Pilot Study to Evaluate Effects of Atorvastatin on Monocyte Activation in HAART-treated HIV Infected Individuals
Phase: PHASE4
Conditions: HIV Dementia
Sponsor: University of Pennsylvania

TITLE OF STUDY 
 
 
Statin modulation of monocyte activation: a pilot study for potential use 
in NeuroAIDS 
 
 
 
IRB PROTOCOL # 
 
812196 
 
 
 
NCT # 
 
NCT01263938 
 
 
Document Date: October 5, 2011 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

                                                        Study Protocol 
Abstract  
Neurological complications of HIV infection are common and persist even in treated 
people. Monocytes play a major role in the initiation and progression of neurocognitive 
dysfunction. Monocytes get activated and migrate to the brain where they produce 
soluble factors that cause inflammation. They also carry virus to the brain. During the 
course of the disease several factors contribute to immune activation (including monocyte 
activation), many of which play a role in progression of neurocognitive disease. We 
hypothesize that if immune activation is controlled in these chronic HIV-infected 
individuals, progression to neurological injury can be stopped. Therefore, we propose to 
study the effects of a statin drug with anti-inflammatory functions, on the monocyte 
activation status of these individuals. Eligibility: Subjects enrolled in the study will be on 
anti-HIV therapy, viral load less than 200 copies/ml for more than 6 months. 
Intervention: 1) Subjects on PI-based HAART will be administered 10 mg/day X 2weeks 
and then 20 mg/day; subjects on non-PI-based HAART will receive 20 mg/day X 2 
weeks and then 40 mg/day Atorvastatin. 2) At specific time points during the study 
period blood will be drawn to study various immune and virological parameters.  
Objectives  
Our objectives are based on the hypothesis that statin treatment will reduce the 
inflammatory monocyte phenotype and negatively regulate the inflammatory cytokines 
that have been linked to neuropathogenesis and may have potential a adjunctive therapy 
in HIV-associated neurological disease. In this pilot project we propose to 1) Determine 
the effects of Atorvastatin on peripheral blood monocyte populations in a 12-week plot 
study in chronically HIV-infected people. 2) Determine the relationship between changes 
in monocyte phenotype following Atorvastatin treatment, and soluble markers of 
activation/ inflammation linked to neuropathogenesis, as well as activation status of T 
cells.  
Primary outcome variable(s)  
1) Peripheral blood monocyte population and surface markers: CD16, CD163, and CCR2. 
2) Plasma levels of monocyte-associated inflammatory cytokines and chemokines: MCP-
1, soluble CD163, and soluble CD14  
Background  
Two principal populations of human peripheral blood monocytes are recognized. The 
CD14+CD16- population comprises ~90% of the total monocytes in a healthy person and 
a CD14+CD16+ population the remaining 10% of the monocytes. The latter are also 
called the pro-inflammatory / non-classical subset, based on expression of inflammatory 
cytokines such as TNF-, IL-1 and IL-6, and enhanced antigen presentation capacity. 

Several lines of evidence suggest that this inflammatory monocyte population, 
characterized by CD16 expression, plays a particularly important role in the pathogenesis 
of HIV-associated neurological disease including HIV encephalopathy (HIVE). This 
population is increased in chronic HIV-1 disease, and in particular is expanded in people 
with HIV encephalopathy or dementia. This monocyte population has a greater 
propensity for invasion into tissues. Furthermore several reports indicate that even though 
monocytes are not generally considered major reservoirs of infection in blood, there is 
preferential infection of this particular subset by the virus, and these cells may help virus 
traffic into tissues including brain. Finally HIVE is associated pathologically with the 
accumulation in brain of monocyte/macrophage cells that express CD16, CD163 and 
several other activation markers, and it is believed that release of both inflammatory 
mediators from these macrophage lineage cells, as well as viral products from infected 
cells within this population, are mainly responsible for the neuronal injury that occurs . 
Since CD163 is not normally expressed by resident microglia in normal brain tissue, the 
numerous CD163+ cells observed in SIVE / HIVE suggest emigration of monocytes into 
the CNS from the peripheral blood. Increased CD163+/CD16+ monocyte/ macrophages 
in the CNS have been associated with increased frequency of CD163+/CD16+ monocytes 
in the periphery and correlates significantly with increased viral load and CD4+ T-cell 
decline. Monocyte neuroinvasion is preceded by a dramatic increase in the number of 
peripheral blood monocytes as well as peak levels of viral load and proinflammatory 
mediators in plasma. These observations highlight the importance of this monocyte 
subset as a link between HIV / SIV CNS disease and peripheral immune pathogenesis 
and suggest that events in the periphery are central to the development of HIV-associated 
neuropathogenesis. Recent findings suggest a role for elevated LPS levels, a result of 
translocation of bacterial products from a leaky gut, in triggering monocyte activation 
during HIV infection, thereby contributing to HIV-dementia pathogenesis via trafficking 
of activated monocytes into brain (6, 70). Furthermore, elevated LPS levels compromise 
the integrity of the blood brain barrier which enhances monocyte transmigration into the 
CNS . LPS triggers the release of many inflammatory cytokines and other effectors 
including IL-1, IFN-, TNF-, IL-8, along with platelet activating factor and arachidonic 
acid metabolites and is a likely cause of immune activation in HIV infection. Of note, 
LPS up-regulates the expression of MCP-1, which is essential for the recruitment of 
monocytes into sites of inflammation. This is especially relevant to HIV-
neuropathogenesis since elevated MCP-1 is associated with increased risk of progressing 
to HIV-dementia. Interestingly CD14 +CD16+ monocytes are a major source of MCP-1 
and other proinflammatory proteins that recruit T cells and additional monocytes into the 
CNS. In addition, CD14+CD16+ monocytes promote highly efficient viral replication on 
differentiation into macrophages and also activate resting T cells for HIV-1 infection. It 
has been proposed that T-cell activation in HIV disease is an indirect consequence of 
monocyte activation and is of significance since it suggests that altered interactions 
between monocytes and T cells may contribute to dysregulated responses, which is a 
feature of HIV infection. Since HIV- dementia is partly a result of an inflammatory 
environment in the periphery over an extended period of time, it is important to identify 

potential treatment methods of reducing systemic immune activation / inflammation in 
the setting of chronic HIV disease. In recent years since the advent of HAART, though 
the incidence of HAD has decreased, its prevalence has increased, leading to efforts at 
identifying adjunctive treatment options. Several studies report the neuroprotective 
effects of statins in neurodegenerative diseases like Alzheimer's and Parkinson's disease, 
and further trials are currently underway to test its effects in multiple sclerosis treatment. 
Since statins are well tolerated and have relatively few side effects, they may be 
considered for treatment of neurological complications in HIV infection. Statins (HMG-
CoA reductase inhibitors), are cholesterol lowering drugs with pleiotropic 
immunomodulatory / anti-inflammatory properties which are independent of their 
cholesterol lowering ability. These effects include inhibition of cytokine / chemokine 
expression, inhibition of T- cell activation, inhibition of leukocyte adhesion and down-
regulation of MHC II, to name a few. Preliminary animal studies have demonstrated the 
ability of statins to enhance bacterial clearance and attenuate pro-inflammatory responses 
to LPS . Other studies suggest that statins can attenuate the pro- inflammatory effects of 
endotoxin in astrocytes, microglia and endothelial cells. Based on in vitro data, another 
proposed effect of statins might be to lower viral load -an important factor in monocyte 
activation, although the limited studies that have evaluated that in vivo have reached 
different conclusions. Interestingly, activated CD16+ monocytes are also associated with 
an increased risk of coronary artery disease (CAD) and macrophages (in the vascular 
wall) are believed to play an important role in vascular injury, but whether statins alter 
the CD16+ population of monocytes as part of their well documented beneficial effects in 
CAD, has received little attention. Evidence of the immunomodulatory / anti-
inflammatory effects of statins on the monocyte function is limited. No in vivo studies 
have yet been carried out to assess the effects of statins on the pro-inflammatory 
monocyte sub-population in chronic HIV disease. Moreover, it is unclear how 
modulation of the monocyte function by statins correlates with T cell responses. Recent 
evidence of a positive correlation between elevated plasma LPS levels, immune 
activation, disease progression, monocyte activation and CD4+ T cell depletion, suggests 
that all these parameters are inter-dependent. The inflammatory phenotype of monocytes 
makes them a potential therapeutic target for reducing disease burden. We hypothesize 
that treatment of chronic HIV-1 positive individuals with statins, would modulate 
monocytes from a pro- inflammatory towards an anti-inflammatory phenotype 
functionally by decreasing the number of CD14 +CD16+CD163+ monocytes. Statin 
treatment would also strongly reduce plasma MCP-1 levels (along with other anti-
inflammatory effects), as demonstrated in our preliminary data and by others and would 
reduce systemic immune activation, which has direct implications for neuroAIDS 
pathogenesis. We also hypothesize that statin treatment would result in decreased viral 
load which would also result in decreased immune activation. In the long term, decreased 
immune activation as a result of statin treatment, would slow down the progression to 
AIDS and decrease the prevalence of HAD.  
 

Design  
This is an open label pilot to study the monocyte-related anti-inflammatory effects of 
Atorvastatin drug in chronic HIV-1 infected individuals who are on HAART. On-
treatment data was compared with pre-treatment data.  
Study duration  
The duration of the study is one year. The time duration of treatment is 12 weeks, with a 
total duration of subject participation of 18 weeks.  
Characteristics of the Study Population  
Target population  
We will enroll chronic HIV infected individuals who are on anti-retroviral HAART 
therapy (without changes in HAART drug combination for at least 3 months). 
Additionally, they will not be on any prior statin therapy. These individuals will have 
plasma viral loads less than 200copies/ml for 6 months. The effects of Atorvastatin 
treatment will be determined by comparing the various parameters during the treatment 
period to baseline levels.  
Subjects at UPenn  
15  
Subjects at Sites Other than UPenn  
0  
Accrual  
We will have access to the study subjects through the CFAR Clinical core. The initial 
proposed sample size was 20 subjects based on having sufficient power to detect a 
change in each of the outcome variables from baseline to week 12. Because of the 
modifications in the protocol involving: a) the use of Atorvastatin rather than 
Simvastatin, and b) the inclusion of hs-CRP as a screening test, both factors leading to 
increased costs, we have decided to decrease the total enrollment to 15 subjects. We 
believe this will not negatively impact the value of the study as this pilot is meant to 
generate data for future definitive studies.  
Key inclusion criteria  
1. Chronic HIV-1 infected individuals on HAART (no change in drug combination for at 
least 3 months prior to enrollment in the study), and be willing to continue on therapy for 

the duration of the study.  
2. HIV viral load less than 200 copies/ml for more than 6 months.  
3. hs-CRP levels above the upper limit of normal (3 mg/L).  
3. Willingness to use a method of contraception during the study period.  
4. Willingness to have blood drawn.  
5. If female, willingness to undergo pregnancy testing on a monthly basis and are not 
breastfeeding.  
6. Ability to understand and willingness to sign the informed consent. 
Key exclusion criteria  
1. Concomitant use of fibric acid derivatives or other lipid lowering agents including 
patients on statins and Ezetimibe. 
2. Use of any anti-inflammatory drugs (OTC or prescription) on a daily basis.  
3. Pregnancy or breastfeeding.  
4. Active drug use or alcohol abuse/dependence, which in the opinion of the investigators 
will interfere with the patient's ability to participate in the study.  
5. Allergy or hypersensitivity to Atorvastatin or any of its components.  
6. History of myositis or rhabdomyolysis with use of any statins.  
7. Patients who are on concurrent immunomodulatory agents, including systemic 
corticosteroids (including nasal or inhaled steroids) will be ineligible for 3 months after 
completion of therapy with the immunomodulating agents.  
8. History of inflammatory muscle disease such as poly or dermatomyositis.  
9. Serious intercurrent illness requiring systemic treatment and/or hospitalization within 
30 days of entry.  
10. Evidence of active opportunistic infections requiring treatment or neoplasms that 
require chemotherapy during the study period. 
11. Creatinine phosphokinase elevations (CPK) greater than 3 times the upper limit of 
normal.  
12. Known active liver disease or AST/ALT greater than 2x the upper limit of normal.  

13. Renal insufficiency indicated by serum creatinine 2 mg/dl.  
14. Absolute neutrophil count (ANC) 1000/mm3, hemoglobin less than 10.0 g/dL for 
males or 9.0 g/dL for females, platelet count 100,000/mm. 
15. HCV co-infection.  
16. Ischemic heart disease.  
17. NYHA Class III or IV congestive heart failure.  
Subject recruitment  
The CFAR Clinical Core will assist us in the identification and recruitment of subjects 
required for the study from the Clinical Core Cohort.  
Subject compensation  
Yes, subjects will be compensated a total of $300 by the end of the study. They will be 
paid $5 at the screening visit. Following enrollment in the study, they will be paid $20 at 
each of the subsequent study visits in cash and the balance of $195 will be paid at the end 
of the study by check.  
Procedures  
SAMPLE COLLECTION: Informed consent will be obtained from all individuals 
enrolled in the study. As shown in the ‘study schedule’, subjects will undergo screening 
tests for enrollment into the study at week -2. The treatment period will be for a total of 
three months, blood samples will be collected before starting statin treatment on day 0, 
and at 2 weeks, 6 weeks and 12 weeks through the duration of the treatment. Finally, 
blood samples will be collected 4 weeks after the end of treatment (washout period).  
 
 
 
 
 
 
 

 
 
 
 
 
Study Schedule 
  
  
T=-2wk 
T=0 
T=2 wk 
T=6 wk 
T=12 wk 
T=16 wk 
  
Atorvastatin 
treatment: 
  
begin 
+ 
+ 
end 
  
  
Sample type: 
screen 
baseline 
treatment treatment treatment Wash-out 
Safety labs 
CBC  
X 
 
X 
X 
X 
 
  
Metabolic panel (Cr, 
AST/ALT) & CPK 
X 
 
X 
X 
X 
 
Treatment 
monitoring labs 
Lipid panel (non-
fasting) 
X 
 
X 
 
X 
 
Clinical outcome 
labs (plasma) 
D-dimer 
 
X 
X 
 
X 
 
  
hsCRP 
X 
 
X 
 
X 
 
Lymphocyte 
subset panel 
CD3/CD4/CD8 
X 
X 
 
 
X 
 
HIV VL 
HIV viral load 
 
X 
 
 
X 
 
Urine Pregnancy   
X 
 
X 
X 
X 
 
PBMC FACS 
CD14/16/163; 
CD3/4/8; 
CCR5/CXCR4/CX3C
R1/CCR2; 
CD25/38/DR;TF 
 
X 
 
X 
X 
X 
Monocyte 
transcriptiome 
analysis 
Purified monocyte 
RNA (store, run if 
funds permit) 
 
X 
 
X 
X 
X 
Plasma luminex 
Soluble mediators 
(store)* 
X 
X 
X 
X 
X 
X 
Plasma ELISA 
sCD14/sCD163 
/Tissue Factor 
(store)* 
X 
X 
X 
X 
X 
X 
 
 
  
  
  
  
  
  
Total vol bld 
/time point 
  
21.5 
100 
26 
96.5 
111.5 
85 
 
 
 
 

Analysis Plan  
STATISTICAL ANALYSIS: Because this is a pilot study with a small sample size, any 
statistical test applied to the data will only be able to detect large changes or trends over 
time. Instead, our main goal will be to look for trends occurring in the data and to 
estimate means needed for accurate sample size calculations for a full study. The data 
will be analyzed as follows.  
DESCRIPTIVE STATISTICS: Standard descriptive statistics will be used to summarize 
the sample characteristics at baseline. For continuous variables means, medians and 
ranges will be calculated, while frequencies and percentages will be calculated for 
categorical variables. The outcome variables (marker levels, viral loads, and cell counts) 
will also be summarized at baseline and each follow-up time point. In addition, the 
change in outcomes from baseline to week 12 will be calculated for each subject and 
statistically described. Further, graphical tests, such as stem-and-leaf plots, will be used 
to test for normality in the continuous variables, and guide in the choice of transformation 
where warranted. 
 CHANGES IN OUTCOME: The change in the outcomes from baseline to week 12 will 
be compared using paired t-tests (normally distributed) or Wilcoxon rank-sum tests (non-
normal). Bivariate comparisons will be used to determine if the changes in the outcomes 
are associated with any of the baseline demographic or clinical characteristics. For 
categorical variables, t-tests, ANOVA, or non-parametric equivalents will be used to 
compare changes in outcomes between groups, while correlation coefficients will be 
calculated with continuous characteristic variables. Also, since outcome variables are 
taken at multiple times, they will be graphed to visually inspect for trends across times 
via Spaghetti plots. Longitudinal data analysis methods will be applied to test for changes 
over time, and if those changes are affected by any of the subject characteristic variables.