Clinical translation of liver regeneration therapies: a conceptual road map

Journal Pre-proofs Clinical translation of liver regeneration therapies: a conceptual road map Linda E. Greenbaum, Chinweike Ukomadu, Jan S.Tchorz PII: DOI: Reference:

S0006-2952(20)30068-X https://doi.org/10.1016/j.bcp.2020.113847 BCP 113847

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

6 November 2019 4 February 2020

Please cite this article as: L.E. Greenbaum, C. Ukomadu, J. S.Tchorz, Clinical translation of liver regeneration therapies: a conceptual road map, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp. 2020.113847

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Clinical translation of liver regeneration therapies: a conceptual road map Linda E. Greenbaum1,4*, Chinweike Ukomadu2,4*, Jan S.Tchorz3,4* 1Novartis

Institutes for Biomedical Research, Novartis Pharma AG, East Hanover, NJ

2Novartis

Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, MA

3Novartis

Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

4these

authors contributed equally to this work

*Correspondence:

[email protected], [email protected],

[email protected] Running title: liver regeneration drug development Document statistics: Words: 6881 (excl. references), Figures and tables: 3, References: 108 Non-standard abbreviations: ALPPS, associating liver partition and portal vein ligation for staged hepatectomy BEC, biliary epithelial cell CRC, colorectal cancer DDI, drug drug interaction eHx, extended hepatectomy FLR, future liver remnant GRWR, graft versus recipient weight ratio HCC, hepatocellular carcinoma HTN, hypertension LDLT, living-donor liver transplantation MoA, mechanism of action NASH, Non- alcoholic steatohepatitis PD, pharmacodynamic pHx, partial hepatectomy PK, pharmacokinetic PoC, proof of concept SFSS, small for size syndrome YAP, yes-associated protein

Abstract The increasing incidence of severe liver diseases worldwide has resulted in a high demand for curative liver transplantation. Unfortunately, the need for transplants by far eclipses the availability of suitable grafts leaving many waitlisted patients to face liver failure and often death. Routine use of smaller grafts (for example left lobes, split livers) from living or deceased donors could increase the number of life-saving transplants but is often limited by the graft versus recipient weight ratio defining the safety margins that minimize the risk of small for size syndrome (SFSS). SFSS is a severe complication characterized by failure of a small liver graft to regenerate and occurs when a donor graft is insufficient to meet the metabolic demand of the recipient, leading to liver failure as a result of insufficient liver mass. SFSS is not limited to transplantation but can also occur in the setting of hepatic surgical resections, where life-saving large resections of tumors may be limited by concerns of post-surgical liver failure. There are, as yet no available pro-regenerative therapies to enable liver regrowth and thus prevent SFSS. However, there is optimism around targeting factors and pathways that have been identified as regulators of liver regeneration to induce regrowth in vivo and ex vivo for clinical use. In this commentary, we propose a roadmap for developing such pro-regenerative therapy and for bringing it into the clinic. We summarize the clinical indications, preclinical models, pro-regenerative pathways and safety considerations necessary for developing such a drug. Main article 1. Introduction It is known that hepatocytes have near infinite capacity to regenerate [1]. This has enabled lifesaving therapies such as liver transplantation from deceased living donors, and curative resections especially of large liver masses [2-4]. In these examples, therapy relies on the fact that the transplanted or remnant liver will regrow and restore function. Nevertheless, this process is not perfect and is impaired when the transplanted organ or the post-resection remnant is too small, when there is marked acute cellular injury or when there is pre-existing chronic fibrotic liver disease [5-9]. There has been an explosion of knowledge on the molecular pathways that regulate liver regeneration and proof exits that modulation of these pathways either genetically or pharmacologically can induce restoration of functional and architecturally intact liver mass (e.g. YAP/Hippo pathway, WNT/β-Catenin and nuclear hormone receptor signaling, growth factors; see dedicated section in this commentary and Table 1 for details). Despite this new understanding

of molecular mechanisms that govern regeneration, which are key to development of any potential therapy, candidates which support small size liver regrowth are still missing in the clinic. Hurdles include lack of insight into how pharmacological therapies can be incorporated for diseases that are currently treated surgically, identification of ideal indications, and unclear pre-clinical efficacy and safety studies required to enable regulatory approval for clinical trials. Here we outline possible approaches to enable the development of a liver regenerative drug for clinical use. This review will concentrate on our understanding of pathways that regulate regeneration and highlight how regenerative therapies can support surgical approaches for SFSS by accelerating liver regeneration. Additional highlights include pre-clinical models to gauge efficacy and potential safety concerns and mitigations. Approaches to liver regeneration in the setting of chronic liver injury have recently been reviewed elsewhere [10] and will not be the subject of this review. 2. Unmet medical need and therapeutic approaches 2.1 Indications and approaches A significant area of unmet medical need that could be supported by regenerative therapies is the prevention of liver failure resulting from insufficient functional hepatic mass either following hepatic transplantation or after resection. This syndrome is referred to as small for size syndrome (SFSS). In general, SFSS is associated with a liver remnant or graft to body weight ratio < 0.8% or a remnant to total liver volume of less than 25%-30% [11, 12] . However, SFSS may be best defined as a liver mass that is insufficient to meet the post- operative metabolic needs of the recipient. While liver mass is clearly a variable in SFSS, other factors can increase the risk of SFSS even in the presence of a normal liver to body weight ratio including increased portal inflow, neo-adjuvant chemotherapy and graft quality and recipient portal hypertension [13, 14].

The most frequent clinical manifestations of SFSS are those reflective of liver failure, e.g. ascites, coagulopathy, hyperbilirubinemia and encephalopathy [12, 14]. SFSS is usually detected within the first two weeks post-transplant but may occur as late as one month post-transplant. The role of impaired regeneration in SFSS can be modeled in pre-clinical studies where accelerating the restoration of liver mass and function by pharmacological means reduces the risk of SFSS [15]. Reducing vascular shear stress has also improved survival in preclinical studies, but has not yet been tested in clinical trials [14]. 2.2. Regenerative Therapies to support surgical liver transplantation and hepatic resections

Transplantations of organs from deceased and living donors and resection of hepatic masses especially those resulting from metastatic colorectal carcinoma are now routine [2, 4]. However, candidates for surgery are carefully selected to ensure a low risk of post-surgical liver failure. Many who do not meet rigorous criteria for selection are not considered because of concerns that the residual liver mass will not be adequate to support metabolic demands. In the setting of hepatic resections for colorectal cancer for example, approximately 10%-20% of these carefully selected surgical patients will still develop liver failure [2, 16]. The current understanding of molecular pathways that can accelerate recovery of liver mass and restore associated function suggests that pharmaco-therapeutic approaches can augment these exceptional surgical advancements. Such therapies may be developed to enable early regrowth of functionally intact liver in vivo or ex vivo, thus allowing for a reduction in the risk of post-surgical liver failure and even potentially expand the breadth of patients eligible for surgical intervention (Figure 1A-C). The goal of such therapies should be to expand the population of patients who can benefit from surgery and not to replace surgical approaches. There is a shortage of suitable livers for transplantation globally and many patients die as a result. Potential regenerative therapies could support surgical transplantation, expand organ usage and limit concerns about SFSS. In the USA, only a third of the subjects on the transplant list annually receive organs (https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/). Many patients are not even listed, having been excluded from the wait list due to stringent criteria for candidacy, driven by the inadequate supply of organs [17]. Therefore, approaches to increase efficient use of organs or improve access to organs should be sought. Specific areas where regenerative therapies might improve efficiency and access to organs include; 1) Increased use of small for size grafts from deceased donors. For example, the potential to transplant the left lobe despite concerns of SFSS in non-pediatric populations may be feasible if early restoration of functional mass is achievable through pharmacological approaches. This can be either i) in vivo through earlier restoration of liver mass, by the use of pharmacologic agents that enable earlier and accelerated hepatocyte cell cycle entry or ii) ex vivo through novel perfusion techniques, especially normothermic devices [18, 19], which may be adapted to support pharmacologically aided liver regeneration outside of the body prior to transplantation (Figure 1AC). 2) An increase in the proportion of transplants resulting from living donors can also be envisioned if safe and effective regenerative drugs are available. While pre-treatment of donors is not proposed nor likely to be supported, pro-regenerative drugs can play a role by allowing for the

use of small segmental grafts, for example, the left hepatic lobe. This would reduce the risk associated with living donation by avoiding the larger and more complex right lobe resections as currently done [20]. In both of these cases availability of regenerative therapies might support the faster volume and functional restoration of the liver even when the graft is small for the body size. Insufficient hepatic remnant after hepatic resection also leads to SFSS [11, 21] and concerns about insufficient future liver remnant (FLR) limit candidacy for a potentially curative surgery in the setting of metastatic colorectal cancer (CRC), the most common reason for segmental resection of the liver [22]. In the United States, more than 145,000 new cases of CRC are diagnosed with over 50,000 deaths occurring yearly with CRC as the third most common cause of death in women and the second in men [23]. Globally there are more than 1.8 million new cases of CRC diagnosed each year (10.2% of all cancers) and more than 880,000 deaths (9.2% of cancer deaths) [24]. In this setting resection of hepatic masses has led to 5-year survival rates of over 50% among patients with CRC [25, 26] . It is estimated that 25-30% of all patients with CRC will have colorectal liver metastases at some point during the course of their disease [27, 28]. Concerns of FLR however limit curative resections even though negative surgical margins at resection improve long-time outcomes [29]. This suggests that when possible robust resections with extended margins should be performed. As in the case for liver transplantation, therapies that can accelerate restoration of functional mass from the remnant liver might expand the pools of subjects eligible for surgery (Figure 1D,E) and improve post resection outcomes. 3. Pathways and promising therapeutic targets Many excellent review articles have extensively discussed the pathways involved in and required for liver regeneration [10, 30]. In most cases, inactivation of individual genes has been shown to partially impair or delay liver regrowth, suggesting that concerted action of multiple signaling pathways mediates diverse aspects of liver regeneration. In addition, crosstalk between and contribution of most liver cell types is critical for functional liver regrowth [10, 30]. While diverse therapeutic approaches have the potential to ameliorate SFSS in patients, such as modulation of portal hypertension and hepatic microcirculation [14], we focus on pathways and associated exploratory preclinical therapeutics that have been shown to enhance liver regrowth following partial (pHx; 70% removed) or extended hepatectomy (eHx; 86-90% removed) and thus may provide possible candidate pro-regenerative therapies to prevent SFSS (summarized in Table 1). In addition, we describe their potential to enhance liver function and prevent apoptosis – key mechanisms believed to have pro-regenerative potential in the indications discussed above.

3.1. YAP/HIPPO pathway Nuclear YAP (and its homolog TAZ) assembles with transcription factors (such as TEADs) to promote liver growth by enhancing hepatocyte proliferation. Transgenic YAP increased liver mass by several fold suggesting that YAP is a central player in controlling liver size [31]. The HIPPO complex restricts YAP activity by promoting its cytoplasmic retention and the YAP/HIPPO complex is a central growth regulator in many organs [32]. MST kinases are central components of the HIPPO complex and have been targeted to promote YAP signaling and enhance liver regeneration [33, 34]. Importantly, insufficient YAP activity was associated with impaired liver regeneration during SFSS in mice and man [35]. Lipid nanoparticle (LNP)-formulated siRNAs targeting MST1 and MST2 enhanced liver regrowth following pHx in aged mice [34]. The MST inhibitor XMU-MP-1 significantly improved liver regrowth following pHx and promoted tissue repair in response to acute injury in liver and intestine [33]. However, no clinical development of XMUMP-1 has been reported to date and independent validation of its efficacy is pending. Besides the important role of YAP in promoting hepatocyte proliferation [31], YAP also controls expansion of biliary epithelial cells (BECs) [36] and angiogenesis [37]. Since functional liver regrowth requires expansion of all hepatic cell types, this multicellular activity of YAP might be beneficial. Importantly, YAP activation also protects from liver damage and hepatocyte apoptosis [38]. While YAP activation induces SOX9+ hybrid hepatocytes that harbor increased regenerative potential [39, 40], transgenic YAP expression induced dedifferentiation of hepatocytes into biliary epithelial cells (BECs) [41], which would likely reduce the number of available metabolically functional hepatocytes. Sustained YAP activation is involved in hepatic tumor formation and progression [32]. Similar to almost all growth promoting pathways listed below, YAP activation might therefore increase the risk for tumor formation. However, limiting target engagement of potential therapeutic drugs to the short period during which liver regrowth enhancement in SFSS patients would be needed, could likely mitigate this risk. Many of the pro-regenerative pathways including some discussed below converge into the YAP/HIPPO pathway, suggesting that YAP is a key mediator of liver regeneration [42]. Given the importance of YAP in homeostasis of multiple organs, mitigation of systemic proliferative effects will need to be implemented in preclinical evaluation of candidate compounds and liver-specific drug delivery strategies should be considered (see also section on safety considerations). 3.2. WNT/β-Catenin pathway

A porto-central WNT/β-Catenin activity gradient is required to establish and maintain metabolic liver zonation [43]. Moreover, WNT/β-Catenin signaling plays an essential role during liver development, regeneration and tumor formation by regulating hepatocyte proliferation [44]. This dual role in regulating the expression of key metabolic enzymes and promoting liver growth renders WNT/β-Catenin signaling as an attractive target for improving liver regeneration and function in SFSS patients. WNT/β-Catenin pathway activation in patient livers following acetaminophen-overdose positively correlated with hepatocyte proliferation and spontaneous liver regeneration [45]. RSPO proteins bind LGR4 and LGR5 receptors to promote WNT/βCatenin activity [46, 47] by sequestering the ubiquitin ligases ZNRF3 and RNF43 that mediate FRIZZLED turnover and thereby restrict WNT/β-Catenin signaling [48]. In the liver, the RSPOLGR4/5-ZNRF3/RNF43 module not only enhances WNT/β-Catenin signaling but is also essential for metabolic function. Combined deletions of LGR4 and LGR5 resulted in hypoplastic livers and abrogated liver zonation [49]. Likewise, LGR4/5 deletion impaired hepatocyte proliferation and regeneration following pHx [49] or liver drug-induced liver injury [36]. Conversely, injections of recombinant RSPO protein significantly increased liver size and accelerated liver regrowth following pHx [49]. RSPO treatment extended the pericentral metabolic gradient and thus increased the number of hepatocytes expressing key metabolic enzymes [49]. Moreover, WNT/βCatenin activity protects from hepatocyte apoptosis [50] and promotes angiogenesis [37]. Interestingly, WNT/β-Catenin activity was reduced in small-sized grafts in rats, which was restored following

treatment

with

a

WNT

agonist

(2‐amino‐4‐[3,4‐[methylenedioxy]benzylamino]‐6‐[3‐methoxyphenyl] pyrimidine]: CID 11210285) [15]. The WNT agonist restored cyclin D1 expression and ATP production, attenuated hepatocellular injury and increased hepatocyte proliferation and survival rates after transplantation of a 30% liver graft [15]. Likewise, hydrodynamic delivery of Wnt-1 naked DNA accelerated the proliferative response post pHx in mice by activating WNT/β-Catenin signaling [51]. WNT/β-Catenin signaling also controls tissue homeostasis in several other organs [52] and excessive activation promotes tumor formation [44]. Similar to the YAP/HIPPO pathway, systemic effects of WNT/β-Catenin activation would therefore have to be time restricted. There are several mechanisms and experimental drugs described which activate WNT/β-Catenin signaling that could be tested for their ability to prevent SFSS [53]. 3.3. Nuclear hormone receptors 1,4‐bis [2‐(3,5‐dichloropyridyloxy)] benzene (TCPOBOP) is one of the most potent mitogens in the liver and an agonist of the constitutive androstane receptor (CAR) [54]. Importantly, CAR

activation also promotes xenobiotic metabolism [55] and its activation might thereby accelerate restoration of hepatic metabolism in SFSS patients. A single dose of TCPOBOP not only accelerated liver regrowth following pHx [56], it also prevented SFSS in a subset of mice following eHx [57]. TCPOBOP suppressed the cell cycle inhibitor p21, restored hepatocyte proliferation in a FOXM1-dependent manner, accelerated liver weight gain, and normalized metabolic SFSS features after eHx [57]. Interestingly, deficiency in the CAR-FOXM1 axis is evident in human SFSS and activation of human CAR mitigates SFSS in humanized CAR mice [57]. Agonists for mouse CAR (TCPOBOP) do not show significant potency on human CAR and the human CAR agonist

CITCO

(6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehydeO-(3,4-

dichlorobenzyl)oxime showed less efficacy when tested in mice with humanized CAR [57]. While optimizing potency of CITCO or developing alternative human CAR agonists might offer an attractive strategy to prevent SFSS, the significant differences between rodent and human CAR remain a challenge. Mouse and human CAR showed differential activation of pro-proliferating genes [58], raising the possibility that the dramatic mitogenic effects in rodents might not translate into the clinic. Moreover, the fact that even TCPOBOP, despite its favorable efficacy profile, only rescued 40% of the mice from eHx [57] suggests that activation of additional mechanisms might be required to enable safe and efficient liver regeneration during SFSS. However, the mostly liverspecific expression of CAR [59] renders it an attractive potent CAR agonist as it may be associated with minimal systemic adverse effects. Two additional members of the nuclear hormone receptor family, PXR [40] and FXR [60] have been proposed as targets to improve liver regeneration. PXR agonism using the mouse PXR agonist pregnenolone‐16α‐carbonitrile enhanced liver regeneration following pHx [40]. Interestingly, PXR was shown to mediate liver regeneration via YAP signaling [40]. Similar to the CAR study [57], the authors tested human PXR agonism in PXR-humanized mice [40]. However, the drug used (Rifampicin) is rather unspecific and given the limited homology between the human and mouse ligand binding sites on the respective PXR, a selective and potent human PXR agonist with efficacy in preventing SFSS would need to be developed. FXR was also shown to be essential for liver regeneration and its activation via bile acids significantly promoted liver regeneration following pHx [60]. FXR regulates various aspects of metabolism and inflammation which have made it an attractive drug target for several diseases, such as obesity, metabolic syndrome, non-alcoholic steatohepatitis (NASH), cholestasis and chronic inflammatory diseases of the liver and intestine. Consequently, several FXR agonists are

currently being tested in clinical trials for several of these indications [61]. A regenerative effect in damaged livers or promotion of liver regrowth to prevent SFSS might represent promising additional indications for patients. However, more research is required to assess the proregenerative potential of FXR agonists currently in the clinic. 3.4. Thyroid hormone signaling Pre-treatment of rodent livers before pHx with thyroid hormone (T3) injections substantially increased liver mass and allowed for normal liver regrowth [62]. T3 treatment induced hepatocyte proliferation by protein kinase A-dependent beta-catenin activation with a trend towards increased survival in the eHx model [63]. Similarly, the selective T3 hormone receptor β1 (TRβ1) agonist GC-1 showed enhanced WNT/β-Catenin-dependent hepatocyte proliferation and regeneration following pHx [64]. Although these results are promising, additional work is required to substantiate this finding. It is important to note that pretreatment of the donor with a primary mitogen such as T3, as proposed by the authors, or any therapy that would require donor pretreatment, would likely impose several regulatory challenges. 3.5. Growth factors and cytokines Several growth factors play a crucial role during liver regrowth including EGF, FGF, IGF, VEGF, and BMP, whereas TGFβ blocks liver regeneration (reviewed in [65]). Inhibition of TSP1-mediated TGFβ1 signaling using a leucine-serine-lysine-leucine (LSKL) peptide accelerated liver regeneration after pHx [66]. The most prominent growth factor in the liver, hepatocyte growth factor (HGF) was identified in the serum of rats in response to partial hepatectomy [67] and is the ligand for cMET receptors [68]. Recombinant HGF enhanced liver regrowth following pHx [69]. Moreover, transfusion of HGF-overexpressing mesenchymal stem cells (MSCs) rescued rats from small for size liver transplantation injury [70]. Recombinant IL-6 improved recovery and survival from eHx by accelerating liver regrowth, reducing oxidative stress, and maintaining mitochondrial function [71]. The designer cytokine Hyper-IL-6 consisting of soluble IL-6R covalently linked to IL6 accelerated liver regeneration following pHx [72]. However, all these growth factors have pleiotropic roles in other tissues. Possibly, stabilized growth factors targeted to the liver could be an option to promote liver regrowth and prevent SFSS. 3.6. Complement inhibition Activation of complement promotes the production of the effector molecules C3A, C5A, and the membrane attack complex (MAC). C3A and C5A are cleaved soluble bioactive peptides and play

an important role during liver regeneration by promoting hepatocyte proliferation [73]. CR2-CD59, a site-targeted mouse complement inhibitor that specifically inhibits the terminal MAC, not only ameliorated hepatic ischemia reperfusion injury but also rescued 70% of treated mice from SFSS in an eHx model [74]. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and AKT activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores [74]. Although differences in the eHx model used in the complement inhibitor study [74] to the one used for the CAR agonist studies [57] might not allow a direct comparison, the potential of CR2-CD59 to prevent SFSS was most remarkable. Developing a human version of this complement inhibitor to validate the potential to prevent SFSS in patients could be an attractive strategy. 3.7. New targets on the horizon Several genetic screens using shRNA- or CRISPR-mediated loss of function have revealed novel potential targets for improving liver regeneration. These screens used the Fah-/- model that has a genetic defect in the tyrosine catabolism pathway resulting in accumulation of the toxic metabolite fumarylacetoacetate (FAA) and hepatocyte cell death upon the removal of the protective drug NTBC [75]. Widespread hepatocyte death in this model provides a competitive advantage to hepatocytes that have a restored Fah gene as well as loss of function of a potential inhibitor of liver regeneration. Such in vivo screens identified Map2k4 [76], Tnfr1 [77], as well as Pkd1, Kmt2d and Arid1a [78] as genes encoding the proteins that repress regeneration, whereas Foxa3 was essential for liver regeneration in the Fah-/- model [77]. TRAP-seq screening identified cystine/glutamate antiporter Slc7a11 as a key regulator of liver regeneration and its overexpression promoted hepatocyte repopulation in the Fah-/- model [79]. Together, these screens provide novel targets for pro-regenerative therapies to be evaluated further. It is conceivable that epigenetic modification of hepatocyte chromatin during SFSS at least in part prevents their regenerative response, although no experimental drug has confirmed this hypothesis to date. Recent work highlights the importance of epigenetic mechanisms to enable pro-proliferative gene programs. ARID1A is part of the SWI/SNF complex, whose members have helicase and ATPase activities and regulate transcription of certain genes by altering their chromatin structure. ARID1A deletion in mice resulted in substantially increased liver regeneration by promoting hepatocyte proliferation without excessive overgrowth and tumor formation [80]. Mechanistically, ARID1A mediates the complex formation between YAP/TAZ and the SWI/SNF components, which competes with the association of YAP/TAZ with the DNA-binding platform

TEAD. Likewise, deletion of ARID1A restores YAP/TAZ-TEAD interactions and thereby enables the growth promoting gene program induced by this complex [81]. In addition, YAP/TAZ require the general coactivator bromodomain-containing protein 4 (BRD4) to enable proliferation [82]. More research is required to better understand the epigenetic regulation of the key pathways regulating liver regeneration and their involvement in SFSS. 4. Preclinical models Studies in preclinical models have identified candidate pathways that may enhance liver regeneration in SFSS and could be developed as pharmacological therapies. In the following section, the advantages and potential challenges of each of these models to test drug candidates are discussed. A proposed flowchart for developing a drug promoting liver regeneration is outlined in Figure 2. 4.1. Cellular assays for identification and validation of pro-regenerative drugs For a target-centric drug development program, biochemical and/or cellular and in vitro potency need to be determined for any given target/drug combination (Figure 2A). It is important to assess cellular toxicity and/or cell survival since cell death and thereby reduced confluency can trigger pro-regenerative pathways and thus may result in false-positive screening hits (e.g. YAP is regulated by mechanosensing/confluency [83]) (Figure 2B). For testing the efficacy of the compound in vitro to promote hepatocyte proliferation, the choice of the right cells is important. While primary hepatocytes can be triggered to proliferate [84], their proliferative capacity in vitro is generally very limited. Moreover, they cannot be propagated in culture for long and the need to frequently prepare or purchase new cells will likely introduce batch-to-batch variation. While hepatocyte-derived cell lines proliferate in culture, most of them carry mutations and exhibit differential dependency on growth-promoting pathways [85, 86], likely not providing a nearphysiological system. Recently described hepatocyte liver organoids are primary liver cells that can be propagated in culture and might offer a better model system [87, 88]. Such organoid systems, when adapted to a format fit for large scale screens, could combine the advantage of cell lines with those of primary cells. However, their characteristics and potential to predict proproliferative effects of tested molecules in vivo remains to be shown. A cellular or organoid-based proliferation assay could also be used directly to perform a phenotypic screen for compounds promoting hepatocyte proliferation, without knowing the target (Figure 2C). 4.2. Pharmacokinetic (PK) and pharmacodynamics (PD) profiling

Pre-clinically, liver regeneration is typically studied in hepatectomised mice or rats. To ensure the most efficient use of these complex models and to increase the likelihood of success it is important to first understand the PK/PD relationship of the investigational therapy in these species. Typically this means initially measuring drug exposure in blood with time following either intravenous (i.v.) dosing (biological and low molecular weight entities) and/or orally (low molecular weight therapies) in animals with intact livers and relating the exposure to some PD marker of drug target engagement. Studies in hepatectomised animals can then be designed so that a drug dosing regimen delivers the drug at both the desired level and duration to engage the target. Large excursions in drug concentration as sometimes seen with bolus dosing can be ameliorated by using constant rate i.v. infusion via cannulated blood vessels. While PK profiles in hepatectomised animals can be predicted from PK/PD studies in healthy animals, additional PK/PD studies in hepatectomised animals will more accurately inform about the differential PK profiles in animals with smaller livers (Figure 2D). Before testing therapeutic candidates in efficacy models, early safety assessment is necessary to identify tolerated dosing regimens for the anticipated treatment duration (Figure 2E). Candidates that do not achieve sufficient target engagement at the tolerated dose may still have a developmental path forward, providing that no liver toxicity is identified and liver targeting is possible to avoid dose-limiting systemic toxicity. 4.3. 70% partial hepatectomy (pHx) model The 70% pHx incorporates some but not all of the alterations seen in SFSS and, therefore, its primary utility during the preclinical stages of drug development is to validate promising targets and to assess acceleration of key growth parameters including hepatocyte and nonparenchymal cell proliferation and growth and to confirm that liver architecture and liver function are maintained (Figure 2F). In response to a coordinated response of cytokine, growth factor and metabolic signals that are induced and/or mobilized rapidly after pHx, normally quiescent hepatocytes rapidly reenter the cell cycle with the peak of hepatocyte DNA synthesis occurring 24 hours posthepatectomy in the rat and 36-48 hours in the mouse [10, 30]. Nonparenchymal cells including cholangiocytes, endothelial cells and hepatic stellate cells proliferate with a more delayed peak of DNA synthesis. A significant strength of the 70% pHx model is that it is reproducible. Because regeneration is robust and morbidity/mortality is principally a reflection of surgical technique, this model can be used to screen and identify targets and promising drug candidates that accelerate hepatocyte proliferation. Enhanced liver growth can be assessed as early as 4 days post pHx with near complete restoration of liver mass achieved by 7-10 days. The differences in the timing of peak DNA synthesis in the rat vs. the mouse pHx model, reflects the shorter G1 phase in the rat.

One of the potential challenges to using the rat model to screen drug candidates is this short G1 phase, since it presents a very narrow time window to detect a shift to earlier hepatocyte proliferation. Given the complexity of the regenerative process, systems analysis approaches represent a powerful tool to understanding the relative contribution of driving factors from parenchymal vs non-parenchymal cells, impact of cell cycle duration, replication and cell growth on the recovery of the liver mass [89, 90]. Using this approach, Cook and colleagues have shown that a combination of metabolic demand and cell growth were beneficial for enhancing liver mass recovery. However, when metabolic demand significantly exceeded cell growth, mass recovery was reduced [89]. Incorporating human data into these profiles will no doubt provide important insights into the translatability of the rodent model data. 4.4. Partial liver graft transplantation and extended hepatectomy (eHx) models Several models have been employed to evaluate potential therapeutic interventions to treat SFSS. These models include partial liver graft transplantation [91], 86% eHx [92]), 90% eHx [10] and the 90% eHx with associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) [93, 94]. The 86% and 90% eHX models, developed in both mouse and rat closely reflect the pathophysiology of SFSS in the clinical setting, however because they require a high level of surgical expertise, their use has been limited to surgical laboratories. The 90% eHx model in which all lobes are resected, with the exception of the caudate lobe, is associated with high mortality and assessment of efficacy of therapeutic interventions is based on improvement in survival. A variation on this model is the 86% eHx in which the right posterior lobe rather than the caudate lobe is retained [92]. This model, while technically more difficult than the 90% eHx model, is associated with a higher survival rate than the 90% eHx model and allows for evaluation of cell cycle parameters in addition to histologic injury [92]. The ALPPS procedure, involves portal vein ligation with the goal to stimulate hypertrophy of the contralateral lobe increase functional liver reserve and thus allow for resection of previously unresectable liver tumors [94]. Rodent and pig models of ALPPS have been developed. However, to date, there has been limited characterization of the modes of injury and regenerative pathways in ALPPS that mediate the proliferative response of the FLR. Additional basic research to identify modes of injury and regenerative pathways in the ALPPS will likely be most useful as an intermediate step to identification of novel therapies to improve ALPPS rather than as a primary model to evaluate preclinical efficacy for drug candidates [95].

In the 90% eHx rodent model a reduction in mortality is the generally accepted endpoint to support that the intervention tested has a positive effect on liver regeneration (Figure 2G). The 90% eHX model reflects many but not all manifestations of SFSS, i.e. kinetics of liver regeneration, extent of liver function and injury markers alterations as well as the extent of liver resection compatible with survival in patients differ between rodents and patients with SFSS [96]. Nevertheless, this model can be highly useful to confirm survival benefit with promising drug candidates that have been first identified in the 70% pHx model (Figure 2F). Studies in larger species to evaluate PK/PD, efficacy, safety and human dose predictions will need to be performed (Figure 2H) prior to evaluating a candidate therapy in the clinic (Figure 2I). 5. Preclinical safety considerations 5.1 Adverse effects and cancer risk There are multiple other factors that need to be taken into consideration for preclinical selection of molecules in order to proceed to clinical trials. For example, confirmation that liver architecture, zonation and function are maintained in the face of accelerated hepatocyte proliferation can be achieved with histological staining and functional assays. Similarly, while stimulation of hepatocyte proliferation is the desired effect, sustained activation of growth promoting pathways has the potential to induce tumors. Therefore, it will be essential to confirm in preclinical toxicology studies that proliferation is self-limited, both within the liver and in extrahepatic tissues. Dedicated carcinogenicity studies, such as evaluation of colorectal cancer cell lines and/or xenograft assays may be particularly relevant if considering a therapy for patients who will undergo resection for metastatic liver lesions. Even with a clean carcinogenicity assessment, limiting the duration of treatment to 1-2 weeks post tumor resection would be a prudent approach to minimize safety risks in the clinic. In some cases, where a therapeutic window is not achievable with systemic therapy, delivery approaches that target the drug to the liver may be considered. For example, LNPformulated siRNAs [34] could also be considered as these might therefore have an advantage over systemic activation (e.g. using XMU-MP-1 [33]). Alternatively, regeneration supported in the ex vivo setting for transplantation may enable organ specific cell proliferation and regeneration prior to transplantation [19]. 5.2 Drug-drug interactions Patients who have received a transplanted liver or have undergone hepatic resection for metastatic lesions are likely to be taking multiple anti-rejection or chemotherapeutic medications. Therefore, the potential for significant drug-drug interaction (DDI) that could result in either lower

or higher than expected drug exposures should be evaluated. Particularly in the transplant population, selection of candidate compounds that have lower DDI potential with frequently used co-medications (e.g. mTOR inhibitors and calcineurin inhibitors, anti-lipidemics and diabetic medications) will be important and dedicated DDI studies will reduce the risk of clinically significant DDI [97]. 6. Biomarker development When feasible, identification of serum and/or imaging biomarkers during preclinical development will help to confirm target engagement in clinical trials and inform dose selection by allowing identification of the dose that achieves maximum target engagement. In early clinical trials, biomarkers of target engagement are equally important when the drug is not efficacious. Demonstration of target engagement in this situation eliminates the possibility that the lack of efficacy was the result of insufficient drug access to the target. Identification of biomarkers of target engagement should ideally be correlated with noninvasive measurements of both liver growth and recovery of liver function. Liver mass recovery posttransplant or resection can be monitored in patients with CT, MRI [10] with SPECT (Single-photon emission CT) also providing assessment of liver metabolic function. The 13C breath test [98] and real time imaging of metabolic function using intravital dye [99] represent two promising techniques that have been used to noninvasively detect changes in liver metabolic function in the rodent 70% hepatectomy model and could be translatable to the clinic for assessment of recovery of liver metabolic function during clinical trials. System biology `omics` technologies will aid the discovery of efficacy and toxicity biomarkers. Transcriptomic analyses of regenerating liver samples from ALPPS surgery could be mined for markers of regrowth and graft function [100]. Serum proteomics profiling technologies, such as SOMAscan [101], could detect secreted hepatic factors indicating efficacy or adverse effects in patient serum. As an example, YAP signaling controls the expression of several secreted proteins (e.g. CYR61, CTGF) [102] which could be used to ascertain target engagement non-invasively. While this approach cannot distinguish between factors derived from the liver or other organs, circulating exosomes derived from liver cells [103] might contain suitable biomarkers of liver regeneration, function and toxicity with cellular resolution . Metabolomics may not only enable monitoring of vital liver functions but can also detect changes in metabolic processes (metabolic remodeling) associated with regenerative processes in mouse livers [99]. Translating such findings into the clinic and identifying metabolic or proteomic profiles associated with functional liver regrowth may be used in the future to monitor the success of hepatic regenerative therapies.

7. Planning a clinical proof of concept (PoC) study Identification of the optimal patient population to evaluate therapies in the clinic will be guided by the mechanism of action of the drug, preclinical safety profile and feasibility of identifying patients to enroll in a clinical trial. Patients with large cavernous hemangiomas, focal nodular hyperplasia or adenomas that require surgical resection could be candidates. The advantage of these patient populations include a nondiseased liver remnant, lack of prior use of pre-resection neo-adjuvant chemotherapy and lessened concern about accelerating malignant cancer growth with a pro-proliferative drug. The significant obstacle is that studies will be challenging to recruit given the low prevalence of benign hepatic lesions especially those large enough to require resection [104, 105]. Studies will undoubtedly be expensive given the requirement for a large number of clinical sites and prolonged duration of study conduct. The more clinically impactful indication is to evaluate pro-regenerative therapies in patients with metastatic CRC to the liver. Here not only are there concerns of SFSS due to FLR but the risk of post-operative liver failure is heightened in patients who undergo extended hepatectomy for malignancy as a consequence of neo-adjuvant chemotherapy induced hepatotoxicity [13]. Concerns about driving cancer growth regrowth within and outside of the liver will require careful assessment. Currently, strategies to minimize SFSS include surgical procedures to limit portal blood flow (ALPSS), application of stringent selection criteria to avoid use of high-risk livers and avoiding transplanting small graft size livers into high risk patients such as those who have high MELD scores and other co-morbid conditions have resulted in improved surgical outcomes. Mismatch between liver demand and recovery of functional liver cell mass also plays a role in the SFSS. Acceleration of hepatocyte replication and cell growth may provide clinical benefit and therefore mitigate SFSS. Because SFSS occurs over a short period of days to weeks following surgery and is invariably defined by emergence of poor liver function and failure, proof of concept studies can be short and performed over a period of 4 weeks. Given what are likely recruitment hurdles in these early studies, approaches such as use of historic controls for placebo may be necessary. Shorter

recovery time (i.e. reduced hospital stay) could be considered as a primary endpoint for this indication with supportive PD endpoints such as lack of signs of early allograft dysfunction (defined as one or more of ALT or AST≥ 2000 IU within the first 7 days or bilirubin≥10 mg/dl on day 7 or INR≥ 1.6 on day 7) [106]. In addition, kinetics of liver mass restoration by serial imaging and where possible histologic analysis for pathway activation, hepatic architecture, cellular proliferation can be added as exploratory objectives The PoC approach outlined above focuses on regrowth as the core component for preventing SFSS. However, other factors have been implicated in SFSS such as portal hyperperfusion, liver graft status, and recipient status including cirrhotic pathophysiology, particularly portal hypertension [14].Thus, acceleration of hepatocyte proliferation alone may not be the only factor to consider when developing pharmacological therapies to enhance liver regeneration in patients. Evidence from preclinical models suggests that optimizing the balance between metabolic demand, hepatocyte proliferation and cell growth may be required to achieve improved clinical outcomes [89]. An approach that may limit these concerns may be to initiate the regrowth of the organ for transplantation ex vivo. Ex vivo repair of marginal organs and/or expansion of the size and function of a left lobe segment prior to transplantation may reduce risk to the donor, expand the number of available livers for transplantation, and reduce transplant waiting list time and mortality [18]. 8. Next Steps To advance any drug to the clinic will require the collaborative effort of academicians, particularly liver biologists, hepatologists and hepatobiliary surgeons, health authorities and the pharmaceutical industry. This approach has been used to gain consensus for development of therapies in a number of indications in hepatology and recently for NASH where the Liver Forum has been used to gain insights into the identification of patient populations, indications and aiding in outlining regulatory pathways for NASH drugs [107, 108]. Given the advances in scientific understanding of liver regeneration and its potential role to address unmet needs in liver diseases such dialog should start sooner than later and will be essential to enable the development of regenerative therapies for our patients. Footnotes Acknowledgements

We would like to thank all researchers who substantially moved this field within the past decades and apologize for not being able to cite all the work in this Commentary. We further thank PierreAlain Clavien, Seth Karp, Scott Friedman, Gary Levy, Bindi Sohal, Sukhdeep Sahambi, Juergen Maibaum, Andreas Sailer, Ralf Glatthar and Elizabeth George for helpful discussions. Conflict of interest statement L.E.G., C.U. and J.S.T. are employees and/or shareholders of Novartis Pharma AG. Legends to Figures and Tables Figure1: Indications with unmet medical need to improve liver regeneration for prevention of small for size syndrome (SFSS). (A) A living or deceased donor donates a liver segment which then has to regrow in the recipient. (B, C) The size of a small donated liver graft is being increased ex vivo in a perfusion device (B) before transplantation into the recipient (C). (D, E) A tumor is resected (D), requiring the remnant to regrow within the patient (E). (F) A healthy liver segment is removed and increase in liver mass achieved ex vivo in a perfusion device before transplantation back into the recipient with removal of the tumor-containing segment. Table 1: Pathways and targets with experimental drugs that have shown efficacy in either partial hepatectomy (pHx) or extended hepatectomy (eHx) models. Trivial names of the drugs (chemical compounds or biologics) have been provided; the full names can be found in the respective literature listed in the table. *, transplantation model; **, no statistical significance; MSC, mesenchymal stem cells; n.t., not tested. Figure 2: Proposed flowchart for screening pro-regenerative liver drugs. (A) Target engagement has to be assessed to identify drugs with in vitro potency. (B, C) Toxicity/cell survival (B) as well as efficacy in promoting proliferation in vitro (C) will be tested in hepatocyte-derived cell lines or liver organoids. (D) Pharmacokinetic profiles and target engagement (in vivo pharmacodynamic (PD) effect) will be assessed for selected candidates. (E-G) Early safety assessment (E) is important to determine tolerated doses for efficacy studies in partial hepatectomised (pHx) rodents (F) or extended hepatectomy (eHx) models associated with SFSS. (G) Larger species will likely be needed to for testing selected candidates with efficacy and acceptable safety profile in rodents. (F-G) Efficacious drug candidates which meet safety criteria (I) can finally be tested in patients (H).

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Schabel, V. Miller, A.J. Sanyal, G. Liver Forum Case Definitions Working, Case definitions for inclusion and Experimental drug

Efficacy

Pathway

Target

Class

Drug name

pHx

eHx

Reference

YAP/HIPPO pathway YAP/HIPPO pathway WNT/β-Catenin pathway WNT/β-Catenin pathway WNT/β-Catenin pathway HGF-cMET pathway HGF-cMET pathway TGFβ pathway

MST1/2

small molecule inhibitor

XMU-MP-1

Yes

n.t.

Fan et al., 2016

MST1/2

LNP-formulated siRNA

n/a

Yes

n.t.

Loforese et al., 2017

LGR4/5ZNRF3/RNF43 WNT pathway

RSPO1-fc biologic

RSPO1-fc

Yes

n.t.

Planas-Paz et al., 2016

WNT agonist

CID 11210285

(Yes)*

n.t.

Ma et al., 2016

WNT pathway

naked Wnt-1 DNA

n/a

Yes

n.t.

Nejak-Bowen et al., 2010

cMET

Recombinant HGF

n/a

Yes

n.t.

Ishii et al., 1995

cMET

HGF-overexpressing MSCs TSP1-blocking peptide

n/a

Yes

Yes

Yu et al., 2007

LSKL

Yes

n.t.

Kuroki et al., 2015

IL6 pathway

IL6R

Hyper IL6

Yes

n.t.

Peters et al., 2000

IL6 pathway

IL6R

soluble IL-6R covalently linked to IL-6 Recombinant IL6

n/a

n.t.

Yes

Jin et al., 2007

Nuclear hormone receptor Nuclear hormone receptor Nuclear hormone receptor Nuclear hormone receptor Nuclear hormone receptor Complement pathway Thyroid hormone pathway

CAR

CAR agonist (mouse)

TCPOBOP

Yes

Yes

CAR

CAR agonist (human)

CITCO

Yes

Yes

Tschuor et al., 2016; Orsini et al., 2016 Tschuor et al., 2016

FXR

FXR agonist

Bile acid

Yes

n.t.

Huang et al., 2006

PXR

PXR agonist (mouse)

Yes

n.t.

Jiang et al., 2019

PXR

PXR agonist (human)

pregnenolone‐1 6α‐carbonitrile Rifampicin

Yes

n.t.

Jiang et al., 2019

MAC

C2-C59 biologic

C2-C59

n.t.

Yes

Marshall et al., 2014

Thyroid hormone receptor T3 hormone receptor β1 (TRβ1)

Thyroid hormone (T3) biologic

T3

n.t.

(Yes)**

Malik et al., 2005*: Fanti et al., 2014

TRβ1 agonist

GC-1

Yes

n.t.

Alvarado et al., 2016

Thyroid hormone pathway

TSP1

analysis of endpoints in clinical trials for nonalcoholic steatohepatitis through the lens of regulatory science, Hepatology 67(5) (2018) 2001-2012.

CRediT author statement

Linda E. Greenbaum, Chinweike Ukomadu and Jan S. Tchorz equivally contributed to this Commentary: Conceptualization, Writing – Original draft, review and editing, Visualization.

Table 1