The Effect of Rosuvastatin on Immune Activation Markers in Treatment-naïve HIV-Positive Patients; a Randomized Placebo-Controlled Trial

Despite the enormous success of highly active antiretroviral therapy (HAART) in
the Western world, life expectancy of HIV infected patients still lags behind
the general population. Nowadays, excess mortality is largely due to non-AIDS
defining diseases such as cardiovascular diseases and non-AIDS defining
malignancies. Many factors can be held responsible for this increase in
non-AIDS defining morbidity and mortality, such as smoking, toxicity of HAART,
or socioeconomic factors. Recently, a number of studies have indicated that
ongoing immune activation may play an important role in the pathogenesis of
these non-AIDS defining conditions. Early in HIV infection, mucosa-associated
lymph nodes in the gut are largely destroyed. Decreased host defense at the
level of the gut mucosa with ensuing low-grade translocation of bacteria and
bacterial products such as lipopolysaccharide (LPS) contributes to ongoing
immune activation in HIV infected patients. In a recent small study, microbial
translocation was associated with sustained failure in CD4+ T-cell
reconstitution in HIV infected patients on HAART. LPS induces a broad
activation of macrophages and circulating monocytes with increases in the
production of proinflammatory cytokines and activation of coagulation.
Circulating LPS was significantly increased in chronically HIV-infected
individuals and in pathogenic simian immunodeficiency virus (SIV)-infected
rhesus macaques, and interestingly also in non-HIV infected patients with
idiopathic CD4 lymphopenia. In a recent nested case control study derived from
the SMART study, mortality of HIV patients correlated strongly with increased
concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6) and
D-dimer, a marker of coagulation activation. It has been postulated that
modulating this residual inflammatory activation in HIV infected patients
receiving HAART may be beneficial for countering the increased risk of non-AIDS
defining conditions.
After binding to lipopolysaccharide binding protein (LBP), LPS stimulates CD14
positive monocytes and macrophages through its receptor Toll-like receptor 4
(TLR4). This complex process leads to intracellular signal transduction and the
subsequent production of pro-inflammatory cytokines such as interleukin-1
(IL-1), tumor necrosis factor alpha (TNF alpha) and IL-6. HIV infection is
associated with increased TLR expression and responsiveness. Polymorphisms in
the TLR4 gene have been described to influence signal transduction and thus
modulate the inflammatory cascade. In HIV patients, the TLR4 Asp299Gly
polymorphism seems to increase the risk of active tuberculosis.

Statins are potent lipid lowering agents that are extensively used in the
primary and secondary prevention of atherosclerotic diseases. They are
generally well tolerated. There is an ongoing debate on the possible
pleiotropic effects of statins. A number of studies have shown
anti-inflammatory effects of statins and two recent studies found that statins
have an inhibitory effect on the TLR4-mediated inflammatory response.
Moreover, retrospective studies found that prior statin use was associated with
improved outcome of pneumonia, and this finding was recently confirmed in one
prospective study. In these studies, C-reactive protein (CRP) levels were lower
in statin-treated patients. In the recently published JUPITER study, high-dose
rosuvastatin resulted in significant decreases in vascular events in patients
with slightly elevated high sensitivity CRP (hsCRP) levels without further risk
factors. Rosuvastatin caused a significant decrease in hsCRP levels compared to
placebo-treated patients, indicating an anti-inflammatory effect. A recent
study showed that high-dose atorvastatin decreased cellular immune activation
markers in treatment-naïve HIV patients without affecting HIV viral load.
Taken together, these studies point to an antiinflammatory effect of statins
that does not impair host response to infections, and that may be explained by
influencing TLR4-mediated inflammatory responses. In the present study, we want
to investigate the effect of high-dose rosuvastatin on markers of immune
activation in treatment-naïve HIV patients. Immune activation can be expected
to be more pronounced in treatment-naïve patients than in patients on HAART. A
number of markers will be studied that represent subsequent steps in the
process of immune activation: circulating LPS (LAL-assay),TLR-messenger RNA in
whole blood, the pro-inflammatory cytokine IL-6, D-dimer, hsCRP, markers of
T-cell activation (CD38 and HLA-DR expression on CD4 and CD8 cells), and
microparticles and endogenous thrombin potential as indicators of endothelial
damage. Also, TLR4 polymorphisms will be determined and related to the levels
of immune activation. Since both immune activation and statin use may cause
fatigue, a standardized questionnaire (the EuroQol-6D questionnaire) estimating
the quality of life will be used. Other parameters studied will include CD4-
and CD8-positive T lymphocytes, HIV viral load, total cholesterol and
cholesterolsubfractions, apolipoprotein

Primary Objective: to investigate the effect of rosuvastatin 20 mg qd on
subsequent immune activation markers in treatment-naïve HIV-patients:
circulating LPS (LAL assay), TLR mRNA expression in whole blood, circulating
IL-6, D-dimer, hsCRP, CD38 and HLA-DR expression on lymphocytes, and
microparticles and endogenous thrombin potential as indicators of endothelial
damage.

Secondary Objective: to investigate the effect of rosuvastatin 20 mg qd on HIV
viral load, CD4 cell count, total cholesterol and cholesterol subfractions,
ApoB/ApoA1 ratio, CK, liver enzymes, renal function, and complete blood count
as well as on markers of quality of life measured with the EuroQol-6D
questionnaire in treatment-naïve HIV-patients throughout the study period.

This is a double blind, randomized, placebo-controlled therapeutic intervention
study with a cross-over design. The patients will be assigned to random groups:
one receives rosuvastatin 20 mg daily, and the other receives placebo.
Patients remain in their treatment groups for 8 weeks. After 8 weeks all
patients, in both study groups, will be required to discontinue all
study-related medications for 4 weeks. After that period, patients receiving
placebo will take rosuvastatin, and vice versa. The study will proceed for
another 8 weeks, followed by a period of stopping study-related medications and
patients being observed for 4 weeks. Throughout the study, patients will have
regularly scheduled visits at the clinic every 4 weeks. At those visits there
will be collection of blood samples, assessments of symptoms, physical
examinations, and questionnaires to complete.
To obtain reliable reference values for the experimental laboratory
investigations, ten healthy, age and sex-matched volunteers will be asked to
donate blood samples twice for all the parameters investigated in the patients.
Laboratory investigations include complete blood count, sodium, potassium,
creatinine, BUN, glucose, ALAT, ASAT, LDH, alkaline phosphatase, gammaGT,
bilirubin, CK, total cholesterol, HDL-, LDL-cholesterol, triglycerides
ApoB/ApoA1 ratio. Moreover, circulating IL-6 , D-dimer, microparticles and
endogenous thrombin potential, hsCRP, LPS (LAL-assay), TLRmRNA and TLR4
polymorphism, T-cell activation markers (CD 38; HLA-DR), CD4- and CD8-cell
count, and HIV viral load are measured.
Blood is drawn for baseline measurements on28 days and one day before the start
of study medication. After the start of study medication, blood is drawn on
days 28, 56, 84, 112, 140 and 168 (weeks 4, 8, 12, 16, 20 and 24). The study
medication consists of rosuvastatin 20 mg daily.
To ensure the double blind character of the study, the treating physician will
not be informed about the results of blood tests during the study period. A
local supervising committee will weekly review laboratory results throughout
the study period, and will intervene in study participation of individual
patients whenever serious adverse events occur.

rosuvastatin 20 mg qd, or placebo

Burden: the main burden for the patients concerns the fact that they will have
to visit the outpatient clinic every 4 weeks for a total of 24 weeks. During
these visits, blood will be drawn, the treating physician will be visited, en
three times a questionaire will have to be filled in. The aliquot of blood
drawn will not exceed 50 ml per visit, which cannot be considered a burden or
risk.

Risk for the patients concerns the possible side effect of rosuvastatin. The
investigators will closely monitor the occurence of any side effect by history
taking, controlling CK and liver enzymes, and by evaluating questionaires.

Burden and risk are considered to be acceptable in light of the potential
benefit to the patient of finding a therapeutic approach that could possibly
postpone the moment to start the patient on HAART and/or partly neutralize the
increased cardiovascular rist of HIV-infeced patients. Moreover, rosuvastatin
is a widely used registered drug, that is taken by tens of thousands of
patients without major side effects.