Introduction

In recent years, there has been a rapid increase in the number of patients with liver cirrhosis. According to statistical data from the World Health Organization, from 2020 to the present day, this disease is among the top ten leading causes of death worldwide, characterizing this pathology as a formidable medical, social, and economic problem [1]. In the Russian Federation, the number of patients diagnosed with liver cirrhosis for the first time in their lives is also increasing every year, and by 2023, this number had steadily reached 80.1 per 100,000 population, exceeding the same indicator for 2020 by 6 times! [2].

A key link in the pathogenesis of liver cirrhosis is portal hypertension syndrome. The manifestations of this symptom complex are diverse, the most urgent and life-threatening of which is varicose veins of the esophagus and stomach complicated by bleeding. In this regard, the problem of preventing bleeding from varicose veins is currently very relevant. For this purpose, modern treatment regimens for patients with liver cirrhosis include non-selective β-blockers (β-BBs), one of which is propranolol. The clinical recommendations of the Russian Gastroenterological Association for the treatment of patients with liver cirrhosis recommend prescribing propranolol at a dose that reduces the resting heart rate by 25% or to 55 beats per minute with an initially low pulse. Thus, the dose of the drug can vary from 10 to 320 mg per day and is selected individually [3].

Foreign guidelines also still do not offer a clear algorithm for prescribing propranolol. The latest clinical practice guidelines from the European Association for the Study of the Liver (EASL) state that the use of non-selective β-blockers in patients with liver cirrhosis should be based on an assessment of the risk/benefit ratio for the patient and their central hemodynamic parameters [4]. Furthermore, in some cases, β-blockers do not exert a positive hemodynamic effect.

Cytochrome 2D6 (CYP2D6) is an isoenzyme of cytochrome P450. It has been established that CYP2D6 is responsible for the metabolism of a wide range of drugs. These drugs also include non-selective β-blockers, including propranolol [5]. The metabolism of the latter is carried out via a 4-hydroxylation reaction [6]. One of the features of CYP2D6 is the significant variability of its activity in the population. The main reason for this variability is genetic polymorphism, i.e., the existence of different alleles of the CYP2D6 gene. The most common CYP2D6 alleles are represented by the following functional groups: normal function alleles (e.g., CYP2D6*1, *2, and *35), decreased function alleles (e.g., CYP2D6*9, *10, *17, *29, and *41), and non-functional allelic variants (CYP2D6*3, *4, *6, *7, *8, *11, *12, *14, *15, *19, *20) [7].

In 2020, we conducted a prospective randomized study aimed at optimizing the effectiveness of pharmacotherapy with β-blockers for portal hypertension syndrome in patients with liver cirrhosis using pharmacogenetic technologies [8]. This article presents two clinical examples of patients who participated in this research study.

Objective

To demonstrate, through clinical examples, the differences in the efficacy of propranolol, determined by the dynamics of the mean linear velocity of portal vein blood flow (MLPV), in patients with liver cirrhosis and different genotypes for the polymorphic marker CYP2D6*4.

Materials and methods

The prospective randomized study, conducted at the clinical base of the Department of Gastroenterology of the Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation, involved 60 patients diagnosed with liver cirrhosis.

Inclusion criteria for the study: confirmed diagnosis of liver cirrhosis of various etiologies, Child-Pugh class A, B, and C, age under 75 years, and the patient's signed informed voluntary consent to participate in the study and for the processing of personal data. 

Non-inclusion criteria were the presence of contraindications to the use of propranolol, as regulated in the instructions for medical use of the drug approved by the Ministry of Health of the Russian Federation; concomitant use of CYP2D6 isoenzyme inhibitors or drugs metabolized via CYP2D6; patient history of bleeding from esophageal varices during the current hospitalization; and concomitant oncological disease.

To verify the diagnosis of liver cirrhosis, clinical methods (collection of complaints and medical history, general visual examination, measurement of abdominal volume, percussion and palpation of the abdomen with determination of liver and spleen sizes by Kurlov) and laboratory research methods (complete blood count and urinalysis, coagulogram, biochemical blood test with determination of total and direct bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, total protein, albumin, glucose, creatinine, and urea) were used. To assess the presence or absence of complications and determine their severity, instrumental research methods were used: ultrasonography of the abdominal organs using Dopplerography of the portal vein. Measurements were taken of the liver and spleen sizes with a description of their echo-structure, the diameter and mean linear velocity of portal vein blood flow, and the amount of ascitic fluid. The reference value for the mean linear velocity of portal vein blood flow was taken as 22.9±4.2 cm/s [9]. To detect or exclude esophageal varices, portal hypertensive gastropathy, and signs of bleeding, esophagogastroduodenoscopy was performed.

To determine the dynamics of portal hypertension syndrome during propranolol therapy at a dose of 10 mg three times a day after 14 days, the following ultrasonographic parameters were reassessed: portal vein diameter and linear velocity of blood flow in the portal vein (Toshiba Aplio 500 ultrasound diagnostic device). We considered any increase in MLPV compared to baseline as a criterion for response to propranolol therapy. To assess the impact of the CYP2D6*4 (1846G>A) gene polymorphism on the hemodynamic effect of propranolol within the scientific research, an increase in MLPV by ≥10% and ≥20% compared to baseline was taken as a response criterion.

Patients' venous blood was collected on the first day of the study into vacuum tubes with EDTA-K3 IMPROVACUTER (Guangzhou Improve Medical Instruments Co., Ltd, China). Genomic DNA was isolated from whole blood using the S-Sorb reagent kit for DNA isolation on a silicon sorbent (Syntol LLC, Russia). The carriage of the CYP2D6*4 (1846G>A) polymorphic marker was determined using commercial reagent kits for detecting the corresponding polymorphisms (Syntol LLC, Russia and TaqMan® SNP Genotyping Assays and TaqMan Universal Master Mix II, no UNG, Applied Biosystems, USA) by real-time PCR on a CFX96 Touch Real Time System instrument with CFX Manager software version 3.0 (BioRad, USA). This study was conducted at the Research Institute of Molecular and Personalized Medicine of the Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation.

To assess the influence of the CYP2D6*4 (1846G>A) gene polymorphism on the efficacy of propranolol therapy in patients with liver cirrhosis, a univariate logistic regression model was constructed. Calculations were performed on a personal computer with the Windows 10 Home operating system using the licensed software product STATISTICA v10.0 ("StatSoft Inc.", USA).

Clinical case

Clinical case No. 1. Patient B., 63 years old. Hospitalized due to decompensation of liver cirrhosis.

Upon admission, he complained of an increase in abdominal volume, decreased appetite, and mixed dyspnea occurring at rest.

Medical history: One year prior to this hospitalization, he was diagnosed with alcoholic liver cirrhosis. Past medical history includes ligation of esophageal veins due to grade 3 varices. He intermittently takes ademethionine and spironolactone. He noted a deterioration in his health over the week, manifested by increased lower extremity edema, an increase in abdominal size, and progressive mixed dyspnea at rest, worsening in the supine position.

Objective examination findings revealed pallor of the skin, icteric sclera, palmar erythema, symmetric pastosity of both lower legs and feet, sinus tachycardia up to 96 beats per minute, and tachypnea with a respiratory rate of 20 per minute. The tongue was dry, moderately coated with a white coating over the entire surface. The abdomen actively participated in breathing, was enlarged (101 cm) due to ascites, and was not tense. Palpation was painless in all areas. Liver sizes by Kurlov and spleen size could not be reliably determined due to ascites.

Laboratory tests revealed impaired protein synthetic function of the liver in the form of hypoalbuminemia, and hyperbilirubinemia indicative of hepatocellular insufficiency. Markers for viral hepatitis B and C were not detected in the blood (Table 1).

Table 1. Patient's laboratory test data

Complete Blood Count     RBC, 1012/LHb, g/LMCV, unitsWBC, 109/LPLT, 109/LESR, mm/h4.09128899.421950

  Urinalysis       ColorSpecific GravitypHProtein, g/LBilirubinUrobilinogenLeukocytesRBCYellow10265.00.1++1.0++

 Biochemical Blood Test      Total Protein, g/LAlbumin, g/LALT, U/LAST, U/LTotal Bilirubin, µmol/LAlpha-Amylase, U/LAlkaline Phosphatase, U/L8123195033.390234

 Coagulogram    INRThrombin Time, secProthrombin (Quick), %aPTT, secFibrinogen, g/L1.3516.745.136.62.4

 Viral Hepatitis Markers HbsAganti-HCVNot detectedNot detected

According to abdominal ultrasound, an increase in liver size was found: right lobe 165 mm, left lobe 88 mm. Liver characteristics: contours clear, smooth, echogenicity significantly increased, echostructure homogeneous, no masses. Common bile duct not dilated. Intrahepatic ducts not dilated. Portal vein diameter 15 mm. Mean linear velocity of portal vein blood flow 7.4 cm/s. Resistivity index 0.17. Spleen size 105x58 mm. Spleen area 45 cm². Spleen volume 215 cm³. Free fluid in the abdominal cavity is detected in large amounts in all areas.

Thus, the patient's ultrasonography reveals signs of hepatomegaly, portal hypertension (including a significant decrease in the mean linear velocity of portal vein blood flow), and ascites.

Esophagogastroduodenoscopy: Status post ligation of esophageal varices.

Based on the medical history, complaints, physical examination, laboratory, and instrumental findings, the patient was diagnosed with:

Primary disease: Liver cirrhosis of nutritional etiology, Child-Pugh class B (8 points), MELD score 19. Maddrey's Discriminant Function 25.87.

Complications of the primary disease: Portal hypertension: Status post endoscopic ligation of esophageal varices due to grade 3 esophageal varices. Hepatocellular insufficiency: hypoalbuminemia. Grade 2 ascites.

The patient underwent pharmacogenetic testing — detection of the CYP2D6*4 (1846G>A) polymorphic marker by real-time polymerase chain reaction. A heterozygous G/A1846 genotype was determined.

The patient was prescribed complex therapy: hepatoprotective (ursodeoxycholic acid 500 mg/day), infusion (5% glucose solution + 20% riboxin solution in a volume of 500 ml/day), diuretic (furosemide 80 mg/day, spironolactone 150 mg/day).

To correct portal hypertension, propranolol was prescribed at 10 mg three times a day per os.

Two weeks after starting propranolol therapy, a follow-up ultrasonographic measurement of the mean linear velocity of portal vein blood flow showed a slight increase to 8.0 cm/s, i.e., by 0.6 cm/s or 8.1% compared to the baseline MLPV (Fig. 1).

Fig. 1. Dynamics of patient mean linear velocity of portal vein blood flow and the reference value, cm/s

This clinical case demonstrates an insufficient therapeutic effect (increase in MLPV less than 20% from baseline) of propranolol in a patient with the heterozygous *G/A1846* genotype, which necessitated an increase in the drug dose considering central hemodynamic parameters.

Clinical case No. 2. Patient G., 42 years old.

Complaints on admission: Increase in abdominal volume, decreased appetite, nausea, severe general weakness.

Anamnesis morbi: Previously, he did not suffer from chronic digestive diseases and was not followed by a gastroenterologist. The current deterioration occurred within 10 days prior to this hospitalization after a week-long period of alcohol abuse.

Physical examination of the patient revealed icteric sclera, palmar erythema, telangiectasias on the skin of the back and anterior chest wall, and dilatation of the subcutaneous venous network of the anterior abdominal wall. The patient's speech was slowed (answering in short sentences with pauses). The tongue was crimson in color, coated with a white coating at the base. The abdomen actively participated in breathing, was of normal shape, and not distended. Palpation was soft, painless in all areas. Liver size by Kurlov 12x11x10 cm, the edge was firm, painless on palpation. Spleen size 10x7 cm, on palpation its edge was soft, painless.

Laboratory tests revealed grade 1 thrombocytopenia, decreased red blood cell levels, and a moderate biochemical syndrome of cytolysis, reflecting hepatocellular insufficiency and hypersplenism syndrome. Macrocytosis of erythrocytes indicates an alcoholic genesis of cirrhosis. Markers for viral hepatitis B and C were not detected in the blood (Table 2).

Table 2. Patient's laboratory test data

Complete Blood Count     RBC, 1012/LHb, g/LMCV, unitsWBC, 109/LPLT, 109/LESR, mm/h3.96134102.99.712156

 Urinalysis       ColorSpecific GravitypHProtein, g/LBilirubinUrobilinogenLeukocytesRBCDark yellow10265.50++2.0+-

 Biochemical Blood Test      Total Protein, g/LAlbumin, g/LALT, U/LAST, U/LTotal Bilirubin, µmol/LAlpha-Amylase, U/LGGTP, U/L85386515068.3632931

 Coagulogram    INRThrombin Time, secProthrombin (Quick), %aPTT, secFibrinogen, g/L1.3213.647.737.23.07

 Viral Hepatitis Markers HbsAganti-HCVNot detectedNot detected

Abdominal ultrasound: Liver — right lobe 218 mm, left lobe 124 mm. Liver characteristics: contours clear, smooth, echogenicity significantly increased, echostructure homogeneous, no masses. Common bile duct and intrahepatic ducts not dilated. Portal vein diameter 12.3 mm. Mean linear velocity of portal vein blood flow 6.4 cm/s. Resistivity index 0.52. Splenic vein diameter 10 mm. Spleen size 163x103 mm. Spleen volume 863 cm³. Free fluid in the abdominal cavity is detected interloop.

Thus, the patient has ultrasonographic signs of hepatosplenomegaly, portal hypertension (including a significant decrease in the mean linear velocity of portal vein blood flow), and grade 1 ascites.

Esophagogastroduodenoscopy: Grade 2 esophageal varices, duodenogastric reflux.

Based on the medical history, complaints, physical examination, laboratory, and instrumental findings, the patient was diagnosed with:

Primary disease: Liver cirrhosis of nutritional etiology, Child-Pugh class B (8 points), MELD score 18. Maddrey's Discriminant Function 13.65.

Complications of the primary disease: Portal hypertension: splenomegaly with signs of hypersplenism, grade 1 thrombocytopenia. Grade 2 esophageal varices. Hepatocellular insufficiency: stage 2 hepatic encephalopathy. Grade 1 ascites.

The patient underwent pharmacogenetic testing — detection of the CYP2D6*4 (1846G>A) polymorphic marker by real-time polymerase chain reaction. A homozygous G/G1846 genotype was determined.

The patient was prescribed complex therapy: hepatoprotective (ursodeoxycholic acid 1000 mg/day), infusion (0.9% sodium chloride solution in a volume of 500 ml/day + 5% potassium chloride solution 10 ml), diuretic (furosemide 20 mg/day, spironolactone 100 mg/day), and hypoammonemic (lactulose suspension 30 ml/day per os).

To correct portal hypertension, propranolol was prescribed at 10 mg three times a day per os.

A follow-up ultrasonographic measurement of the mean linear velocity of portal vein blood flow showed a significant increase to 13.2 cm/s, i.e., by 6.8 cm/s or 106.2% compared to the baseline MLPV (Fig. 2).

Fig. 2. Dynamics of patient mean linear velocity of portal vein blood flow and the reference value, cm/s

Thus, a patient with the homozygous G/G1846 genotype showed a significant positive therapeutic effect of propranolol (increase in MLPV by more than 20% from baseline). The patient continued therapy at the same dose.

Figure 3 presents a visual comparison of the efficacy of propranolol, determined by the dynamics of the mean linear velocity of portal vein blood flow, in patients with liver cirrhosis and different CYP2D6*4 genotypes.

Fig. 3. Dynamics comparison of the blood flow average linear velocity in the portal vein of patients with different CYP2D6*4 genotypes, cm/s

Results

As a result of our study, it was found that positive dynamics of portal hypertension manifestations in the form of an increase in MLPV by 20% or more from baseline during propranolol therapy is more often observed in carriers of the homozygous CYP2D6 G/G1846 genotype (89.7%) than in patients with the heterozygous G/A1846 genotype (10.3%) in the Russian population (p <0.05).

Furthermore, we have for the first time defined an ultrasonographic criterion for positive dynamics of portal hypertension in the form of an increase in MLPV by 20% or more from baseline during propranolol therapy (p <0.05).

Based on the results of the pharmacogenetic study, an algorithm for personalizing the treatment of patients with liver cirrhosis with non-selective β-blockers was developed (Fig. 4).

Fig. 4. Treatment personalization algorithm of patients with liver cirrhosis by non-selective β-blockers.

Note: *Propranolol should be prescribed in a dose that reduces the heart rate by 25% at rest or up to 55 beats per minute in bradycardia.

To achieve a positive hemodynamic effect, patients with the homozygous G/G1846 genotype should initiate propranolol therapy at a dose of 10 mg three times a day. Carriers of the heterozygous G/A1846 genotype should be prescribed a dose higher than 30 mg per day. To monitor therapy, ultrasonographic assessment of the dynamics of the mean linear velocity of portal vein blood flow is required. When observing a positive hemodynamic effect based on the change in MLPV, it is necessary to continue propranolol at the same dose; otherwise, increase the dose individually, considering central hemodynamic parameters according to the current clinical recommendations approved by the Ministry of Health of the Russian Federation for the diagnosis and treatment of liver cirrhosis (2021) [3].

Conclusion

These clinical examples clearly demonstrate the differences in the efficacy of propranolol in patients with liver cirrhosis and different genotypes for the polymorphic marker CYP2D6*4.

Adherence to the proposed algorithm for personalizing the treatment of patients with liver cirrhosis with non-selective β-blockers is relevant for the prevention of bleeding from esophageal varices, as one of the most formidable complications of liver cirrhosis. The results of our study have been implemented into clinical practice.