PET/CT Imaging Companion Study To ACTG A5314

HIV infection is associated with a substantially increased risk of CVD that is not fully
accounted for by traditional risk factors. Several lines of evidence suggest that chronic
immune cell activation may be complicit. In support of this concept, we recently used
18F-FDG-PET/CT to demonstrate that HIV-infected individuals have increased arterial
inflammation compared to non-HIV, FRS-matched controls and that the degree of arterial
inflammation is related to markers of monocyte activation. Further, we and others have shown
that increased arterial FDG uptake predicts future CVD events in non-HIV cohorts. Together,
these observations support the concept that targeting arterial inflammation may provide
benefit for HIV infected individuals.

The NHLBI recently provided funding for a clinical trial targeting the excess inflammation in
HIV. The "Parent Study" is being performed as a collaboration with the National Institute of
Allergy and Infectious Diseases (NIAID) AIDS Clinical Trials Group and the NHLBI. That
randomized, double-blind, placebo-controlled trial will assess whether 24-week treatment with
very low-dose methotrexate (LDMTX): i) is safe, ii) reduces levels of circulating
inflammatory biomarkers and activated immune cells and iii) improves brachial artery
reactivity (BART). However, neither the biomarkers nor the BART studies measured as part of
Parent Study will report on atherosclerotic inflammation, (the desired pathobiological target
of LDMTX therapy in HIV). As such, the direct evaluation of arterial inflammation would
substantially enhance the scientific value of the Parent Study.

Atherosclerotic inflammation can be non-invasively and reproducibly measured with FDG-PET/CT
imaging, a well-validated quantitative technique that can sensitively detect changes in
atherosclerotic inflammation. FDG-PET/CT imaging has been employed in several multi-center
trials to measure changes in arterial inflammation in response to anti-inflammatory
treatments. Accordingly, we propose a time sensitive ancillary imaging study to determine if
treating virologically suppressed, HIV-infected individuals with LDMTX reduces
atherosclerotic inflammation, assessed by FDG-PET/CT. Our central hypothesis is that
persistent immune cell activation results in chronic arterial inflammation, which
subsequently contributes to the CVD risk observed in HIV. This fully integrated ancillary
study would, in a subset of patients enrolled in the parent trial: (i) assess the impact of
LDMTX on arterial inflammation, and (ii) identify the immune cell subtypes whose changes
(with LDMTX) are associated with changes in arterial inflammation. Accordingly, the proposed
ancillary study would provide unique and highly complementary information that would greatly
increase the knowledge and mechanistic insights gained from the Parent Study. The ancillary
study has two specific aims:

Specific Aim 1: To determine the impact of anti-inflammatory treatment with LDMTX on arterial
inflammation, as assessed by FDG-PET/CT imaging, in virally suppressed HIV-infected
individuals. [Primary and secondary outcomes]

Knowledge Gap/Need: The parent study does not provide for assessment of the target pathology
(arterial inflammation) hence insights regarding effects of LDMTX treatment on arterial
inflammation are needed.

Hypothesis 1: LDMTX therapy will reduce arterial inflammation (to a greater extent than
placebo).

To test hypothesis 1: We will determine in 91 patients (a subset of the parent study) if
arterial inflammation (measured with PET/CT at 0 and 24 weeks) is reduced by LDMTX (relative
to placebo).

Specific Aim 2: To evaluate the cellular and biochemical mediators associated with arterial
inflammation in HIV and to explore the potential mechanism of the effect of LDMTX therapy on
arterial inflammation in HIV. [Exploratory outcomes]

Knowledge Gap/Need: The parent study, on its own, does not provide the tools to directly
evaluate arterial inflammation in HIV. Evaluation of the relationships between circulating
inflammatory mediators and arterial inflammation in the context of this study would yeild
mechanistic insights regarding atherosclerotic disease in HIV and provide a greater
understanding of the effect of LDMTX on arterial inflammation in HIV.

Hypothesis 2.1:At baseline, arterial inflammation is closely associated with measures of
activated immune cells (CD14+/CD16+ monocytes). Additional relationships might be seen
between arterial inflammation and other cellular subsets (e.g. T-cells), inflammatory
cytokines, and endothelial function.

Hypothesis 2.2: Post-treatment changes in arterial inflammation are positively correlated
with changes in measures of activated immune cells (especially activated monocytes).

To test these hypotheses: We will evaluate if arterial inflammation (by PET) correlates with
inflammatory cell activation and other measured biomarkers separately. We will then compare
the strength of the associations and test their independence. The evaluation of associations
will be performed: comparing baseline measurements (SA 2.1), and comparing post-randomization
changes (SA2.2).

We expect that LDMTX therapy will improve arterial inflammation and that this mechanistic,
proof-of-concept study will demonstrate the importance of inflammation and immune activation
in HIV. This would thus form the basis for event-driven trials to evaluate whether
anti-inflammatory strategies reduce CVD risk in individuals with treated HIV infection.
Accordingly, the study has the potential to shift the paradigm in the approach to treating
atherosclerosis in HIV-infected individuals, and potentially in other populations as well.

1. Inclusion Criteria:

1.1 HIV-1 infection, documented by any licensed rapid HIV test or HIV enzyme or
chemiluminescence immunoassay (E/CIA) test kit at any time prior to study entry and
confirmed by a licensed Western blot or a second antibody test by a method other than the
initial rapid HIV and/or E/CIA, or by HIV-1 antigen, plasma HIV-1 RNA viral load.

NOTE: The term "licensed" refers to a US FDA-approved kit, which is required for all IND
studies.

WHO (World Health Organization) and CDC (Centers for Disease Control and Prevention)
guidelines mandate that confirmation of the initial test result must use a test that is
different from the one used for the initial assessment. A reactive initial rapid test
should be confirmed by either another type of rapid assay or an E/CIA that is based on a
different antigen preparation and/or different test principle (e.g., indirect versus
competitive), or a Western blot or a plasma HIV-1 RNA viral load.

1.2 Currently on continuous ART for ≥24 weeks prior to study entry. This is defined as
continuous active therapy for the 24-week period prior to study entry with no treatment
interruption longer than 7 consecutive days and a total duration off treatment of no more
than 14 days in the 90 days prior to study entry.

1.3 CD4+ T-cell count ≥400 cells/mm3 obtained within 60 days prior to study entry by any US
laboratory that has a Clinical Laboratory Improvement Amendments (CLIA) certification or
its equivalent.

1.4 HIV-1 RNA level below the limit of quantification using a FDA-approved assay for at
least 24 weeks prior to study entry and confirmed within 60 days prior to study entry. The
assay used for eligibility can be performed by any US laboratory that has a CLIA
certification or its equivalent.

NOTE: Single determinations that are between the assay quantification limit and 200
copies/mL are allowed as long as the preceding and subsequent determinations are below the
level of quantification.

1.5 The following laboratory values obtained within 60 days prior to study entry by any US
laboratory that has a CLIA certification or its equivalent;

- Fasting glucose <180 mg/dL

- ALT [serum glutamic pyruvic transaminase (SGPT)] <2 times upper limit of normal (ULN)

- AST [serum glutamic oxaloacetic transaminase (SGOT)] <2 x ULN

- Estimated creatinine clearance (CrCl) ≥50 mL/min by Cockcroft-Gault (see section 6.3.5
for CrCl instructions) NOTE: Candidates who are taking TDF as part of their ART
regimen must have an estimated CrCl ≥60 mL/min.

- White blood cell (WBC) >3000/mm3

- Hemoglobin >12.0 g/dL

- Platelets >150,000/mm3

1.6 Females subjects who are postmenopausal (i.e., of non-childbearing potential), defined
as having either:

1. Appropriate medical documentation of prior hysterectomy and/or complete bilateral
oophorectomy (i.e., surgical removal of the ovaries, resulting in "surgical menopause"
and occurring at the age at which the procedure was performed), OR

2. Permanent cessation of previously occurring menses as a result of ovarian failure with
documentation of hormonal deficiency by a certified healthcare provider (i.e.,
"spontaneous menopause"). Hormonal deficiency should be properly documented in the
case of suspected spontaneous menopause as follows:

1. If age >54 years and with the absence of normal menses for at least 24
consecutive months: serum follicle stimulating hormone (FSH) level elevated to
within the post-menopausal range based on the laboratory reference range where
the hormonal assay is performed;

2. If age ≤ 54 years and with the absence of normal menses for at least 24
consecutive months: Negative serum or urine (β-HCG) performed within 48 hours
prior to study entry with concurrently elevated serum FSH level in the
post-menopausal range, depressed estradiol (E2) level in the post-menopausal
range, and absent serum progesterone level, based on the laboratory reference
ranges where the hormonal assays are performed.

1.7 Male subjects must agree not to participate in a conception process (i.e., active
attempt to impregnate, sperm donation). If participating in sexual activity that could lead
to pregnancy, the male subject must agree to the use of TWO reliable forms of
contraceptives simultaneously while on study and for a minimum of 3 months after therapy.

At least TWO of the following contraceptives MUST be used appropriately by male subjects
and their female partners:

- Condoms (male or female) with or without a spermicidal agent

- Diaphragm or cervical cap with spermicide

- Intrauterine device (IUD)

- Tubal ligation

- Hormone-based contraceptive

NOTES: Sexual activity that could lead to pregnancy is not applicable to sexual activity
with an infertile partner.

Acceptable documentation for the use of two methods of contraceptives is the male subject's
report of use of a second form of contraceptive by his female partner.

1.8 Subjects who are not of reproductive potential (defined as women who have been
postmenopausal for at least 24 consecutive months or men who have documented vasectomy) are
eligible for the study without requiring the use of contraceptives. In order to verify lack
of reproductive potential, written documentation or verbal communication by the clinician
or clinician's staff of one of the following must be in the source documents:

- Physician report/letter.

- Operative report or other source documentation in the patient record.

- Discharge summary.

- Follicle-stimulating hormone (FSH) measurement elevated into the menopausal range as
established by the reporting laboratory.

NOTES: Any statement of self-reported sterility or infertility (or that of a partner's)
should be entered into the source document.

Females who report being postmenopausal for at least 24 consecutive months but who do not
have documentation of postmenopausal status from a clinician will have the hormonal
assessments performed prior to study entry.

1.9 Moderate or high CVD risk defined as:

A) Documented CVD as assessed by meeting at least 1 of 3 criteria below:

1. Coronary artery disease (CAD): prior MI due to atherosclerosis, coronary artery bypass
graft surgery, percutaneous coronary intervention, or angiographic CAD with luminal
diameter stenosis of at least one coronary artery at least 50%.

2. Cerebrovascular disease: prior ischemic stroke of carotid origin, carotid
endarterectomy or stenting, or angiographic carotid stenosis of at least 50%.

3. Peripheral arterial disease: prior lower extremity arterial surgical or percutaneous
revascularization procedure, or angiographic lower extremity arterial stenosis of at
least 50%.

OR

B) Controlled type II diabetes mellitus (HbA1C ≤8.0% within the past 90 days prior to study
entry, regardless of use of medications)

OR

C) Any one of the following CVD risk factors below:

1. Current smoking: subject report of smoking at least a half a pack of cigarettes a day,
on average, in the past month.

2. Hypertension (HTN): two consecutive BP readings with either systolic >140 mmHg or
diastolic >90 mmHg; or on antihypertensive medications.

3. Dyslipidemia: defined as non-HDL-C >160 mg/dL, regardless of medication use.

4. hsCRP ≥2mg/L

1.10 Men and women age ≥40 years.

1.11 Ability and willingness of subject to provide informed consent.

1.12 For subjects taking tenofovir (TFV) as part of their ART regimen, willingness to
conside participate in the PK component of the study.

NOTE: Participation in the PK component is optional but strongly encouraged.

1.13 Completion of the pre-entry FMD assessment.

NOTE: At least one FMD must be performed at the site and confirmed as acceptable by the
University of Wisconsin Atherosclerosis Imaging Research Program (UW AIRP) core lab prior
to study entry.

1.14 Additional Inclusion criteria for the Imaging sub-study:

To be eligible for the PET/CT Imaging sub-study, the subject must:

A) Be a participant in the Parent Study B) Not yet have received study drug C) Be able to
undergo imaging prior to randomization

2. Exclusion Criteria:

2.1 Acute or serious illness requiring systemic treatment and/or hospitalization within 60
days prior to study entry.

NOTE: Treatment must have ended at least 60 days prior to study entry for eligibility.

2.2 Documentation of any CDC category C AIDS-indicator condition [78] or oropharyngeal
candidiasis (thrush) within 90 days prior to study entry.

2.3 Receipt of antibiotic therapy within 30 days prior to study entry.

2.4 Latent TB infection (defined as a positive PPD ≥5 mm, positive interferon-gamma release
assay, or positive T-spot test at any time in the past) or evidence of latent TB on the
screening chest x-ray that has not been completely treated or was treated within the past 6
months prior to study entry .

2.5 TB disease requiring treatment within 48 weeks prior to study entry.

2.6 Life-threatening fungal infection requiring treatment, in the opinion of the site
investigator, within 48 weeks prior to study entry.

2.7 Herpes-zoster viral infection requiring treatment within 90 days prior to study entry.

2.8 A history of or current, active hepatitis B infection defined as positive hepatitis B
surface antigen test or positive HBV DNA in subjects with isolated HBcAb positivity,
defined as negative HBsAg, negative HBsAb, and positive HBcAb within 24 weeks prior to
study entry.

NOTE: Subjects who are positive for hepatitis B surface antigen but who are HBV DNA
negative are permitted in the study.

2.9 Chronic hepatitis C infection defined as a positive hepatitis C antibody and positive
hepatitis C RNA at any time prior to study entry.

NOTE: Subjects who are positive for hepatitis C antibody but who are HCV RNA negative are
permitted in the study.

2.10 Previously diagnosed myelodysplasia syndrome.

2.11 Treated lymphoproliferative disease ≤5 years prior to study entry.

2.12 Clinically significant lung disease on the screening chest x-ray that, in the opinion
of the site investigator, places the subject at increased risk of lung toxicity (e.g.,
history of pulmonary fibrosis, interstitial lung disease, or pulmonary lymphoproliferative
disease).

2.13 Use of immunomodulators (e.g., interleukins, interferons, cyclosporine), HIV vaccine,
systemic cytotoxic chemotherapy, or investigational therapy within 30 days prior to study
entry.

2.14 Change in the ART regimen in the 12 weeks prior to study entry or intended
modification of ART during the study.

NOTE: Modifications of ART doses during the 12 weeks prior to study entry are permitted. In
addition, the change in formulation (e.g., from standard formulation to fixed-dose
combination) is allowed within 12 weeks prior to study entry. A within class single drug
substitution (e.g., switch from nevirapine to efavirenz or from atazanavir to darunavir) is
allowed within 12 weeks prior to study entry, with the exception of a switch from any other
NRTI to abacavir. No other changes in ART in the 12 weeks prior to study entry are
permitted.

2.15 Known allergy/sensitivity or any hypersensitivity to components of study drug(s) or
their formulation.

2.16 Average daily consumption of three or more alcoholic beverages for 4 weeks prior to
study entry or intention to consume an average of two or more alcoholic beverages a day
during the study.

NOTE: An alcohol-containing beverage is defined as 12 ounces of beer, 4 ounces of wine, or
1 ounce of spirits.

2.17 Active drug or alcohol use or dependence that, in the opinion of the site
investigator, would interfere with adherence to study requirements.

2.18 Changes in lipid-lowering or antihypertensive medication within 90 days prior to study
entry or expected need to modify these medications during the study.

NOTE: Lipid-lowering medication includes: statins, fibrates, niacin (dose ≥250 mg daily),
and fish-oil/omega 3 fatty acids (dose >1000 mg of marine oils daily).

2.19 Vaccination (e.g., influenza, pneumococcal polysaccharide) within 14 days prior to
study entry.

2.20 Anticipated need to receive vaccination (e.g., influenza, pneumococcal polysaccharide)
within 1 week prior to week 4, 12, 24, or 36 study visits.

2.21 Excess extracompartmental fluids including ascites, pericardial fluid, and pleural
effusions which, in the opinion of the study investigators, would result in difficulty in
monitoring the dose of MTX.

2.22 Use of drugs that alter folic acid metabolism such as trimethoprim/sulfamethoxazole or
reduce tubular excretion such as probenecid within 14 days prior to study entry.

2.23 New York Heart Association Class IV congestive heart failure.

2.24 Diabetes mellitus with HbA1C >8.0% within the past 90 days prior to study entry.

2.25 Patients who report any significant radiation exposure over the course of the year
prior to randomization. Significant exposure is defined as:

i) More than 2 PCI within 12 months of randomization ii) More than 2 myocardial perfusion
studies within the past 12 months iii) More than 2 CT angiograms within the past 12 months
iv) Any subjects with history of radiation therapy.

2.26 Current daily use of insulin.