John Michael Beals

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate, LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.

Novel PEGylated Basal Insulin LY2605541 Has a Preferential Hepatic Effect on Glucose Metabolism

The impact of the novel basal insulin LY2605541 (LY) on hepatic and nonhepatic glucose uptake (non-HGU) was evaluated. Conscious dogs underwent euglycemic clamps with tracer and hepatic balance measurements. Clamp period infusions were peripheral venous regular insulin (0.1 nmol ⋅ kg−1 ⋅ h−1 [control], n = 6) or LY (bolus [nmol/kg], continuous [nmol ⋅ kg−1 ⋅ h−1]: 0.5, 0.5 [n = 6]; 0.375, 0.375 [n = 5]; 0.25, 0.25 [n = 4]), somatostatin, and glucose, as well as intraportal glucagon (basal). During the clamp, the dogs switched from net hepatic glucose output to uptake (rates reached 2.1 ± 1.2, 0.9 ± 2.1, 8.6 ± 2.3, and 6.0 ± 1.1 µmol ⋅ kg−1 ⋅ min−1 within 5 h in control, LY0.25, LY0.375, and LY0.5, respectively). Non-HGU in LY increased less than in control; the ratio of change from basal in non-HGU to change in net hepatic glucose balance, calculated when glucose infusion rates (GIRs) were ~20 µmol ⋅ kg-1 ⋅ min−1 in all groups, was higher in control (1.17 ± 0.38) versus LY0.25 (0.39 ± 0.33), LY0.375 (−0.01 ± 0.13), and LY0.5 (−0.09 ± 0.07). Likewise, the change from baseline in glucose Rd-to-Ra ratio was greatest in control (1.4 ± 0.3 vs. 0.6 ± 0.4, 0.5 ± 0.2, and 0.6 ± 0.2 in LY0.25, LY0.375, and LY0.5, respectively). In contrast to exogenously administered human insulin, LY demonstrated preferential hepatic effects, similar to endogenously secreted insulin. Therefore, the analog might reduce complications associated with current insulin therapy.

Assembly and Dissociation of Human Insulin and LysB28ProB29-Insulin Hexamers: A Comparison Study

AbstractPurpose. Investigations into the kinetic assembly and dissociation of hexameric LysB28ProB29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog. Methods. The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R6 hexamer disruption were studied by extraction of Co(II) with EDTA. Results. Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T6 → T3R3 and T3R3 → R6 transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol. Conclusions. The extraction data support the formation of an R6-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.

Enhancing exposure of protein therapeutics

Therapeutic proteins have made a major impact on medicine, with significant expansion in the past two decades. The medicinal attributes of these agents, particularly their efficacy and often their safety profile, make protein therapeutics attractive, despite the general necessity of invasive (parenteral) delivery. This perceived hurdle has been a primary component in limiting expansion of this class of drug therapies. Strategies that reduce the frequency of administration directly provide greater convenience to the patient, and potentially greater efficacy, that can yield a significant treatment advantage.:

Reversible adsorption of soluble hexameric insulin onto the surface of insulin crystals cocrystallized with protamine: an electrostatic interaction.

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 104 M−1 to 107 M−!, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.

Recommendations regarding technical standards for follow-on biologics: comparability, similarity, interchangeability

Abstract Background: Policy makers around the world are currently considering the creation of a regulatory pathway for follow-on biologics (FOB), which will have to account for the substantial technical challenges associated with FOB development. These challenges will likely involve more complexity than comparability assessments of process changes made by the same manufacturer. The history of industry–regulator comparability discussions helps explain why the same degree of testing and flexibility now applied to change-control within a manufacturers own process, at this time, cannot be extrapolated to the observed and possibly unknown differences between two manufacturing processes that are independently developed by different (non-collaborating) parties. Objectives: This commentary provides recommendations on the technical aspects that should be considered in the creation of an approval pathway for FOB products. Conclusions: In the authors’ view, analytical methodology in its current state cannot alone provide full assurance that the FOB is sufficiently similar to the innovator product. Moreover, the FOB manufacturer will not have access to the extensive knowledge accumulated by the innovator manufacturer from early development through marketing. Thus, extensive clinical evaluation will likely be necessary to provide assurance that the FOB is safe and efficacious. If such testing demonstrates the FOB is safe and efficacious per existing regulatory standards, the product should receive marketing approval as a ‘similar’ product. Since ‘similarity’ is a fundamentally different determination than establishing interchangeability between the two products, an interchangeability determination must be based on additional testing and market experience to ensure patient safety. Post-marketing surveillance of the FOB should be conducted to ensure that the approved molecule has similar clinical safety and efficacy as the innovator product, prior to any consideration of interchangeability.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor–dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.

Amide hydrogen exchange of the central B-chain helix within the T- and R-states of insulin hexamers

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

Addition of 20-kDa PEG to insulin lispro alters absorption and decreases clearance in animals

ABSTRACTPurposeDetermine the pharmacokinetics of insulin peglispro (BIL) in 5/6-nephrectomized rats and study the absorption in lymph duct cannulated (LDC) sheep.MethodsBIL is insulin lispro modified with 20-kDa linear PEG at lysine B28 increasing the hydrodynamic size to 4-fold larger than insulin lispro. Pharmacokinetics of BIL and insulin lispro after IV administration were compared in 5/6-nephrectomized and sham rats. BIL was administered IV or SC into the interdigital space of the hind leg, and peripheral lymph and/or serum samples were collected from both LDC and non-LDC sheep to determine pharmacokinetics and absorption route of BIL.ResultsThe clearance of BIL was similar in 5/6-nephrectomized and sham rats, while the clearance of insulin lispro was 3.3-fold slower in 5/6-nephrectomized rats than in the sham rats. In non-LDC sheep, the terminal half-life after SC was about twice as long vs IV suggesting flip-flop pharmacokinetics. In LDC sheep, bioavailability decreased to <2%; most of the dose was absorbed via the lymphatic system, with 88% ± 19% of the dose collected in the lymph after SC administration.ConclusionThis work demonstrates that increasing the hydrodynamic size of insulin lispro through PEGylation can impact both absorption and clearance to prolong drug action.