Efficacy Of Bovine Colostrum In The Treatment Of Severe Alcohol-Associated Hepatitis

Severe alcohol-associated hepatitis is associated with hepatocellular necrosis, inflammation, hyperactivated immune system, paradoxical immune exhaustion, leaky gut, and alteration in the gut microbiome(1). The leading cause of mortality is a bacterial infection with multi-organ failure. (2) Pentoxifylline was ineffective (3,4) and Interleukin-1-based therapies - Anakinra (5,6) and Canakinumab (7)have not improved survival rates. The granulocyte colony-stimulating factor has shown mixed results. Indian studies (8-10) improved 90-day survival, while Western studies on Pegfilgrastim have been negative (11) Corticosteroids decrease the mortality for only a month (3-5,12-14) Improved survival was noted at 1 month among patients with severe acute alcohol-associated hepatitis who received combination therapy with prednisolone and N-acetylcysteine, as compared with those who received prednisolone only, but 6-month mortality, the primary outcome, was not improved with combination (15) In a recent study on Severe alcohol-associated hepatitis, Larsucosterol, a DNA methyltransferase inhibitor, was associated with non-significant improvement in 90-day mortality: 14.7 % (30 mg/day) and 16.67 % (90 mg/day) versus 24.27% on Methylprednisolone. (16) Thus, a more durable treatment of severe alcohol-associated hepatitis is needed. (17) Bovine Colostrum contains many bioactive components such as immunoglobulin G, A, and M (70 - 80 % of total protein), (18,19) Lactoferrin and Short-chain fatty acids. Bovine Colostrum has 30-100 times higher Lactoferrin concentration than milk. (18)IgG and Lactoferrin synergistically neutralise lipopolysaccharide/ Endotoxin (20) and act on mucosa-associated lymphoid tissue of the leaky gut, transforming it into healthy mucosa. (21) Fewer bacteria and Endotoxins - Pathogen-associated molecular patterns enter the portal circulation to interact with Toll-Like Receptor - 4 of the Liver Kupffer cells. Proinflammatory cytokines such as interleukins-1,6,8 and tumour necrosis factor-alpha, generation decreases, mitigating hepatocyte inflammation, necrosis, and cell death. In this study, we aimed to assess the effectiveness and safety of Bovine Colostrum in treating patients with severe alcohol-associated hepatitis. ClinicalTrials.gov ID NCT02473341

INTRODUCTION

Background and Rationale Alcohol-Associated Hepatitis is a clinical entity with rapid onset of jaundice within the prior 8 weeks with elevated serum aspartate transaminase (AST), arising on the background of heavy alcohol use. Liver biopsy usually reveals macrovesicular steatosis, with 1 of the following: neutrophil infiltration, hepatocyte injury (ballooning), Mallory-Denk bodies, cholestasis, and severe fibrosis. An average consumption of more than 3 drinks (\~40 g) per day for women and 4 drinks (\~50-60 g) per day for men are reasonable minimal thresholds for the diagnosis of Alcohol-Associated Hepatitis. Patients typically have been drinking heavily for \>5 years but may be intermittently abstinent. Usually, heavy alcohol use should have occurred for \>6 months, with \<60 days of abstinence before the onset of jaundice. Jaundice is often accompanied by malaise, tender hepatomegaly, and decompensation (ascites, encephalopathy, bacterial infection, and variceal bleeding). Serum bilirubin is usually elevated (\>3 mg/dL \[\>50 μmol/L\]), as is the Asparate Aminotransferase (AST) (\>50 IU/mL), and Asparate Aminotransferase to Alanine aminotransferase (ALT) ratio of \>1.5. The AST and ALT do not typically exceed 400 IU/mL. Imaging should exclude biliary obstruction; viral hepatitis, severe autoimmune liver disease, and Wilson disease should be tested for and ruled out. (22) Natural History of Severe Alcohol-Associated Hepatitis in the West In the STOPAH study (3) , 60% - 67% patients were males; baseline Maddrey's Discriminant Function was 61.9 + 25.7 and the Model For End-Stage Liver Disease was 20.7 + 5.5 in the Placebo arm. Out of the total 418 deaths, 168(40%) occurred before day 29, 28% occurred between days 28 and 90 , 32% occurred between day 91 and 1 year.

Background cirrhosis was in 86.2-93.7% patients with Severe Alcohol Associated Hepatitis treated with prednisone who had undergone liver biopsies (23) Although cirrhosis is strictly speaking a histologic diagnosis, a combination of clinical, laboratory, and imaging features help to confirm this diagnosis. (24,25) Natural History of Severe Alcohol-Associated Hepatitis in India In all Indian studies (9,12,26,27) patients had a very severe disease as evidenced by the high Maddrey's Discriminant Function scores and Model For End-Stage Liver Disease scores. The majority of deaths occurred within the first month, with only a few deaths in the second and third months. The vast majority of these patients had underlying cirrhosis and were males. Corticosteroids could be offered in only 12%, due to contraindications in the rest of the patients - Acute Kidney Injury (37%) , infection (45%), Gastrointestinal bleed in (18%), patient's refusal in 17%.(26)

Treatment Abstinence from alcohol This is the most important factor in predicting the outcome after surviving the acute Alcohol-Associated hepatitis (AH) episode.

Nutrition A careful evaluation of nutritional status and energy intake is done, with the aim to deliver 35-40 kcal/kg of body weight and a daily protein intake of 1.2-1.5 g/kg of body weight orally.(28) Given the reduced glycogen storage and accelerated gluconeogenesis, frequent, small meals and a bedtime snack is advised. (29) Supplementation of Thiamine, other B vitamins, Vitamin D, and trace elements, including Zinc, should be done.

Pharmacotherapy of Severe Alcohol-Associated Hepatitis Corticosteroids All recent Western studies (3-5,13,14,30) have concluded that corticosteroid use decreases the 30-day, but not 90- or 180-day mortality rate in patients with Severe Alcohol-Associated Hepatitis. A more durable treatment of severe alcohol-associated hepatitis is needed. (17) The Maddrey's Discriminant Function in the Western studies (7,12-14,16) ranged from \[Median (Inter Quartile Range)\] 54.4 (39.2 - 76.1) to 69.7(53.7 - 89.4). The Model For End-Stage Liver Disease score ranged from \[median (Inter Quartile Range)\] 21.49 (19.95 - 22.89) to 25(22 - 27). The 90- day mortality rate was 30% in the STOPAH trial(3), 30% in the DASH trial (5) and 26% in the AntiBiocor trial (13)compared to 10% in the Gawrieh S, et al study (6) due to younger patients in the last study. The pooled 90-day survival was 73.61% (95% CI: 68.18%-78.69%) in placebo-treated patients in above studies.(31) Combining four contemporary Indian studies on Severe Alcohol-Associated Hepatitis (8-10,26,27) the median Maddrey's Discriminant Function scores ranged from 70 (32 - 320) to 84 (56 - 185) and the median Model For End-Stage Liver Disease scores ranged from 26 (15 - 40) to 27.5 (19 - 41). The 90-day survival ranged from 22% - 56% on standard medical treatment.

The inference is that Severe Alcohol-Associated Hepatitis in Indian patients is more severe than in Western patients.

Hence, in light of the National Institute on Alcohol Abuse and Alcoholism recommendations, it is clear that Corticosteroids are not a therapeutic option for Indian patients as Maddrey's Discriminant Function and Model For End-Stage Liver Disease scores are very high.

Although our pilot study conducted in 2015 (32) had shown that treatment with a combination of Bovine Colostrum and Corticosteroids had improved the Maddrey's Discriminant Function level and survival rate at 3 months significantly, we would only use Bovine Colostrum as a therapeutic option versus standard medical treatment, rather than use corticosteroids as a active comparator arm which has no impact on the 90-day mortality.

N-Acetylcysteine A trial to evaluate the efficacy of glucocorticoids plus N-acetylcysteine, as compared with glucocorticoids alone, in patients with severe acute alcoholic hepatitis. (15) reported improved one-month survival in the combined treatment group. However, the 6-month survival was not different in the two groups Larsucosterol Larsucosterol, an endogenous oxysterol, is an epigenetic inhibitor of DNA methyltransferases, mitigating apoptosis. In a recent study (Model of End-Stage Liver Disease score-24, Maddrey's discriminant function scores 57.2 - 63), Larsucosterol was associated with non-significant improvement in 90-day mortality: 15.2 % (30 mg/day) and 16.8 % (90 mg/day) versus 24.5% on Methylprednisolone. (16)However, the severity of hepatitis was milder compared to Indian our studies.y Failed Treatments Pentoxifylline was ineffective,(3,4) and Interleukin-1-based therapies - Anakinra(5,6) and Canakinumab(7) have not improved survival rates. The granulocyte colony-stimulating factor has shown mixed results. Indian studies (8-10) improved 90-day survival, while Western studies on Pegfilgrastim have been negative. (11) Imm 124-E, hyperimmune bovine Colostrum, and Prednisolone were administered for 28 days to severe alcohol-associated hepatitis patients, however, it did not reduce the circulating levels of lipopolysaccharides. (33)

Bovine colostrum Composition and Rationale for its Treatment Potential in Severe Alcohol-Associated Hepatitis The Bovine Colostrum has the following components:(18) fat, protein, peptides, lactose ,immunoglobins, lactoferrin, lysozyme, lactoperoxidase, insulin growth factor-I (34) Many factors, such as calving interval, breed, age, genetic factors, heat, humidity, technological processes,time of harvesting and hygiene conditions of production, influence the quality of Bovine Colostrum.(35,36) Colostrum contains elevated levels of IgG, IgA, and IgM, which make up 70-80% of the total protein in colostrum(35). Short-Chain Fatty Acids in Colostrum improve the integrity of the intestinal inner cell membrane(37).

The lactoferrin concentration in colostrum is 30 - 100 fold higher than that in milk. (18) Lactoferrin binds to lipid A of Lipopolysaccharide (LPS) to neutralize it. IgG and lactoferrin synergistically neutralize LPS. IgG and lactoferrin interact with mucosa-associated lymphoid tissue of leaky mucosal barrier to convert into healthy mucosal barrier. It increases the growth and proliferation of enterocytes.(20) Additionally, lactoferrin has an impact on the levels of cytokines and chemokines that are produced by Gut-Associated Lymphoid Tissue and creates an environment for the growth of beneficial bacteria in the gut.(21) Fewer bacteria and LPS /Endotoxin, - pathogen-associated molecular particles enter the Portal vein resulting in decreased trafficking and interaction of bacteria and LPS with Toll-like receptors-4 of the macrophages and Kupffer Cells in the liver. The production of pro-inflammatory cytokines such as interleukin-1 beta,6,8,10, (IL-1 beta,6,8,10), Reactive Oxygen Species (ROS) and Tumour necrosis factor-alpha (TNF-alpha) are decreased and hepatocellular necrosis is mitigated.

Thermal processing of colostrum In studies conducted by Donahue and Godden et al., heating colostrum at 60 °C for 60 min decreased total plate counts, coliform counts and other pathogens, but did not affect native IgG concentration(38,39). This was associated with increased efficiency of IgG absorption and, consequently, higher serum IgG concentration in these calves fed heat-treated Colostrum. (38)

Derivation of Dose of Bovine Colostrum Oral bovine colostrum preparation (Lactobin®) 56g/day was given prophylactically to 20 patients for 3 days preoperatively who underwent abdominal surgeries, and (Placebo) standard milk 56 g/day was given for 3 days to 20 similar patients. The daily dose of Lactobin or Placebo was divided into four parts (before breakfast, lunch, supper, and night).

The course of the plasma endotoxin / LPS levels and the endotoxin neutralization capacity were measured daily up to the 10th postoperative day. The results showed that the LPS levels in the Lactobin group, were significantly lower than those in the control group (p \< 0.05). The difference between the two groups was apparent on the day of the operation and the day after. There was a significantly greater increase in endotoxin-neutralizing capacity in the patients treated with lactobin than in the control group (p\<0.006). Thus, Bovine Colostrum has been successfully used to decrease the level of Endotoxemia / Lipopolysaccharides.

In the second placebo-controlled randomized study(40), 60 patients who underwent coronary bypass operations with either 42 g of a Bovine colostrum milk preparation per day or placebo for 2 days preoperatively. There was no reduction in Endotoxin levels and no increase in endotoxin-neutralizing capacity levels in patients receiving the lower dose of Colostrum. Thus, the correct dose of Bovine Colostrum appears to be 60 grams/day. (41) The protein content of the Bovine Colostrum used in our study was 52.75% and the IgG content was 34.25% (342.5 mg IgG/gram) of protein The protein content was 80% and the IgG/gram of the total Protein content was 52 mg in Lactobin® Bovine Colostrum. Thus, the Colostrum used in our study has a much higher content of IgG than the Lactobin Colostrum used by Bolke E et al. (41) This is because the Spray dried technique used in Lactobin preparation decreases the IgG much more than the freeze-dried technique used in the preparation of Bovine Colostrum in the current study.

Trial design This is a multicentre, parallel, double-blind, randomised (1:1), placebo-controlled trial. This is a superiority trial where the hypothesis is that the treatment intervention (Bovine Colostrum) is superior to the control (Placebo) in the treatment of Severe Alcohol-Associated Hepatitis.

Methods: Participants Study setting The study will recruit patients admitted with "probable Severe Alcohol-Associated Hepatitis " at five academic centers and Hospitals in India. The accuracy of a clinical diagnosis of "probable Alcohol-Associated Hepatitis using the Crabbe et al criteria is 96%. Thus patients can be started on initiating treatment promptly on presentation rather than to rely upon histology which may delay assessment .Hence , no patients underwent liver biopsy. (42,43)

Relevant concomitant care given during the trial Standard of care treatment will be given in both arms are as follows- Oral / Enteral nutrition (if oral intake is insufficient: Protein 1.2 -1.5 gm/kg/day, energy (kcal) 35-40/day, small frequent feeds with a bedtime snack of 50 g of complex carbohydrates with 13.5 g of proteins and B complex vitamins daily. Antibiotics for sepsis; Salt restriction and Diuretics for Ascites; Lactulose / Rifaximin / L Ornithine L Aspartate for Hepatic Encephalopathy; Terlipressin and Albumin for Type 1 Hepato Renal Syndrome; Carvedilol for prophylaxis against variceal hemorrhage; Terlipressin with Endoscopic Variceal Ligation for variceal bleed, if indicated.

Criteria for discontinuing or modifying allocated interventions for a given trial participant Occurrence of adverse effects of Bovine colostrum (Experimental arm) and pasteurised milk powder (placebo comparator): Allergy, Lactose Intolerance Strategies to improve adherence to intervention protocols All patients will be initially admitted to the hospital ICU / wards. After discharge from the hospital, the patients will be contacted telephonically and reminded to shall return for outpatient visits after 7 days, 14 days, 21 days, 28 days, and then bimonthly for two months. At the outpatient visit, history, physical examination, and laboratory tests will be done. The patients will return the empty sachets (which will be counted), and a new batch of sachets will be given until the next outpatient visit

Investigations

Each patient shall have an:

1. Abdominal ultrasonography.
2. Upper gastrointestinal endoscopy would be done if indicated.
3. Liver biopsy: This is needed in patients with confounding factors.
4. Laboratory tests: Hemogram, prothrombin time, Procalcitonin levels, blood glucose, liver function tests, blood urea, serum creatinine, and serum electrolytes will be done at baseline, and at 7 days, 14 days, 21 days, 28 days, and then bimonthly for two months or earlier, if indicated.
5. Microbiologic tests: Blood, urine, sputum cultures, and cultures of aspirates from endotracheal tubes in ventilated patients shall be performed at admission. At baseline, a diagnostic paracentesis will be performed in all patients with ascites
6. Chest radiograph
7. Cytokine levels: Endotoxin, Tumor Necrosis Factor-alpha, Interleukin 6 \& 8 levels will be done at baseline and end of treatment
8. Tests of Aetiology : To rule out viral, autoimmune hepatitis, Wilson disease, Hemochromatosis and Non-alcoholic steatohepatitis will be done.
9. Assessment of effect of Intervention on Intestinal Barrier function To confirm the effect of bovine colostrum in improving the intestinal barrier function, we will measure both the Seum Endotoxin levels (34) as well as the TNF -alpha levels (an important downstream pro-inflammatory mediator).

Sample size determination

In an Indian study of patients suffering from Severe Alcohol-Associated Hepatitis, published in 2014 by Singh et al (8) the the Maddrey Discriminant Function score was 77.4 (range: 37- 235), the Model for End-Stage Liver Disease score was 27.5 (range:19- 41), and the survival rate was 22% at 90 days.

Hence, in our study of patients with Severe Alcohol-Associated Hepatitis, we calculated the sample size based on the above study.

We considered a 20% increase in survival rate (80% power at 5% alpha), to be clinically meaningful and sufficient to change practice. To detect a 20% increase in survival (from 22% to 42%) in 3 months \[the sample size was based on survival at a fixed time point (22% vs 42% at 3 months)\], we required 79 patients in each group. Assuming, 10% dropouts. the total sample size required for the study is 174.

Randomisation Consecutive patients diagnosed with Severe Alcohol-Associated hepatitis will be then registered by the site medical team onto the trial site via Trans European Network for clinical trials services, a web-based registration and randomisation system, and randomised into two groups (groups A and B) to receive active drug or placebo 1:1.

Blinding Treatment allocation will be blinded to patients, investigators, data collectors, and statisticians by providing each patient with a unique number from the Trans European Network for clinical trials services registration system

Statistical methods Statistical methods for primary and secondary outcomes

Subject population (S) for analysis:

For primary survival (efficacy) analysis, we will use intention-to-treat analysis, in such a way that all study subjects who are randomized, regardless of adherence to study medication, will be used in the analysis in the treatment arms to which they are originally assigned to at randomization ("as randomized"). Significance will be assessed using a two-sided alpha level of 0.05.

Baseline Patients' Characteristics The baseline patients' characteristics will be compared between the two treatment groups to demonstrate the actual study group balance and to ensure whether a proper randomization is established or not. We will measure central tendency and variability with means and standard deviations or medians and interquartile range for continuous characteristics and frequencies with percentages for categorical characteristics.

Primary Outcome Unadjusted Log-Rank Test Of The Kaplan-Meier Survival Estimates For the primary outcome 'survival at 3 months', we will first, use this test for the two treatment arms at a two-sided alpha level of 0.05 with survival censored at 3 months.

Hazard Ratios Further, a Cox proportional hazards regression model (with survival censored at 3 months) will be used to estimate the hazard ratio with a 95% confidence interval associated with the treatment arm (A) compared to the Placebo (B).

Scaled Schoenfeld Residuals plots The pattern against the time in these plots will be observed. If a nonrandom pattern is observed, this will be considered a violation of the Proportional Hazards assumption.

Restricted Mean Survival Time To address the violation of the proportional hazards assumption, the restricted mean survival time for both treatment arms over 90 days for the Actual Observed outcome and the Worst Case Scenario will be calculated.

Secondary Outcomes A similar unadjusted log-rank test of the Kaplan-Meier survival estimates and Cox proportional hazards regression model analysis will be planned for secondary outcome survival at 1 month. In the graphical Scaled Schoenfeld tests, the pattern against the time in these plots will be observed. If a non-random pattern is observed ,we will then calculate the restricted mean survival time for both treatment arms over 30 days for the Actual Observed outcome and the Worst Case Scenario.

Other secondary outcomes measures such as Change in Maddrey Discriminant Factor levels/ Model For End-Stage Liver Disease score, Endotoxin levels, and Cytokines levels between baseline and 4 weeks assessment will be assessed for normal distribution by graphical (Quantile-Quantile Plot) interpretation and numerical method (Kolmogorov-Smirnov test). A paired t-test will be used for all those outcomes which will follow approximately normal distribution and a Mann-Whitney U test will be used for skewed parameters. The proportionality assumption will be assessed for the considered ordinal outcome number of episodes of sepsis (Pneumonia, Spontaneous Bacterial Peritonitis, cellulitis, and Urinary Tract Infection, Bacteremia). Partial proportional odds ratio regression will be used to assess the effect of treatment on the number of episodes of sepsis (Pneumonia, Spontaneous Bacterial Peritonitis, Cellulitis, Urinary Tract Infection, Bacteremia) over 4 weeks.

Subgroup analysis Subgroup analysis may be conducted based on the clinical age group criteria, grams of alcohol consumed criteria, alcohol relapse (recidivism) and the Model for End-Stage Liver Disease scores.

Analysis of baseline variables in patients who dropped out versus those who did not drop out from the study.

It is important to document and investigate the reasons for patients who dropped out of the study. We will examine the distribution of baseline characteristics between patients who completed the treatment with those who dropped out within each treatment group to identify the known factors associated with poor treatment compliance.

Sensitivity Analysis Sensitivity analyses will be performed to assess the robustness of the results to protocol deviations.

Unadjusted and Adjusted (For Centre, Acute Kidney Injury, and Ascites grade) Relative Risk of mortality- will be performed for :

* Actual Observed Outcomes
* Best-case scenario assumption - participants lost to follow-up are considered alive in the Colostrum arm and deceased in the Placebo group
* Worst-case scenario assumption - participants lost to follow-up are considered deceased in the Colostrum arm and alive in the Placebo group

Handling missing data in the primary outcome Multiple Imputation (Missing At Random assumption) We will use Rubin's multiple-imputation method to deal with the missing data. Although the multiple-imputation method is developed based on the missing at-random assumption, this method can handle both missing completely at random and missing not at random.

Tipping point analysis (Missing Not At Random assumption) Another approach is a tipping point analysis, a multiple imputations technique under missing not at random assumption and will be used to impute the missing data not at random. Under this analysis, a tipping point can be defined as the difference of means for continuous data and the difference of event numbers for binary data where the p-values get changed. This method does not require assessing the missing data mechanism and does not involve model uncertainty and assumptions.

Interim analyses No formal interim analysis is planned for this trial. Monitoring for Adverse Events A treatment-emergent Adverse Event will be defined as an Adverse Event that begins or that worsens in severity after at least one dose of the study drug has been administered. Any adverse event occurring during the study will be documented in the subject's Case Report Form specifying the time of onset, the duration, the severity, and the relationship to the test medication

Grades of Severity of the Adverse Event as per the Common Terminology Criteria for Adverse Events (CTCAE) v 5.0:

* Grade 1 Mild Adverse Event
* Grade 2 Moderate Adverse Event
* Grade 3 Severe Adverse Event
* Grade 4 Life-threatening or disabling Adverse Event
* Grade 5 Death related to Adverse Event

Eligibility Criteria:
Inclusion Criteria:

I. Onset of jaundice within prior 8 weeks II. Ongoing consumption of \> 40 (female) or 60 (males) g alcohol/day for 6 months or more, with less than 60 days of abstinence before the onset of jaundice III. Aspartate aminotransferase \> 50, aspartate aminotransferase/alanine aminotransferase \> 1.5, and both values \< 400 IU/L IV. Serum bilirubin (total) \> 3.0 mg/dL V. Liver biopsy confirmation in patients with confounding factors including possible ischemic hepatitis (eg, severe upper gastrointestinal bleed, hypotension, or cocaine use within 7 days); possible DILI; uncertain alcohol use assessment (eg, patient denies excessive alcohol use); and atypical laboratory tests (eg, AST \< 50 IU/mL or \> 400 IU/mL, AST/ALT ratio \< 1.5), antinuclear antibody \> 1:160 or SMA \> 1:80 VI. Maddrey's discriminant function ≥ 32 assuming a control prothrombin time of 12 seconds VII. Model for End-stage Liver Disease score \> 20

Exclusion Criteria I. Uncontrolled infections II. Multiorgan failure III. Uncontrolled upper gastrointestinal bleeding. However, recent UGI bleeding that is controlled for \>48 hours should not exclude the patient.

IV. Preexisting kidney injury with serum creatinine \> 2.5 mg/dL V. Other underlying liver diseases including hepatitis B infection,\* autoimmune liver diseases, Wilson disease, suspected drug-induced liver injury\* VI. Hepatocellular carcinoma or other active malignancies except skin cancer VII. Pregnancy VIII. Underlying diseases that might be exacerbated by proposed treatments (eg, hepatitis C, hemochromatosis, latent tuberculosis) IX. Uncontrolled drug addiction X. Cow milk allergy or severe lactose intolerance XI. Treatment for alcoholic hepatitis within 1 month of study entry with corticosteroids use\>1 week.