Effect of Supplementation With Alpha-lipoic Acid and Silybum Marianum (LUDLEV®) in Association With a Mediterranean Diet for the Improvement of Metabolic Dysfunction-associated Fatty Liver Disease
Phase: NA
Conditions: Metabolic Associated Fatty Liver Disease
Sponsor: Saluz Investigación S. C.

1 
 
STUDY OR PROJECT TITLE: Effect of alpha-lipoic acid and Silybum marianum 
supplementation (LUDLEV®) in conjunction with a Mediterranean diet for the 
improvement of metabolic-associated fatty liver disease. 
 
 
 
RESPONSIBLE INVESTIGATORS: 
Dr. José María Remes Troche 
Dr. Ana D. Cano Contreras 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Febrary 8, 2023. Veracruz, Mexico. 

2 
 
BACKGROUND 
Metabolic-associated fatty liver disease (MAFLD) is currently the most common 
chronic liver disease worldwide, affecting approximately 25-30% of the population, 
making it a significant public health problem. 1 It is characterized by the presence of 
hepatic steatosis in more than 5% of hepatocytes, as demonstrated by biopsy, 
imaging, or biomarkers, and at least one of the following criteria 1) overweight or 
obesity, 2) diabetes mellitus, 3) at least two metabolic abnormalities (waist 
circumference greater than 90 cm in men and 80 cm in women; blood pressure 
≥130/85 mmHg or specific pharmacological treatment; triglycerides ≥150 mg/dL or 
specific pharmacological treatment; high-density lipoprotein cholesterol <40 mg/dL 
in men and <50 mg/dL in women or specific pharmacological treatment; fasting 
glucose ≥100 mg/dL; insulin resistance index ≥2. 5; C-reactive protein ≥2 mg/dL). 2 
The increase in the prevalence of MAFLD is favored by sedentary lifestyles and poor 
dietary habits, such as excessive calorie and saturated fat intake, which coincide 
with the rise in overweight and obesity. On the other hand, the new definition of 
MAFLD has moved away from the dichotomy of simple steatosis and steatohepatitis, 
as it has been demonstrated that fatty liver disease should not be seen as a duality, 
but as an evolving process in which tissue accumulation of triglycerides can be 
initially benign with subsequent progression to chronic inflammation and tissue 
damage (steatohepatitis). The presence of fibrosis is considered a prognostic marker 
of MAFLD and may be present to varying degrees, with or without inflammation, 
characterized by the formation of collagen fibers due to the transformation of stellate 
cells into fibroblast-like cells. 3 
The hepatic steatosis observed in patients with MAFLD is secondary to excessive 
synthesis of hepatic triglycerides from fatty acids derived from adipose tissue, de 
novo lipogenesis, and endocytosis of triglyceride-rich lipoproteins. Furthermore, the 
excessive triglyceride content in the liver tissue leads to secondary VLDL secretion, 
which is characteristic of the atherogenic dyslipidemia in patients with MAFLD. 
These patients also have elevated serum triglycerides, elevated LDL cholesterol, 
and low HDL cholesterol, explained by the activity of the plasma protein CETP, which 

3 
 
transfers cholesterol from HDL or LDL to triglyceride-rich lipoproteins (TRL). 4,5 
Remnant lipoproteins from lipid metabolism and systemic transport have the ability 
to passively deposit in arterial walls, triggering atherosclerosis. For this reason, the 
dyslipidemia present in fatty liver is associated with the risk of cardiovascular 
diseases and mortality. 6,7 
In addition to alterations in hepatic triglyceride metabolism, patients with MAFLD 
experience systemic metabolic dysregulation consistent with components of the 
metabolic syndrome. The most frequently associated comorbidities, now considered 
diagnostic criteria, are the presence of diabetes mellitus and obesity. 
Currently, there is no approved pharmacological treatment for MAFLD. However, it 
has been demonstrated that intervention with Mediterranean and hypocaloric dietary 
styles, with or without exercise regimens, improves intrahepatic fatty acid content 
and hepatic inflammation, as reflected by a reduction in transaminase levels, when 
weight loss of 5 to 7% is achieved. Improvement in fibrosis and inflammation can be 
observed with a 10% weight loss. 8,9 
Milk thistle (Silybum marianum) is a medicinal plant native to North Africa, Southern 
Europe, and Southern Russia, which contains flavonolignans including silybin, 
isosilybin, silidianin, and silicristin. It has been used in traditional Chinese medicine 
for the treatment of liver and biliary diseases. Currently, it has demonstrated 
antioxidant, anti-inflammatory, and antifibrotic effects in liver diseases, particularly in 
fatty liver disease. Its therapeutic effects are attributed to the active component of 
the plant's seeds, known as silymarin. 11,12 Silibin, the main active component of 
silymarin, inhibits oxidative stress in the endoplasmic reticulum of hepatocytes and 
the assembly of the NLRP3 inflammasome through the NAD+/SIRT2 pathway in 
mice with fatty liver. 13 
On the other hand, alpha-lipoic acid is a cofactor of mitochondrial enzymes that has 
been considered an antioxidant. Its reduced form, dihydrolipoate, reacts with 
reactive oxygen species, thereby protecting the cell membrane.14 Administration of 
alpha-lipoic acid has been shown to have beneficial effects on adipokine levels and 

4 
 
metabolic disorders such as diabetes and obesity, which are commonly present in 
patients with MAFLD. It has been demonstrated that the administration of 1200 mg 
of alpha-lipoic acid in patients with fatty liver disease reduces insulin resistance and 
serum leptin levels while increasing adiponectin concentration. However, it did not 
influence liver histology and biochemical markers. 15 
Therefore, our objective is to demonstrate that the administration of the combination 
of alpha-lipoic acid and Silybum marianum (LUDLEV®) can improve steatosis in 
patients with MAFLD when associated with a Mediterranean-style diet, while 
ensuring a favorable safety profile. 
 
 
 
RESEARCH QUESTION 
Can supplementation with alpha-lipoic acid and Silybum marianum (LUDLEV®) in 
combination with a Mediterranean diet improve steatosis and fibrosis in patients with 
MAFLD? 
HYPOTHESIS 
The administration of the combination of alpha-lipoic acid and Silybum marianum 
(LUDLEV®) in combination with a Mediterranean diet reduces the degree of fibrosis 
and steatosis in patients with MAFLD as assessed by kPa and CAP measurements 
after 24 weeks of treatment. 
OBJECTIVES 
Overall objective: 
To evaluate the effects of administering alpha-lipoic acid and Silybum marianum 
(LUDLEV® 300 mg/46.2 mg) in combination with a Mediterranean diet compared to 

5 
 
placebo plus a Mediterranean diet on fibrosis and steatosis in patients with MAFLD 
after 24 weeks of treatment. 
Specific Objectives: 
·         To assess the effects of administering alpha-lipoic acid and Silybum marianum 
(LUDLEV® 300 mg/46.2 mg) in combination with a Mediterranean diet versus 
placebo plus a Mediterranean diet on fibrosis and steatosis in patients with MAFLD 
after 12 weeks of treatment. 
·         To evaluate changes in fibrosis measured by kPa using FibroScan® 
elastography after 12 and 24 weeks of administering alpha-lipoic acid and Silybum 
marianum (LUDLEV® 300 mg/46.2 mg) every 12 hours in combination with a 
Mediterranean diet, compared to placebo plus a Mediterranean diet in patients with 
MAFLD. 
·         To assess changes in the controlled attenuation parameter (CAP) measured 
by FibroScan® elastography after 12 and 24 weeks of administering alpha-lipoic acid 
and silybum marianum (LUDLEV® 300 mg/46.2 mg) every 12 hours in combination 
with a Mediterranean diet, compared to placebo plus a Mediterranean diet in patients 
with MAFLD. 
·         To evaluate biochemical improvement after 12 and 24 weeks of administering 
alpha-lipoic acid and Silybum marianum (LUDLEV® 300 mg/46.2 mg) every 12 
hours in combination with a Mediterranean diet, compared to placebo plus a 
Mediterranean diet in patients with MAFLD. 
·         To assess weight loss and changes in body composition after 12 and 24 weeks 
of administering alpha-lipoic acid and Silybum marianum (LUDLEV® 300 mg/46.2 
mg) every 12 hours in combination with a Mediterranean diet, compared to a 
Mediterranean diet alone in patients with MAFLD. 
METHODOLOGY 
Research Design 

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Study type: Randomized, controlled, double-blind clinical trial. 
Population Definition 
Patients regularly attending the outpatient clinic of the Gastroenterology Department 
of the Instituto de Investigaciones Médico-Biológicas de la Universidad 
Veracruzana, diagnosed with MAFLD. 
 
Inclusion criteria: 
·         Subjects treated at the Instituto de Investigaciones Médico-Biológicas de la 
Universidad Veracruzana, diagnosed with fatty liver. 
·         Willingness to voluntarily participate in the study and signing of informed 
consent. 
·         Age over 18 years, both sexes. 
·         Patients with evidence of hepatic steatosis and overweight or obesity. 
·         Patients with evidence of hepatic steatosis, diabetes mellitus, and overweight 
or obesity. 
·         Patients with evidence of steatosis plus metabolic risk abnormalities (waist 
circumference >102 cm in men, 88 cm in women, blood pressure >130/85 mmHg or 
on specific treatment, triglycerides >150 mg/dL or on specific treatment, HDL 
cholesterol <40 mg/dL in men, <50 mg/dL in women or on specific treatment, HOMA-
IR >2. 5, CRP >2 mg/dL, or prediabetes defined by fasting plasma glucose 100-125 
mg/dL, 2-hour postload glucose 140-199 mg/dL, or HbA1c between 5.7% and 6.4%). 
·         Agree not to start a diet program during the study. 
·         Agree not to change their current exercise plan during the study. 
Exclusion criteria: 

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• 
Patients who meet diagnostic criteria for MAFLD with normal weight. 
• 
Patients with terminal illness, cancer, or end-stage renal disease on renal 
replacement therapy. 
• 
Pregnant women. 
• 
Patients who are unable to understand or follow the study protocol. 
• 
Sensitivity to the components of the formula. 
Elimination criteria: 
• 
Patients who do not comply with the follow-up time and drug administration. 
• 
Patients who do not comply with proper drug intake. 
• 
Patients with intolerance or severe adverse effects to the components of the 
drug. 
• 
Patients who are unable to undergo FibroScan® examination due to 
technical limitations. 
Sample size calculation 
It is calculated based on the previous study by Martinez Rodriguez et al.( Clinical 
and Experimental PharmacologyClin Exp Pharmacol 2014, 5:1) where forty patients 
with NAFLD were randomized into two groups to receive metformin 1500 mg q.a.d. 
or metformin 1500 mg q.a.d. + (15 mcg)-methionine (3 mg)-alpha lipoic acid (200 
mg) group for 24 weeks, and showed a 70% versus 15% decrease in ultrasound-
graded steatosis (p<0.001) in patients receiving alpha lipoic acid. Taking into 
account this difference in proportions, considering a power of 80%, alpha error of 
20% and confidence interval of 95%, 20 pairs (40 patients) are required to be 
randomized. If we consider a loss of 25% (n=10), a total sample of 50 patients would 
be required. 
Interventions  
Patients included in the study will be randomly distributed in a 1:1 ratio to receive 
the two treatment regimens. 

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1. Initial assessment and follow-up 
Initial medical assessment including: 
1. Complete medical history 
2. Hepatic ultrasound showing evidence of hepatic steatosis. The degree of 
hepatic steatosis will be determined according to Hamaguchi score (≥3 
points) classifying it as follows: absent (0 points), mild (1 point), moderate (2 
points) and severe (3 points). This scoring system has a maximum sum of 6 
points and considers hepatorenal echo contrast, hepatic brightness, deep 
attenuation and vascular visualization with a specificity of 100% and 
sensitivity of 91.7%. 16 
3. The risk of fibrosis will be determined according to FIB-4, NAFLD score or 
APRI. FIB-4 is considered high risk of fibrosis score ≥3.25, indeterminate 
1.45-3.25 and absent ≤1.45; in NAFLD score high risk with ≥0.675, 
indeterminate -1.455 to 0.675 and absent ≤-1.455; in APRI high risk of 
significant 
fibrosis 
with 
score 
≥1.5, 
indeterminate 
0.5-1.5 
and 
absent≤0.5.17,18,19 
4. Transitional liver elastography with FibroScan® will be performed at baseline, 
12 and 24 weeks follow-up to estimate hepatic fatty infiltration according to 
the controlled attenuation parameter (CAP) and hepatic stiffness in 
kilopascals (kpa). Considering fibrosis stages the following cut-off points F0 
≤6.4kpa, F1 6.5 to 6.9 kpa, F2 7 to 8.6kpa, F3 8.7 to 10kpa, F3-F4 10.1 to 
15 kpa and F4 ≥15kpa.    For hepatic fatty infiltration is considered by CAP 
S0 ≤247, S1 between 248 to 267, S2 268 to 279 and S3 ≥280. 10 
representative valid measurements with success rate (IQR) 30% are 
considered. All measurements will be taken by the same operator.21,22 
5. At baseline,12 and 24 weeks follow-up the following laboratory studies will be 
performed: blood biometry,blood chemistry, liver function tests, lipid profile 
and C-reactive protein, serum insulin, HOMA calculation. 
6. The study of viral hepatitis type B and C will be performed at the beginning of 
the study.  

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2. 
Nutritional assessment and follow-up  
The nutritional assessment will consist of: 
1. Nutritional medical history: anthropometric measurements, basic electrical 
impedance and nutritional diagnosis. 
2. A diet plan will be established with a Mediterranean style diet according to 
nutritional diagnosis and sex (caloric plans of 1400, 1500, 1600 or 1700 
kcal/day). The composition of the diet is designed to receive 20% protein, 
50% carbohydrates, 15% monounsaturated fats, 10% polyunsaturated fats, 
5% saturated fats, less than 250 mg/day cholesterol and 20-30 g/day fiber. 
3. Subsequent nutritional follow-up appointments: will be carried out in person 
on a monthly basis for 24 weeks. 
4. Anthropometric measurements and basic electrical impedance at 12 and 24 
weeks of follow up. 
5. Test of adherence to the dietary plan at 12 and 24 weeks of follow-up. 
3. 
Randomization by groups  
1. Patients will be randomly distributed by systematic sampling into 2 groups 
(A and B).  
• 
Group A will receive placebo in combination with a personalized low-calorie 
Mediterranean diet adjusted for age and sex (between 1400 to 1700 kcal/day) 
for 24 weeks. 
• 
Group B will receive alpha-lipoic acid ethyl esters and silybum marianum 
(LUDLEV® 300 mg/46.2 mg) every 12 hours in association with a 
personalized low-calorie Mediterranean diet adjusted for age and sex 
(between 1400 to 1700 kcal/day) for 24 weeks.  
 

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4. 
Follow-up and surveillance  
1. Physical activity: It will be evaluated by means of the international physical 
activity questionnaire (IPAQ) that categorizes the levels of physical activity 
in: low (category 1), moderate (category 2) and high (category 3) according 
to the intensity, frequency and duration of the training. The recommendation 
will be made not to modify the usual training plan. 
2. Nutritional plan: The monitoring will be carried out in a personalized way 
through a monthly face-to-face nutritional consultation. 
3. Pharmacological treatment: The adherence to the pharmacological 
treatment will be monitored through a monthly telephone interview. 
Telephone assistance will be available to resolve doubts and report adverse 
effects.  
 
5. 
Analysis of the results  
The analysis of the results, elaboration of figures and tables will be carried out with 
the statistical program IBS SPSS version 22.0. Nominal and ordinal variables will be 
described with frequencies and percentages; continuous and discrete variables with 
measures of central tendency and dispersion according to their distribution. The 
normality of data distribution will be evaluated with the Kolmogorov-Smirnov test, 
and nonparametric statistics will be used to evaluate intergroup differences between 
nominal variables. Non-parametric statistics will be used for ordinal variables. A p-
value of less than 0.05 will be considered statistically significant.  
 
 
ETHICAL ASPECTS  
During its development, this medical research will comply with international 
standards in accordance with the ethical principles for medical research on human 

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beings established in the Declaration of Helsinki of the World Medical Association 
(64th General Assembly, Fortaleza, Brazil October 2013) and with the national 
legislation in force indicated in the General Health Law: Title Five; Research for 
Health; Sole Chapter, to the Regulation of the General Health Law on Research: 
Title Two; Of the Ethical Aspects of Research on Human Beings; Chapter I; Articles 
16 and 23.  
This is a low risk research and will not be carried out on vulnerable population groups 
according to the General Health Law Regulations on Research.  
It will be controlled by clinical record in total privacy respecting the guidelines 
established in the Mexican Official Standard NOM-012-SSA3-2012 that establishes 
the criteria for the execution of research projects for health in human beings and 
safeguarding the privacy of patients in compliance with the Federal Law for the 
Protection of Personal Data in Possession of Individuals; Chapter II and the Federal 
Law of Transparency and Access to Public Governmental Information; Chapters 20 
to 22. 
Measures will be taken to protect the confidentiality of such data, omitting information 
that could reveal the identity of individuals, limiting access to the data only to the 
research team and using a numerical code to identify patients without using names. 
 
 
 
 
  
SCHEDULE OF ACTIVITIES 2022-2023 

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ACTIVITIES 
APRIL 
MAY 
JUNE 
JULY 
AGO 
SEP 
OCT 
NOV 
DIC 
ENE 
FEB 
MARCH 
APRIL 
MAY 
JUN 
JUL 
AGO 
SEP 
Elaboration and 
approval of the 
research project 
 
Elaboration of 
placebo  
X 
X 
Patient recruitment  
- Medical and 
nutritional 
evaluation.  
- Initial laboratory 
studies 
- Nutritional 
counseling to 
establish a 
Mediterranean style 
dietary plan. 
- Initial FibroScan® 
study 
 
 
X 
X 
X 
 
 
 
 
Medical and 
nutritional follow-up 
and monitoring (12 
weeks) 
- Monitoring of 
compliance with the 
nutritional plan and 
treatment.  
- Laboratory studies 
- FibroScan® study 
 
 
 
 
X 
X 
X 
 
 

13 
 
Medical and 
nutritional follow-up 
and monitoring (24 
weeks) 
- Monitoring of 
compliance with the 
nutritional plan and 
treatment.  
- Laboratory studies 
- FibroScan® study 
 
 
 
 
 
X 
X 
X 
 
Data processing 
X 
Statistical analysis of 
results 
X 
Reporting the 
results  
Manuscript writing  
X 
Dissemination 
X 
 
 
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