A Double-Blind, Placebo Controlled, Randomized Phase 1 Cross-Over Study with ALLN-177, an Orally Administered Oxalate Degrading Enzyme.

BACKGROUND: Hyperoxaluria may result from increased endogenous production or overabsorption of dietary oxalate in the gastrointestinal tract leading to nephrolithiasis and, in some, to oxalate nephropathy and chronic kidney disease. ALLN-177 is an oral formulation of a recombinant, oxalate specific, microbial enzyme oxalate decarboxylase intended to treat secondary hyperoxaluria by degrading dietary oxalate in the gastrointestinal tract, thereby reducing its absorption and subsequent excretion in the urine. METHODS: This double-blind, placebo controlled, randomized, cross-over, phase 1 study of ALLN-177 evaluated the tolerability of ALLN-177 and its effect on urinary oxalate excretion in 30 healthy volunteers with hyperoxaluria induced by ingestion of a high oxalate, low calcium (HOLC) diet. The primary end point was the difference in the mean 24-hour urinary oxalate excretion during the ALLN-177 treatment period compared with the placebo treatment period. RESULTS: The daily urinary oxalate excretion increased in the study population from 27.2 ± 9.5 mg/day during screening to 80.8 ± 24.1 mg/day (mean ± SD) on the HOLC diet before introducing ALLN-177 or placebo therapy for 7 days. Compared to placebo, ALLN-177 treatment reduced urinary oxalate by 11.6 ± 2.7 mg/day, p = 0.0002 (least squares mean ± SD). CONCLUSIONS: In healthy volunteers, with diet-induced hyperoxaluria treatment with ALLN-177, when compared to placebo, significantly reduced urinary oxalate excretion by degrading dietary oxalate in the gastrointestinal tract and thereby reducing its absorption. ALLN-177 may represent a new approach for managing secondary hyperoxaluria and its complications.

Hyperoxaluria is reduced and nephrocalcinosis prevented with an oxalate-degrading enzyme in mice with hyperoxaluria.

BACKGROUND/AIMS: Hyperoxaluria is a major risk factor for recurrent urolithiasis and nephrocalcinosis. We tested an oral therapy with a crystalline, cross-linked formulation of oxalate-decarboxylase (OxDc-CLEC) on the reduction of urinary oxalate and decrease in the severity of kidney injury in two models: AGT1 knockout mice (AGT1KO) in which hyperoxaluria is the result of an Agxt gene deficiency, and in AGT1KO mice challenged with ethylene glycol (EG). METHODS: Four different doses of OxDc-CLEC mixed with the food, or placebo were given to AGT1KO mice (200 mg/day, n = 7) for 16 days and to EG-AGT1KO mice (5, 25, and 80 mg, n = 11) for 32 days. RESULTS: Oral therapy with 200 mg OxDc-CLEC reduced both urinary (44%) and fecal oxalate (72%) in AGT1KO mice when compared to controls. Similarly, in EG-AGT1KO mice, each of the three doses of OxDc-CLEC produced a 30-50% reduction in hyperoxaluria. A sustained urinary oxalate reduction of 40% or more in the 80 mg group led to 100% animal survival and complete prevention of nephrocalcinosis and urolithiasis. CONCLUSION: These data suggest that oral therapy with OxDc-CLEC may reduce hyperoxaluria, prevent calcium oxalate nephrocalcinosis and urolithiasis, and can represent a realistic option for the treatment of human hyperoxaluria, independent of cause.

Protein crystals for the delivery of biopharmaceuticals.

The year 2002 marked the 20th anniversary of the first successful product of modern biotechnology, the regulatory approval of recombinant insulin for biopharmaceutical applications. Insulin is also the first crystalline protein to be approved for therapeutic use. Over the past two decades, almost 150 biopharmaceuticals have gained marketing authorisation; however, insulin remains the only crystalline protein on the market. Significant research and development efforts have focused on the engineering of protein molecules, efficacy testing, model development, and protein production and characterisation. These advances have dramatically boosted the therapeutic applications of proteins, which now include treatments against acute conditions, such as cancer, cardiovascular disease and viral disease, and chronic conditions, such as diabetes, growth hormone deficiency, haemophilia, arthritis, psoriasis and Crohn's disease. Despite these successes, many challenges normally associated with biopharmaceuticals, such as poor stability and limited delivery options, remain. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high concentration, low viscosity formulation and controlled release protein delivery. This review will discuss challenges related to the broader utilisation of protein crystals in biopharmaceutical applications, as well as recent advances and valuable new directions that protein crystallisation-based technologies present.

Injectable controlled release formulations incorporating protein crystals.

Development of ready-to-inject in situ formable controlled release gel systems for proteins is extremely challenging due to poor stability of proteins in the organic solvents typically used to fabricate these systems and because of the need of initial drying of proteins. The focus of the present study was to develop and characterize injectable controlled release systems composed of crystals of amylase, a model protein, suspended in solutions of polymeric and non-polymeric matrix materials in organic solvents. In this study, alpha-amylase derived from Aspergillus oryzae was crystallized and crystals were suspended in a poly(DL-lactide-co-glycolide) (PLGA) solution in acetonitrile (PLGA/acetonitrile), or in sucrose acetate isobutyrate (SAIB) plasticized with ethanol (SAIB/ethanol) systems. The results indicate that the protein crystals could be incorporated in these in situ formable gels without the need for initial drying. The crystals withstand organic solvents and water/organic solvent interfaces, and provide high protein loading (>30%) in these systems. Moreover, changing the morphology of the amylase crystals successfully modulated amylase release profiles. Study of long-term stability at 4 degrees C revealed a greater stability of crystalline protein compared to amorphous amylase. The above-mentioned data suggest that protein crystals might offer greater feasibility in developing sustained release injectable in situ formable protein depot systems.

Protein Crystals as Novel Catalytic Materials.

In this era of molecular biology, protein crystallization is often considered to be a necessary first step in obtaining structural information through X-ray diffraction analysis. In a different light, protein crystals can also be thought of as materials, whose chemical and physical properties make them broadly attractive and useful across a larger spectrum of disciplines. The full potential of these protein crystalline materials has been severely restricted in practice, however, both by their inherent fragility, and by strongly held skepticism concerning their routine and predictable growth, formulation, and practical application. Fortunately, these problems have turned out to be solvable. A systematic exploration of the biophysics and biochemistry of protein crystallization has shown that one can dependably create new protein crystalline materials more or less at will. In turn, these crystals can be readily strengthened, both chemically and mechanically, to make them suitable for practical commercialization. Today, these novel materials are used as industrial catalysts on a commercial scale, in bioremediation and "green chemistry" applications, and in enantioselective chromatography of pharmaceuticals and fine chemicals. In the near future, their utility will expand, to include the purification of protein drugs, formulation of direct protein therapeutics, and development of adjuvant-less vaccines.

Novel long-acting crystal formulation of human growth hormone.

PURPOSE: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. METHODS: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. RESULTS: Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. CONCLUSIONS: The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

Crystalline monoclonal antibodies for subcutaneous delivery.

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity in vitro. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.