2. Nonalcoholic Fatty Liver Disease
(“insulin-resistance associated steatosis”)
69%
14%
7%
3%
1%
6%
NAFLD Definition*
• liver fat (by histology) >5-10%
• Fewer than 2-3 drinks per day
• No evidence of other liver disease
• Prevalence 10-24% (as common as
the metabolic syndrome)
• Histologically covers a range of liver
disease from benign steatosis to NASH
• is the most common cause of elevated
LFTs
Causes of elevated LFTs
(steatosis)-NHANES III
NAFLD
Alcohol
Hepatitis
Hemochromat
Hepatitis B
Combination
2
3. 20 % population (UTZ)
Steatosis/Fatty Liver
1-9% ( liver biopsy)
Cirrhosis
Liver related Death (10 years)
Type of NAFLD(III and IV)
Diabetes Type II
Matteoni, Gastroenterology, 1999
Natural History of NAFLD
??
3
4. How is NAFLD diagnosed
• Risk factors may be helpful e.g. type 2
diabetes, central obesity, age> 45 years
• Often increase ALT incidental finding (e.g.
insurance medical)
• AST/ALT<1, non specific antibodies
positive
• HOMA (insulin resistance marker)
increased
• Ultrasound – echogenic liver with vascular
blurring, otherwise NAD
• Diagnostic liver biopsy – but patchy
disease and subject to variation in
interpretation 4
5. Therapy for NASH should prevent or reverse
hepatic injury induced by lipotoxicity
One strategy is to correct
IR and hyperinsulinemia
and to reduce fat mass, in
particular visceral
adiposity.
A second strategy is to
prevent/reverse hepatic
cellular damage induced
by lipotoxicity.
Inhibiting lipid
peroxidation and oxidative
stress, or use of anti-
inflammatory, anti-
apoptotic or
epatophrotective
agents.
STEATOSIS NASH
INSULIN
RESISTANCE
Insulin
sensitizers
HYPERLIPIDEMIA
Lipid
lowering
drugs
Physical
activity
Body
weight
ENDOTOXEMIA
OXIDATIVE
STRESS
Blockade
of TNF
Antioxidants
6. Management Algorithm
Trial of weight reduction, exercise, dietary changes, improved lipid or
diabetic control, complete cessation of alcohol, discontinue all
potential hepatoxic drugs
Consider antioxidant therapy and entry
into clinical drug trial
Referral to
Hepatologist
Probable NAFLD Definite (biopsy confirmed NASH)
Improved
LFT’s ↑ LFT’s
Observe
Steatosis Liver Biopsy Cirrhosis
NASH
6
7. Silymarin is a mixture of 4 isomeric flavonoids
extracted from the fruits of Silybum marianum
HO
OH
O
O
OH
O
O
CH2OH
OCH3
OH
HO
OH
O
O
OH
O
O CH2OH
OCH3
OH
HO
OH
O
O
OH
O
CH2OH
OCH3
OH
OH
O
OCH3
O
H
H
OH
2'
3'
Silibinin Isosilibinin
Silicristin Silidianin
C25H22O10: mol. wt. 482
7
8. Mechanism of Action of
Silymarin
Main Effect
Drug Cellular Level
Molecular
Level
Silymarin
(Silybin)
Cytoprotective
and cell-
regenerating
action
Free Radical
Scavenger
GSH-sparing
action
Reversible
Inhibition of
CP450
Free Radical
Scavenger
Ribosomal RNA
synthesis
Increase of
Protein Synthesis
Valenzuella A, Garride A: Biochemical bases of the pharmacological
action of the flavonoid silymarin and of its structural isomer silibinin.
Biol Res 1994, 27, 105-112.
Membrane –
stabilizing action
Microsomal
Xenobiotic
metabolism
Nuclear and
nucleolar
action
8
9. Effect of silybinin on liver metabolic processes
related to glycemia regulation
9
10. Membrane Effect
Silibinin is incorporated in
the cell membrane and inhibits
toxin transport
Receptor blockade
n. of pts. with amatoxin intoxication
treated survived
treatments
Mengs U Current Pharmaceutic Biotechnology 13: 2012
10
11. Lieber CS et al.:J Clin Gastroenterol (2003) 37: 336-339
Silymarin reduces lipid peroxidation in ethanol-intoxicated baboons.
Silymarin reduces lipidperoxidation
in ethanol -intoxicated baboons
Silymarin prevents accumulation of
collagen I in alcohol-induced liver
fibrosis
Antifibrotic Effect
11
12. Silymarin and Liver Cirrhosis
Main findings in the placebo-controlled trials
Analysis Variable Effect of
Silymarin Significance
Pooled Primary
Endpoints
Total mortality
Liver related mortality
Reduced 4.4%
Reduced 7.3%
NS
P < 0.01
Pooled Secondary
Endpoints
He Hospitalization
UGIB
HCC
Reduced 6.3%
Reduced 6.1%
Reduced 1.6%
P < 0.1
P < 0.05
NS
Individual studies
Secondary Endpoints
Encephalopathy
Cirrhosis + DM (IR)
Reduced 8.7%
Reduced 25%
P = 0.06
P < 0.01
13
13. Phosphatidylcholine (PC)
They are a major component
of biological membranes and the
principal PL circulating in plasma,
where it is an integral component of
the lipoproteins, especially HDL.
It can be easily obtained from a variety
of readily available sources such
as egg yolk or soy beans from which
they are mechanically extracted or
chemically extracted using hexane.
14
14. Essential Phospholipids
✦ Essential Phospholipids
(EPL) is the highly
purified fraction of
phosphatidylcholine with
polyunsaturated fatty
acids in positions C1 and
C2, whereby 1,2
dilinoleyphosphatidylchol
ine is the main active
ingredient.
✦ Taken orally PC is very
well absorbed, up to
90% per 24 hrs when
take with meals.
E. Kuntz, H.-D. Kuntz: HEPATOLOGY Textbook and Atlas, 3rd Edition 15
16. EPL has effective antioxidative properties
Aleynik SI, Lieber CS: Alcohol & Alcoholism 2003; 38: 208-212
SAMe,
nmol/g;
Mean
+/-
SEM
***
*
**
0
10
20
30
40
50
60
70
80
90
100 Control (n=8)
Ethanol (n=8)
EPL(n=6)
Ethanol+EPL (n=9)
GSH,
µmol/g;
Mean
+/-
SEM
**
*
#
0
1
2
3
4
5
6
7
8
Control(n=8)
Ethanol (n=8)
EPL (n=6)
Ethanol+EPL (n=9)
Effect of alcohol and/or EPL on hepatic S-
adenosylmethionine (SAMe) in rats. EPL significantly
attenuated the ethanol induced decrease of SAMe
* P <0.05, *** P <0.001 vs control; ** P <0.01 vs
alcohol
Effect of alcohol and/or EPL on hepatic reduced
glutathione (GSH). EPL decreased ethanol induced
oxidative stress by fully restoring hepatic GSH
* P <0.05, ** P <0.01 vs control; # P <0.01 vs alcohol
17
17. EPL in NAFLD and ALD:
A Systematic Review of Double-Blind Studies
Hu G et al: Liver 2005; 10: 5-7
1. Knüchel F et al: Med. Welt 1979; 30: 411-416 n= 25/25
2. Schüller Pérez A et al: Med. Welt 1985; 36: 517-521 n= 20/20
3. Gonciarz Z et al: Med. Chir. Digest. 1988; 17: 61-65 n= 15/15
4. Marios Z et al: Eur. J. Gastroenterol. Hepatol. 1990; 2: 351-355 n= 53/51
5. Li J et al: Infect. Dis. Information 2000; 13: 180-182 n= 24/12
6. Lieber CS et al: Clin. Exp. Res. 2003; 27: 1757-1764 n= 202/210
n = 339 NAFLD; n = 333 ALD
Effect on overall clinical efficacy (clinical
symptoms and biochemical variables)
4 studies
(1-3, 5)
P=0.03
Effect on histology
3 studies
(3, 4, 6)
P=0.02
Effect on mortality 1 study (4) P=0.07
18
19. Silybin has one limiting
factor
Silybin low solubility in water ,
low availability and poor
intestinal absorption
20
20. SILIPHOS
1:2 COMPLEX OF PHOSPHATIDYL CHOLINE
protects the liver by
conserving glutathione
in the parenchymal cells
repair and replace cell
membranes.
21
21. PC, the principal molecular building block of cell membranes, is miscible both in
water and in oil/ lipid environments, and is well absorbed when taken by mouth.
22
22. Silybin first combines with the polar head group of PPC, forming a SLB-PPC
complex.
Using the co-precipitation method, the hydrocarbon tails of the PPC bind to the
hydrophobic surface of SDC, forming spheres and exposing the hydrophilic
surfaces of SDC to contact with the aqueous solvent.
Dual Acta Pharmacologica Sinica 2011 23
23. an SDC concentration of 20 mg/mL, at which
concentration the PPC-SDC molar ratio was 1 and
solubilized silybin could be up to 10.14±0.36 mg/mL
After reaching a concentration of 60 mg/mL, continuously increasing
the PPC concentration resulted in a relatively stable solubilization of SLB
and eventually decreased solubility
Thermogram of SLB,
PPC complex
24
24. In rats, after oral administration of 200
mg/kg of silybin, the plasma levels of this
drug were below the analytical detection
limit, while, after oral administration of
SILIPHOS® (200 mg/kg as silybin) the
plasma levels of silybin (free and total)
were easily measurable
after oral administration of
SILIPHOS®, the biliary elimination
of silybin was not complete at 24 h
and accounted for about 3.7% of
the administered dose
The compound was rapidly
excreted in urine where at
72 h the amount recovered
accounted for about 3.3%
Pharmacokinetic studies of free silybin and silymarin,
SILIPHOS® represents the most absorbable form of silybin
25
25. Plasma levels of unconjugated and total
silybin in 3 subjects on the first and last
day of treatment with Silyphos 120 mg
BID for 8 consecutive days
Biliary concentration s of SilyBin following
administration of as single 120 mg oral dose
of Siliphos and silymarine in a
cholecystectomy patient
26
27. steatohepatitis characterized by
panacinar steatosis
predominantly as
macrovesicular fat with lobular
inflammation
macrovesicular liver
steatosis with
minimal neutrophil
recruitment
moderate-severe steatohepatitis
With diffuse lobular and
portal inflammatory infiltrate with the
presence of microgranulomas,
neither
portal
inflammation nor
microgranulomas
SILIPHOS
• preserved mitochondrial
bioenergetics and prevented
mitochondrial proton leak and ATP
reduction.
• limited the formation of HNE- and
MDA-protein adducts.
Servidio, J OF PHARMACOLOGY AND EXPERIMENTAL
THERAPEUTICS 2011
28
28. Effects of silymarin and silibinin: phosphatidylcholine complex on plasma
and lipoprotein cholesterol, and Cu2+-mediated oxidation of LDL in rats
fed on high-fat (lard fat or currant oil), high-cholesterol diet
Silymarin was effective in prevention of development of dietary induced
hypercholesterolemia but SPC supressed more effectively than silymarin
LDL oxidizability. 29
29. The Effect of a Silybin-Vitamin E-Phospholipid Complex
on Nonalcoholic Fatty Liver Disease: A Pilot Study
Loguercio, GUT 2006 30
Editor's Notes
Therapy for NASH should prevent or reverse hepatic injury
induced by lipotoxicity. One strategy is to correct IR and hyperinsulinemia
and to reduce fat mass, in particular visceral adiposity.
Weight loss and physical exercise, diet and lifestyle changes,
insulin-sensitizing agents and anti-obesity surgery are all aimed
at this objective. A second strategy is to prevent/reverse hepatic
cellular damage induced by lipotoxicity. This can be achieved
by inhibiting lipid peroxidation and oxidative stress, or through
the use of anti-inflammatory, anti-apoptotic or other hepatoprotective
agents. These two strategies may be at best combined, and
future therapeutic research in NASH should focus on tailoring this
dual approach to the individual patient. The treatment and monitoring
of metabolic and cardiovascular comorbidities should be
implemented alongside the hepatologic management.
Structurally related to flavonoids, queretin and fisetin which have been previously demonostrated to be bery active on liver metabolic processes related to glycemia regulation. A a concentration range of 50 to 300 uM, silibinin inhibited flucegenersis in the fasted state and ingibited flucogenolysis and glycolysis in the fed condition. The mechanisms by which silibimnin exerted these activeions were multiple and complex. It inhibited the activity of flucose 6 phosphatase inhibited pyruvate carrier and reduced the efficiency of mitochondrial energytransduction. Ti also act vy reducing the supply of NADH for fulconogenesis and mitochondria throug ti prooxidative actions.
Alos exeted some dostomct effects pm omgjobptpru effect on oxygen consumption athe fed condition an a change in the eneergy status of the prefused livers.
a. Histopathology of liver in normal animals
b. Histopathology of liver in carbon tetrachloride treated animals
c. Histopathology of liver in animals treated with silymarin lipid
emulsion (SLE) (after carbon tetrachloride treatment).
d. Histopathology of liver in animals treated with plain lipid
emulsion (PLE) (after carbon tetrachloride treatment).
e. Histopathology of liver in animals treated with silymarin solution
(after carbon tetrachloride treatment
Class of phospholipids that incorporate choline as a headgroup.
SAME
GS
Most of the bioactive constituents of phytomedicines are flavonoids (e.g., anthocyanidins from bilberry, catechins from green tea, silymarin from milk thistle). However, many flavonoids are poorly absorbed.1 The poor absorption of flavonoid nutrients is likely due to two factors.
First, they are multiplering molecules too large to be absorbed by simple diffusion, while they are not absorbed actively, as occurs with some vitamins and minerals. Second, flavonoid molecules typically have poor miscibility with oils and other lipids, severely limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine. pharmacokinetic studies in comparison with free silybin and silymarin, SILIPHOS® represents the most absorbable form of silybin
In 1990, Malandrino succeeded in improving the bioavailability of silymarin extract by complexing it with soy PC – a phytosome.8 Subsequently, a more purified silybin was complexed withPC. The intermolecular bonding of silybin with PC proved to be specific and stable, and the resulting molecular complex is more soluble in lipophilic, organic solvents.9 This property predicts the enhanced ability of phytosomes to cross cell membranes and enter cells.Silybin is actually a flavonolignan, probably produced within the plant by the combination of a flavonol with a coniferyl alcohol. It is now known that silybin is the most potent of the three.5 Silybinprotects the liver by conserving glutathione in the parenchymal cells, while PC helps repair and replace cell membranes.7 These constituents likely offer thesynergistic benefit of sparing liver cells from destruction. In its native form within the milk thistle extensively researched and found to have impressive bioactivity, albeit limited by poor bioavailability. In 1990, Malandrino et al succeeded in improving the bioavailability of silymarin extract by complexing it with soy PC – a phytosome.8 Subsequently, a more purified silybin was complexed withPC. The intermolecular bonding of silybin with PC proved to be specific and stable, and the resulting molecular complex is more soluble in lipophilic, organic solvents.9 This property predicts the enhanced ability of phytosomes to cross cell membranes and enter cells.
Water-soluble flavonoid molecules can be converted into lipid-compatible molecular complexes, aptly called phytosomes. Phytosomes are better able to transition from a hydrophilic environment into the lipid-friendly environment of the enterocyte cell membrane and from there into the cell, finally reaching the blood.2 The lipid-phase substances employed to make flavonoids lipid-compatible are phospholipids from soy, mainly phosphatidylcholine (PC). PC, the principal molecular building block of cell membranes, is miscible both in water and in oil/ lipid environments, and is well absorbed when taken by mouth. Precise chemical analysis indicates a phytosome is usually a flavonoid molecule linked withat least one PC molecule. A bond is formed between the two molecules, creating a hybrid molecule. This highly lipid-miscible hybrid bond is better suited to merge into the lipid phase of the enterocyte’s outer cell membrane. Phosphatidylcholine is not merely a passive “carrier” for the bioactive flavonoids of the phytosomes,
but is itself a bioactive nutrient with documented clinical efficacy for liver disease, including
Despite the promising biological effects of SLB, its poorwater solubility (about 40 μg/mL) and oral bioavailability (about 0.73%) in both rodents and humans, as reported by several researchers[12–15], has restricted its clinical application. To improve this situation, there have been many contributions to the development of SLB formulations[15–17]. However, few have produced a marked effect, and a majority of them are based on oral drug delivery systems. Because no proper formulation of silybin has been approved for intravenous administration, our goal was to develop a novel mixed micelle system for its parenteral Upon consideration of its physiological compatibility and solubilizing capacity, the phosphatidylcholine-bile saltsmixed micelle system (PC-BS-MM) seems to be a good candidate for intravenous drug administration[18, 19]. Bile salts (BS),
detergent-like chemicals produced by the liver and stored in the gall bladder, are able to solubilize phosphatidylcholine (PC) to a large extent, forming a clear mixed micellar solution 20]. Previous studies have shown that the hemolytic effect of bile salts can be neutralized by solubilized phospholipids[21]. In addition, PC-BS-MMs can be locally and systemically tolerated with no embryotoxic, teratogenic or mutagenic effects[22].
Therefore, these PC-BS-MMs might be the ideal drug carrier of SLB for parenteral administration.
The influence of the PPC concentration on SLB solubility and drug loading efficiency was also investigated. SLB solubility was enhanced by incorporating increasing amounts of PPC in the MM system. After reaching a concentration of 60 mg/mL, continuously increasing the PPC concentration resulted in a relatively stable solubilization of SLB and eventually decreased solubility. However, a further increase in the PPC amount (after 40 mg/mL) led to a dramatic decrease in drug loading efficiency. Hence, to ensure higher solubilization efficiency and lower viscosity, a PPC concentration of 40 mg/mL was chosen for further investigations.
At this concentration, the PPC-SDC molar ratio was 1, which agrees with the results regarding the influence of the SDC concentrationAfter reaching a concentration of 60 mg/mL, continuously increasing the
PPC concentration resulted in a relatively stable solubilization of SLB and eventually decreased solubility
As demonstrated by pharmacokinetic studies in comparison with free silybin and silymarin, SILIPHOS® represents the most absorbable form of silybin known until now. In rats, after oral administration of 200 mg/kg of silybin, the plasma levels of this drug and its conjugated metabolites were below the analytical detection limit, while, after oral administration of SILIPHOS® (200 mg/kg as silybin) the plasma levels of silybin (free and total) were easily measurable (Fig. 2).
Furthermore, after oral administration of SILIPHOS®, the biliary elimination of silybin was not complete at 24 h and accounted for about 3.7% of the administered dose (Fig. 3). The compound was rapidly excreted in urine where at 72 h the amount recovered accounted for about 3.3% (Fig. 4). After administration of uncomplexed silybin, biliary and urinary elimination accounted for only 0.001% and 0.032%, respectively.The improvement of the oral bioavailability for SILIPHOS® is mainly dependent on a marked increase of its absorption in the gastrointestinal tract, most likely due to the lipophilic character of the complex. Also in
comparison with silymarin, SILIPHOS® demonstrated a superior bioavailability which, as calculated for cumulative biliary excretion, resulted to be about 10-fold higher than that of the extract.8ILIPHOS® shows the same pharmacokinetic profile in man. After oral treatment, the bioavailability in healthy volunteers, in cholecystectomised patients or in patients suffering from hepatic cirrhosis is comparable to that demonstrated in animal models.After oral intake of SILIPHOS®, silybin appears rapidly in the bloodstream and is eliminated from the plasma with a relatively short half-life. Silybin ismetabolized extensively, most of the drug recovered by the systemic circulation being present as sulphate and/or glucuronide conjugates. nly a small fraction of the dosage could be recovered in urine indicating that silybin is mostly eliminated by
biliary excretion.
Silymarin, butnot SPC, was effective in prevention of development of dietary induced hypercholesterolemia in the both dietary
fats with slightly better result in diet containing the currant oil. On the other hand, SPC supressed more effectively
than silymarin LDL oxidizability. The results suggest that antihypercholesterolemic effect of SM in rats fed on HCdiet
is improved by dietary currant oil, but the currant oil induces an increased oxidizability of LDL. This can be
supressed by improvement of bioavailability of silibinin, as shown here for silibinin-phosphatidylcholine complex
