Preclinical investigation of Pegylated arginase 1 as a treatment for retina and brain injury.

Arginase 1 (A1) is the enzyme that hydrolyzes the amino acid, L-arginine, to ornithine and urea. We have previously shown that A1 deletion worsens retinal ischemic injury, suggesting a protective role of A1. In this translational study, we aimed to study the utility of systemic pegylated A1 (PEG-A1, recombinant human arginase linked to polyethylene glycol) treatment in mouse models of acute retinal and brain injury. Cohorts of WT mice were subjected to retinal ischemia-reperfusion (IR) injury, traumatic optic neuropathy (TON) or brain cerebral ischemia via middle cerebral artery occlusion (MCAO) and treated with intraperitoneal injections of PEG-A1 or vehicle (PEG only). Drug penetration into retina and brain tissues was measured by western blotting and immunolabeling for PEG. Neuroprotection was measured in a blinded fashion by quantitation of NeuN (neuronal marker) immunolabeling of retina flat-mounts and brain infarct area using triphenyl tetrazolium chloride (TTC) staining. Furthermore, ex vivo retina explants and in vitro retina neuron cultures were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R) and treated with PEG-A1. PEG-A1 given systemically did not cross the intact blood-retina/brain barriers in sham controls but reached the retina and brain after injury. PEG-A1 provided neuroprotection after retinal IR injury, TON and cerebral ischemia. PEG-A1 treatment was also neuroprotective in retina explants subjected to OGD/R but did not improve survival in retinal neuronal cultures exposed to OGD/R. In summary, systemic PEG-A1 administration is neuroprotective and provides an excellent route to deliver the drug to the retina and the brain after acute injury.

Safety, PK/PD and preliminary anti-tumor activities of pegylated recombinant human arginase 1 (BCT-100) in patients with advanced arginine auxotrophic tumors.

Background The study determined the safety, pharmacokinetics/pharmacodynamics (PK/PD), and recommended Phase II dose of BCT-100 for arginine auxotrophic tumours in a non-Chinese population. Methods This is a Phase I, 3 + 3 dose-escalation, open-label, multi-centre study in two arginine auxotrophic cancers-Malignant Melanoma (MM) and Castration Resistant Prostate Cancer (CRPC). Patients were enrolled to receive weekly intravenous BCT-100. The dose cohorts were respectively 0.5 mg/kg, 1.0 mg/kg, 1.7 mg/kg and 2.7 mg/kg. Results There were 14 MM and 9 CRPC patients, 16 males and 7 females with a median age of 71. No dose-limiting toxicities were reported. Among all the AEs, 18 were drug-related (mostly were Grade 1). Although there were individual variations in PKs amongst the patients in each cohort, the median arginine level was maintained at 2.5 µM (lower limit of quantification) in all 4 cohorts of patients after the second BCT-100 injection. Therapeutic Arginine Depletion was found in the 1.7 and 2.7 mg/kg/week cohorts when anti-tumor activities were observed. The two cohorts had a similar AUC (20,947 and 19,614 h*µg/ml respectively). Since the 2.7 mg/kg/week cohort had a more sustained arginine depletion for 2 weeks, the 2.7 mg/kg/week dose is chosen as the future phase II dose. There were two complete remissions (1 MM & 1 CRPC), 1PR (MM) and 2 stable diseases with a disease control rate (CR + PR + SD) of 5/23 (22%). Conclusions BCT-100 is safe in a non-Chinese population and has anti-tumor activities in both MM and CRPC. Weekly BCT-100 at 2.7 mg/kg is defined as the optimal biological dose for future clinical phase II studies.

Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn2+ and Mg2+ ions enhanced arginase activity while, Li+, Cu2+, and Al3+ ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml-1) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Preliminary efficacy, safety, pharmacokinetics, pharmacodynamics and quality of life study of pegylated recombinant human arginase 1 in patients with advanced hepatocellular carcinoma.

This study was designed to evaluate the efficacy, safety profile, pharmacokinetics, pharmacodynamics and quality of life of pegylated recombinant human arginase 1 (Peg-rhAgr1) in patients with advanced hepatocellular carcinoma (HCC). Patients were given weekly doses of Peg-rhAgr1 (1600 U/kg). Tumour response was assessed every 8 weeks using RECIST 1.1 and modified RECIST criteria. A total of 20 patients were recruited, of whom 15 were deemed evaluable for treatment efficacy. Eighteen patients (90%) were hepatitis B carriers. Median age was 61.5 (range 30-75). Overall disease control rate was 13%, with 2 of the 15 patients achieving stable disease for >8 weeks. The median progression-free survival (PFS) was 1.7 (95% CI: 1.67-1.73) months, with median overall survival (OS) of all 20 enrolled patients being 5.2 (95% CI: 3.3-12.0) months. PFS was significantly prolonged in patients with adequate arginine depletion (ADD) >2 months versus those who had ≤2 months of ADD (6.4 versus 1.7 months; p = 0.01). The majority of adverse events (AEs) were grade 1/2 non-hematological toxicities. Transient liver dysfunctions (25%) were the most commonly reported serious AEs and likely due to disease progression. Pharmacokinetic and pharmacodynamic data showed that Peg-rhAgr1 induced rapid and sustained arginine depletion. The overall quality of life of the enrolled patients was well preserved. Peg-rhAgr1 is well tolerated with a good toxicity profile in patients with advanced HCC. A weekly dose of 1600 U/kg is sufficient to induce ADD. Significantly longer PFS times were recorded for patients who had ADD for >2 months.

A phase 1 dose-escalating study of pegylated recombinant human arginase 1 (Peg-rhArg1) in patients with advanced hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) cells are auxotrophic for arginine, depletion of which leads to tumour regression. The current study evaluated safety, pharmacokinetics (PK)/ pharmacodynamics (PD) parameters, and potential anti-tumor activity of pegylated recombinant human arginase 1 (peg-rhArg1) in advanced HCC patients. METHODS: Eligibility criteria included advanced HCC with measurable lesions, Child-Pugh A or B, and adequate organ function. Initial single IV bolus was followed by weekly doses of peg-rhArgI escalated from 500 U/kg to 2500 U/kg in a 3 + 3 design. RESULTS: Fifteen patients were enrolled at weekly doses of 500 U/kg (n = 3), 1000 U/kg (n = 3), 1600 U/kg (n = 3) and 2500 U/kg (n = 6). The median age was 57 years (33-74); 87% were hepatitis B carriers and 47% had prior systemic treatment. The most commonly reported drug-related non-haematological adverse events (AEs) were diarrhea (13.3%), abdominal discomfort (6.7%) and nausea (6.7%). No drug-related haematological AEs were seen. Only 1 of the six patients that received 2500U/kg peg-rhArg1 experienced DLT (grade 4 bilirubin elevation) and thus the maximum tolerated dose was 2500 U/kg. PK and PD analysis indicated that peg-rhArg1 was efficacious in inducing arginine depletion in a dose-dependent manner. Adequate arginine depletion dose was achieved in the 1,600-2,500 U/kg range and therefore the optimal biological dose was at 1600 U/kg, which was chosen as the recommended dose. The best response was stable disease for >8 weeks in 26.7% of the enrolled patients. CONCLUSION: Peg-rhArg1 has manageable safety profile and preliminary evidence of activity in advanced HCC patients.

Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy.

Systemic L-arginine depletion following intravenous administration of l-arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t(1/2)=4.8h) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn(2+) metal center in human Arginase I with Co(2+) (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12-15 fold reduction in the IC(50) towards a variety of malignant cell lines and, importantly a t(1/2)=22h in serum. To investigate the utility of Co-hArgI for L-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20kDa PEG-maleimide (Co-hArgI-C(PEG-20K)); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-K(PEG-5K)). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only Co-hArgI amine conjugated to 5kDa PEG remained in circulation for sufficiently long durations. Using Co-hArgI-K(PEG-5K) labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6mg/kg weight, a well tolerated dose, the circulation t(1/2) of the protein in Balb/c mice is 63±10h. Very low levels of serum L-Arg (<5µM) could be sustained for over 75h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn(2+)-containing hArgI conjugated to 5kDa PEG-NHS ester (Mn-hArgI-K(PEG-5K)). The favorable pharmacokinetic and pharmacodynamic properties of Co-hArgI-K(PEG-5K) reported here, coupled with its human origin which should reduce the likelihood of adverse immune responses, make it a promising candidate for cancer therapy.

Enzyme modification by MPEG with an amino acid or peptide as spacer arms.

A method for the modification of enzymes by MPEG carrying an amino acid or peptide as a spacer arm is described and tested with aliphatic or aromatic side chains amino acids. The procedure involves MPEG activation by p-nitrophenylchloroformate for the amino acid or peptide coupling that is in turn activated for the protein binding. The advantage of the method resides in the possibility to introduce proper reporter groups between the polymer and the protein as norleucine for a direct evaluation of the bound polymer chains, tryptophan for structural studies of the polymer-protein adduct, and radioactive amino acid for pharmacokinetic investigations. The method was positively tested with arginase, ribonuclease, and superoxide dismutase as enzymes of therapeutic value.