Lee E. Kirsch

The degradation pathways of glucagon in acidic solutions

OBJECTIVE Glucagon is a 29 amino acid peptide hormone that exhibits degradation via both chemical and physical pathways. The objective of the studies reported herein was to identify the degradation products and scheme for glucagon hydrolysis in acidic solutions. METHODS Solutions of glucagon in 0.01 N HCl (pH 2.5) were degraded at 60 degrees C for 70 h. One isocratic and two gradient RP-HPLC methods were developed to separate the degradation products. Structure elucidation of the separated peaks was achieved using amino acid sequencing, amino acid analysis, and mass spectrometry. Degradation was carried out in the pH range 1.5-5 to check for changes in degradation scheme with pH. Authentic samples of degradation products were degraded under similar acidic conditions to confirm precursor successor relationships in the degradation scheme. RESULTS Sixteen major degradation products were isolated and identified. The major pathways of degradation were found to be aspartic acid cleavage at positions 9, 15, and 21 and glutaminyl deamidation at positions 3, 20, and 24. Cleavage occurred on both sides of Asp-15 but only on the C-terminal side of Asp-9 and Asp-21. Deamidation of the Asn residue at position 28 was not detected.

Preformulation Studies on the S-Isomer of Oxybutynin Hydrochloride, an Improved Chemical Entity (ICE™)

(S)-Oxybutynin HCl (S-OXY) is a white crystalline solid powder with an acicular particle morphology. Differential scanning calorimetry (DSC) thermograms revealed one characteristic endotherm at 116.2°C. On rescanning a sample heated to 120°C, no thermal events were distinguished in the temperature range 25°C to 150°C. Weight loss curves determined by thermogravimetric analysis showed a continuous, gradual weight loss of about 0.15% over the temperature range 30°C to 110°C, followed by a change in slope and more rapid weight loss beginning at 150°C. Observation by hot-stage microscopy confirmed the melting endotherm observed by DSC. Equilibrium moisture uptake studies indicated low water vapor uptake at low relative humidities (< 52.8%). At relative humidities of 75.3% and 84.3%, S-OXY first deliquesced and then converted to a lower melting point crystal form. X-ray powder diffraction (XRPD) data supported the DSC findings. S-OXY underwent degradation by ester hydrolysis at alkaline pHs. The kinetics of this reaction were studied at 25°C in carbonate-bicarbonate buffers. Observed rate constants of 0.008 h−1 and 0.0552 h−1 were determined at pH 9.69 and 10.25, respectively. The pKa of S-OXY was 7.75. The aqueous solubility of S-OXY was described as a function of pH and the free-base solubility. The mean partition coefficient log P was 3.33 using 1-octanol. The surface tensions of aqueous solutions of S-OXY decreased with increasing concentration, but no concentration-independent region was observed, indicating that S-OXY does not form micelles in aqueous solution. The dissolution rate of S-OXY from a compressed disk in 0.1 N HCl was rapid, whereas it was considerably slower at pH 7.4. Addition of 1% hexadecyltrimethylammonium bromide (CTAB) at pH 7.4 significantly improved the dissolution rate. S-OXY displayed very poor flow properties when compared to standard pharmaceutical excipients. XRPD results indicated that S-OXY exhibited a loss in crystallinity following ball milling. Hiestand tableting indices indicated that S-OXY has good bonding properties and forms strong compacts, but is likely to be susceptible to capping on ejection from the die. This indicated the need for a plastically deformable excipient such as Avicel PH-101 in tablet formulations.

Development of a Lyophilized Formulation for (R,R)-Formoterol (L)-Tartrate

(R,R)-formoterol is a β-agonist for inhalation. Aqueous instability suggested the need for a reconstitutable lyophilized dosage form. The objective of these studies was to devise a stable, rapid-dissolving, therapeutically compatible dosage form. The effects of diluents and residual moisture on the stability of thermally stressed formoterol formulations were investigated. Drug and various excipients (acetate, lactose, and mannitol) were lyophilized and placed in humidity chambers (0 to 90% relative humidity) at 25 to 50°C. Stability was characterized by time-dependent changes using HPLC, pH, and XRD. Residual moistures were determined by Karl Fisher methods. Regression models were developed to quantify the effects of formulation and environmental variation on drug stability. Solid-state instability was observed as a function of high residual moisture and diluent type. Although the residual moistures in mannitol formulations were typically below 1%, the degradation rate (50°C) varied from 2 to 10 mcg/day, which was 1.3- to 20-fold high than observed for lactose formulations under the same relative humidity conditions. At high relative humidity, the presence of acetate significantly increased the degradation rate (p < 0.04). The critical residual moisture content for lactose formulations was 3%. The amount of lactose was optimized by evaluating the degradation over the temperature range 25 to 50°C. Mannitol and acetate were shown to be unsuitable excipients, and an optimal lactose amount was 50 mg for vials containing 50 mcg of drug.

Population pharmacokinetics of artesunate and dihydroartemisinin following single- and multiple-dosing of oral artesunate in healthy subjects

BackgroundThe population pharmacokinetics of artesunate (AS) and its active metabolite dihydroartemisinin (DHA) were studied in healthy subjects receiving single- or multiple-dosing of AS orally either in combination with pyronaridine (PYR) or as a monotherapy with or without food.MethodsData from 118 concentration-time profiles arising from 91 healthy Korean subjects were pooled from four Phase I clinical studies. Subjects received 2-5 mg/kg of single- and multiple-dosing of oral AS either in combination with PYR or as a monotherapy with or without food. Plasma AS and DHA were measured simultaneously using a validated liquid chromatography- mass spectrometric method with a lower limit of quantification of 1 ng/mL for both AS and DHA. Nonlinear mixed-effect modelling was used to obtain the pharmacokinetic and variability (inter-individual and residual variability) parameter estimates.ResultsA novel parent-metabolite pharmacokinetic model consisting of a dosing compartment, a central compartment for AS, a central compartment and a peripheral compartment for DHA was developed. AS and DHA data were modelled simultaneously assuming stoichiometric conversion to DHA. AS was rapidly absorbed with a population estimate of absorption rate constant (Ka) of 3.85 h-1. The population estimates of apparent clearance (CL/F) and volume of distribution (V2/F) for AS were 1190 L/h with 36.2% inter-individual variability (IIV) and 1210 L with 57.4% IIV, respectively. For DHA, the population estimates of apparent clearance (CLM/F) and central volume of distribution (V3/F) were 93.7 L/h with 28% IIV and 97.1 L with 30% IIV, respectively. The population estimates of apparent inter-compartmental clearance (Q/F) and peripheral volume of distribution (V4/F) for DHA were 5.74 L/h and 18.5 L, respectively. Intake of high-fat and high-caloric meal prior to the drug administration resulted in 84% reduction in Ka. Body weight impacted CLM/F, such that a unit change in weight resulted in 1.9-unit change in CLM/F in the same direction.ConclusionsA novel simultaneous parent-metabolite pharmacokinetic model with good predictive power was developed to study the population pharmacokinetics of AS and DHA in healthy subjects following single- and multiple-dosing of AS with or without the presence of food. Food intake and weight were significant covariates for Ka and CLM/F, respectively.

Evaluation of Lipopeptide (Daptomycin) Aggregation Using Fluorescence, Light Scattering, and Nuclear Magnetic Resonance Spectroscopy

The aggregation behavior and critical aggregation concentration (CAC) values of daptomycin in aqueous solutions were evaluated under the external factors of pH, temperature, daptomycin concentration, and calcium ions concentration by using the complementary characterization techniques, fluorescence, dynamic and static light scattering, and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the intrinsic fluorescence resonance energy transfer of daptomycin, the CAC values were identified by an upward inflection of the fluorescence emission from Kyn-13 at 460 nm. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM at pH 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy.

Studies on the instability of chlorhexidine, part I: Kinetics and mechanisms

The objectives of the studies presented herein were to determine the pH-dependent chlorhexidine (CHD) degradation scheme, to determine the rate laws, and to propose reasonable mechanisms for CHD hydrolysis in aqueous solutions. A series of degradation kinetic studies was conducted at 90.0 °C using reaction mixtures containing 0.10 mM CHD prepared in the pH range of 0.5-9.0 using hydrochloric acid, sodium hydroxide, acetate, phosphate, or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffers at a constant ionic strength of 0.500 M. Concentration-time profiles for all degradation products, intermediates, and substrates were determined by high-performance liquid chromatography (HPLC). Degradation products and intermediates were identified using a combination of liquid chromatography-mass spectrometry, kinetic analysis, and HPLC comparison with authentic compounds. pH-dependent degradation scheme and rate laws were parameterized using nonlinear regression. The direct formation of p-chloroaniline (PCA) from CHD is the major pathway in acidic conditions, whereas the indirect formation of PCA via the formation of p-chlorophenylurea is the main pathway in alkaline conditions.

The effect of polyvinylpyrrolidine on the stability of taurolidine.

In an aqueous solution, taurolidine reversibly degrades to taurultam, hydroxymethyltaurultam, taurineamide, and formaldehyde. The objectives of this research were to investigate the mechanism of polyvinylpyrrolidine (PVP)-induced taurolidine stabilization by measuring the effect of PVP on the rate and extent of formaldehyde release from taurolidine and its decomposition products, the ionization of taurineamide, and the NMR and FT-IR spectra of taurineamide. PVP was found to a) increase the rate constant for taurultam formation from taurineamide and formaldehyde, b) decrease the apparent pKa of taurineamide, and c) alter the NMR and FT-IR taurineamide spectra. The effect of PVP on the stability of taurolidine was to increase the stability of its primary degradation product, taurultam, which, in turn increased taurolidine stability because taurultam was reversibly formed from taurolidine. Furthermore, the increased stability of taurultam in acidic, aqueous PVP solutions was due to an increased rate of taurultam formation from its primary degradation products: taurineamide and formaldehyde. The rate constant for taurultam formation increased three-fold in the presence of 5% PVP. The rate increase was caused by the interaction of unprotonated taurineamide with PVP. The estimated taurineamide-PVP association constant was 1800 M-1. This interaction decreased the concentration of unreactive/protonated taurineamide while increasing the concentration of unprotonated/associated substrate which was found to be fivefold less reactive than the free/unprotonated amine.

Estimated pKa values for specific amino acid residues in daptomycin

Daptomycin is a cyclic lipopeptide antibiotic. The ionization constants of daptomycin have not been individually elucidated. The objective of this research is to determine the sequence-specific ionization constants of daptomycin in the monomeric state. The pH titrations of daptomycin were performed by nuclear magnetic resonance (NMR) spectroscopy. The sequence-specific pKa values for the four acidic residues and one aromatic amine (Kyn-13) in daptomycin were determined by two-dimensional total correlation spectroscopy (1) H NMR. From the NMR pH titration, the estimated pKa values for Asp-3, Asp-9, and methylglutamic acid (mGlu-12) were determined to be 4.2, 3.8, and 4.6 in the absence of salt, and 4.1, 3.8, and 4.4 in the presence of 150 mM NaCl, respectively. The pKa value for Asp-7 is estimated to be approximately 1.0 in the absence of salt and 1.3 in the presence of salt. The estimated Hill coefficients for Asp-7 were 0.72 and 1.31 in the absence and presence of salt, respectively. The increase in Hill coefficients from 0.72 to 1.31 with increasing salt concentration is consistent with the estimated lower pKa in the absence of salt, and suggests that a salt bridge is formed in solution possibly between Asp-7 acidic group and the neighboring Orn-6 basic group.

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

BackgroundGlucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.ResultsHigh molecular weight aggregates of glucagon were detected in otherwise monomeric solutions using light scattering techniques. These aggregates were detected upon initial mixing of glucagon powder in dilute HCl and NaOH. In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process. Field-flow fractionation was used to separate aggregates from monomeric glucagon and determine relative mass. The molar mass of the large aggregates was shown to grow appreciably over time as the glucagon solutions gelled.ConclusionThe results of this study indicate that initial glucagon solutions are predominantly monomeric, but contain small quantities of large aggregates. These results suggest that the initial aggregates are seed nuclei, or intermediates which catalyze the aggregation process, even at low concentrations.

Kinetic model for solid-state degradation of gabapentin

Gabapentin degrades directly to gabapentin-lactam (gaba-L) in the solid state. The objective of this study was to formulate a drug degradation model that accounted for the environmental storage conditions and mechanical stress (prior to storage) on lactamization kinetics. The effects of mechanical stress on drug degradation kinetics were determined by milling gabapentin in a FRITSCH Planetary Micro Mill for 0 and 60 min. The resultant gabapentin powder was stored at 40 °C-60 °C and 5%-30% relative humidity. The rate of gaba-L formation was measured by high-performance liquid chromatography. An irreversible two-step autocatalytic reaction scheme was fit using nonlinear regression methods. The resultant kinetic model was used to predict the time-dependent concentration of degradant of gabapentin tablets prepared under various exemplary manufacturing conditions, thereby demonstrating the ability of the model to link manufacturing variation and chemical stability in solid-state gabapentin formulations.