Safety and Efficacy of (α1Proteinase Inhibitor, α1PI) in HIV Disease

This study protocol is designed to investigate the benefit of a well-tolerated, FDA approved
biological product that has been extensively used in a different patient population.

For more than 20 years, α1proteinase inhibitor (α1PI or α1antitrypsin) therapy has been the
standard treatment for people with insufficient α1PI blood levels. This disorder, also known
as α1antitrypsin deficiency, was previously thought to be caused only as an inherited trait
due to the gene PIzz. Recent evidence shows that it can also be an acquired disease.
Specifically, individuals with HIV-1 disease have been found to have severely low α1PI blood
levels (Bristow et al., 2001; Bristow et al., 2010; Bristow et al., 2012). Many people with
the inherited version of α1PI deficiency eventually develop emphysema, and among individuals
with HIV-1 disease, 90% have acquired α1PI deficiency. Whether they go on to develop
emphysema is still unresolved.

HIV-1 infected individuals are the first patient population identified so far in which severe
α1PI deficiency is acquired through infection rather than being inherited.

It was found that a decrease in α1PI blood levels is directly correlated to a decrease in CD4
lymphocytes (Bristow et al., 2001; Bristow et al., 2012): In a pilot study to determine
whether α1PI therapy might benefit the CD4 counts in HIV-1 infected patients
(Clinicaltrials.gov NCT01370018), HIV-1 patients with infection-related α1PI deficiency
received weekly α1PI therapy (120mg/kg) for a period of 8 weeks and results were compared
with those of patients having the inherited version of α1PI deficiency who were
simultaneously receiving weekly α1PI therapy (60mg/kg). None of the patients in the study had
ever previously received α1PI therapy. It was found that CD4 cells rose to normal levels
following 2 weeks of intravenous α1PI therapy with no adverse effects observed in the HIV-1
patients (n=3) or the control group of HIV-1 uninfected, α1PI deficiency patients (n=2). The
new crop of CD4 cells were functional, capable of fighting infection, and appeared to be
generated from bone marrow-derived stem cells (Bristow et al., 2010). As a bonus, it was
found in the HIV-1 patients that LDL levels (bad cholesterol) decreased and HDL levels (good
cholesterol) increased {Bristow et al., in review).

The information gained from the initial pilot study will be incorporated into this study
design to further characterize the mechanism of lymphocyte renewal and to increase the number
of patients observed. Only minor modifications to the pilot protocol are proposed including
the sample size, addition of volunteers not receiving therapy, and a double-blind design: Ten
(10) HIV-1 infected patients will be dosed with PROLASTIN®-C (Grifols Therapeutics Inc.),
five (5) will be dosed with placebo, and five (5) uninfected volunteers not receiving therapy
will be monitored.

It has been observed that CD4 counts and cholesterol levels are correlated and that there is
cyclic variation in individuals with and without HIV. To examine whether there are
differences due to HIV infection in cyclic variation of CD4 counts, cholesterol levels, and
other values monitored during this study, five (5) volunteers, who do not have HIV, will be
included in the study for blood collection only.

In a previous study, we determined that in individuals with abnormally low active α1PI
levels, CD4 counts exhibit sinusoidal cycling with 23 day periodicity. Wave form appearance
of CD4 cells with a peak every 23 days is a pattern that is indicative of adult thymopoiesis.
We showed that in response to α1PI therapy, HIV infected and uninfected individuals exhibited
an increased CD4 counts axis of oscillation, but no change in periodicity. In contrast, one
uninfected individual who was not receiving therapy, exhibited no change in the axis of
oscillation, but periodicity was 27 days. Human adult thymopoiesis is not well characterized,
and based on these results, we have two hypotheses.

One hypothesis is that in response to α1PI therapy, thymopoiesis will increase, manifested by
an increased axis of oscillation and increased numbers of recent thymic emigrants. In the
proposed study, we will perform weekly TREC analysis which measures recent thymic emigrants
and is definitive for thymopoiesis. We will test whether the changes in TREC numbers
corresponds with changes in the CD4 counts axis of oscillation in HIV infected individuals
receiving α1PI therapy as compared with HIV infected individuals with abnormally low active
α1PI levels not on α1PI therapy, and HIV uninfected individuals with normal active α1PI
levels not on α1PI therapy.

The second hypothesis is that in individuals with abnormally low active α1PI levels, the
periodicity of CD4 counts is shorter than in individuals with normal active α1PI levels.
Although we will measure periodicity in only 5 individuals with normal active α1PI levels and
15 with abnormal active α1PI levels, this will allow us to determine whether active α1PI is a
linear determinate of periodicity or whether there are other variables that regulate
periodicity. In an 8-week study, only 2 periods can be observed. Thus, following an 8-week
regimen of therapy, one study subject may be asked to continue for a second 8-week regimen of
therapy to allow the observation of 5 periods.

In addition to these two primary hypotheses regarding CD4 counts, we have found that HDL and
LDL levels are linearly dependent on the balance of active and inactive α1PI levels
(manuscript in review). This phenomenon is due the transport of HDL and LDL by CD4 cells. We
found that in HIV infected individuals with abnormally low active α1PI and abnormally high
inactive α1PI, HDL and LDL increased or decreased differently from individuals with normal
active α1PI. Based on these results, our hypothesis is that in HIV infected individuals with
abnormally low active α1PI receiving α1PI therapy, LDL levels will decrease and HDL levels
will increase as active α1PI levels increase in contrast to HIV uninfected individuals with
normal active α1PI levels not on therapy and in contrast to HIV infected individuals with
abnormally low active α1PI levels not on therapy.

Prolastin-C treatment will be given weekly for eight (8) to sixteen (16) weeks by intravenous
infusion. Blood will be collected immediately prior to each infusion. As in the previous
study, complete blood count, lymphocyte phenotype, extended lymphocyte phenotype, lymphocyte
function; HIV-1 viral load, lipid levels, blood chemistry, and markers of inflammation will
be measured. The present study will also include measurements for HIV-1 tropism and markers
for recent thymic emigrants.

Inclusion Criteria:

1. HIV-1 patients must have confirmed HIV-1 disease, diagnosed using the standard
criteria and be on antiretroviral therapy. Uninfected volunteers will be age and
gender matched.

2. HIV-1 patients must have measurable disease, defined as HIV-1 infected patients on
antiretroviral therapy with undetectable HIV RNA (<1000 HIV RNA copies/ml) and CD4
counts more than 200 and less than 600 cells/uL.

3. Not have previously received α1PI augmentation therapy

4. Age at least 18 years and under 65 years

5. Capacity for and commitment to attend all protocol scheduled visits at ACRIA

6. Life expectancy of greater than 5 years

7. Patients must have lab values within the limits defined below:

- WBC >4,1000/uL

- ANC >1,000/uL

- platelets >100,000//uL

- total bilirubin 2-12 mg/dL

- AST(SGOT)/ALT(SGPT) < or = 2.5 X upper limit of normal

- creatinine Male : 0.50-1.30 mg/dL Female: 0.40-1.20 mg/dL

8. HIV-1 patients must have active α1PI below 11 uM (normal is 18-53 uM)

9. HIV-1 patients must have one year history (prior to the study) with CD4+ lymphocytes
at levels greater than 200 and less than 400 cells/uL

10. HIV-1 patients must have absence of symptoms suggestive of HIV-1 disease progression

11. HIV-1 patients must have adequate suppression of virus (<400 HIV RNA/mL)

12. HIV-1 patients must have a history of compliance with antiretroviral medication based
on undetectable virus levels

13. No evidence of malignancy

14. The effects of Prolastin-C on the developing human fetus at the recommended
therapeutic dose are unknown: At any time throughout the study, from the signing of
the informed consent form until after the last study visit, all female and male
subjects who are biologically capable of having children must agree and commit to use
a reliable method of birth control.

15. Ability to understand and the willingness to sign a written informed consent document

Exclusion Criteria:

1. Recent illness that will prevent the patient from participating in required study
activities

2. Patients receiving other investigational agents

3. Patients with known malignancies

4. History of allergic reactions attributed to compounds of similar chemical or biologic
composition to Prolastin-C

5. IgA deficient patients

6. Patients with ≥1000 HIV-1 RNA copies/ mL

7. Patients with >600 CD4 cells/uL

8. Uncontrolled illness including, but not limited to, ongoing or active infection,
myeloid dysplastic syndrome, anemia, bone marrow failure, DiGeorge Syndrome, thymic
disorders, or psychiatric illness/social situations that would limit compliance with
study requirements

9. Pregnant and breastfeeding women

10. Refusal to give informed consent