Effect of Oral L-arginine 3.32 g a Day on Oxidative Stress Influencing Beta Cell Function and Insulin Resistance. A Phase 3, Randomized, Double-blind, Placebo-controlled Explorative Study in Overweight and Obese Patients With Pre-diabetes.
Phase: NA
Conditions: Diabetes, Pre-diabetes, Metabolic Syndrome
Sponsor: Federico II University

1 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
 
Title: 
Effect of oral L-arginine 3.32 g a day on oxidative stress influencing beta cell function and 
insulin resistance. A phase  3, randomized, double-blind, placebo-controlled explorative 
study in overweight and obese patients with pre-diabetes. 
Acronym Protocol: L-BIOARG 
Versione: 2.0 
Date: 4/11/2024 
 
 
Principal Investigator: 
Professore Andrea Giustina 
UO Endocrinologia 
IRCCS Ospedale San Raffaele, Milano 
Co-Experimenters: 
Dott. Luigi di Filippo 
UO Endocrinologia 
IRCCS Ospedale San Raffaele, Milano 
Promoter 
DAMOR farmaceutici 
Corso Milano, 46, 20900 Monza MB 

2 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
 
SIGNATURE PAGE 
 
 
I declare that this protocol has been read carefully and fully understood. I agree to follow the 
study procedures as described in this protocol in accordance with Good Clinical Practice and 
all other regulatory requirements. 
 
 
Prof. Andrea Giustina 
 
4/11/2024 
Primario U.O. per conto del 
Promotore/ Sperimentatore Principale 
Firma 
Data 
 
 
 
 
 
 
 
 
 
 
 

3 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
 
 
INTRODUCTION 
 
Insulin resistance and its mechanisms 
Insulin resistance (IR) is a complex condition characterized by impaired insulin ability to 
activate glucose transport in various tissues, as well as an inability to suppress glucose 
production in the liver through gluconeogenesis (Zhao et al., 2023). This phenomenon extends 
beyond muscle cells and adipose tissue, affecting the kidney, gastrointestinal tract, brain, 
vascular system, and the pancreas endocrine function (DeFronzo et al., 2015). In healthy 
conditions, during fasting, the liver releases glucose to supply peripheral tissues, including the 
central nervous system, while adipocytes release free fatty acids and glycerol for muscle energy 
and hepatic NADH production. However, a meal stimulates insulin secretion by pancreatic β- 
cells, inhibiting lipolysis and gluconeogenesis, facilitating glucose uptake in muscle and 
adipose tissue. The majority of ingested glucose is stored in muscles as glycogen, while fraction 
of glucose accumulates in adipocytes to generate glycerol 3-phosphate for triglyceride 
synthesis. The liver absorbs glucose and becomes a substrate for glycogen synthesis under 
insulin stimulation, also initiating de novo lipogenesis using products from peripheral fasting 
catabolism (James et al., 2021). 
 
At the molecular level, insulin binds to its receptor (INSR), leading to the phosphorylation of 
insulin receptor substrates (IRS), notably IRS1 and IRS2. This activation triggers intracellular 
signalling pathways, such as PI3K, which increases the expression of glucose transporter 
GLUT4 and inhibits transcription factor FOXO1, promoting glucose uptake and suppressing 
hepatic glucose production (DeFronzo et al., 2015). PI3K activation is a key mediator of insulin 
sensitivity, and alterations in this pathway are responsible for insulin resistance (DeFronzo et 
al., 2015; James et al., 2021; Zhao et al., 2023). Excessive phosphorylation of IRS proteins 
appears to be a contributing factor, inhibiting the phosphorylation of insulin receptor- 
associated tyrosine dimers (Bouzakri et al., 2006; Copps & White, 2012). Additionally, 
excessive serine-phosphorylation of IRS can lead to their degradation, reducing their 
availability as substrates for the receptor (Hiratani et al., 2005). These processes collectively 
lead to reduced receptor activation in response to insulin, with causes including ectopic lipid 
accumulation, mitochondrial dysfunction, hypoxia, inflammation, lipotoxicity, and 
endoplasmic reticulum stress (DeFronzo et al., 2015; James et al., 2021; Zhao et al., 2023). 
 
These mechanisms are often present in conditions like obesity and type 2 diabetes, which are 
closely linked to IR. Indeed, IR is a risk factor for various metabolic diseases, including type 2 
diabetes, cardiovascular diseases, non-alcoholic fatty liver disease, certain cancers, 
neurodegenerative diseases and frailty (Zhao et al., 2023). However, it's important to note that 
IR alone is not sufficient to cause type 2 diabetes; β-cell failure, responsible for insulin 
synthesis and secretion, is another critical factor. Multiple factors contribute to β-cell failure, 
including aging, genetic predisposition, resistance to or deficiency of incretins like GLP-1 and 

4 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
 
GIP, lipotoxicity, glucotoxicity, hypersecretion of Islet Amyloid Polypeptide (IAPP), oxidative 
stress, and inflammation (DeFronzo et al., 2015). 
 
Inflammation and oxidative stress 
Insulin resistance, inflammation and oxidative stress are highly related to metabolic syndrome 
(MetS) and visceral obesity. Indeed, these two conditions are linked with chronic low-grade 
systemic inflammation, characterized by a considerable increase in the systemic levels of 
cytokines and adipo-cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta 
(IL-1β), interleukin-6 (IL-6) and Leptin. Increased TNF- α levels and IL-6 levels are associated 
with insulin resistance mainly through the impairment of hepatic signalling and affection of 
IRS1 phosphorylation and GLUT-4 activity. Moreover, the impaired Leptin/Adiponectin ratio 
observed in obesity and metabolic syndrome is another well-known factor inducing insulin 
resistance. Furthermore, a synergistic relationship between low-grade systemic inflammation 
and oxidative stress is well-documented. Cytokines act as recruitment factors for immune cells 
along with adhesion molecules leading to their migration in the extracellular and subendothelial 
space. Here cytokines and immune cells are able to trigger the production of reactive oxygen 
(ROS) and nitrogen species (RNS) to cope with defense activities. On the opposite, persistent 
and unbalanced cytokine release results in increased pro-oxidative state (Colak et al, J Med. 
Biochem., 2021).  
 
The reactive species can be free radicals and non-radical oxidants. The free radicals are unstable 
because of unpaired electrons presence in their outer electron orbit. Since free radicals are 
highly unstable and reactive, tend to neutralize themselves by reacting with other molecules 
causing their oxidation. Therefore, by reacting with important biological molecules, they can 
cause damage to lipids, proteins and DNA. Indeed, free radicals can induce cell membranes 
lipid peroxidation, DNA bonds ruptures and protein modifications, as the unfolding or 
alteration of protein structure, modification of key enzymes or their regulatory sites. These 
events are crucial for cells survival and usually trigger cell signalling alteration and 
programmed cell death pathways (Cooke et al., The FASEB J, 2003; Vona R., Antioxidants, 
2021). 
 
Superoxide, hydrogen peroxide and hydroxyl radical are the major components of ROS, 
generated in many biological processes. Superoxide is very toxic as it can act as a precursor to 
other ROS, such as hydrogen peroxide and the hydroxyl radical, leading to biological 
molecules damage (Li et al., Gene, 2016). Specifically, superoxide plays a key role in the 
initiation of lipid peroxidation. (Fang et al. Nutrition, 2002). Superoxide dismutase (SOD), 
catalase (CAT) and glutathione peroxidases (GPx) represent the first endogenous antioxidant 
defense against oxidative stress. SOD plays an important role in protecting from the toxic 
effects of superoxide radical through production of hydrogen peroxide (H2O2) and molecular 
oxygen (O2). CAT is an enzyme responsible for the removal of hydrogen peroxide preventing 
from the oxidative-stress-related damage (Demirci-Çekiç et al, J. of Pharm. and Biomed. 
Analysis, 2021). Glutathione (GSH) is one of the most important antioxidant molecules. GSH 

5 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
directly reacts with ROS and other reactive species resulting in the formation of thiyl radicals 
(GS•), leading to different products such as glutathione disulfide (GSSG), mixed disulfides or 
glutathione sulphonamide. GSH is mainly used as a co-substrate by GPx reducing hydrogen 
peroxide or lipid peroxides, such as malonic dialdehyde (MDA) and 4-hydroxy-2-nonenal, with 
the production of glutathione disulfide (GSSG), water and alcohols.  Both GSH and GSSG are 
substrates for the extracellular membrane-bound enzyme γ-L-Glutamyl transpeptidase (GGT) 
which catalyzes the production of L-glutamate   and   L-cysteinyl-glycine, providing the basis 
for excreted GSH and GSSG recycling by the cells. Upregulation of this process provides an 
additional mechanism for GSH maintenance, enhancing cellular antioxidant potential. 
(Lutshack, J. of Amino Acids, 2012). 
 
Oxidative stress leads to endothelial dysfunction, reducing vasodilators bioavailability, 
especially NO, activating endothelial cells and providing a pro-inflammatory, proliferative and 
pro-coagulant state. This condition favours LDL-oxidation, immune cells recruitment and 
atherogenensis (Hadi et al., Vasc. Health Risk Manage., 2005). Oxidative stress is a well-
known contributor to β-cells damage and progressive destruction (Demirci-Çekiç et al, J of 
Pharm. and Biomed. Analysis, 2021). Many studies have described the association between 
pro-oxidant status and β-cells function (R. Alghazeer, J. Med, Biomed. Sci., 2018; P. Codoñer-
Franch, Pediatr. Diabetes, 2011). Moreover, excess free fatty acids (FFAs) are always present 
in MetS due to insulin resistance and increased lipolysis. FFAs accumulate in extra-adipose 
tissues such as the liver and the muscle. In this condition a higher concentration of FFAs is 
delivered from mitochondria leading to excessive production of superoxide anion through an 
increased β-oxidation rate, impaired switching to carbohydrate substrate, decreased 
tricarboxylic acid cycle activity and impaired mitochondrial electron transport chain. Increased 
β-oxidation increases mitochondrial NADH/NAD+ ratio, resulting in increased activation of 
protein kinase C (PKC), advanced glycosylation end products (AGEs) and NF-kB. All these 
mechanisms contribute to ROS production, nitric oxide synthase (eNOS) inhibition in 
endothelial cells, reduction in nitric oxide (NO) production in vascular smooth muscle cells 
and to the activation of nuclear transcription factor NF-kB maintaining and worsening 
inflammation pathways (Colak et al, J. Med. Biochem., 2009). 
 
L-arginine 
L-arginine, a conditionally essential amino acid, plays a crucial role in the production of Nitric 
Oxide (NO). Under normal conditions, L-arginine is metabolized by NOS, producing NO and 
L-citrulline, which have favorable effects on endothelial function, cardiovascular health, 
insulin secretion, and sensitivity. L-arginine is also involved in the synthesis of various 
essential compounds and has diverse functions, including promoting growth hormone 
secretion, T cell proliferation, and immune responses (Forzano et al., 2023). Several preclinical 
and clinical studies have demonstrated the beneficial effects of L-arginine supplementation in 
insulin-resistant, prediabetic, and diabetic patients. It has a protective role on β-cell function 
and neogenesis, as well as improving insulin sensitivity in obese and type 2 diabetic patients. 
One study has shown that L-arginine supplementation enhances insulin secretion through a 
GLP-1 mediated mechanism (Clemmensen C, Endocrinology, 2013). Additionally, L-arginine 

6 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
supplementation has been reported to affect adiposity by reducing white adipose tissue in 
favour of brown adipose tissue (Shi XY, Endocrinology, 2008). Importantly, long-term follow-
up studies suggest that L-arginine supplementation may significantly reduce the cumulative 
incidence of type 2 diabetes in high- risk individuals with impaired glucose tolerance and 
metabolic syndrome (Monti L et al., Eur J Nutr 2018). 
Furthermore, L-arginine has been reported to reduce oxidative stress and improve endothelial 
function leading to the production of NO, stimulating glutathione reductase and glutathione 
(GSH) de novo synthesis and enhancing antioxidant enzymes transcription against oxidative 
stress. A study on rats by Liang et al. (Liang et al., Food and Chem. Toxicology, 2015) 
demonstrated the enhancement of nuclear factor erythroid 2(NF-E2)-related factor 2 (Nrf2), a 
key transcription factor which positively regulates GSH metabolism and promotes the cellular 
defense mechanisms against oxidative stress through inducing and up-regulating superoxide 
dismutase (SOD) and catalase (CAT) gene expressions and other molecules involved in 
antioxidant pathways. GSH is a tripeptide constituted by L-glutamate, L-cysteine, and L-
glycine. GSH synthesis depends also upon nutritional intake. L-Arginine is a well-established 
substrate for glutamate synthesis. (Bansal and Ochoa, 2003; Cynober et al., 1995; Morris, 
2004, 2006; Wu and Morris, 1998). Farther, Liang et al (Liang et al., Food and Chem. 
Toxicology, 2015) described a positive correlation between hepatic GSH content and arginase 
expression in rats, highly suggesting L-Arginine as one of the precursors in GSH synthesis. 
This evidence highlights the role of L-Arginine not only as a mere NO donator but as a possible 
key part involved in prooxidant-antioxidant balance. 
 
Side effects 
No side effects have been reported in previously conducted studies. 
 
Rationale 
Recent studies have demonstrated beneficial effects of the use of L-arginine supplementation 
as a nutrient treatment in diabetes and prediabetes, with documented beneficial effects on β- 
cell function (Mendez JD, Biomed Pharmacol., 2005; Vasilijevic A, J Physiol. 2007) and 
insulin sensitivity (Wascher TC, Eur J Clin Investig, 1997; Lucotti P, Metabolism, 2009). 
 
A long-term follow-up randomized clinical trial also demonstrated a reduced cumulative 
incidence of type 2 diabetes among subjects at high risk of developing the disease (Monti L et  
al., Eur J Nutr, 2018). The same study reported a significantly AOPP (Advanced oxidation 
protein products) levels reduction at 18 months in L-arginine arm compared to placebo and this 
effect was maintained through the 108 months of follow up. Indeed, AOPP levels decreased by 
26.1% at 18 months, remaining lower during the post intervention period in L-arginine group 
(Figure1. AOPP at 108 months: 773.2 ± 244.4 vs. 1045.7 ± 282.9 μmol/l, p < 0.05) (Monti L et 
al., Eur J Nutr, 2018).  
However, in this study, AOPP levels were firstly evaluated at baseline and then only 18 months 
after starting treatment. Therefore, to date, no data are currently available regarding the effects 
of a short-term treatment on AOPPs levels. In addition, data in vivo on rats suggest a rapid 
improvement in antioxidant defense and in total antioxidative capacity in plasma and liver after 

7 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
a 14 days treatment period with L-arginine (Liang et al., Food and Chem. Toxicology, 2015). 
 
Advanced oxidation protein products are formed during oxidative stress by myeloperoxidase 
action in activated neutrophils through chloraminated oxidants production. AOPPs molecular 
structure is similar to advanced glycation endproducts (AGEs) as they have similar biological 
activities. Precisely, AOPPs exert induction of proinflammatory cytokines and adhesion 
molecules maintaining inflammatory pathways activated and favoring pro-oxidant state 
(Witko-Sarsat V, Kidney Int 1996; Catakay U, Diabetes Metab 2005). In addition, many reports 
highlighted a strict correlation between AOPPs and glucose metabolism (Kalousová M, Physiol 
Res 2002; Martín-Gallán P, Free Radic Biol Med 2003; Piwowar A, Diabetes Res Clin Pract, 
2007;) suggesting being an early marker of diabetes mellitus and metabolic syndrome 
(Zurawska-Plaksej E, J Endocrinol Invest., 2014; Martín-Gallán P, Free Radic Biol Med 
2003). The prooxidant-antioxidant index (PAI) has been proposed as possible marker of MetS. 
It is expressed as the ratio between the AOPP levels and the total radical-trapping antioxidant 
capacity (TRAP), a surrogate for the total antioxidant defenses in the plasma.  An interesting 
study by Venturini et al. evidenced the relationship between AOPPs and MetS also highlighting 
that PAI progressively increased (P <0.05) according to the number of MetS components, 
whereas AOPPs and total radical-trapping antioxidant parameter increased (P <0.05) when 5 
components were compared with 3 and 4 components (Venturini et al., Nutr Research, 2015).  
 
Lipid peroxidation products such as malondialdehyde (MDA), and 4-hydroxynonenal (HNE) 
have been reported to be highly related to inflammation and MetS. These molecules are 
considered important biomarkers of oxidative stress because of the high susceptibility of 
polyunsaturated fatty acids-rich cell membranes to lipid peroxidation when exposed to ROS or 
other free radicals (Nair, Free Radic Biol Med, 2007; Demirci-Çekiç et al, J of Pharm. and 
Biomed. Analysis, 2021). A positive correlation has been described by several studies among 
obesity, IR, MetS, glycaemic control and MDA and HNE levels (Srikanthan, Molecules, 2016, 
Picklo MJ, Nutr Rev 2015).  
 
Regarding the antioxidant enzymes, a study by Lang et al. on rats fed with L-arginine for 14 
days demonstrated a significant stimulation in GSH synthesis enzymes, CAT, SOD and GPx 
activity and mRNA expression enhancing the total antioxidative capacity in plasma (T-AOC 
or TRAP) and in the liver strongly suggesting that L-arginine oral administration can 
ameliorate antioxidant defenses (Liang et al., Food and Chem. Toxicology, 2015). 
 
To conduct this study, we selected a L-arginine dosage of 3,32 g/day, based on evidence that 
this dosage was associated to an improvement of the inflammatory response and beneficial 
modulating of oxidative stress in favor of an antioxidant state (G. Fiorentino et al., 
Eclinicalmedicine, 2021).  

8 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
 
 
 
 
 
Objective 
The objective of this clinical trial is to explore whether L-arginine supplementation with 
3.32g/day may enhance antioxidant defenses in overweight and obese patients with pre-
diabetes and metabolic syndrome, possibly providing beta cell function and insulin resistance 
improvement. The primary efficacy endpoint will be to explore the effects of a short-term L-
arginine supplementation on AOPP levels and PAI after three months of treatment, based on the 
rationale explained above. Secondary, the effects of L-arginine supplementation on lipid 
peroxidation products and antioxidant defenses will be explored. Tertiary, the effects of L-
arginine supplementation on insulin resistance and glucose metabolism parameters will be also 
evaluated. 
 
METHODS 
Study design 
The study is a prospective, double-blind, placebo-controlled single-center, phase 3 study. It 
will involve 42 patients with BMI ≥25 kg/m2 and pre-diabetes. They will all receive a calorie- 
restricted Mediterranean diet, a physical activity program, and will be randomly (1:1) assigned 
to either L-arginine 1.66 g twice a day for 90 consecutive days (treatment group) or placebo 
(control group). To record eating and exercise habits, a food and physical activity diary will be 
kept by the patient throughout the study period. An individualized nutrition regimen will be 
prescribed to the patient based on characteristics like gender, age, BMI, physical activity and 
preferences.  
Figure1: Mean AOPP levels in the L-arginine (black boxes) and placebo (white boxes) 
groups; *p < 0.05 vs. placebo; #p < 0.01 vs. placebo. 
Adapted from Monti L et al., Eur J Nutr, 2018. 

9 
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
Patients undergoing metabolic assessment at the Endocrinology Unit of IRCCS Ospedale San 
Raffaele, willing to participate in the trial and meeting the inclusion criteria, will be enrolled 
in this study. Patients will be screened at our “dysmetabolic diseases” outpatient clinic. The 
patients with Metabolic Syndrome according to WHO definition and with BMI ≥25 kg/m2, 
glycemia between 100 mg/dl and 125 mg/dl and/or HbA1c between 5.7% and 6.4%, according 
to ADA definition of pre-diabetes status (ADA Standards of Care 2023) will be enrolled and 
subsequently randomized during the baseline study visit. During this visit, the participants will 
undergo evaluation of anthropometric measures, in accordance with standard clinical practices, 
including weight, height, waist circumference, and waist-hip ratio. Furthermore, 1600 meal 
plan and physical activity will be evaluated. Additionally, blood samples will be collected for 
AOPP, TBARs, TRAP, total glutathione, Catalase and SOD activity, urate, IL-6, TNF-a, VES, 
CRP, total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides, blood glucose, 
insulin, C-peptide levels and glycated hemoglobin (HbA1c) measurement at the central 
laboratory of IRCCS Ospedale San Raffaele (measured through standardized HPLC technique 
(TOSOH HLC723®-G11 Analyzer, Tosoh Europe NV). Patients will be randomized to receive 
L-bioarginine or placebo for 90 days. The follow-up visit will be performed within 1 and 5 
days after the last dose of study treatment. At day 91-95 a Visit 1 will be performed to collect 
the same parameters collected at the Baseline study visit and the patient will undergo a second 
blood sample collection for the same parameters determined at baseline visit. 
 
Sample size 
The main endpoint of the study is to analyze the differences in the reduction of AOPP levels in 
patients treated with L-arginine versus placebo. Based on results of a previous study (Monti L 
et al., Eur J Nutr 2018) showing a reduction of AOPP levels in patients treated with L-arginine 
by 20-30% as compared to negligible effect in the placebo group, for the present study we 
hypothesized that 50% of patients treated with L-arginine will achieve a reduction of at least 
20% in AOPP levels compared to only 10% (or less) of patients in the placebo group. Based 
on these assumptions, 17 patients in each group would be needed to obtain a statistical power 
of 80% with α = 0.05 (two tailed). Estimating a drop-out rate of about 10%, the total number 
of enrolled patients will be 40 (20 in each group).  
Number of patients: 40 patients with BMI ≥25 kg/m2, pre-diabetes or MetS will be enrolled 
and randomized in 2 groups, i.e. 20 treated with L-arginine supplementation 1.66 g twice a day 
and 20 with placebo for 90 consecutive days. 
 
 
Study period 
Projected starting date (first-patient-in):  
December 2024 
Projected completion of patient accrual (last-patient-in): December 2025 
Projected study end date (last-patient-last-visit): 
March 2026 
 
Eligibility criteria 
Inclusion criteria: consented male and female patients aged 20-70 years with Body Mass Index 

10
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
(BMI) >25 kg/m2, pre-diabetes (fasting glucose 100-125 mg/dL and HbA1c 5.7-6.5%) or 
metabolic syndrome, defined according to modified NCEP-ATP III criteria (Grundy SM et al., 
Circulation, 2005) as the presence of three or more of the following clinical features: blood 
glucose levels >100 mg/dL, HDL-cholesterol <40 mg/dL in males and <50 mg/dL in females, 
triglycerides levels >150 mg/dL, waist circumference >102 cm in males and >88 cm in females 
and hypertension, defined as repeated blood pressure measurements >130/85 mmHg. 
Exclusion criteria: 1. moderate to severe renal impairment (calculated creatinine clearance 
(CrCl) <60 mL/min according to the Cockcroft-Gault formula); 2. hepatic dysfunction 
(ALT/AST >3 x upper limit of normal and total bilirubin >3 mg/dL); 3. hypoalbuminemia 
(serum albumin <3 g/dL); 4. history of any past or current clinically significant cardiovascular 
diseases; 5. monogenic, secondary and pharmacological causes of diabetes and obesity; 6. any 
other clinical condition/disease that the Principal Investigator believes might confound study 
outcome; 7. patients on treatment with insulin or any anti-diabetic drugs or medications known 
to influence glucose tolerance will also be excluded; 8. pregnant or breast-feeding women. 
 
Efficacy endpoints 
Efficacy endpoints will be: 
- Changes from the baseline in AOPPs, TRAP (Total plasma radical-trapping 
antioxidant capacity) as a measure of all the hydrosoluble and the liposoluble 
antioxidants present in plasma (Primary endpoints) and PAI (Prooxidant-
antioxidant index), calculated as AOPPs (μmol/L) divided by TRAP (μM 
Trolox). These are the primary composite endpoint; 
- TBARs quantitative assays as a measure of oxidative stress and lipid 
peroxidation products (Secondary endpoint); 
- Catalase (CAT) and Superoxide-dismutase (SOD) quantitative assays 
(Secondary endpoint); 
- Total glutathione quantitative assay and GSH/GSSH ratio (Secondary 
endpoint); 
- Serum IL-6, TNF-a, VES, CRP, GGT, urate, total cholesterol, HDL 
cholesterol, LDL cholesterol and triglycerides (Tertiary endpoint); 
- HbA1c (mmol/mol) (Tertiary endpoint);  
- Proportion of patients with Impaired fasting glucose (IFG) (Tertiary 
endpoint);  
- Changes in Body weight and BMI (Tertiary endpoint); 
- Abdominal obesity (waist and waist-hip ratio circumference, measured by 
the same  operator) (Tertiary endpoint); 

11
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
-  HOMA-B%, as a simplified one-sample β-cells function assessment model, 
calculated  by the following formula equation: 
 
(𝒇𝒂𝒔𝒕𝒊𝒏𝒈 𝒊𝒏𝒔𝒖𝒍𝒊𝒏 𝒙 𝟐𝟎) 
(𝒇𝒂𝒔𝒕𝒊𝒏𝒈 𝒈𝒍𝒖𝒄𝒐𝒔𝒆 − 𝟑. 𝟓) 
 
with glucose expressed in mmol/L and insulin mU/l ) (Matthews et al., 1985) 
(Tertiary endpoint); 
- HOMA-IR, as a simplified one-sample insulin-resistance assessment model, 
calculated by the following formula equation: 
(𝒇𝒂𝒔𝒕𝒊𝒏𝒈 𝒃𝒍𝒐𝒐𝒅 𝒈𝒍𝒖𝒄𝒐𝒔𝒆 𝒙 𝒇𝒂𝒔𝒕𝒊𝒏𝒈 𝒊𝒏𝒔𝒖𝒍𝒊𝒏𝒆𝒎𝒊𝒂 
𝟐𝟐. 𝟓 
 
with glucose expressed in mmol/L and insulin mU/l (normal range 0.23-2.5)  
(Matthews et al., 1985) (Tertiary endpoint). 
 
 
Patient management and treatment 
Patients enrolled in the study will be randomized to receive either L-arginine treatment or 
placebo. Randomization will be performed through an excel file to favor the allocation 
concealment. Indeed, the company will provide clinicians with an excel table showing 
preparations A and B by patient numbers ensuring randomization using the program's 
''random'' function (see attached). 
Blindness of the study is ensured because Farmaceutici Damor will provide the hospital 
with identical packages of vials of the preparation under study distinguished only by the 
indication ''preparation A'' and ''preparation B.'' The vials will be identical in both 
aesthetics and palatability (see attached data sheets). 
Only the company will be aware of which of the two preparations corresponds to 
Bioarginina®zero and which to placebo. 
It is remarkable that L-arginine treatment is already commercially available and although 
no notable adverse events have been reported, there are limited and insufficient data 
regarding its efficacy in improving clinical parameters concerning insulin resistance and 
beta- cell function. For this reason, both treatments (L-arginine vs. placebo) can be 
considered ethically accepted in this clinical setting. The two groups will be managed with 
the same modalities and their management will be carried out as part of standard clinical 
practice visits. 
 
Sides effects management 
No side effects are expected for this study as the L-arginine was not reported to induce side 
effects. The occurrence of side effects will be monitored on an ongoing basis and reported with 
narratives, allowing for the collection of additional information, as warranted. 
 

12
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
Description of data management and statistical analysis 
Data will be extracted from the electronic patient data management system and will be obtained 
through medical interviews and visits at our outpatient clinic. Data will be analyzed using 
descriptive statistics for all study variables. Categorical variables will be summarized as counts 
and percentages. The normality of continuous variables will be assessed using the 
Kolmogorov–Smirnov test (p > 0.05). For normally distributed data, means and standard 
deviations (SD) will be reported. In cases where the data distribution deviates from normality, 
medians and inter-quartile ranges will be utilized. Statistical significance testing will be carried 
out using appropriate methods: Fisher exact test or χ2 test (for categorial variables) and Student 
T-test or Mann-Whitney test, (for continuous variables). These tests will be applied to discern 
significant differences in proportions, means, or medians between the two groups at baseline. 
Furthermore, paired samples T-test or Wilcoxon signed-rank test will be employed to evaluate 
changes from baseline to follow-up for all endpoints. Multiple logistic regression analyses will 
be used to estimate the effects of L-arginine treatment on the protocol efficacy endpoints 
evaluating the impact of the other covariates and variables collected in the study as the 
demographics and anthropometric data, or dietary and physical activity adherence (collected 
as binary categorical variables). 
A p-value of <0.05 will be considered statistically significant. Statistical analysis will be 
conducted using IBM SPSS Statistics (IBM SPSS Statistics for Windows, Version 23.0. 
Armonk, NY: IBM Corp.). 
 
 
ETHICAL ASPECTS AND DISSEMINATION 
Ethical aspects and informed consent  
This protocol, the information attached to the informed consent, the informed consent itself, 
the privacy protection information, and the privacy consent have been submitted to the 
Territorial Ethics Committee of Lombardia (CET Lombardia). Subjects will be informed 
about the clinical study both in written and verbal form by physicians of the Endocrinology 
Unit of IRCCS Ospedale San Raffaele. Ample opportunity will be given to decide whether to 
participate in this study and to ask questions regarding the study. It will also be clearly 
explained that the patient's participation in the study can be discontinued at any time, without 
providing a reason, and that the patient will not be penalized in any way for this decision. All 
information in the informed consent will be communicated to patients in an understandable 
language. The physician conducting the study and the patient will personally sign and date 
the informed consent form with a declaration regarding data confidentiality. The informed 
consent will be stored in accordance with Good Clinical Practice (GCP) standards, current 
regulations, and in line with OSR internal policies. A copy will be given to the patient. 
 
The research project will be conducted in accordance with national and international norms 
and legislation, as well as European standards of research ethics, as expressed in the applicable 
legislation/norms: 

13
 
 
L-BIOARG Protocol 
Data: 4/11/2024 
Version 2.0 
• The Declaration of Helsinki; 
• Ethical principles for medical research involving human subjects; 
• Council of Europe Convention on Human Rights and Biomedicine (Oviedo Bioethics 
Convention); 
• Directive 95/46/EC (amended 2003) and Regulation (EC) No 45/2001 of the European 
Parliament and of the Council, dated December 18, 2001, on the protection of individuals 
concerning the processing of personal data; 
• National legislation and regulations applicable to the specific research activity: 
Legislative Decree No. 158/2012; Ministerial Decree 8/2/2013 published in the Official 
Gazette No. 96 of 24/4/2013; Regional Decree of Lombardy No. 5493 of 25/6/2013; 
Chapter on fundamental rights of the EU. 
 
Procedures for the collection, storage, protection, preservation, and destruction of sensitive 
data include: 
• 
Obtaining informed consent; 
• 
Confidentiality of personal data; 
• 
Measures for the coding and storage of samples; 
 
This study will be financially supported by the DAMOR Farmaceutici. The study will be 
covered by a specific insurance policy, undersigned by the sponsor.  
 
Data confidentiality 
Guaranteed according to current regulations. Data will be processed using computerized and 
non-computerized tools and stored in paper archives and electronic databases and will be 
disseminated only in strictly anonymous form, for example through scientific publications, 
statistics and scientific conferences. Only the medical staff of the Endocrinology Unit will be 
aware of patient personal data. In addition, the Ethics Committee and the Italian Health 
Authorities will have access to the personal data of patients enrolled in this study, including 
information contained in original medical documentation, exclusively for the purposes of 
checking the procedures of the Practice and the correctness and accuracy of the data collected. 
In any case, all precautions to ensure the necessary confidentiality of patients identity within 
the limits set by applicable regulations, in particular in strict compliance with the principles of 
correctness, competence, accuracy, relevance and completeness of treatment (art.11 D.Lgs. 
196/2003 - Code for the protection of personal data). 
 
Data ownership and methods of publication 
The sponsor of this study declares that all data from this study belong to the sponsor. No 
specific rule is established for the publication of study data, which is intended to be 
disseminated anonymously at scientific congresses and in publications in journals.