The oral delivery of protein therapeutics offers numerous advantages for patients but also presents significant challenges in terms of development. Currently, there is limited knowledge available ...regarding the stability and shelf life of orally delivered protein therapeutics. In this study, a comprehensive assessment of the stability of an orally delivered solid dosage variable domain of heavy-chain antibody (VHH antibody) drug product was conducted. Four stability related quality attributes that undergo change as a result of thermal and humidity stress were identified. Subsequently, these attributes were modeled using an accelerated stability approach facilitated by ASAP
software. To the best of our knowledge, this is the first time that this approach has been reported for an antibody drug product. We observed overall good model quality and accurate predictions regarding the protein stability during storage. Notably, we discovered that protein aggregation, formed through a degradation pathway, requires additional adjustments to the modeling method. In summary, the ASAP approach demonstrated promising results in predicting the stability of this complex solid-state protein formulation. This study sheds light on the stability and shelf life of orally delivered protein therapeutics, addressing an important knowledge gap in the field.
Genotoxic impurities (GTIs) are potential carcinogens that need to be controlled down to ppm or lower concentration levels in pharmaceuticals under strict regulations. The static headspace gas ...chromatography (HS-GC) coupled with electron capture detection (ECD) is an effective approach to monitor halogenated and nitroaromatic genotoxins. Deep eutectic solvents (DESs) possess tunable physico-chemical properties and low vapor pressure for HS-GC methods. In this study, zwitterionic and non-ionic DESs have been used for the first time to develop and validate a sensitive analytical method for the analysis of 24 genotoxins at sub-ppm concentrations. Compared to non-ionic diluents, zwitterionic DESs produced exceptional analytical performance and the betaine : 7 (1,4- butane diol) DES outperformed the betaine : 5 (1,4-butane diol) DES. Limits of detection (LOD) down to the 5-ppb concentration level were achieved in DESs. Wide linear ranges spanning over 5 orders of magnitude (0.005–100 µg g−1) were obtained for most analytes with exceptional sensitivities and high precision. The method accuracy and precision were validated using 3 commercially available drug substances and excellent recoveries were obtained. This study broadens the applicability of HS-GC in the determination of less volatile GTIs by establishing DESs as viable diluent substitutes for organic solvents in routine pharmaceutical analysis.
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•Trace level analysis of genotoxic impurities (GTIs) in drug substances is discussed.•Deep eutectic solvents (DESs) offer low chromatographic background in HS-GC.•Twenty-four GTIs containing halogens and nitro functional groups are examined.•Zwitterionic DESs are excellent diluents for pharmaceutical analysis.
GDC-0941 is an orally administered potent, selective pan-inhibitor of phosphatidylinositol 3-kinases (PI3Ks) with good preclinical antitumor activity in xenograft models and favorable ...pharmacokinetics and tolerability in phase 1 trials, and it is currently being investigated in phase II clinical trials as an anti-cancer agent. In vitro solubility and dissolution studies suggested that GDC-0941, a weak base, displays significant pH-dependent solubility. Moreover, preclinical studies conducted in famotidine-induced hypochlorhydric dog suggested that the pharmacokinetics of GDC-0941 may be sensitive to pharmacologically induced hypochlorhydria. To investigate the clinical significance of food and pH-dependent solubility on GDC-0941 pharmacokinetics a four-period, two-sequence, open-label, randomized, crossover study was conducted in healthy volunteers. During the fasting state, GDC-0941 was rapidly absorbed with a median Tmax of 2 h. The presence of a high-fat meal delayed the absorption of GDC-0941, with a median Tmax of 4 h and a modest increase in AUC relative to the fasted state, with an estimated geometric mean ratio (GMR, 90% CI) of fed/fasted of 1.28 (1.08, 1.51) for AUC0-∞ and 0.87 (0.70, 1.06) for Cmax. The effect of rabeprazole (model PPI) coadministration on the pharmacokinetics of GDC-0941 was evaluated in the fasted and fed state. When comparing the effect of rabeprazole + GDC-0941 (fasted) to baseline GDC-0941 absorption in a fasted state, GDC-0941 median Tmax was unchanged, however, both Cmax and AUC0-∞ decreased significantly after pretreatment with rabeprazole, with an estimated GMR (90% CI) of 0.31 (0.21, 0.46) and 0.46 (0.35, 0.61), respectively for both parameters. When rabeprazole was administered in the presence of the high-fat meal, the impact of food did not fully reverse the pH effect; the overall effect of rabeprazole on AUC0-∞ was somewhat attenuated by the high-fat meal (estimate GMR of 0.57, with 90% CI, 0.50, 0.65) but unchanged for the Cmax (estimate of 0.43, with 90% CI, 0.37, 0.50). The results of the current investigations emphasize the complex nature of physicochemical interactions and the importance of gastric acid for the dissolution and solubilization processes of GDC-0941. Given these findings, dosing of GDC-0941 in clinical trials was not constrained relative to fasted/fed states, but the concomitant use of ARAs was restricted. Mitigation strategies to limit the influence of pH on exposure of molecularly targeted agents such as GDC-0941 with pH-dependent solubility are discussed.
Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior ...and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable
and
payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-
-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (
, proteins) and was suitable for composition analysis as well as for evaluating
release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.
We evaluated the effects of dam removal on fish assemblage structure and spatial distributions after four low‐head dam removals in the Baraboo River, Wisconsin, using data collected at 35 study sites ...over 7 years. After dam removal, biotic integrity scores (possible range = 0–100) increased by 35–50 points at three of the four former impoundments as a result of decreases in percent tolerant species, increases in the number of intolerant species, and in some cases, increases in species richness. Fish assemblage shifts were muted at a fourth, lower‐gradient impoundment site, indicating that responses differ among dam sites within a river system. In tailwater areas, postremoval assemblage shifts were transient; biotic integrity and species richness declined initially but then recovered at two of the three sites within 2 years after dam removal. An analysis of spatial distributions before dam removal revealed 11 fish species that were found below, but rarely or never above, the downstream‐most dam. After dam removal, 10 of the 11 species were collected at new sites upstream from the dam, indicating that recolonization of reconnected upstream sites had occurred. Some species recolonized rapidly and in large numbers. For example, emerald shiners Notropis atherinoides recolonized 16 upstream sites and were collected 123 km upstream from the dam within the first year after removal. One of the nine recolonizing species, the spotted sucker Minytrema melanops, was only detected during spring, suggesting that these fish recolonized seasonally, perhaps for spawning. Recolonizing species were generally lacustrine or large‐river fishes known to undergo overwintering and spawning migrations. Our study suggests that dam removal is a viable option for restoring lotic fish communities, but further study is needed on recovery patterns as they relate to channel morphology, hydrologic characteristics, impoundment sediment storage capacity, and the distance to source populations of recolonizing taxa.
The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A ...dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.
2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is a thermodynamically ...stabilized product analogue that binds tightly to oxidized MCAD (K(dox) = 3.5 +/- 0.1 microM, pH 7.6) and elicits a redox potential shift that is 78% of that observed with the natural substrate/product couple Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715. The midpoint potential of the MCAD.HD-CoA complex exhibits a pH dependence that is consistent with the redox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a,ox) approximately 9.3) and Glu99-COOH (pK(a,ox) approximately 7.4) in the oxidized MCAD.HD-CoA complex indicate that while binding of the C(6) analogue makes Glu376 a stronger catalytic base (pK(a,ox) approximately 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximately 7.5, free MCAD) Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605-14612. This finding is in agreement with the apparent pK of 9.2 determined for Glu376 in the human MCAD.4-thia-octenoyl-CoA complex Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445. The pK(a)s estimated for Glu376 and Glu99 in the reduced pig kidney MCAD.HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonated in the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK(a) and redox potential shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman difference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized pi-electron polarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectively polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.
The SVX4 integrated circuit Garcia-Sciveres, M.; Krieger, B.; Walder, J-P. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
09/2003, Letnik:
511, Številka:
1
Journal Article
Recenzirano
A first prototype of the SVX4 readout IC with enclosed transistor layout for radiation tolerance has been fabricated in a commercial
0.25
μm
bulk CMOS process. The SVX4 is intended to instrument the ...CDF and D0 Run IIB silicon strip detector upgrades at Fermilab. The design and test results are discussed.
We have conducted a beam test of a three-station telescope composed of silicon hybrid pixel detectors developed at the University of California, Davis. Each detector comprises a 6 /spl times/ 90 ...sensor array of 64 /spl times/ 256 /spl mu/m/sup 2/ pixels bump-bonded to a radiation-hard sparsified readout application-specific integrated circuit with unit cell performance suitable for use in hadron collider experiments. We describe the pixel arrays, the telescope and its data-acquisition system, the telescope alignment optimization procedure using tracks, and the performance of the system in a 15-GeV electron beam at the Stanford Linear Accelerator Center.
