Assessment of the Pharmacokinetics, Disposition, and Duration of Action of the Tumour-Targeting Peptide CEND-1.

CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.

Dual αV-integrin and neuropilin-1 targeting peptide CEND-1 plus nab-paclitaxel and gemcitabine for the treatment of metastatic pancreatic ductal adenocarcinoma: a first-in-human, open-label, multicentre, phase 1 study.

BACKGROUND: CEND-1 is a novel cyclic peptide that targets αV integrins and neuropilin-1 and enhances tumour delivery of co-administered anticancer drugs. We investigated the safety, tolerability, and biological activity of CEND-1 in patients with metastatic pancreatic ductal adenocarcinoma in combination with nab-paclitaxel and gemcitabine. METHODS: This open-label, multicentre, phase 1 study, conducted at three hospitals in Australia, enrolled participants aged 18 years or older with histologically confirmed metastatic pancreatic ductal adenocarcinoma who had one or more lesions measurable on MRI or CT, an Eastern Cooperative Oncology Group performance status score of 0 or 1, and a life expectancy of at least 3 months. Exclusion criteria included previous chemotherapy and brain metastases or other malignancy (unless receiving curative intent). There was no randomisation or masking. CEND-1 monotherapy was given as an intravenous fluid bolus on day 1 of a run-in phase of 7 days (0·2-3·2 mg/kg) followed by CEND-1 plus intravenous gemcitabine (1000 mg/m2) and nab-paclitaxel (125 mg/m2) on days 1, 8, and 15 of 28-day treatment cycles until disease progression. The primary safety endpoints were incidence, severity, and duration of treatment-emergent and treatment-related adverse events; overall survival; and clinical laboratory results, which were all assessed in the safety population. This study is registered with ClinicalTrials.gov, NCT03517176, and the Australian New Zealand Clinical Trials Registry, ACTRN12618000804280. FINDINGS: Between Aug 13, 2018, and Nov 30, 2019, 31 patients were enrolled (eight in the dose-escalation phase [cohort 1a] and 23 in the expansion phase [cohort 1b]). Two patients were excluded from the efficacy population. No CEND-1 dose-limiting toxicities were observed in the safety population (n=31). The most common grade 3 or 4 events were neutropenia (17 [55%] patients), anaemia (eight [26%]), leukopenia (five [16%]), and pulmonary embolism (four [13%]). Serious adverse events occurred in 22 (71%) patients, mostly related to disease progression. Ten deaths occurred during the study due to progression of metastatic pancreatic cancer (n=9) and a left middle cerebral artery stroke (n=1). In the efficacy population (n=29), 17 (59%) patients had an objective response, including one complete response and 16 partial responses. After a median follow-up of 26 months (IQR 24-30), median overall survival was 13·2 months (95% CI 9·7-22·5). INTERPRETATION: CEND-1 with nab-paclitaxel and gemcitabine has an acceptable safety profile, with no dose-limiting toxicities and encouraging activity. Adverse events were generally consistent with those seen with nab-paclitaxel and gemcitabine. Further randomised trials to determine the efficacy of CEND-1 are warranted. FUNDING: DrugCendR Australia Pty.

Chronic systemic endotoxin exposure: an animal model in experimental hepatic encephalopathy.

Plasma levels of gut-derived endotoxins (lipopolysaccharides, LPS) are often elevated in cirrhotics and are thought to contribute to hepatic encephalopathy. Circulating LPS activates macrophages to produce tumor necrosis factor alpha (TNF-alpha) and other potentially cytotoxic proinflammatory mediators. A pathogenic role for endotoxins is supported by studies showing that treatment with Lacto-bacillusor antibiotics, both of which reduce LPS-producing intestinal Gram-negative bacteria, alleviates experimental liver damage. To mimic the "leaky gut" syndrome with endotoxin translocation into the circulation in cirrhotics, a new animal model was developed. Rats were chronically exposed to ethanol and for the four last weeks also infused with endotoxin into the jugular vein from subcutaneously implanted osmotic minipumps. Animals receiving endotoxin had elevated hepatic expression of both pro- and anti-inflammatory cytokines, but compared to ethanol treatment alone hepatic steatosis and inflammatory changes were only marginally increased. This demonstrates marked endotoxin tolerance, probably as a consequence of a counteracting anti-inflammatory cytokine response. The role of gut-derived endotoxin in hepatic encephalopathy has recently received considerable attention. To further delineate the role and actions of endotoxin and its extrahepatic effects, studies applying both acute challenge and chronic infusion seem warranted. The chronic endotoxin model, mimicking the "leaky gut," may best be combined with more robust ways to impair liver function, such as carbon tetrachloride treatment, bile duct ligation, or galactosamine administration.

Protective function of complement against alcohol-induced rat liver damage.

The complement system can promote tissue damage or play a homeostatic role in the clearance and disposal of damaged tissue. We assessed the role of the terminal complement pathway in alcohol-induced liver damage in complement C6 (C6-/-) genetically deficient rats. C6-/- and corresponding C6+/+ rats were continuously exposed to ethanol by feeding ethanol-supplemented liquid diet for six weeks. Liver samples were analyzed for histopathology and complement component deposition by immunofluorescence microscopy. Prostaglandin E receptors and cytokine mRNA levels were analyzed by RT-PCR and plasma cytokines by ELISA. Deposition of complement components C1, C3, C8 and C9 was observed in C6+/+ rats, but not in C6-/- animals. The histopathological changes, the liver weight increase and the elevation of the plasma pro-/anti-inflammatory TNF-alpha/IL-10 ratio were, on the other hand, more marked in C6-/- rats. Furthermore, ethanol enhanced the hepatic mRNA expression of the prostaglandin E receptors EP2R and EP4R exclusively in the C6-/- rats. Our results indicate that a deficient terminal complement pathway predisposes to tissue injury and promotes a pro-inflammatory cytokine response. This suggests that an intact complement system has a protective function in the development of alcoholic liver damage.

Hepatic expression of multiple acute phase proteins and down-regulation of nuclear receptors after acute endotoxin exposure.

Acute systemic lipopolysaccharide (endotoxin, LPS) exposure, which can lead to septic shock, enhances the hepatic expression of inflammatory and acute-phase proteins (APPs). To better understand how LPS aggravates damage, changes in hepatic gene expression after a single LPS dose was screened by using microarrays for 1176 rat genes. We detected more than 20 new potential LPS-induced APPs. Following acute LPS challenge, significant up-regulation of the steady-state mRNA levels of several important early transcription factors, such as c-jun and STAT3, and cytokine-associated genes, was observed. In contrast, RT-PCR analysis revealed marked down-regulation of the nuclear receptors RXRalpha, PXR, FXR, LXR, PPARalpha and CAR. Also genes encoding lipolytic, antioxidant as well as drug- and alcohol-metabolizing enzymes were down-regulated. These data suggest that acute LPS treatment induces important early transcription factors and co-ordinately down-regulates nuclear receptors, and that this results in altered expression of a large number of downstream genes.

Caspase-1 activation by Salmonella.

Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system.

Activation of complement components and reduced regulator expression in alcohol-induced liver injury in the rat.

The purpose of this study was to evaluate the possible contribution of complement-mediated inflammation to the development of alcoholic liver disease. Male Wistar rats were fed ethanol by liquid diet in a model that results in continuous ethanol intoxication and induces early signs of alcoholic liver injury. After a six-week study period liver samples were analyzed for the deposition of complement components (C1, C3, and C8) and expression of cell membrane-bound regulators (Crry and CD59). Activation of the homologous complement system in vitro was tested by treating frozen liver sections with normal rat serum (NRS). Immunohistochemical analysis showed deposits of C8 in the liver sections of ethanol-treated rats. When frozen liver sections from these rats were treated with NRS, periportal deposition of both C3 and C8, but only slight C1 deposition, was observed. Immunohistochemical and Western blot analysis both revealed a reduced expression of the complement regulators Crry and CD59. These results suggest an induction of complement-activating capacity in the liver after chronic ethanol treatment. Lack of C1 deposition in the lesions suggests that complement activation occurs primarily via the alternative pathway. The reduced expression of the critical complement regulatory proteins Crry and CD59 may sensitize the liver to complement-mediated damage.

Acinar distribution of rat liver arylamine N-acetyltransferase: effect of chronic ethanol and endotoxin exposure.

The expression of most drug-metabolising enzymes is highest in the perivenous region of the liver, where drug-induced damage is commonly initiated. Arylamine N-acetyltransferase plays an important role in activation or detoxification of many drugs, carcinogens, pesticides and other xenobiotics, but its acinar distribution is unknown. In this study we have analysed the activity of N-acetyltransferase in cell lysates obtained from the periportal or perivenous region by digitonin treatment during in situ liver perfusion. Livers from control animals were compared with rats chronically exposed either to ethanol by liquid diet or to lipopolysaccharide (endotoxin) by intravenous administration. The activity of N-acetyltransferase in the perivenous region was slightly (+ 20%) higher than in the periportal region. Although chronic ethanol exposure did not change total activity, the acinar distribution was reversed to a higher activity in the periportal region. In contrast, chronic endotoxin significantly increased N-acetyltransferase activity, but did not affect the acinar distribution. This increase was counteracted by simultaneous ethanol treatment. N-Acetyltransferase activity in perivenous lysates was significantly reduced after the co-administration of ethanol and endotoxin compared to that after endotoxin alone. Thus, the perivenous zonation of liver N-acetyltransferase is moderate compared to other transferases or P450 isozymes, and the cellular capacity for N-acetylation in the perivenous region, where xenobiotic activation to reactive intermediates dominates, may be insufficient.