Preclinical investigation of PEGylated tumor necrosis factor alpha in dogs with spontaneous tumors: phase I evaluation.

PURPOSE: Tumor necrosis factor-alpha (TNF) is a cytokine with potent antitumor activity; however, toxicity and short half-life have limited its utility. Polyethylene glycol (PEG) conjugation of biotherapeutics can decrease immunogenicity while improving bioactivity and half-life. PEGylation of TNF (PEG-TNF) significantly improved half-life and toxicity in mice, resulting in enhanced antitumor activity. This study characterized toxicity, biological effect, and antitumor activity of PEG-TNF in pet dogs with spontaneous cancer. EXPERIMENTAL DESIGN: A phase I clinical trial enrolled dogs with measurable tumors in which standard therapy had failed or been declined. Physiologic, hematologic, and biochemical parameters were evaluated and tumor biopsies obtained serially. A subset of patients underwent serial dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Fifteen dogs were enrolled at doses from 20.0 to 30.0 microg/kg. Dose-limiting toxicity at 30.0 microg/kg consisted of vascular leak in one and hypotension/coagulopathy in one, establishing 26.7 microg/kg as the maximum tolerated dose. Mean elimination half-life was 15.3 +/- 4.9 hours. Biological activity (transient fever and leukopenia, increased tumor inflammation, and necrosis) was observed at all dosages. A significant increase in tumor blood flow was observed with dynamic contrast-enhanced magnetic resonance imaging. Minor/transient antitumor responses were observed in dogs with melanoma, squamous cell carcinoma, and mammary carcinoma, and a partial response was observed in a dog with angiosarcoma. CONCLUSIONS: Using a clinically relevant, spontaneous large animal model of neoplasia, we have shown that biologically effective doses of PEG-TNF can be administered safely, and that PEG-TNF administration is associated with encouraging biological activity. These results justify the clinical evaluation of PEG-TNF in human cancer.

Pegylated arginine deiminase lowers hepatitis C viral titers and inhibits nitric oxide synthesis.

BACKGROUND: The arginine-degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI-SS PEG 20,000 mw reduces HCV viral titers through nitric oxide (NO)-dependent effects. METHODS: The effects of ADI-SS PEG 20,000 mw (dose, 160 IU/m2; three cycles of four once-weekly i.m. injections) on HCV titers, serum NO and plasma arginine, were evaluated using archived plasma from patients with HCC and HCV and in vitro cell model measurements of HCV replication. RESULTS: ADI-SS PEG 20,000 mw selectively inhibited HCV replication in vitro (IC50 = 0.027 IU/mL). Fifteen HCC/HCV patients completed treatment. The HCV titers were reduced by up to 99% in five out of 10 (50%) HCV-serotype 1b patients (P = 0.0093). These patients also experienced significant improvements in liver function (P = 0.0091). There were concomitant reductions of plasma arginine and serum NO levels. The HCV titer was not reduced in HCV-type 2c patients. CONCLUSION: Reduction of extracellular arginine by ADI-SS PEG 20,000 mw in HCC patients reduces HCV viral titers and improves liver function, possibly through suppression of NO.

Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies.

PURPOSE: Individuals with metastatic melanoma have a poor prognosis. Many human melanomas are auxotrophic for arginine, and arginine is not an essential amino acid in humans. We hypothesized that this auxotrophy may be therapeutically exploited. A novel amino acid-degrading enzyme (arginine deiminase) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) was used to lower plasma arginine in individuals with metastatic melanoma. PATIENTS AND METHODS: Two cohort dose-escalation studies were performed. A phase I study in the United States enrolled 15 patients, and a phase I to II study in Italy enrolled 24 patients. The Italian patients also received two subsequent cycles of treatment, each consisting of four once-weekly injections of 160 U/m2. The goals of these studies were to determine pharmacokinetics (PK), pharmacodynamics (PD), safety, and the antitumor activity of ADI-SS PEG 20,000 mw. RESULTS: PK and PD studies indicated that a dose of 160 U/m2 lowered plasma arginine from a resting level of approximately 130 micromol/L to less than 2 micromol/L for at least 7 days; nitric oxide levels also were lowered. There were no grade 3 or 4 toxicities directly attributable to the drug. Six of 24 phase I to II patients responded to treatment (five partial responses and one complete response; 25% response rate) and also had prolonged survival. CONCLUSION Elimination of all detectable plasma arginine in patients with metastatic melanoma was well tolerated and may be effective in the treatment of this cancer. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with metastatic melanoma is warranted.

Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies.

PURPOSE: Recently, we reported that a large number of human hepatocellular cancer (HCC) cell lines were auxotrophic for arginine. Here we report the results obtained with the amino acid-degrading enzyme arginine deiminase (ADI) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) as a means of lowering plasma arginine to treat HCC. The study was a cohort dose-escalation phase I/II study. PATIENTS AND METHODS: Pharmacodynamic studies indicated an ADI-SS PEG 20,000 mw dose level of 160 U/m(2) was sufficient to lower plasma arginine from a resting level of approximately 130 micromol/L to below the level of detection (< 2 micromol/L) for more than 7 days, a dose later defined as the optimal biologic dose. All patients were to receive three cycles at the optimum biologic dose. RESULTS: This therapy was well tolerated, even in patients who had no detectable plasma arginine for 3 continuous months of therapy. Of the 19 patients enrolled, two had a complete response, seven had a partial response, seven had stable disease, and three had progressive disease. The median survival for the 19 patients enrolled on this study was 410 days, with four patients still alive at present (> 680 days). CONCLUSION: Elimination of all detectable plasma arginine in patients with HCC was well tolerated and seemed to be effective in the treatment of some patients with HCC. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with HCC as well as other human tumors auxotrophic for arginine is warranted.

Serum uric acid-lowering therapies: where are we heading in management of hyperuricemia and the potential role of uricase.

Although allopurinol has been available for approximately 50 years, hyperuricemia and its sequelae are not only prevalent, but the incidence and costs associated with this disorder continue to increase. However, several new therapies have been developed. Recombinant urate oxidase has been useful in the treatment of tumor lysis hyperuricemia, and pegylated urate oxidase shows promise in patients with hyperuricemia and gout. Febuxostat and Y-700 are new oral xanthine oxidase inhibitors that are in human clinical trials. Tailoring of antilipid therapy in selected hyperuricemic and hyperlipidemic patients with fenofibrate may be of benefit in lowering blood cholesterol and uric acid levels. Similarly, treatment of selected hyperuricemic patients who also are hypertensive with losartan or amlodipine may be beneficial in lowering blood pressure and hyperuricemia. Despite these advances, new treatments for hyperuricemia are needed.

Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation.

BACKGROUND: Argininosuccinate synthetase (ASS) was the first of two enzymes to convert citrulline to arginine. This pathway allowed cells to synthesize arginine from citrulline, making this amino acid nonessential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS. Selective elimination of arginine from the circulation of animals with these tumors is a potentially effective anticancer treatment. The purpose of these experiments was to determine the frequency of ASS deficiency and arginine auxotrophy in a variety of human malignant tumors. METHODS: The authors analyzed the expression of ASS by immunohistochemistry with a monoclonal antibody in a variety of human tumor biopsies. They found that the incidence of ASS deficiency varied greatly with the tumor type and tissue of origin. RESULTS: Melanoma, hepatocellular carcinoma, and prostate carcinoma were most frequently deficient in ASS. Some human cancers were almost always positive for ASS (e.g., lung and colon carcinomas). However, other human cancers, including sarcomas, invasive breast carcinoma, and renal cell carcinoma, also were sometimes ASS deficient. CONCLUSIONS: These data indicated that immunohistochemical detection of ASS may prove an effective means for determining ASS deficiency in malignant human tumors and for identifying patients most likely to respond to arginine deprivation therapy. Based on these results, human clinical trials using arginine-degrading enzyme therapy to treat patients with advanced melanoma or hepatocellular carcinoma have been initiated.

Regression of hepatocellular cancer in a patient treated with arginine deiminase.

We report the first pharmacokinetic and clinical response data from a patient with unresectable hepatocellular cancer treated with a new drug, ADI PEG20,000 mw (arginine deiminase-polyethylene glycol 20,000 molecular weight). A single patient with idiopathic cirrhosis and unresectable hepatocellular cancer was treated with escalating dosages of ADI-PEG20,000 mw. Human hepatocellular cancer has been found to be arginine-dependent for growth because of loss of expression or arginosuccinate synthetase, the rate-limiting enzyme in the conversion of citrulline to arginine. Thus, an arginine-degrading enzyme like ADI-PEG20,000 mw should produce cell death in hepatocellular cancer cells without significantly affecting normal cells. There was a dose-dependent reduction of plasma arginine levels after weekly intramuscular administration of ADI-PEG20,000 mw. Successive treatment cycles at the optimal biologic dose of 160 IU/m2 led to reduction in tumor size and serum alpha-fetoprotein levels. Sufficient tumor cytoreduction was achieved with ADI-PEG20,000 mw treatment to permit surgical treatment. The patient developed no toxicities or side effects related to ADI-PEG20,000 mw treatment. The results in a single patient with unresectable hepatocellular cancer treated with ADI-PEG20,000 mw suggests this may be a promising, low-toxicity treatment. Full-scale clinical trials have been initiated.

Pegylated arginine deiminase (ADI-SS PEG20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo.

Some murine melanomas and hepatocellular carcinomas (HCCs) have been shown to be auxotrophic for arginine. Arginine deiminase (ADI; EC 3.5.3.6.), an arginine-degrading enzyme isolated from Mycoplasma, can inhibit growth of these tumors. We found that ADI was specific for arginine and did not degrade other amino acids. Although arginine is not an essential amino acid for most cells, all human melanomas and HCCs tested were found to be inhibited by ADI in vitro. Arginine is synthesized from citrulline in two steps by argininosuccinate synthetase and argininosuccinate lyase. Melanomas and HCCs did not express argininosuccinate synthetase mRNA but did express argininosuccinate lyase mRNA, suggesting that the arginine auxotrophy of these cells was a result of an inability to produce argininosuccinate synthetase. Human melanomas and HCCs were transfected with an expression plasmid containing argininosuccinate synthetase cDNA. The transfected cells were much more resistant to ADI than the parental cells in vitro and in vivo. Initial attempts to use ADI in vivo indicated that this enzyme had little efficacy, consistent with its short circulation half-life. Formulation of ADI with polyethylene glycol to produce ADI-SS PEG(20,000 mw) resulted in an enzyme with a much longer circulation half-life that, and although equally effective in vitro, was more efficacious in the treatment of mice implanted with human melanomas and HCCs. These data indicate that sensitivity of melanoma and HCC is due to the absence of argininosuccinate synthetase in these cells and that an effective formulation of ADI, which causes a sustained decrease in arginine, may be a useful treatment for arginine auxotrophic tumors including melanoma and HCC.

Uricase formulated with polyethylene glycol (uricase-PEG 20): biochemical rationale and preclinical studies.

OBJECTIVE: Humans have a non-sense codon inserted into the 5 prime end of the open reading frame of urate oxidase, and thus express an enzymatically inactive fragment of this enzyme; and consequently are unable to metabolize uric acid into allantoin and are prone to develop hyperuricemia and gout. Various urate oxidases (uricase) from mammals and microorganisms have been administered to humans with hyperuricemia and gout. Although successful in lowering plasma uric acid, these therapies have had limited application due to undesirable biochemical properties of the enzymes used, the short circulating half-life, and inherent antigenicity of these preparations. METHODS: We compared urate oxidase from a variety of sources for specific enzyme activity, pH optimum, affinity, and retention of enzyme activity under physiological conditions. A variety of polyethylene glycols (PEG) were tested to formulate uricase. RESULTS: Urate oxidase from Candida utilis had more favorable enzymatic properties and PEG of 20,000 MW (termed uricase-PEG 20) had greatly reduced antigenicity and increased circulating half-life as compared to those previously described. CONCLUSION: It is anticipated that uricase-PEG 20 may have utility as a treatment for hyperuricemia and gout.

Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production.

BACKGROUND: Nitric oxide (NO) is a biomediator believed to be synthesized primarily from extracellular arginine. Various methodologies have been used to inhibit NO synthesis so as to elucidate its physiological and pathophysiological functions. Several investigators have utilized various argin ine degrading enzymes as a means of lowering extracellular arginine. Arginase, most commonly derived from mammalian sources, has been most often used. However, arginase has failed to inhibit NO synthesis. Therefore, a systematic biochemical characterization of arginase and arginine deiminase (ADI) derived from M. Hominus was undertaken. MATERIAL/METHODS: The murine macrophage cell line N-9 was treated with either arginase or arginine deiminase to determine the effect on intracellular and extracellular arginine and nitric oxide production. RESULTS: Arginase was found to have an alkaline pH optima(approximately 9.5) with little enzyme activity at physiological pH. In contrast, the pH optima of ADI was approximately 6.5, retaining >70% of its activity at physiological pH. ADI had more than 1000 fold higher affinity for arginine (Km approximately 30 KM for ADI vs approximately 45 mM for arginase), and was able to lower arginine levels to a much greater extent than arginase. ADI, unlike arginase, was effective in lowering extracellular arginine in tissue culture media and inhibit NO production by the murine macrophage cell line N-9 in response to gamma interferon and LPS stimulation. CONCLUSIONS: These data suggest that ADI may be useful for delineating the role of NO in a variety of biological systems as well as determining the role of extracellular arginine in its synthesis.

Enzymic degradation of plasma arginine using arginine deiminase inhibits nitric oxide production and protects mice from the lethal effects of tumour necrosis factor alpha and endotoxin.

Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor alpha (TNFalpha). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFalpha or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of M(r) 20000 (ADI-SS PEG(20000)) eliminated all measurable plasma arginine (from normal levels of approximately 155 microM arginine to 2 microM). In addition, ADI-SS PEG(20000) also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFalpha or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG(20000) resulted in increased resistance to the lethal effects of TNFalpha and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFalpha and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG(20000). Interestingly, pretreatment with ADI-SS PEG(20000) did not inhibit the anti-tumour activity of TNFalpha in vitro or in vivo. This treatment may allow greater amounts of TNFalpha, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death.

Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties.

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI-PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI-PEGs retained approximately 50% of enzyme activity when PEG was covalently attached to approximately 40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI-PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.