and patient follow-up will be continued according to the routine of the Rheumatology and Dermatology 
Divisions of HCFMUSP. 
RISKS AND DISCOMFORT: Thalidomide is considered a very effective medication for the treatment of skin 
lesions caused by lupus, which has been used for many years in the treatment of this disease. Despite this, some 
patients may develop side effects with the use of this medication, with peripheral neuropathy being one of the 
most common. Pain, tingling and / or loss of sensation in legs and arms characterize this adverse event, which 
can occur in about 25% of patients. When this happens, treatment with Thalidomide should be stopped as soon 
as possible, as this problem may be irreversible. For this reason, you will be reassessed frequently by the 
researching doctor and will also perform Electroneuromyography periodically and you can also contact us on the 
phones below. If you develop symptoms or signs of neuropathy on this exam, we will immediately withdrawal 


thalidomide and you will continue to be followed up in the lupus outpatient clinic and medications will be used 
as per the routine of that clinic. In addition, to reduce the possible side effects, we will use low doses of the 
medication (maximum 1 tablet of 100 mg daily) (and this dose will be reduced, as soon as skin lesions are better, 
to half a tablet per day and then for a quarter of a pill a day) and for a maximum of 12 months (as peripheral 
neuropathy, when it appears, is usually after 1 year of Thalidomide`s treatment). Other possible side effects are: 
drowsiness, constipation, headache, bloating, dizziness, increased appetite, nausea, itching and weight gain. To 
reduce sleepiness, thalidomide should be taken at night. Skin biopsies and Electroneuromyography can also 
cause some pain or discomfort. 
VOLUNTARY PARTICIPATION / STUDY EXIT: Your participation is completely voluntary, i.e., you can 
decide to participate or not in this study and you are also free to leave the study at any time, without any loss or 
loss of your follow-up and treatment rights at the Hospital. If you decide to participate, you must sign this 
informed consent (last sheet) and initials (make a small signature) on all other sheets. One copy will be provided 
to you and the researcher will keep the other. If you decide not to participate in or leave the study, that decision 
will have no influence on your future medical care. 
BENEFITS: As already mentioned above, thalidomide is a very effective medication in the treatment of skin 
lesions caused by lupus erythematosus. Therefore, the main benefit will be the possible improvement of the 
lesions on your skin with the use of this medicine. There are no financial benefits either for you or for the 
researchers. You will be informed of all test results. 
CONFIDENTIALITY: This survey is confidential and your personal data will not be revealed, under any 
circumstances, to other people. The confidentiality of the study will only be broken in cases where there is legal 
permission to do so. The results of the study may be presented at congresses or scientific publications, but your 
identity will always be preserved. The data collected in this study will be used only for this research. 
COMMERCIAL INTERESTS: The study will be free of charge. As there are no economic or financial 
interests on the part of the participating institutions, you, your family or any of the researchers are not expected 
to receive any payment for participating in it. 
 
 


MORE INFORMATION: This consent was approved by the Ethics, Teaching and Research Committee 
(institutional review board) of this institution. If you have additional questions regarding the survey or any other 
questions or symptoms, please contact: 
Prof. Eloisa Bonfa, MD; Emily Figueiredo V. Neves, MD or Sandra G. Pasoto, MD at 
the addresses and phone numbers below: 
- Address: Faculdade de Medicina da USP Av. Dr. Arnaldo, No. 455 - 3rd floor, room 3150. Cerqueira César, 
São 
Paulo - SP. Zip code: 01246-903 
- Phones: (+5511) 3061-7492 / (+5511) 3061-7490 (Secretary of the Rheumatology Division, Faculdade de 
Medicina da USP), (+5511) 2661-6105 (Outpatient Clinic of the Rheumatology Division, Hospital das Clinicas, 
Faculdade de Medicina da USP) 
- Email: reumato@usp.br 
If you have any considerations or doubts about the ethics of this research, please contact the Research Ethics 
Committee (REC) at the following address: Ovídio Pires de Campos Street, 225 - 5th floor. Phones: (+5511) 
2661-7585, 2661-1548, and 2661-1549. E-mail: cappesq.adm@hc.fm.usp.br. 
 
I believe I have been sufficiently informed about the report that I have read or that have been read to me, 
describing the study �TREATMENT EVALUATION OF CUTANEOUS LUPUS ERYTHEMATOSUS 
WITH 
THALIDOMIDE: 
CLINICAL, 
LABORATORY 
AND 
HISTOLOGICAL 
FACTORS 
ASSOCIATED WITH CLINICAL RESPONSE AND ADVERSE EVENTS�. I discussed with Prof Eloisa 
Bonfa, MD about my decision to participate in this study. It became clear to me what the purposes of the study 
are, the procedures 
to be performed, their discomforts and risks, the guarantees of confidentiality and permanent clarifications. It 
was also clear that my participation is free of charge and that I am guaranteed access to hospital treatment when 
necessary. I voluntarily agree to participate in this study and I will be able to withdraw my consent at any time, 
before or during the same, without penalties or prejudice or loss of any benefit that I may have acquired, or in 
my follow-up in that Service. 
__________________________________________________________________________________________ 
Patient/ legal representative signature Date: ___/____/_____ 
__________________________________________________________________________________________ 
Witness signature Date: ___/____/_____ 
For cases of patients under 18 years old, illiterate, semi-illiterate or with hearing or visual impairment. 
(Only for the researcher) 
I declare that I have appropriately and voluntarily obtained the Informed Consent of this patient or legal 
representative to participate in this study. 
__________________________________________________________________________________________ 
Researcher in charge signature Date: ___/____/_____