A phase I/II study of ARO-HSD, an RNA interference therapeutic, for the treatment of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Loss-of-function HSD17ß13 mutations protect against the development of chronic liver disease. HSD17ß13 inhibition represents a potential approach to treat liver diseases, such as non-alcoholic steatohepatitis (NASH). ARO-HSD is an RNA interference (RNAi) therapeutic designed to selectively reduce expression of HSD17ß13 mRNA in hepatocytes. In this study, we evaluated the effects of ARO-HSD in normal healthy volunteers (NHVs) and patients with confirmed or clinically suspected NASH. METHODS: The safety, tolerability, and pharmacodynamics of ARO-HSD were evaluated in 32 NHVs and 18 patients with confirmed/clinically suspected NASH. Double-blind NHV cohorts received single escalating doses of ARO-HSD (25, 50, 100, or 200 mg) or placebo subcutaneously on Day 1. Open-label patient cohorts received ARO-HSD (25, 100, or 200 mg) subcutaneously on Days 1 and 29. Liver biopsy was performed pre-dose and on Day 71 to evaluate expression levels of HSD17ß13 mRNA and protein. RESULTS: ARO-HSD treatment was well tolerated with no treatment-related serious adverse events or drug discontinuations. The most frequently reported treatment-emergent adverse events were mild injection site reactions, which were short in duration. Mean changes in hepatic HSD17ß13 mRNA from baseline to Day 71 were: -56.9% (25 mg), -85.5% (100 mg), and -93.4% (200 mg). The mean HSD17ß13 mRNA reduction was 78.6% (p <0.0001) across pooled cohorts. Hepatic HSD17ß13 protein levels were similarly reduced across doses. In patients, mean changes in alanine aminotransferase from baseline to Day 71 were -7.7% (25 mg), -39.3% (100 mg), and -42.3% (200 mg) (p <0.001 for pooled cohorts). CONCLUSIONS: ARO-HSD was well tolerated at doses ≤200 mg. This proof-of-concept study demonstrated that short-term treatment with ARO-HSD reduces hepatic HSD17ß13 mRNA and protein expression, which is accompanied by reductions in alanine aminotransferase. GOV NUMBER: NCT04202354. IMPACTS AND IMPLICATIONS: There is an unmet medical need for new therapies to treat alcohol-related and non-alcoholic liver disease. ARO-HSD is a small-interfering RNA designed to silence HSD17ß13 expression and hence to phenocopy the protective effect seen in individuals with HSD17ß13 loss-of-function. The reductions in HSD17ß13 expression and in transaminases seen with ARO-HSD administration represent an initial step towards clinical validation of HSD17ß13, a drug target with substantial genetic validation, as an important modulator of human liver disease.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

Hepatic-targeted RNA interference provides robust and persistent knockdown of alpha-1 antitrypsin levels in ZZ patients.

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder causing pulmonary and liver disease. The PiZ mutation in AAT (SERPINA1) results in mis-folded AAT protein (Z-AAT) accumulating in hepatocytes, leading to fibrosis and cirrhosis. RNAi-based therapeutics silencing production of hepatic Z-AAT might benefit patients with AATD-associated liver disease. This study evaluated an RNAi therapeutic to silence production of AAT. METHODS: Part A of this double-blind first-in-human study randomized 54 healthy volunteers (HVs) into single dose cohorts (two placebo: four active), receiving escalating doses of the investigational agent ARC-AAT from 0.38 to 8.0 mg/kg or placebo. Part B randomized 11 patients with PiZZ (homozygous for Z-AAT) genotype AATD, who received up to 4.0 mg/kg of ARC-AAT or placebo. Patients with baseline FibroScan® >11 kPa or forced expiratory volume in one second (FEV1) <60% were excluded. Assessments included safety, pharmacokinetics, and change in serum AAT concentrations. RESULTS: A total of 36 HVs received ARC-AAT and 18 received placebo (part A). Seven PiZZ individuals received ARC-AAT and four received placebo (part B). A dose response in serum AAT reduction was observed at doses ≥4 mg/kg with similar relative reductions in PiZZ patients and HVs at 4 mg/kg and a maximum reduction of 76.1% (HVs) vs. 78.8% (PiZZ) at this dose. The time it took for serum AAT to return to baseline was similar for HV and PiZZ. There were no notable differences between HV and PiZZ safety parameters. The study was terminated early because of toxicity findings related to the delivery vehicle (ARC-EX1) seen in a non-human primate study. CONCLUSION: PiZZ patients and HVs responded similarly to ARC-AAT. Deep and durable knockdown of hepatic AAT production based on observed reduction in serum AAT concentrations was demonstrated. LAY SUMMARY: Accumulation of abnormal proteins in the livers of patients with alpha-1 antitrypsin deficiency may lead to decreased liver function and potentially liver failure. Therapeutics targeting the production of these abnormal proteins may be used to prevent or treat liver disease in patients with alpha-1 antitrypsin deficiency. CLINICAL TRIAL REGISTRATION NUMBER: NCT02363946.

Targeting the interleukin-11 receptor α in metastatic prostate cancer: A first-in-man study.

BACKGROUND: Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors ("vascular zip codes") within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS: The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα-based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS: BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m(2) ). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS: These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients.

Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

A peptidomimetic targeting white fat causes weight loss and improved insulin resistance in obese monkeys.

Obesity, defined as body mass index greater than 30, is a leading cause of morbidity and mortality and a financial burden worldwide. Despite significant efforts in the past decade, very few drugs have been successfully developed for the treatment of obese patients. Biological differences between rodents and primates are a major hurdle for translation of anti-obesity strategies either discovered or developed in rodents into effective human therapeutics. Here, we evaluate the ligand-directed peptidomimetic CKGGRAKDC-GG-(D)(KLAKLAK)(2) (henceforth termed adipotide) in obese Old World monkeys. Treatment with adipotide induced targeted apoptosis within blood vessels of white adipose tissue and resulted in rapid weight loss and improved insulin resistance in obese monkeys. Magnetic resonance imaging and dual-energy x-ray absorptiometry confirmed a marked reduction in white adipose tissue. At experimentally determined optimal doses, monkeys from three different species displayed predictable and reversible changes in renal proximal tubule function. Together, these data in primates establish adipotide as a prototype in a new class of candidate drugs that may be useful for treating obesity in humans.

Combinatorial targeting and nanotechnology applications.

The development of improved methods for targeted cell detection is of general interest in many fields of research and drug development. There are a number of well-established techniques for the study and detection of biomarkers expressed in living cells and tissues. Many of them rely on multi-step procedures that might not meet ideal assay requirements for speed, cost, sensitivity, and specificity. Here we report and further validate an approach that enables spontaneous molecular assembly to generate biologically active networks of bacteriophage (phage) assembled with gold (Au) nanoparticles (termed Au-phage nanoshuttles). Here, the nanoshuttles preserve the cell binding and internalization attributes mediated by a displayed peptide targeted to a cell surface receptor. The organization of such targeted assemblies can be further manipulated to be used as a multimodal detection assembly, and they can be characterized as fractal nanostructures by angle-dependent light scattering fractal dimension analysis. Targeted Au-phage nanoshuttles offer multiple functionalities for nanotechnology-based sensing and reporting, including enhanced fluorescence and improved contrast for darkfield microscopy.

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Techniques to decipher molecular diversity by phage display.

Combinatorial phage display technology may be applied to decipher the molecular diversity of peptide binding specificity to isolated proteins, purified antibodies, cell surfaces, intracellular/cyto-domains, and blood vessels in vivo. The application of such a strategy ranges from identifying receptor-ligand pairs and antigen binding sites to understanding the progression of diseases by their differential expression patterns and developing therapeutic targeting strategies. Different strategies can be used to isolate peptides from diverse libraries displayed on the surface of bacteriophage by exposing the library to a target molecule or organ, washing away nonbinding phage, eluting and amplifying the bound phage for multiple round use, and then analyzing the peptide sequences of the enriched phage. The following methods first outline the construction of a phage library and then delineate various in vitro and in vivo biopanning applications to probe isolated integrins, purified antibodies, cell surface molecules, and vascular endothelial cells.

Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents.

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.