N-Acetylcysteine and Milk Thistle for Treatment of Diabetic Nephropathy

Oxidative stress and GSH imbalance are major contributors to the pathogenesis of diabetic
nephropathy. Current options for the treatment of oxidative stress in diabetic nephropathy
are limited and only partially effective, thus interest in the development of new strategies
is high.

The study intends to test the hypothesis that combined oral supplementation of the
antioxidants N-acetylcysteine (NAC) and milk thistle flavonolignan silibin (as
silibin-phosphatidylcholine) will reduce proteinuria and urinary and systemic manifestations
of oxidative stress and inflammation, which are characteristically observed in patients with
type 2 diabetes mellitus and related nephropathy. We expect these effects to be achieved
with minimal or no side effects, and with good patient tolerance.

The trial is designed as a two-center, double-blind, placebo-controlled, randomized,
modified-factorial dose-ranging design, five-arm pilot study in patients with Type 2
diabetes mellitus and advanced diabetic nephropathy with proteinuria.

Intervention consists of three-month oral administration of NAC, silibin, and/or respective
placebos for three months. Subjects are randomized to the following five intervention arms:
(A) placebo; (B) NAC; (C) silibin; (D) NAC + silibin; and (E) NAC + double-dose silibin.

The primary outcome measure is urinary excretion of albumin, a marker of glomerular injury.
Secondary outcome measures are alpha-1 microglobulin, a marker of tubular injury, and
urinary excretion of inflammatory cytokines and C-C chemokines, i.e. markers of renal
inflammation. In addition, peripheral blood monocytes from the same patients are analyzed
for glutathione (GSH) content and activity of GSH metabolizing enzymes. All outcome measures
are monitored in relation to both treatment allocation and prevalent blood and urine levels
of the active treatment. Safety and tolerability of this combination treatment are monitored
throughout the trial.

Inclusion Criteria:

- Males or females, age 18-70 years old.

- Type 2 diabetes mellitus

- Diabetic nephropathy, as defined by:

- estimated GFR between 60 and 15 ml/min,

- presence of proteinuria.

- Current medical treatment with low dose aspirin

- Treatment of hypertension with (but not limited to) one diuretic, one beta-
blocker and one medication from the classes ARBs or ACE inhibitors.

- Treatment of hyperglycemia with (but not limited to) glipizide and the medication
class insulin.

- Treatment of hypercholesterolemia with (but not limited to) one medication from the
class statins.

Exclusion Criteria:

- Type 1 diabetes mellitus.

- Glycosylated hemoglobin (HbA1C) > 10%

- >20% variation in estimated GFR, during last 6 months

- SBP >170 mmHg or DBP >100 mmHg on medications

- Other secondary forms of hypertension (endocrine, renovascular)

- History of intolerance to:

- Both ACE-I and ARBs;

- The investigational supplements;

- Iodinated radiologic contrast material.

- Known non diabetic renal disease, or history of solid organ transplantation.

- Hepatitis virus or Human Immunodeficiency virus infections

- Use of one of the following medications within 2 months prior to enrollment in the
study:

- Metformin.

- Thiazolidinediones (pioglitazone or rosiglitazone);

- Prescription-grade vitamin E, vitamin C, systemic steroids, and/or non-steroidal
anti-inflammatory agents;

- Over-the-counter vitamin E, vitamin C, and/or non-steroidal anti-inflammatory
agents.

- Over-the-counter antioxidants supplements including: Lipoic acid, Coenzyme Q10,
N-acetyl-cysteine (NAC), Glutathione (GSH), Chromium, Fish-oil extracts (omega-3
fatty acids), Soy extracts (isoflavones), Milk thistle extract (silymarin),
Green-tea preparations, Pomegranate extracts, Grape extracts, and Prickly pear
extract.

- Active coronary artery disease or cerebral vascular disease within 3 months prior to
signing the informed consent.

- Hepatic dysfunction as defined by abnormal total bilirubin or liver enzymes (ALT,
AST) >2 times upper limit of normal range.

- Active malignancy.

- History of drug or alcohol dependency.

- Psychiatric or neurological condition, preventing aware consent to the study and/or
adherence to the study protocol

- Unwillingness to practice birth control throughout the study.

- Participation to another clinical study within 1 month prior to signing the informed
consent form.

- Planned move to outside the study area, surgery or radiographic studies utilizing
iodine-based contrast material within the next one year