Recent advances in managing hepatitis D

Hepatocyte entry inhibitors

Myrcludex B, a myristoylated lipopeptide comprising 47 amino acids of the pre-S1-domain of the HBV L-surface protein, represents the first hepatocyte entry inhibitor. As pointed out earlier, myristoylation of glycine in the pre-S1-domain and 77 amino acids in the pre-S1-domain appears to be important for cellular attachment^2,53. Myrcludex B was tested both in intravenous and in subcutaneous formulations (Table 2)⁵⁴. A transient, asymptomatic, serum lipase elevation was observed in 5 out of 36 healthy volunteers. Conjugated bile acids increased without clinical consequences. It needs to be mentioned that bile acids have been implicated in cardiac arrhythmias⁵⁵. In addition, mild alterations of hematological parameters have been reported, but overall the administration of Myrcludex B was reported to be well tolerated (Table 3)^54,56. The subcutaneous route displayed 85% bioavailability⁵⁴. In the proof-of-concept study, 24 patients received either 48 weeks of peg-IFN monotherapy or 24 weeks of 2 mg/kg subcutaneous Myrcludex B daily for 6 months (after that, patients received 48 weeks of peg-IFN monotherapy) or 6 months of Myrcludex B plus peg-IFN followed by 6 months of peg-IFN. Myrcludex B led to a >1 log₁₀ reduction in HDV RNA in 6 out of 7 patients at the end of 24 weeks’ treatment⁵⁶. One patient became HDV RNA negative on monotherapy with Myrcludex B. Combination of Myrcludex B with peg-IFN was more effective, and 5 out of 7 patients became HDV RNA negative. The delta decline in HDV RNA was 1.67, 2.17, and 2.59 log₁₀ with Myrcludex B, peg-IFN monotherapies, and their combination, respectively. HDV RNA negativity with combination treatment in 5 out of 7 patients after 6 months of treatment is promising, and a synergistic effect for combination treatment has been suggested⁵⁶. The primary endpoint of the study, a decrease in HBsAg levels of 0.5 IU/mL at any time during the study⁵⁶, was not reached, however. Further studies with Myrcludex B with different dosing and treatment duration regimens are to be expected, and such a study is already ongoing.

Myrcludex B is a promising new drug for both hepatitis B and D. That new treatment is a much urgent need in CHD does not need further explanation. The following issues remain to be resolved: (i) optimal dose and duration for use in human CHD needs to be established; (ii) superiority of combination treatment of Myrcludex B with peg-IFN over peg-IFN monotherapy needs to be convincingly shown; (iv) safety appears to be of less concern, but the effect on bile acids has to be explored in a larger patient group; (v) efforts for replacing a daily subcutaneous formulation with an oral formulation should be seriously considered – this would enable long-term use.

Farnesyltransferase inhibitors

Prenylation of large hepatitis delta antigen is required for HDV virion assembly. Farnesyltransferase inhibitors are specific for HDV⁵⁷, and preclinical studies revealed that they abolished HDV-like particle production in vitro⁵⁸ and in vivo⁵⁹. In the proof-of-concept study, the farnesyltransferase inhibitor lonafarnib (LNF) was tested for a duration of 28 days in 14 CHD patients. A dose-dependent reduction of HDV RNA was observed⁶⁰. LNF serum concentrations correlated with HDV RNA change. Some characteristics of LNF are provided in Table 2. This study was followed by ongoing studies from the University of Ankara Medical School, Hannover Medical School, and National Institutes of Health (NIH) in Turkey, Germany, and the US, respectively. Three different approaches have been explored in these sites. In Ankara, optimal dosing in combination with ritonavir (RTN) with or without peg-IFN was explored. In Hannover, a dose-escalating study for LNF in combination with RTN was conducted, and at the NIH, once-daily dosing of LNF with RTN was explored. In Ankara, first in the LOW-R1 (LNF with and without RTN) study, LNF was tested at the higher doses of 200 and 300 mg, bid, and in combination with peg-IFN or RTN⁶¹. Boosting with RTN increased serum concentrations of LNF 4- to 5-fold. The combinations of LNF–RTN and LNF–peg-IFN led to serum HDV RNA declines of more than 3 logs₁₀ IU/mL from baseline after 8 weeks of treatment⁶¹. Studies on the optimal combination treatment regimen with LNF and RTN and triple therapy with LNF, RTN, and peg-IFN have been tested for different durations in the LOW-R2 study. In this latter study, the triple combination treatment consisting of 25 mg LNF bid with RTN 100 mg bid and peg-IFN appeared to combine best efficacy with tolerability⁶². Three out of 5 patients were HDV RNA negative after 24 weeks of treatment. LNF at 25 or 50 mg bid dosing with RTN was as effective as all oral combination treatment. Five out of 14 (36%) patients had HDV RNA levels below the limit of quantification at 24 weeks of treatment. Combination of LNF at doses of 75 mg bid and higher as dual or triple therapy was not tolerated because of side effects and also did not lead to better antiviral efficacy⁶². In the dose-escalation study in Hannover, patients started with a LNF/RTN dose of 50 mg/100 mg bid and the LNF dose was increased first to 75 mg and then to 100 mg bid at roughly 4-week intervals when patients tolerated the administered doses. Dose was not further increased after reaching LNF/RTN (100 mg/100 mg bid). Escalation to this ultimate dose was possible in 10 out of 15 patients. One patient became HDV RNA negative at the end of treatment and another had HDV RNA levels below the quantification level⁶³. Overall, it appears that off-treatment virologic response is lacking in the majority of patients in these studies. An LNF–RTN once-daily dosing combination regimen for 24 weeks was tested in a double-blind approach at the NIH⁶⁴. Three regimens were tested: LNF 50/75/100 mg qd with RTN 100 mg qd. After 24 weeks of treatment, 6 out of 21 patients had HDV RNA levels below 250 IU/mL. Interestingly, the low-dose group appeared to have better antiviral efficacy compared to higher doses. ALT normalized in all of these studies with LNF in 47 to 75% of patients. LNF is associated with dose-limiting side effects, mainly related to gastrointestinal toxicity. These side effects comprise anorexia, nausea, diarrhea, and weight loss (Table 3). Finally, in the LOW-R2 study, 5 out of 27 patients receiving treatment with LNF-based regimens for 12 to 24 weeks experienced a post-treatment ALT flare, which led to HDV RNA suppression to levels below the level of quantification or to undetectable levels⁶⁵.

These studies performed in the US, Germany, and Turkey have provided substantial data regarding optimal dosing and duration and the optimal combination treatments as well as safety. A larger study using low-dose LNF in combination with RTN with or without peg-IFN now needs to be conducted. The above-mentioned studies have confirmed previous data in oncology patients that LNF is associated with dose-limiting gastrointestinal adverse events. Hence, based on the data mentioned above, when combined with RTN, an LNF dose exceeding 50 mg bid should probably not be considered. Strategies for efficacy off-treatment need to be further explored. Along this line, the superiority of LNF and RTN in combination with peg-IFN over peg-IFN monotherapy has to be explored in a very well-designed study. The notion that patients treated with LNF-RTN as all oral combination may have sustained viral efficacy also has to be explored. Finally, the possibility of inducing a post-treatment beneficial immunological flare has to be investigated in a patient cohort with compensated and less-advanced liver disease.

Nucleic acid polymers

NAPs are sequence-independent phosphorothioated oligonucleotides. Their mechanism of action is not clear. They may affect virus secretion from hepatocytes, although earlier steps of the viral cycle may also be targeted by NAPs. They bind with high affinity to amphipathic protein structures. In preclinical studies in duck HBV infection, the NAP REP9-AC was effective both in vitro⁶⁶ and in vivo⁶⁷. The first phase I/II study in humans was performed in Bangladesh. Seven out of 8 HBeAg-positive CHB patients receiving REP-2055 were reported to have 2 to 7 log reductions in HBsAg and 3 of them to have cleared HBsAg. All 7 patients had developed anti-HBsAg titers. Serum HBV DNA decreased by 3 to 7 logs after 20 to 27 weeks of treatment⁶⁸. Three patients who met protocol defined early termination criteria (e.g. HBsAg loss or HBeAg loss) discontinued treatment at weeks 20 to 27. On long-term off-treatment, only 1 patient had viral and serologic rebound, whereas the other 2 patients continued to be HBV DNA and HBsAg negative. However, REP-2055 intravenous infusion was associated with fever, chills, and headache. REP 2139-Ca, reported to be more stable, was given once-weekly by intravenous infusion to 12 HBeAg-positive CHB patients. Nine responder patients (decrease in HBsAg and HBV DNA by 2 logs) received add-on peg-IFN or thymosin after week 20. During monotherapy, 9 patients had an HBsAg drop of 2 to 7 logs, with 3 becoming HBsAg negative. Similar declines were observed in serum HBV DNA levels. However, off-treatment viral rebound occurred in 7 out of 9 patients within 3 to 24 months⁶⁸. REP-2139-Mg and REP-2165-Mg, a 2139 derivative with reportedly improved tissue clearance, is being tested in combination with peg-IFN and tenofovir in 20 HBeAg-negative CHB patients pre-treated with a 6-month course of tenofovir alone. Initial data suggest similar declines in HBsAg levels⁶⁹. All of these studies have relevance to CHD, since HBsAg appears to be selectively targeted. In the only study in CHD with NAPs, REP-2139-Ca was given once weekly with add-on peg-IFN starting at week 15 for another 15 weeks in 12 CHD patients⁷⁰. Peg-IFN alone was then continued as monotherapy for another 33 weeks. Nine patients displayed declines of >3 logs in HBsAg levels. Post-treatment results were presented at the EASL meeting this year. Five out of 12 patients continued to be HBsAg negative 1 year off-treatment⁷¹. Besides administration route-related side effects such as fever, chills, and peripheral hyperemia, other reported side effects include anorexia, hair loss, dysphagia, and dysgeusia, which were attributed by the investigators as being secondary to heavy metal exposure at the trial site (Table 3). Large randomized controlled trials are now awaited where the efficacy and durability of treatment response can be assessed as monotherapy as well as combination treatment.

NAPs led to the most dramatic response rates in terms of HBsAg declines in both CHB and CHD patients after treatment durations not exceeding 6 months in most cases. HBsAg declines in CHD patients were associated with substantial HDV RNA declines. They reported on- and off-treatment HBsAg clearance with development of antibody to HBsAg in a subset of patients. However, their spectacular results also raised some concerns, not confined to some unusual adverse events such as dysphagia. Deficiencies of pilot studies may be inevitable and understandable. They need to be addressed in better-prepared and -conducted larger studies with clear stopping rules where monotherapy with the selected NAP is compared to NAP–peg-IFN combination treatment as an example. The route of administration of NAPs (intravenous infusion) also appears to be a hurdle for this treatment approach.

Additional treatment approaches

An exciting concept is the use of small interfering RNAs (siRNAs). ARC-520, a siRNA designed to reduce all HBV transcripts via RNA interference, dose-dependently decreased HBsAg levels after one single injection in HBeAg-negative CHB patients in a phase IIa clinical trial⁷². A multi-dose extension study (up to 12 doses once-monthly) of the same compound has been conducted. With multiple dosing, additional decline of HBsAg levels was observed, more so in HBeAg-positive than in HBeAg-negative patients⁷³. It was reported that ARC-520 was well tolerated, but the study was put on hold owing to toxicity problems related to the carrier molecule.

Immune system-targeted approaches

Such approaches may have regained interest in CHB, but a breakthrough clinical success is still awaited. Therapeutic vaccination strategies have not been successful so far. New approaches include DNA vaccines⁷⁴, anti-HB immune complexes⁷⁵, and the use of immunologically active adjuvants such as beta-glucosylceramide⁷⁶. Toll-like receptors (TLRs) as inducers of type 1 IFN responses have always raised interest. Several preclinical studies confirmed their key role in the induction of the innate immune system⁷⁷. The oral TLR-7 agonist GS-9620 was well tolerated and led to the induction of peripheral ISG15 production in CHB patients⁷⁸. No effect on HBV DNA was observed, which may be attributable to short treatment duration.

Drugs targeting programmed death protein 1 (PD1) and its ligand (PD-L1) are receiving attention in both cancer immunotherapy and chronic viral hepatitis. Immune checkpoints are exploited by viral pathogens, as shown by T-cell exhaustion in these conditions. Blocking PD1 appears to be a rational goal to activate the HBV-specific T-cell response. In a phase Ib clinical study, the immune checkpoint inhibitor nivolumab was well tolerated and was associated with a significant decline in HBsAg levels after a single dose of nivolumab⁷⁹. Hepatotoxicity and off-target immune activation are concerns. The future of immune therapy may be using combination immune therapy such as a PD1 blocker with DNA immunization and NA treatment. Such an approach may deserve serious consideration⁸⁰.

siRNAs and various immunological treatment approaches represent scientifically sound treatment vehicles. They are based on a solid scientific background. We know, for example, from hepatitis B that off-treatment viral clearance necessitates tight immune control, hence an immune-based approach in controlling CHD makes sense. However, data showing viral efficacy are needed. The lack of success so far points to the complexity of the immunological process. Maybe better surrogate markers of immunological activity are needed beforehand. Similarly, siRNAs deserve attention, as with one formulation several siRNAs targeting different pre-genomic RNAs can be used. However, for both the immunological and the oligonucleotide approach, time is needed for further development of these approaches.