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Papers by Michael Hageman
Journal of Pharmaceutical Sciences, 1995
The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somato... more The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somatotropin (rbSt) and lysozyme as seen via differential scanning calorimetry were evaluated with respect to moisture and excipients. The denaturation temperature, Tm, of rbSt and lysozyme decreased with increasing moisture irrespective of the excipient. However, the magnitude of the decrease elicited by moisture was dependent on the type of excipient. Furthermore, the effect of the excipient was dependent on the moisture content; excipients decreased Tm in low moisture solids (i.e., <5% moisture) and increased it in hydrated solids (i.e., >15% moisture). In the dry state (<1% moisture), the addition of 50% sucrose, sorbitol, or glycerol lowered the Tm of rbSt from 161 °C to 136, 120, and 83 °C, respectively, indicating a destabilizing mechanism. Likewise, the Tm of lysozyme decreased from 156 °C to 142, 128, and 97 °C due to the addition of sucrose, sorbitol, and glycerol, respectively. At higher moisture contents, the excipients promoted a higher transition temperature at a given moisture content than the pure protein systems, indicating a stabilizing mechanism. An increase in the enthalpy of unfolding for dehydrated lysozyme was noted with increasing levels of moisture and/or excipient, despite the observed decrease in Tm. The thermal stability, or Tm, of the dehydrated proteins appeared to be correlated to the glass transition temperature (Tg) of the excipient, which in turn should be related to the Tg of the system. The lower the Tg of the excipient, the greater was the degree of destabilization. This result suggests different modes of conformational stabilization by excipients may exist for proteins in the solid state depending on the moisture content.
Journal of Food Science, 1996
Dehydrated proteins are frequently subjected to thermal stress in the presence of other component... more Dehydrated proteins are frequently subjected to thermal stress in the presence of other components. The effect such substances may have upon protein structure, and therefore function, has not been fully investigated. Thus, the effect of added polyhydroxy components on the denaturation of lyophilized β-lactoglobulin, ovalbumin, and ribonuclease, as determined by differential scanning calorimetry, was evaluated. The denaturation temperature, Tm, of the globular proteins decreased in the dry state after the addition of sucrose, sorbitol, or glycerol. The thermal stability (based on Tm) of the dehydrated proteins appeared to correlate with the glass transition temperature (Tg) of the polyhydroxy component, which was assumed to be related to the Tg of the mixture. The lower the Tg of the component, the greater was the degree of protein destabilization. Thus, glass transition data may be used to predict the effect that a component would have on denaturation of dehydrated proteins at elevated temperatures.
Journal of Pharmaceutical Sciences, 1999
This paper examines the effect of water content, water activity, and glass transition temperature... more This paper examines the effect of water content, water activity, and glass transition temperature (Tg) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 °C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing Tg. The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or Tg) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of Tg from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 °C, suggesting that the physical state of formulations could be used to predict chemical stability.
Journal of Pharmaceutical Sciences, 1997
The inherent instability of many proteins during freeze-drying and storage necessitates the addit... more The inherent instability of many proteins during freeze-drying and storage necessitates the addition of excipients to protect the proteins. It is emphasized in the literature that lyophilized sugar/protein composites should be stored at temperatures below their glass transition temperature (Tg) to prevent crystallization of excipients. The influence of bovine somatotropin (rbSt) concentration on inhibition of sucrose crystallization at different relative humidities (RH) was of interest. Thermally modulated differential scanning calorimetry (MDSC) was used to measure Tg and sucrose crystallization temperatures (Tc) of the composites. Sorption isotherms of the various sucrose/rbSt mixtures were determined gravimetrically with a controlled atmosphere microbalance (CAM) and verified by Karl Fischer analysis of selected samples. The CAM was also used to determine lag times and sucrose crystal growth rates by monitoring weight losses resulting from water liberation upon crystallization of sucrose at 23 °C. Results obtained by MDSC indicate that the Tc increased linearly from ∼110 °C for pure sucrose to ∼140 °C with 20% rbSt at very low water content (<0.1%). Similarly, at 22% RH (4.4% H2O), Tc increased from ∼70 °C to 120 °C. In neither case was Tg impacted significantly by increasing protein from 0 to 20%. No Tc could be noted for samples with ≥30% rbSt in nonisothermal conditions. Plasticization by water decreased both Tg and Tc quite similarly but did'nt impact the noted effect of protein on Tc. Induction time for sucrose crystallization (i.e. nucleation) at ∼45% RH (23 °C) increased almost 10-fold by addition of 10% rbSt, whereas rates of water loss due to crystallization decreased by no more than 2–3-fold. The overall results strongly indicate that formulations of higher protein concentration will be more resistant to sucrose crystallization and thus more robust when transiently exposed to storage temperatures above their Tg.
Journal of Food Quality, 1995
Recent debates have emerged on whether water activity (aw) or the state of the system as dictated... more Recent debates have emerged on whether water activity (aw) or the state of the system as dictated by the glass transition temperature (Tg) controls the rates of chemical reactions in reduced-moisture solid systems. Previously, model systems in which the effects of water activity and glass transition on chemical reactions could be evaluated independently did not exist. The use of polyvinyl-pyrrolidone (PVP) of different molecular weights allows the water activity and moisture content of the systems to be kept virtually constant while the glass transition temperature varies. At a given water activity and temperature, the equilibrium moisture content for any molecular weight of PVP differed by only 1–2%, while the Tg values differed by 20–30C. Using PVP of different molecular weights as a model system will allow the effects of water activity and glass transition on chemical reactions to be studied independently and at a constant temperature.
Journal of Agricultural and Food Chemistry, 1992
ABSTRACT Lead optimization in drug discovery is the process of taking an initial molecule and opt... more ABSTRACT Lead optimization in drug discovery is the process of taking an initial molecule and optimizing a suite of parameters to arrive at a final drug molecule. This is commonly achieved by making changes to a molecule and tracking the effect on activity. FACT, the fragment activity comparison tool, is a computational tool to help medicinal chemists and molecular modelers navigate mountains of data to facilitate this optimization. The exhaustive calculation to implement FACT is tenable by taking advantage of grid computing.
Journal of Agricultural and Food Chemistry, 1992
Preformulation studies supporting development of sustained-release formulations for bovine (rbSt)... more Preformulation studies supporting development of sustained-release formulations for bovine (rbSt) and porcine (rpSt) somatotropins emphasized the importance of protein stability within the delivery systems following implantation. Intermolecular dimerization reactions via covalent non-disulfide crosslinks were more predominant in the solid state than in solution. Rates of dimerization in the solid increased upon exposure to increasing relative vapor pressure (rvp) and lyophilization from solutions of increasing pH. However, the fractional amount of rbSt lost via dimerization vs other pathways was independent of rvp and pH. The loss of rbSt induced by exposure of the solid to dry heat or MCo irradiation makes the use of terminal sterilization unlikely. The dissolution of rpSt was faster than that of rbSt, consistent with rpSt's greater solubility. However, the relationship between solubility and dissolution rate for somatotropins was unclear, and application of conventional dissolution models/ techniques was limited.
Biopolymers, 1995
The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via d... more The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161°C in the dry state to a plateau of 65°C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy, for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents. © 1995 John Wiley & Sons, Inc.
Journal of Pharmaceutical Sciences, 2003
Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoin... more Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoinduced degradation. The underlying mechanisms of these processes are identified and presented. Lyophilized rbST was photolyzed with near-ultraviolet (UV) light between 305 and 410 nm, and the protein content was analyzed by various bioanalytical techniques, including tryptic mapping, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), amino acid analysis, and fluorescence, UV, Raman and Fourier transform infrared (FTIR) spectroscopy. The solid-state photodegradation of rbST by near-UV light exclusively targets the protein disulfide bonds. The reaction is initiated by photoionization of tryptophan (Trp) and one-electron reduction of the disulfide. However, in contrast to the behavior of other proteins in solution, rbST appears to undergo back electron transfer to restore Trp and yield a pair of cysteine (Cys) thiyL radicals, which add molecular oxygen and ultimately recombine to yield α-disulfoxide, thiosulfinate, and/or thiosulfonate. Photodegradation is strictly dependent on the presence of molecular oxygen, but does not involve singlet oxygen. Between 0.4 and 10%, residual moisture levels do not affect the rate of photodegradation. Our results show a novel mechanism for Trp-mediated photodegradation of protein disulfide bonds via formation of a pair of thiyL radicals followed by addition of molecular oxygen. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1698–1709, 2003
Journal of Pharmaceutical Sciences, 2003
A new, supersaturable self-emulsifying drug delivery system (S-SEDDS) of paclitaxel was developed... more A new, supersaturable self-emulsifying drug delivery system (S-SEDDS) of paclitaxel was developed employing hydroxypropyl methylcellulose (HPMC) as a precipitation inhibitor with a conventional SEDDS formulation. In vitro dilution of the S-SEDDS formulation results in formation of a microemulsion, followed by slow crystallization of paclitaxel on standing. This result indicates that the system is supersaturated with respect to crystalline paclitaxel, and the supersaturated state is prolonged by HPMC in the formulation. In the absence of HPMC the SEDDS formulation undergoes rapid precipitation, yielding a low paclitaxel solution concentration. A pharmacokinetic study was conducted in male Sprague-Dawley rats to assess exposure after an oral paclitaxel dose of 10 mg/kg in the SEDDS formulations with (S-SEDDS) and without HPMC. The paclitaxel S-SEDDS formulation shows ∼10-fold higher maximum concentration (Cmax) and five-fold higher oral bioavailability (F ≈ 9.5%) compared with that of the orally dosed Taxol® formulation (F ≈ 2.0%) and the SEDDS formulation without HPMC (F ≈ 1%). Coadministration of cyclosporin A (CsA), an inhibitor of P-glycoprotein and CYP 3A4 enzyme, at a dose of 5 mg/kg with the S-SEDDS formulation further increased the oral bioavailability (F ≈ 22.6%). This assessment demonstrates that the systemic exposure of paclitaxel following oral administration can be substantially improved via the S-SEDDS approach. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2386–2398, 2003
Pharmaceutical Research, 2002
Purpose. To predict the oxidative stability of a sulfonamide-containing 5,6-dihydro-4-hydroxy-2-p... more Purpose. To predict the oxidative stability of a sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrone in lipid-based delivery systems, N-(3-{1[(3α,6R)-4-hydroxy-2-oxo-6-phenyl-6-propyltetrahydro-2H-pyran-3-yl]propyl}phenyl)-5-(trifluoromethyl)-2-pyridinylsulfonamide (DHP) was oxidized by peroxides and peroxyl radicals in binary mixtures of water and organic cosolvents. Methods. DHP was oxidized by hydrogen peroxide, t-butylhydroperoxide, or peroxyl radicals derived from the thermal decomposition of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) in 40% (v/v) organic cosolvent and 5 mM buffer at or near 40°C. Interactions between DHP and ]propane sulfonic acid and imidazole) and DH− were assessed by 1H-NMR spectroscopy. The formation of CO likely involves a free radical mechanism. Results. The reaction of DHP with peroxides in 40% (v/v) acetonitrile yields epimeric monohydroxylation products, R-OH and S-OH, at C-3 of the pyrone ring, and a keto-derivative (CO). Hydroxylation rates depend on the protonation state of DHP, and the nature of buffer and the organic cosolvent. Organonitriles accelerate the oxidation through formation of peroxycarboximidic acid. Peroxyl radicals do not yield significant amounts of R/S-OH or CO. Conclusions. The hydrogen peroxide-induced degradation of DHP in the presence of acetonitrile involves two reactions, hydroxylation and carbonyl formatin. Hydroxylation proceeds via nucleophilic attack by the monodeprotonated form of DHP (DH−) on peroxycarboximidic acid. The oxidation rate is slowed by ion pairing between nitrogen-containing buffers ([3-N-morpholino]propane sulfonic acid and imidazole) and DH−. The formation of CO likely involves a free radical mechanism.
![Research paper thumbnail of 7Oxo4,7-dihydrothieno[3,2- b]pyridine-6-carboxamides: Synthesis and biological activity of a new class of highly potent inhibitors of human cytomegalovirus DNA polymerase](https://attachments.academia-assets.com/48586781/thumbnails/1.jpg)
](Bioorganic & Medicinal Chemistry Letters, 2007
We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-... more We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.
Journal of Agricultural and Food Chemistry, 1994
... Leonard N. Bell* and Michael J. Hageman ... Roozen, M. J. G. W.; Hemminga, M. A,; Walstra, P.... more ... Leonard N. Bell* and Michael J. Hageman ... Roozen, M. J. G. W.; Hemminga, M. A,; Walstra, P. Molecular Motion in Glassy Water-Mako-Oligosaccharide (maltodex-trin) Mixtures as Studied by Conventional and Saturation-Transfer Spin-Probe ESR Spectroscopy. Carbohydr. ...
At this symposium, as in much of the literature over the last 5-10 years, there has been continue... more At this symposium, as in much of the literature over the last 5-10 years, there has been continued discussion regarding ways to reduce drug candidate attrition during the more costly drug development phases. Much of this discussion has revolved around the recognized importance of selecting drug candidates that have “drug-like” properties, i.e. physical, chemical and structural properties that appear to differentiate nondrug and drug molecules (Kennedy, 1997; Lipinski et al., 2001; Kola and Landis, 2004). As a result, there has been a conscious effort made to move from highly potent ligands toward a molecule that has “drug-like” properties, presumably with a lower risk to attrit during development. In an effort to address these properties sooner, preclinical groups such as pharmaceutics, metabolism and toxicology have been brought into the drug candidate selection process.
Combinatorial Chemistry & High Throughput Screening, 2010
Physicochemical properties of drug molecules impact many aspects of both in vivo and in vitro beh... more Physicochemical properties of drug molecules impact many aspects of both in vivo and in vitro behavior. Poor physicochemical properties can often create a significant impediment to establishing reliable SAR, establishing proof of principle type studies using in vivo models, and eventually leading to added performance variability and costs throughout the development life cycle; in the worst case scenario, even preventing execution of the desired development plan. Understanding the fundamental physicochemical properties provides the basis to dissect and deconvolute experimental observations in such a way that modification or mitigation of poor molecular properties can be impacted at the design phase, insuring design and selection of a molecule which has a high probability of making it through the arduous development cycle. This review will discuss the key physicochemical properties and how they can be assessed and how they are implicated in both discovery enablement and in final product developability of the selected candidate.
Journal of Pharmaceutical Sciences, 2008
A rapid-throughput screening assay was developed to estimate the salt solubility parameter, KSP, ... more A rapid-throughput screening assay was developed to estimate the salt solubility parameter, KSP, with a minimal quantity of drug. This assay allows for early evaluation of salt limited solubility with a large number of counter-ions and biologically promising drug leads. Drugs dissolved (typically 10 mM) in DMSO are robotically distributed to a 96-well plate. DMSO is evaporated, and drugs are equilibrated with various acids at different concentrations (typically <1 M) to yield final total drug concentrations around 2.5 mM. The plate is checked for precipitation. Filtrates from only those precipitated wells were subjected to rapid gradient HPLC analysis. An iterative procedure is employed to calculate all species concentrations based on mass and charge balance equations. The apparent KSP values assuming 1:1 stoichiometry are determined from counter-ion and ionized drug activities. A correlation coefficient >0.975 for eight drugs totaling 16 salts is reported. Intra-day and inter-day reproducibility was <10%. Conventional apparent KSP measurements were translated to 96-well format for increased throughput and minimal drug consumption (typically 10 mg) to evaluate at least eight different counter-ions. Although the current protocol estimates KSP from 10−3 to 10−7 M, the dynamic range of the assay could be expanded by adjusting drug and counter-ion concentrations. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2079–2090, 2008
Drug Development and Industrial Pharmacy, 1988
ABSTRACT The importance of water sorption on solid state stability of proteins can be addressed t... more ABSTRACT The importance of water sorption on solid state stability of proteins can be addressed through an understanding of properties of the sorbed water and its impact on the properties of the protein. Most decomposition reactions are minimal at or below the monolayer level of hydration. Sorption of water beyond that of the monolayer generally results in increased rates of decomposition due to the increased conformational flexibility of the protein and the ability of the less tightly bound water to mobilize reactants. In many cases the rates of decomposition can be influenced by the addition of excipients. An understanding of how such excipients can influence water sorption and stability will allow for improved development strategies to minimize the decomposition of proteins in the solid state.
Pharmaceutical Research, 1995
Purpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume... more Purpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt). Methods. Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed. Results. An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG. Conclusions. Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.
Journal of Pharmaceutical Sciences, 1995
The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somato... more The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somatotropin (rbSt) and lysozyme as seen via differential scanning calorimetry were evaluated with respect to moisture and excipients. The denaturation temperature, Tm, of rbSt and lysozyme decreased with increasing moisture irrespective of the excipient. However, the magnitude of the decrease elicited by moisture was dependent on the type of excipient. Furthermore, the effect of the excipient was dependent on the moisture content; excipients decreased Tm in low moisture solids (i.e., <5% moisture) and increased it in hydrated solids (i.e., >15% moisture). In the dry state (<1% moisture), the addition of 50% sucrose, sorbitol, or glycerol lowered the Tm of rbSt from 161 °C to 136, 120, and 83 °C, respectively, indicating a destabilizing mechanism. Likewise, the Tm of lysozyme decreased from 156 °C to 142, 128, and 97 °C due to the addition of sucrose, sorbitol, and glycerol, respectively. At higher moisture contents, the excipients promoted a higher transition temperature at a given moisture content than the pure protein systems, indicating a stabilizing mechanism. An increase in the enthalpy of unfolding for dehydrated lysozyme was noted with increasing levels of moisture and/or excipient, despite the observed decrease in Tm. The thermal stability, or Tm, of the dehydrated proteins appeared to be correlated to the glass transition temperature (Tg) of the excipient, which in turn should be related to the Tg of the system. The lower the Tg of the excipient, the greater was the degree of destabilization. This result suggests different modes of conformational stabilization by excipients may exist for proteins in the solid state depending on the moisture content.
Journal of Food Science, 1996
Dehydrated proteins are frequently subjected to thermal stress in the presence of other component... more Dehydrated proteins are frequently subjected to thermal stress in the presence of other components. The effect such substances may have upon protein structure, and therefore function, has not been fully investigated. Thus, the effect of added polyhydroxy components on the denaturation of lyophilized β-lactoglobulin, ovalbumin, and ribonuclease, as determined by differential scanning calorimetry, was evaluated. The denaturation temperature, Tm, of the globular proteins decreased in the dry state after the addition of sucrose, sorbitol, or glycerol. The thermal stability (based on Tm) of the dehydrated proteins appeared to correlate with the glass transition temperature (Tg) of the polyhydroxy component, which was assumed to be related to the Tg of the mixture. The lower the Tg of the component, the greater was the degree of protein destabilization. Thus, glass transition data may be used to predict the effect that a component would have on denaturation of dehydrated proteins at elevated temperatures.
Journal of Pharmaceutical Sciences, 1999
This paper examines the effect of water content, water activity, and glass transition temperature... more This paper examines the effect of water content, water activity, and glass transition temperature (Tg) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 °C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing Tg. The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or Tg) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of Tg from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 °C, suggesting that the physical state of formulations could be used to predict chemical stability.
Journal of Pharmaceutical Sciences, 1997
The inherent instability of many proteins during freeze-drying and storage necessitates the addit... more The inherent instability of many proteins during freeze-drying and storage necessitates the addition of excipients to protect the proteins. It is emphasized in the literature that lyophilized sugar/protein composites should be stored at temperatures below their glass transition temperature (Tg) to prevent crystallization of excipients. The influence of bovine somatotropin (rbSt) concentration on inhibition of sucrose crystallization at different relative humidities (RH) was of interest. Thermally modulated differential scanning calorimetry (MDSC) was used to measure Tg and sucrose crystallization temperatures (Tc) of the composites. Sorption isotherms of the various sucrose/rbSt mixtures were determined gravimetrically with a controlled atmosphere microbalance (CAM) and verified by Karl Fischer analysis of selected samples. The CAM was also used to determine lag times and sucrose crystal growth rates by monitoring weight losses resulting from water liberation upon crystallization of sucrose at 23 °C. Results obtained by MDSC indicate that the Tc increased linearly from ∼110 °C for pure sucrose to ∼140 °C with 20% rbSt at very low water content (<0.1%). Similarly, at 22% RH (4.4% H2O), Tc increased from ∼70 °C to 120 °C. In neither case was Tg impacted significantly by increasing protein from 0 to 20%. No Tc could be noted for samples with ≥30% rbSt in nonisothermal conditions. Plasticization by water decreased both Tg and Tc quite similarly but did'nt impact the noted effect of protein on Tc. Induction time for sucrose crystallization (i.e. nucleation) at ∼45% RH (23 °C) increased almost 10-fold by addition of 10% rbSt, whereas rates of water loss due to crystallization decreased by no more than 2–3-fold. The overall results strongly indicate that formulations of higher protein concentration will be more resistant to sucrose crystallization and thus more robust when transiently exposed to storage temperatures above their Tg.
Journal of Food Quality, 1995
Recent debates have emerged on whether water activity (aw) or the state of the system as dictated... more Recent debates have emerged on whether water activity (aw) or the state of the system as dictated by the glass transition temperature (Tg) controls the rates of chemical reactions in reduced-moisture solid systems. Previously, model systems in which the effects of water activity and glass transition on chemical reactions could be evaluated independently did not exist. The use of polyvinyl-pyrrolidone (PVP) of different molecular weights allows the water activity and moisture content of the systems to be kept virtually constant while the glass transition temperature varies. At a given water activity and temperature, the equilibrium moisture content for any molecular weight of PVP differed by only 1–2%, while the Tg values differed by 20–30C. Using PVP of different molecular weights as a model system will allow the effects of water activity and glass transition on chemical reactions to be studied independently and at a constant temperature.
Journal of Agricultural and Food Chemistry, 1992
ABSTRACT Lead optimization in drug discovery is the process of taking an initial molecule and opt... more ABSTRACT Lead optimization in drug discovery is the process of taking an initial molecule and optimizing a suite of parameters to arrive at a final drug molecule. This is commonly achieved by making changes to a molecule and tracking the effect on activity. FACT, the fragment activity comparison tool, is a computational tool to help medicinal chemists and molecular modelers navigate mountains of data to facilitate this optimization. The exhaustive calculation to implement FACT is tenable by taking advantage of grid computing.
Journal of Agricultural and Food Chemistry, 1992
Preformulation studies supporting development of sustained-release formulations for bovine (rbSt)... more Preformulation studies supporting development of sustained-release formulations for bovine (rbSt) and porcine (rpSt) somatotropins emphasized the importance of protein stability within the delivery systems following implantation. Intermolecular dimerization reactions via covalent non-disulfide crosslinks were more predominant in the solid state than in solution. Rates of dimerization in the solid increased upon exposure to increasing relative vapor pressure (rvp) and lyophilization from solutions of increasing pH. However, the fractional amount of rbSt lost via dimerization vs other pathways was independent of rvp and pH. The loss of rbSt induced by exposure of the solid to dry heat or MCo irradiation makes the use of terminal sterilization unlikely. The dissolution of rpSt was faster than that of rbSt, consistent with rpSt's greater solubility. However, the relationship between solubility and dissolution rate for somatotropins was unclear, and application of conventional dissolution models/ techniques was limited.
Biopolymers, 1995
The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via d... more The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161°C in the dry state to a plateau of 65°C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy, for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents. © 1995 John Wiley & Sons, Inc.
Journal of Pharmaceutical Sciences, 2003
Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoin... more Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoinduced degradation. The underlying mechanisms of these processes are identified and presented. Lyophilized rbST was photolyzed with near-ultraviolet (UV) light between 305 and 410 nm, and the protein content was analyzed by various bioanalytical techniques, including tryptic mapping, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), amino acid analysis, and fluorescence, UV, Raman and Fourier transform infrared (FTIR) spectroscopy. The solid-state photodegradation of rbST by near-UV light exclusively targets the protein disulfide bonds. The reaction is initiated by photoionization of tryptophan (Trp) and one-electron reduction of the disulfide. However, in contrast to the behavior of other proteins in solution, rbST appears to undergo back electron transfer to restore Trp and yield a pair of cysteine (Cys) thiyL radicals, which add molecular oxygen and ultimately recombine to yield α-disulfoxide, thiosulfinate, and/or thiosulfonate. Photodegradation is strictly dependent on the presence of molecular oxygen, but does not involve singlet oxygen. Between 0.4 and 10%, residual moisture levels do not affect the rate of photodegradation. Our results show a novel mechanism for Trp-mediated photodegradation of protein disulfide bonds via formation of a pair of thiyL radicals followed by addition of molecular oxygen. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1698–1709, 2003
Journal of Pharmaceutical Sciences, 2003
A new, supersaturable self-emulsifying drug delivery system (S-SEDDS) of paclitaxel was developed... more A new, supersaturable self-emulsifying drug delivery system (S-SEDDS) of paclitaxel was developed employing hydroxypropyl methylcellulose (HPMC) as a precipitation inhibitor with a conventional SEDDS formulation. In vitro dilution of the S-SEDDS formulation results in formation of a microemulsion, followed by slow crystallization of paclitaxel on standing. This result indicates that the system is supersaturated with respect to crystalline paclitaxel, and the supersaturated state is prolonged by HPMC in the formulation. In the absence of HPMC the SEDDS formulation undergoes rapid precipitation, yielding a low paclitaxel solution concentration. A pharmacokinetic study was conducted in male Sprague-Dawley rats to assess exposure after an oral paclitaxel dose of 10 mg/kg in the SEDDS formulations with (S-SEDDS) and without HPMC. The paclitaxel S-SEDDS formulation shows ∼10-fold higher maximum concentration (Cmax) and five-fold higher oral bioavailability (F ≈ 9.5%) compared with that of the orally dosed Taxol® formulation (F ≈ 2.0%) and the SEDDS formulation without HPMC (F ≈ 1%). Coadministration of cyclosporin A (CsA), an inhibitor of P-glycoprotein and CYP 3A4 enzyme, at a dose of 5 mg/kg with the S-SEDDS formulation further increased the oral bioavailability (F ≈ 22.6%). This assessment demonstrates that the systemic exposure of paclitaxel following oral administration can be substantially improved via the S-SEDDS approach. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2386–2398, 2003
Pharmaceutical Research, 2002
Purpose. To predict the oxidative stability of a sulfonamide-containing 5,6-dihydro-4-hydroxy-2-p... more Purpose. To predict the oxidative stability of a sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrone in lipid-based delivery systems, N-(3-{1[(3α,6R)-4-hydroxy-2-oxo-6-phenyl-6-propyltetrahydro-2H-pyran-3-yl]propyl}phenyl)-5-(trifluoromethyl)-2-pyridinylsulfonamide (DHP) was oxidized by peroxides and peroxyl radicals in binary mixtures of water and organic cosolvents. Methods. DHP was oxidized by hydrogen peroxide, t-butylhydroperoxide, or peroxyl radicals derived from the thermal decomposition of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) in 40% (v/v) organic cosolvent and 5 mM buffer at or near 40°C. Interactions between DHP and ]propane sulfonic acid and imidazole) and DH− were assessed by 1H-NMR spectroscopy. The formation of CO likely involves a free radical mechanism. Results. The reaction of DHP with peroxides in 40% (v/v) acetonitrile yields epimeric monohydroxylation products, R-OH and S-OH, at C-3 of the pyrone ring, and a keto-derivative (CO). Hydroxylation rates depend on the protonation state of DHP, and the nature of buffer and the organic cosolvent. Organonitriles accelerate the oxidation through formation of peroxycarboximidic acid. Peroxyl radicals do not yield significant amounts of R/S-OH or CO. Conclusions. The hydrogen peroxide-induced degradation of DHP in the presence of acetonitrile involves two reactions, hydroxylation and carbonyl formatin. Hydroxylation proceeds via nucleophilic attack by the monodeprotonated form of DHP (DH−) on peroxycarboximidic acid. The oxidation rate is slowed by ion pairing between nitrogen-containing buffers ([3-N-morpholino]propane sulfonic acid and imidazole) and DH−. The formation of CO likely involves a free radical mechanism.
Bioorganic & Medicinal Chemistry Letters, 2007
We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-... more We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.
Journal of Agricultural and Food Chemistry, 1994
... Leonard N. Bell* and Michael J. Hageman ... Roozen, M. J. G. W.; Hemminga, M. A,; Walstra, P.... more ... Leonard N. Bell* and Michael J. Hageman ... Roozen, M. J. G. W.; Hemminga, M. A,; Walstra, P. Molecular Motion in Glassy Water-Mako-Oligosaccharide (maltodex-trin) Mixtures as Studied by Conventional and Saturation-Transfer Spin-Probe ESR Spectroscopy. Carbohydr. ...
At this symposium, as in much of the literature over the last 5-10 years, there has been continue... more At this symposium, as in much of the literature over the last 5-10 years, there has been continued discussion regarding ways to reduce drug candidate attrition during the more costly drug development phases. Much of this discussion has revolved around the recognized importance of selecting drug candidates that have “drug-like” properties, i.e. physical, chemical and structural properties that appear to differentiate nondrug and drug molecules (Kennedy, 1997; Lipinski et al., 2001; Kola and Landis, 2004). As a result, there has been a conscious effort made to move from highly potent ligands toward a molecule that has “drug-like” properties, presumably with a lower risk to attrit during development. In an effort to address these properties sooner, preclinical groups such as pharmaceutics, metabolism and toxicology have been brought into the drug candidate selection process.
Combinatorial Chemistry & High Throughput Screening, 2010
Physicochemical properties of drug molecules impact many aspects of both in vivo and in vitro beh... more Physicochemical properties of drug molecules impact many aspects of both in vivo and in vitro behavior. Poor physicochemical properties can often create a significant impediment to establishing reliable SAR, establishing proof of principle type studies using in vivo models, and eventually leading to added performance variability and costs throughout the development life cycle; in the worst case scenario, even preventing execution of the desired development plan. Understanding the fundamental physicochemical properties provides the basis to dissect and deconvolute experimental observations in such a way that modification or mitigation of poor molecular properties can be impacted at the design phase, insuring design and selection of a molecule which has a high probability of making it through the arduous development cycle. This review will discuss the key physicochemical properties and how they can be assessed and how they are implicated in both discovery enablement and in final product developability of the selected candidate.
Journal of Pharmaceutical Sciences, 2008
A rapid-throughput screening assay was developed to estimate the salt solubility parameter, KSP, ... more A rapid-throughput screening assay was developed to estimate the salt solubility parameter, KSP, with a minimal quantity of drug. This assay allows for early evaluation of salt limited solubility with a large number of counter-ions and biologically promising drug leads. Drugs dissolved (typically 10 mM) in DMSO are robotically distributed to a 96-well plate. DMSO is evaporated, and drugs are equilibrated with various acids at different concentrations (typically <1 M) to yield final total drug concentrations around 2.5 mM. The plate is checked for precipitation. Filtrates from only those precipitated wells were subjected to rapid gradient HPLC analysis. An iterative procedure is employed to calculate all species concentrations based on mass and charge balance equations. The apparent KSP values assuming 1:1 stoichiometry are determined from counter-ion and ionized drug activities. A correlation coefficient >0.975 for eight drugs totaling 16 salts is reported. Intra-day and inter-day reproducibility was <10%. Conventional apparent KSP measurements were translated to 96-well format for increased throughput and minimal drug consumption (typically 10 mg) to evaluate at least eight different counter-ions. Although the current protocol estimates KSP from 10−3 to 10−7 M, the dynamic range of the assay could be expanded by adjusting drug and counter-ion concentrations. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2079–2090, 2008
Drug Development and Industrial Pharmacy, 1988
ABSTRACT The importance of water sorption on solid state stability of proteins can be addressed t... more ABSTRACT The importance of water sorption on solid state stability of proteins can be addressed through an understanding of properties of the sorbed water and its impact on the properties of the protein. Most decomposition reactions are minimal at or below the monolayer level of hydration. Sorption of water beyond that of the monolayer generally results in increased rates of decomposition due to the increased conformational flexibility of the protein and the ability of the less tightly bound water to mobilize reactants. In many cases the rates of decomposition can be influenced by the addition of excipients. An understanding of how such excipients can influence water sorption and stability will allow for improved development strategies to minimize the decomposition of proteins in the solid state.
Pharmaceutical Research, 1995
Purpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume... more Purpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt). Methods. Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed. Results. An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG. Conclusions. Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.