Pharmacokinetics and Pharmacodynamics of Insulin Tregopil in Relation to Premeal Dosing Time, Between Meal Interval, and Meal Composition in Patients With Type 2 Diabetes Mellitus.

We evaluated the pharmacokinetics and pharmacodynamics of oral insulin tregopil in relation to premeal dosing time, between-meal interval, and meal composition type in type 2 diabetes mellitus patients in a randomized, placebo-controlled, crossover study consisting of 3 sequential cohorts. In Cohort 1, insulin tregopil administered 10 to 20 minutes before a meal resulted in optimal postmeal exposure and demonstrated better postprandial glucose-lowering effect (glucose area under concentration-time curve [AUC]) compared to the 30-minute group. In Cohort 2, insulin tregopil pharmacokinetic exposure (plasma AUC) showed a progressive increase through 4, 5, and 6 hours of between-meal interval. The 6-hour between-meal interval resulted in better absorption of insulin tregopil in comparison to 4- and 5-hour intervals. However, no significant differences were observed in pharmacodynamic parameters except for higher glucose AUC0-180min in the insulin tregopil 4-hour group during the afternoon meal as compared to the morning meal. In Cohort 3, a high-fiber meal had the least impact on insulin tregopil absorption and resulted in the highest reduction in plasma glucose levels in the afternoon. A high-fat meal reduced insulin tregopil absorption in the afternoon meal; however, pharmacodynamic response was not diminished significantly. Insulin tregopil has a rapid onset of action of approximately 10 minutes and, when administered 10 to 20 minutes before a meal, demonstrated up to 13% to 18% reduction in blood glucose levels compared to baseline. A 5-hour between-meal interval minimizes the impact of a meal on absorption of subsequent (afternoon) insulin tregopil dose, and the pharmacodynamic response of insulin tregopil is not altered by meal composition. Insulin tregopil was well tolerated in patients with type 2 diabetes mellitus.

Impact of Insulin Tregopil and Its Permeation Enhancer on Pharmacokinetics of Metformin in Healthy Volunteers: Randomized, Open-Label, Placebo-Controlled, Crossover Study.

Oral insulin tregopil (IN-105; a new drug under development) may be coadministered with oral antidiabetic drugs, such as metformin in patients with type 2 diabetes mellitus for optimal glycemic control. IN-105 has sodium caprate excipient, a permeation enhancer, for enhancing absorption in the stomach and increasing bioavailability via an oral route. Sodium caprate may increase bioavailability of metformin by a similar mechanism. Therefore, it was necessary to study the effect of IN-105 on pharmacokinetics (PKs) of metformin. In this randomized, open-label, cross-over study, metformin was administered to healthy volunteers receiving IN-105/placebo under fed/fasting conditions. The 90% confidence interval (CI) of the geometric mean ratio of the area under the curve from time zero to infinity (AUC0-inf ; fasting and fed) and peak plasma concentration (Cmax ; fed) of metformin were within 0.80-1.25 acceptance range. Under fasting conditions, the upper bound margin of Cmax was just beyond this range (i.e., 1.27) and was concluded as functionally not relevant. There was no clinically significant effect of sodium caprate/IN-105 on PKs of metformin under fasting/fed conditions, and it was safe.

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics.

We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation. Graphical Abstract ᅟ.

Mass spectrometric distinction of in-source and in-solution pyroglutamate and succinimide in proteins: a case study on rhG-CSF.

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.

Development of a process to manufacture PEGylated orally bioavailable insulin.

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography-tandem mass spectrometry.

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 microm 50 mm x 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 microg/ml when 100 microl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C(max)) 0.40, 0.57, 1.95 microg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31microg/ml on Day 28 for low, mid and high dose treated animals.

Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation.

Aspartic acid formed by nonenzymatic deamidation of asparagine often isomerizes to isoaspartic acid through a succinimide intermediate. Accumulation of isoaspartic acid initiates aggregation and degradation in proteins. Deamidation at the antigen-binding region reduces the efficacy and also upregulates immunogenicity of monoclonal antibodies. We report an improved 'bottom-up' tandem mass spectrometric method to detect and decipher the position of isoaspartate formation in therapeutic immunoglobulin gamma in a single chromatographic run. Differentiation between aspartate and isoaspartate residues through collision-induced tandem mass spectrometry is formidable due to their identical mass. Signature backbone cleavage ions, c(n) + 57 and z(l-n) - 57, produced upon radical-mediated fragmentation, were used to delineate the site of isomerization. It is more conclusive than monitoring the relative peak intensity and the decrease in hydrophobicity of the isoaspartate-containing peptide in a chromatographic elution. Collectively, this methodology provides a useful tool to monitor deamidation and isomerization in biopharmaceuticals during their production, downstream processing and storage.

Oral insulin - a review of current status.

Oral insulin is one of the most exciting areas of development in the treatment of diabetes because of its potential benefit in patient convenience, rapid insulinization of liver, adequate insulin delivery avoiding peripheral hyperinsulinaemia while potentially avoiding adverse effects of weight gain and hypoglycaemia. Growing evidence that earlier initiation of intensive insulin therapy produces sustained tight glycaemic control resulting in substantial delay in complications makes an effective oral insulin product even more vital for the management of patients with diabetes. Despite knowledge of this unmet medical need, oral delivery of insulin has been unsuccessful because of several barriers. For several decades, researchers have tried to develop oral insulin using various technologies without much clinical or commercial success. This review summarizes the development status of oral insulins which are publicly reported to be undergoing clinical studies. Currently, two oral insulin products are in an advanced stage of clinical development and first data from long-term therapy are expected to be available in the second half of 2010.

A tandem mass spectrometric approach to the identification of O-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris.

Glycoforms of glargine expressed in Pichia pastoris were isolated by high-performance liquid chromatography and analyzed by a series of chemical and mass spectrometric methods for the identification of various glycoforms, glycosylation position, nature and structure of glycans. Reduction and alkylation, peptide mapping techniques were used to decipher the amino acid site at which glycosylation had taken place. Chemical methods were coupled with mass spectrometry techniques such as electrospray ionization and matrix-assisted laser desorption/ionization for identification of the glycosylation site.

Process and purification for manufacture of a modified insulin intended for oral delivery.

Oral delivery of insulin is convenient and physiologically desirable in the treatment of diabetes. However, this route of administration has presented substantial challenges as insulin is degraded enzymatically in the gut, resulting in low bioavailability. We have developed a conjugated insulin product (IN-105) that has high bioavailability and is currently undergoing clinical trials for the treatment of diabetes. A process for the manufacture of IN-105 was developed. Initially, recombinant human insulin was conjugated covalently with a monodisperse, short-chain methoxypolyethylene glycol derivative. The desired product, IN-105, was purified from its closely related species using RP-HPLC and cation exchange chromatography to a purity of 98.5%. The elution pool from cation exchange chromatography was crystallized and lyophilized into the dry active pharmaceutical ingredient.