Extracorporeal Immunomodulation Therapy in Acute on Chronic Liver Failure with Multi-Organ Failure: First in Human Use

Abstract

Two patients presented with acute on chronic liver failure and multi-organ failure and, as typical for this disorder, they presented with hyperinflammation and anticipated high mortality rates. Both cases were diagnosed with hepatorenal syndrome (HRS). Under a FDA approved Investigational Device Exemption clinical trial, they underwent treatment with an extracorporeal cell-directed immunomodulatory device, called selective cytopheretic device. Both patients showed rapid clinical improvement associated with a decline in elevated blood cytokine concentrations and diminution of activation levels of circulating leukocytes. On follow up one patient was alive at day 90 after treatment and undergoing liver transplantation evaluation and the other patient had a successful liver transplantation 6 days after SCD therapy ended. These cases represent the first in human evaluation of extracorporeal cell-directed immunomodulation therapy in acute on chronic liver failure with successful clinical outcomes in a disorder with dismal prognosis.

Acute on chronic liver failure (ACLF) is a clinical disorder characterized by acute clinical deterioration in patients with pre-existing chronic liver disease. This disease entity is associated with a precipitating event and results in a high risk of death. ACLF commonly occurs in patients with cirrhosis associated with rapidly evolving multiorgan dysfunction, significant systemic inflammation, and high short-term mortality (1). ACLF appears to develop from systemic inflammation, often due to bacterial infections or alcoholic hepatitis, and progresses to multi-organ failure. Patients with this disorder and acute kidney injury (AKI) either from hepatorenal syndrome (HRS) or acute tubular necrosis (ATN) requiring dialytic support have mortality rates of 85% at 90 days (2). Severe ACLF with ≥ 4 organ failure has even a graver prognosis with a mortality rate at 28 days of 100% (3). The systemic inflammation is dominated by dysregulation of the innate immunological system with activation of neutrophils and monocytes resulting in high blood levels of cytokines and chemokines promoting tissue injury and dysfunction. The therapeutic options for severe AAH and ACLF are limited. Early liver transplant is the treatment of choice for those who are refractory to medical treatment with methyl prednisolone. Intervention to modulate and lessen this systemic inflammatory state in ACLF may alter the progression of multi-organ dysfunction and allow time for liver transplantation. Successful liver transplantation in this disorder improves 90 day survival from 15 to 85% (2).

An immunomodulatory device, called the selective cytopheretic device (SCD), has been shown to reduce systemic inflammation in a number of disorders, including sepsis, acute kidney injury (AKI), acute respiratory distress syndrome (ARDS) associated with COVID-19, hemophagocytic lymphohistiocytosis (HLH), hemolytic uremic syndrome, and cardiorenal syndrome, with improvement in clinical outcomes (4–7). The SCD is a cartridge containing biocompatible hollow fiber membranes with blood flow directed to the outside surfaces of the membranes resulting in a low blood shear stress along the membrane surface. The immunomodulatory effect is dependent upon carefully controlled blood circuit ionized calcium levels less than 0.4mM which is achieved with regional citrate anticoagulation protocols required to also achieve circuit patency. The SCD promotes immunomodulation by selectively binding the most activated circulating neutrophils and monocytes, deactivates these leukocytes in the low iCa environment, and releases the modified cells to promote a reduced systemic inflammatory state. Thus, the SCD is a continuous autologous cell processing device promoting diminution of acute and chronic inflammatory disease states and results in improved clinical outcomes.

The two cases presented in this report documents the first in human SCD treatment of patients with severe ACLF presenting with multi-organ failure. SCD intervention rapidly reduced systemic levels of cytokines, diminished neutrophil activation and shifted circulating monocytes to a lesser inflammatory phenotype. This treatment was associated with survival time to 90 days or successful liver transplantation.

CASE 1.

Patient, a male in his early 30s’ was transferred to the University Hospital for acute alcohol associated hepatitis. He had a 10-year history of large daily alcohol ingestion and had not seen a physician in years. He was transferred from an outside emergency room to our facility and presented with jaundice and confusion. His admitting laboratory blood levels are detailed in Table 1 and were remarkable for: WBC 15.4 thou/mm3, hemoglobin 9.8 g/dL, elevated liver enzymes, albumin 1.9 g/dL, total bilirubin 29.8 mg/dL, ammonia 98 μmol/L, INR 2.7, lactate 2.1 mmol/L, creatinine 8.08 mg/dL. Diagnostic paracentesis was unremarkable. He was admitted to the Intensive Care Unit with mean arterial pressure in the 50s and was started on vasopressors and mechanical ventilation. His abdominal CT scan showed a fatty liver with changes suggestive of chronic hepatocellular disease, portal hypertension with splenomegaly, trace ascites, multiple upper abdominal and pelvic varices. MELD (Model for End Stage Liver Disease) score was 45. Follow-up laboratory evaluation ruled out other causes of liver disease, including viral hepatitis, Budd-Chiari, Wilson’s disease, toxic hepatitis from medications, CMV, HSV, autoimmune, hematologic causes. He was started on lactulose and refaximin. He was diagnosed with acute alcoholic hepatitis. His Maddrey’s discriminant function was 94 but was not started on corticosteroids concern for infection. He was oligo/anuric with a fractional excretion of sodium less than 1% and his urine output did not improve despite volume resuscitation including albumin. He was diagnosed with hepatorenal syndrome and started on continuous renal replacement therapy (CRRT) with regional citrate anticoagulation protocol designed for liver failure disorders with limited citrate metabolism. He met the criteria for ACLS with greater than 4 organ failures (7).

Table 1.

Clinical and laboratory values. Case 1

	Admission	Day 0	Day 2	Day 4	Day 6	24 hours Post-SCD	120 hours Post-SCD	Follow-up Day 30	Follow-up Day 90
Absolute Complete Blood Counts
WBC (103/uL)	15.4	20.8	15.9	32.1	35.4	29.9	23.9	23.3	22.6
Neutrophil (103/uL))	12.5	18.0	12.1	23	26.8	22.2	20.2	16.7	15.1
Monocyte (103/u	1.4	0.4	1.1	4.4	4.1	3.6	2.6	2.6	4.3
Platelet count (103/uL)	177	106	45	47	71	93	171	318	234
Liver Function tests
AST (units/L)	207	316	180	168	161	158	153	148	77
ALT (units/L)	114	115	112	114	38	24	11	105	62
Alkaline Phosphatase (units/L)	112	88	119	146	135	112	105	108	98
Bilirubin (mg/dL)	29.8	30.1	28.6	24.7	22.2	19.7	23.5	18.1	19.2
Albumin (g/dL)	1.9	2.3	2.4	2.4	2.2	2	2.2	1.7	1.7
INR	2.7	1.6	1.6	1.4	1.4	1.5	1.6	1.7	1.8
Immunologic Markers (pg/mL)
IL-6 (normal 0–16)	NA	210	26	19	40	30	40	NA	NA
IL-8 (normal 24–39)	NA	300	152	91	121	67	121	NA	NA
IL-10 (normal 8–16)	NA	3.8	<1	<1	<1	<1	<1	NA	NA
IL-1RA (normal 178–558)	NA	12738	3815	2109	2984	1299	39	NA	NA
MCP-1 (normal 20–80)	NA	48	22	26	33	43	50	NA	NA

NA, Not Available; IL., Interleukin; RA, Receptor Antagonist; MCP, Monocyte Chemoattractant Protein

He was evaluated for enrollment into a FDA approved IDE (G150179) and local IRB approval feasibility trial entitled “Investigator Initiated Pilot Study to Assess the Safety and Efficacy of a Selective Cytopheretic Device (SCD) to Treat ICU Patients with Acute Kidney Injury (AKI) and Hepatorenal Syndrome (HRS) Type I”. Inclusion and Exclusion criteria are detailed in clinicaltrials.gov NCT04898010. The SCD-1.0 and its associated bloodlines (SeaStar Medical, Denver, CO) were integrated into a CRRT blood circuit using a Prismaflex pump system and HF1000 hemofilter (Baxter, Deerfield, IL). The SCD is in series with the hemofilter. The blood circuit was connected to a central venous catheter placed in the jugular vein. SCD and treatment implementation, and CRRT-regional citrate anticoagulation protocol in patients with absent citrate metabolism are detailed in prior publications (6–8). The patient was found to meet all clinical criteria and after written informed consent by his legal authorized representative, he was enrolled into the clinical trial and started on SCD therapy for 7 consecutive days with replacement SCDs every 24 hours, as per protocol. The consent form details that data collected as part of this study may be used for publication purposes.

After starting SCD treatment, the patient stabilized and demonstrated improvement of his multiple organ dysfunctions. He was weaned off vasopressors on day 2 of SCD treatment; he was extubated on day 4 and transitioned to intermittent hemodialysis after the full 7 days of SCD therapy. His hepatic encephalopathy also showed marked improvement during this treatment period. Of importance, no device related adverse events were seen in this period. As displayed in Table 1, his liver enzymes declined with ALT returning to normal levels on day 6 and bilirubin levels declining from 30.1 to 22.1 mg/dL. These improvements coincided with associated reductions in plasma cytokine levels, including pro-inflammatory IL-6 and IL-8 as well as anti-inflammatory IL-10 and IL-1RA. Absolute blood neutrophil, monocyte and platelet counts increased during therapy. Flow cytometry demonstrated SCD promoted removal of the highly activated pools of circulating neutrophils and monocytes (see Supplemental Appendix). He was discharged from the ICU ten days after admission and 7 days after the initiation of SCD treatment. On follow up, he was alive at day 90 on intermittent hemodialysis and being evaluated for liver transplantation.

CASE 2.

A male patient in his 60s was admitted to the University hospital for hypotension and decompensated cirrhosis. He had a past history of chronic liver disease from alcohol and non-alcoholic steatohepatitis (NASH) complicated by ascites. He discontinued alcohol use since this diagnosis was made in late 2021. He was undergoing evaluation for liver transplantation. His admitting laboratory blood levels are detailed in Table 2 and were remarkable for: normal WBC, Hgb 5.9 g/dl, platelet count 80 K/ul, bilirubin 4.0 mg/dl, AST 43 U/L, ALT 19 U/L, alkaline phosphatase 145 U/L, albumin 3.1 g/dl, INR 1.6, BUN 89 mg/dl, creatinine 5.28 mg/dl. Sodium 127 mmol/L and bicarbonate, 18 mEq/dl. His MELD score was 35. He was admitted to the ICU and transfused with packed red blood cells, started on norepinephrine and vasopressin along with broad spectrum antibiotics. Octreotide and proton pump inhibitors were begun. He was oliguric and an intravenous albumin challenge was administered with no improvement with his urine output. Esophagogastroduodenoscopy on the next day demonstrated severe gastric antral vascular ectasia and lesions cauterized. After blood transfusions, he was weaned off vasopressors on day 4 of hospitalization. He continued to be oliguric and not responsive to albumin infusions. He was diagnosed with hepatorenal syndrome (HRS) and started on continuous kidney replacement therapy (CKRT) on day 3. He was evaluated for enrollment into the SCD/HRS clinical study. Similar to case 1, he met all clinical criteria and after written informed consent he was enrolled into the study.

TABLE 2.

Clinical and laboratory values. Case 2

	Admission	Day 0	Day 2	Day 4	Day 6	24 hours Post-SCD	120 hours Post-SCD	Follow-up Day 30	Follow- up Day 90
Absolute Complete Blood Counts
WBC (103/uL)	7.1	45.1	5.4	6.5	5.1	7.1	9.0	5.0	3.0
Neutrophil (103/uL))	5.0	NA	3.5	3.3	2.8	4.7	6.0	NA	1.6
Monocyte (103/uL)	0.7	NA	0.8	0.8	1.0	0.8	1.0	NA	0.5
Platelet count (103/uL)	80	NA	20	28	31	33	22	100	237
Hemoglobin (g/dl)	5.9	7.0	7.4	7.4	7.3	7.1	7.1	8.7	8.3
Liver Function tests
AST (units/L)	42	33	36	42	51	89	150	19	9
ALT (units/L)	26	15	20	24	28	50	65	46	<7
Alkaline Phosphatase (units/L)	115	43	60	67	91	114	136	253	136
Bilirubin (mg/dL)	4.5	9.0	6.5	6.4	6.7	10.3	16.0	1.0	0.2
Albumin (g/dL)	2.7	3.0	2.6	2.5	2.3	2.3	2.4	4.0	3.4
INR	2.0	NA	1.6	1.6	1.6	2.2	2.4	1.0	1.0
Immunologic Markers (pg/mL)
IL-6 (normal 0–16)	NA	67	62	59	52	96	789	NA	NA
IL-8 (normal 24–39)	NA	14	15	30	68	22	44	NA	NA
IL-10 (normal 8–16)	NA	46	15	29	20	41	477	NA	NA
IL-1RA (normal 178–558)	NA	2023	77	1579	91	382	354	NA	NA
MCP-1 (normal 20–80)	NA	151	159	250	135	124	108	NA	NA

NA, Not Available; IL., Interleukin; RA, Receptor Antagonist; MCP, Monocyte Chemoattractant Protein

After starting SCD treatment, the patient stabilized. His laboratory parameters improved (Table 2), including bilirubin, platelet count and INR. During SCD therapy, his MELD score declined to 24. These improvements were associated with a rapid decline in his systemic cytokine levels. He transitioned to intermittent hemodialyis resulting in terminating SCD therapy after 6 days of treatment. After stopping SCD therapy, his liver enzymes steadily increased. His bilirubin rose from 6.7 to 16.0 and his INR went from 1.6 to 2.4. His MELD score increased to 39. This worsening of his clinical condition was associated with a dramatic increase in systemic inflammatory cytokines and his inflammatory monocyte pool. With his rapidly deteriorating status, since he had completed his liver transplant evaluation, he urgently and successfully underwent liver transplantation 6 days after SCD therapy ended. At day 30 after SCD therapy, his liver function was normal and was still requiring intermittent hemodialysis. He was discharged from the hospital 42 days following SCD treatment termination.

DISCUSSION.

Both patients presented with ACLF and HRS. The first case had a severe case of alcohol associated hepatitis. He met the ACLF 3 criteria (greater than 4 organ failures) but despite aggressive supportive therapy including vasopressors, mechanical ventilation and hemodialysis he rapidly deteriorated (10). The second case had NASH cirrhosis and presented with 3 organ failure (liver, renal, cardiovascular). In both cases the clinical decline was associated with systemic hyper-inflammation, as demonstrated by elevated levels of multiple cytokines and markers of neutrophil and monocyte activation. This systemic inflammatory process has been suggested to be the major cause of the high mortality rates of this disorder. Activation of neutrophils and organ microvasculature results in capillary sludging and tissue hypoxia. Migration of leukocytes into tissue and release of degradation enzymes promote toxic tissue injury. The concomitant hypoxic and toxic tissue injury leads to multi organ-failure and poor outcomes.

With this perspective, SCD treatment was undertaken to mitigate the excessive activation of the effector cells of the innate immunologic pathway. In the low ionized calcium (iCa) environment promoted with regional citrate anticoagulation and the low shear force approximating capillary shear, the SCD membranes selectively bind the most activated neutrophils and monocytes due to the calcium dependency of binding reactions of cell surface integrins on the leukocyte. This selective binding of the more activated circulating leukocytes has been seen in both preclinical animal models and clinical application of SCD therapy in various inflammatory disease states (3–7,9). Upon binding the neutrophils degranulate and in the low iCa are promoted to their apoptotic program. After deactivation, they are released back into the systemic circulation and are phagocytosed and degraded by the reticuloendothelial system. Similarly, the most pro-inflammatory monocytes are bound, functionally altered to a less inflammatory state in the low iCa environment and released back to the circulation. Flow cytometry data of cell surface molecule expression on circulating and SCD bound leukocytes support this mechanism of SCD immunomodulation (see Supplemental Digital Content). In this manner, the SCD is a continuous autologous leukocyte processing system which immunomodulates a systemic hyperinflammatory state without immunosuppression. The declines of plasma cytokine levels during SCD treatment, as detailed in Tables 1 and 2, supports this process. The sudden clinical worsening with increases in blood cytokines and the increase in pro-inflammatory monocyte in Case 2 after discontinuing SCD treatment also suggests a cause and effect of SCD treatment in these disorders. The positive clinical outcomes in both cases with 90 day survival and liver transplantation suggest a role of SCD immunomodulation to treat ACLF as a bridge to evaluation or successful intervention for liver transplantation. Of importance, no device related serious adverse events, including infections, were observed in these subjects. This safety profile has been observed in previous SCD clinical trials (4–7,9).

Being cell directed therapy, SCD treatment is different from the commercially available artificial extracorporeal liver support devices which include: (1) molecular adsorbent recirculating system (MARS), (2) fractionated plasma separation and adsorption device (Prometheus) and (3) single-pass albumin dialysis (SPAD). These three devices are based upon albumin dialysis which promotes removal of circulating soluble compounds and toxins to treat patients with acute on chronic liver failure (11,12). The most studied systems have been MARS and Prometheus devices. Elimination of albumin-bound substances, including bilirubin and bile acids, have been clearly demonstrated with these two devices; MARS treatment, but not Prometheus, has been shown to improve the hyperdynamic state of ACLF (13). Both systems increase clearance of circulating cytokines but do not alter the systemic levels of cytokines in this disorder, most likely due to high production rates (14). These findings are in contrast to the results observed with SCD therapy. In both patients, rapid and sustained reductions in systemic levels of multiple cytokines occurred during SCD treatment. These declines were associated with immunomodulation of the hyperinflammatory state of circulating neutrophils and monocytes and is consistent with a decrease in cytokine production. Clinical trials with these commercially available artificial liver support systems have been inconsistent in demonstrating improved clinical outcomes in ACLF patients, most likely due to small sample size and high variability in disease severity of enrolled patients (11,12). Definitive clinical benefit has not been demonstrated. Accordingly, the impact of SCD treatment in the two cases described in this report, although encouraging, needs to be further tested in additional patients and eventually evaluated in randomized control trials.

These cases demonstrate several key points. First, SCD treatment requires carefully regulated ionized calcium (iCa) levels (<0.4 mmoles/L) in the blood circuit to maintain circuit patency to ensure the immunomodulatory effect of the SCD. In patients with severe liver dysfunction, careful control of citrate anticoagulation is required to avoid citrate toxicity due to the loss of liver metabolism of citrate. The use of a specially designed protocol for near absent body metabolism of citrate in this case demonstrated an ability to maintain iCa within the blood circuit below 0.4 mM (in these cases, 0.26–0.36 throughout treatments) and to maintain systemic iCa between the target level of 1.05 to 1.30 mM (in these cases, 1.09–1.27) for effective treatment and avoidance of adverse events (8). Second, the reduction of multiple plasma cytokine levels and flow cytometry data demonstrating removal of highly activated circulating leukocytes were associated with rapid clinical improvement after device intervention and in case 2 rapid deterioration after treatment termination. This correlation is consistent with the primary pathobiology of hyper-inflammation promoting multi-organ failure and poor outcomes in acute on chronic liver failure. Finally, a key point in this case is the recognition that the final common pathway of systemic hyper-inflammation resulting in multi-organ failure is the effector cells of the innate immunologic system. The activation of neutrophils and monocytes is the key driver of the developing hypoxic and toxic tissue damage of solid organs. The immunomodulation of these cellular elements rather than removal or inhibition of soluble cytokines or chemokines of inflammation is the critical target for effective therapy. These results need to be replicated in a larger case series. We believe that there may be a role of SCD for the management of ACLF with multi-organ failure as a bridge to life saving liver transplantation.