2.2.5. Immunomodulatory Strategies

Since PBC is an autoimmune condition characterized by anti-mitochondrial autoantibodies (AMA) and high levels of immunoglobulin M (IgM), many immunosuppressive drugs were studied in PBC, including corticosteroid [54], azathioprine [55], cyclosporine [56], methotrexate [57], and mycophenolate mofetil [58]. However, results were consistently unsatisfactory. Recently, other molecules were studied in PBC.

Rituximab, an anti-CD20 antibody currently used in lymphomas and autoimmune syndromes, was evaluated in PBC due to its promising results in murine models of autoimmune cholangitis [58]. Three clinical trials in PBC patients with an incomplete response to UDCA were reported. In an open label study, Rituximab (two doses of 1000 mg) induced a decrease in AMA and IgM levels, with only a marginal reduction of ALP after 36 weeks [39]. Unfortunately, a similar study including 14 PBC patients showed a significant but only transitory reduction in ALP [40]. Finally, Rituximab was demonstrated not to have an impact on fatigue, assessed by PBC-40 [41].

Ustekinumab is an anti-interleukin (IL)-12/23 monoclonal antibody commonly used in several autoimmune syndromes and inflammatory bowel diseases (IBD). IL-12 and IL-23-mediated Th1/Th17 signaling pathways play a role in the etiopathogenesis of PBC [59]. Unfortunately, a multicenter open label trial did not reach the primary endpoint of reduction in ALP of 40% from the baseline. However, at week 24, a statistically significant decrease of 12.1% in ALP from baseline was observed [42].

Abatacept is a Cytotoxic T-Lymphocyte Antigen 4 IgG antibody used in rheumatoid and psoriatic arthritis. An open-label, 24-week trial was performed in PBC patients, but no significant changes in biochemical enzymes were observed [43].

The efficacy of Baricitinib (LY3009104), a reversible inhibitor of Janus kinase 1 (JAK1) and JAK2 currently used in rheumatoid arthritis, is currently being evaluated in an ongoing, placebo controlled phase 2 trial (NCT03742973) [45].

Other types of molecules are undergoing clinical evaluation in phase 1 and phase 2 trials: FFP104 blocks the CD40/CD40L interaction between CD4+ T helper lymphocytes and B cells that are involved in the pathogenesis of PBC (NCT02193360) [60]; E6011 is an anti-chemokine-adhesion molecule CX3CL1 (fractalkine) antibody, which is elevated in the serum of PBC patients (NCT03092765); Etrasimod is a selective sphingosine-1 phosphate (S1P) receptor (S1PR) modulator targeting S1P receptor subtypes 1, 4, and 5, leading to an inhibition of activated lymphocytes from migrating to sites of inflammation (NCT03155932) [3].

### 2.2.6. Other Treatment

S-adenosyl-L-methionine, added to UDCA in non-cirrhotic PBC patients, was demonstrated to have a positive effect on markers of cholestasis and quality of life, probably due to its hepatoprotective effects [44]. In this open label on 24 PBC patients, there was a significant decrease of ALP, GGT, and total cholesterol over a period of six months. A significant improvement of fatigue and pruritus on the PBC-40 questionnaire was also observed.

#### *2.3. Therapies Evaluated in Pre-Clinical Studies*

24-norursodeoxycholic acid (norUDCA) differs from UDCA due to the resistance in conjugation with taurine or glycine. NorUDCA increases the cholehepatic shunt of bile salts, leading to a supra-physiological secretion of bicarbonate. NorUDCA showed promising results in the treatment of PSC [61], but its efficacy in PBC has yet to be clarified. Up to now, improvements in fibrosis and inflammation were demonstrated in preclinical studies on animal model with cholestatic liver diseases [2].

Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is a hepatocellular uptake transporter of bile salts, and its inhibition by myrcludex B results in hepatoprotective effects, increasing the biliary phospholipid/bile salt ratio. In 3.5-diethoxycarbonyl-1.4 dihydrocollidine-fed mice, a murine model of cholestasis, and in Atp8b1-G308V mice, used for chronic cholestasis, bile salt levels increased in treated animals from 604 ± 277 to 1746 ± 719 μm and from 432 ± 280 to 762 ± 288 μm, respectively, while phospholipid output was maintained, resulting in a higher phospholipid/bile salt ratio. Thus, it may be beneficial in some forms of cholestasis, but further studies need to be performed [62].

### **3. Primary Sclerosing Cholangitis**

Primary sclerosing cholangitis (PSC) is a chronic bile duct disease with a prevalence of 1–16 per 100,000. PSC is more common in men (comprising 60–70% of patients) and is reported more frequently in Northern European countries and in North America. Moreover, 70% of the patients have ulcerative colitis [63]. The diagnosis is based on a combination of clinical, laboratory, imaging, and histological factors. Endoscopic retrograde cholangiopancreatography (ERCP) plays a very limited role in the diagnosis of PSC, while it may be used for the treatment of dominant stenosis [64]. It is well-known that patients affected by PSC have a higher risk of cholangiocarcinoma and gallbladder cancer. Up to now, no pharmacological treatment is universally approved for PSC. The lack of a clear pathogenesis and the absence of consistent endpoints have contributed to the difficulties in unravelling novel molecular targets and in designing effective clinical trials for PSC treatment [45]. The principal promising treatments and ongoing trials will be summarized in this section.
