Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

IntroductionSerum phosphate levels are insufficiently controlled in many patients with end-stage renal disease (ESRD), and novel therapeutic strategies are needed. Blocking intestinal phosphate absorption mediated by sodium-dependent phosphate co-transporter type 2b (NPT-IIb) holds promise; thus, we evaluated the efficacy, safety, tolerability, and pharmacokinetics of the novel and specific small molecule NPT-IIb inhibitor ASP3325 for the first time in humans.MethodsWe conducted a randomized, double-blind, placebo-controlled, phase 1a single (n = 88) and multiple (n = 36) ascending dose study in healthy subjects, and a randomized, open-label, uncontrolled, phase 1b study in hyperphosphatemic ESRD patients on hemodialysis (single oral dose, n = 5; multiple oral doses, n = 17). Primary efficacy measures were urinary phosphate and fecal phosphorous excretion (healthy subjects) and serum phosphate level (ESRD patients).ResultsNo time- or dose-dependent changes in urinary phosphate or fecal phosphorous excretion were observed following single/multiple ASP3325 doses for 7 days in healthy subjects. In ESRD patients, ASP3325 administered 3 times daily for 2 weeks before or after a meal did not reduce serum phosphate levels. ASP3325 was safe and well tolerated in both populations.ConclusionNPT-IIb inhibition with ASP3325 was not effective in reducing serum phosphate levels in ESRD patients. The relevance of NPT-IIb in humans and feasibility of oral NPT-IIb inhibitors for treatment of hyperphosphatemia in ESRD remain uncertain.

1. Uncomplicated insulin-dependent diabetes mellitus is associated with generalized vasodilatation. This vasodilatation is believed to contribute to the development of microvascular complications. The endothelium plays an important role in the regulation of vascular tone. 2. To investigate the role of endothelial mediators, we measured plasma endothelin levels and studied the vascular effects of intravenous L-arginine (the precursor of NO) in 10 male type 1 diabetic patients and 10 non-diabetic subjects. 3. The baseline plasma endothelin level was significantly lower in the diabetic patients [mean 1.7 (SD 0.5) versus 2.1 (0.4) pmol/l; P < 0.05] than in the control subjects. 4. During L-arginine infusion, plasma cyclic GMP (the second messenger for NO) increased in the control subjects [from 5.1 (2.9) to 6.9 (2.9) nmol/l; P < 0.05 versus saline] and in the diabetic patients [from 4.6 (1.8) to 5.7 (2.2) nmol/l; P = 0.09]. L-Citrulline (a by-product of NO synthesis from L-arginine) increased in both groups. The responses to L-arginine were not significantly different between the control subjects and the diabetic patients. The plasma atrial natriuretic peptide level did not change in either group during infusion of L-arginine or of an equal volume of isotonic saline. 5. Blood pressure decreased slightly during L-arginine administration in both groups. In control subjects, the extracellular fluid volume in the lower leg increased during L-arginine infusion as compared with saline; in the diabetic patients both L-arginine and saline increased the extracellular fluid volume.(ABSTRACT TRUNCATED AT 250 WORDS)

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OBJECTIVEIpragliflozin, a sodium-glucose cotransporter 2 inhibitor, stimulates glycosuria and lowers glycemia in patients with type 2 diabetes (T2DM). The objective of this study was to assess the pharmacodynamics of ipragliflozin in T2DM patients with impaired renal function.RESEARCH DESIGN AND METHODSGlycosuria was measured before and after a single ipragliflozin dose in 8 nondiabetic subjects and 57 T2DM patients (age 62 ± 9 years, fasting glucose 133 ± 39 mg/dL, mean ± SD) with normal renal function (assessed as the estimated glomerular filtration rate [eGFR]) (eGFR1 ≥90 mL · min–1 · 1.73 m−2), mild (eGFR2 ≥60 to <90), moderate (eGFR3 ≥30 to <60), or severe reduction in eGFR (eGFR4 ≤15 to <30).RESULTSIpragliflozin significantly increased urinary glucose excretion in each eGFR class (P < 0.0001). However, ipragliflozin-induced glycosuria declined (median [IQR]) across eGFR class (from 46 mg/min [33] in eGFR1 to 8 mg/min [7] in eGFR4, P < 0.001). Ipragliflozin-induced fractional glucose excretion (excretion/filtration) was 39% [27] in the T2DM patients (pooled data), similar to that of the nondiabetic subjects (37% [17], P = ns). In bivariate analysis of the pooled data, ipragliflozin-induced glycosuria was directly related to eGFR and fasting glucose (P < 0.0001 for both, r2 = 0.55), predicting a decrement in 24-h glycosuria of 15 g for each 20 mL/min decline in eGFR and an increase of 7 g for each 10 mg/dL increase in glucose above fasting normoglycemia.CONCLUSIONSIn T2DM patients, ipragliflozin increases glycosuria in direct, linear proportion to GFR and degree of hyperglycemia, such that its amount can be reliably predicted in the individual patient. Although absolute glycosuria decreases with declining GFR, the efficiency of ipragliflozin action (fractional glucose excretion) is maintained in patients with severe renal impairment.