beta A4 peptide (beta AP) accumulates in amyloid plaques of Alzheimer's disease and may contribute to neuronal degeneration. Conflicting observations have been reported regarding the direct in vitro ...and in vivo neurotoxicity of beta AP. We have assessed in vitro beta AP toxicity in high density primary rat hippocampal cultures and found marked lot-to-lot differences in the neurotoxic properties of beta AP. One lot of beta AP from a commercial supplier resulted in significant direct neurotoxicity at 10 microM, while 2 other lots from the same supplier were essentially nontoxic. Three additional lots of beta AP from unrelated sources were also nontoxic at 10 microM. Initial biochemical characterization has not yet revealed any marked differences among the various lots of beta AP. Low levels of endotoxin (ca., 1 EU/ml) were detected in several beta AP preparations but did not correlate with neurotoxicity. Our observation that lot-to-lot variability of beta AP occurred even under identical in vitro culture conditions may account for part of the present controversy in this area.
The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was
studied. Denaturation was measured by ultraviolet absorbance, intrinsic ...fluorescence, far ultraviolet circular dichroism,
and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating
a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs
free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165
(large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid
or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated
hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism.
The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding
of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These
results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium
intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the
denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking
native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of
oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation
is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide
bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior
is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium
folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state.
The folding results with hGH and alkylated hGH substantiate this.
This chapter contains sections titled:
Introduction
History of Biologics Regulation in United States
Regulatory Classification of Proteins
Regulation of Generic Drugs
Legal Arguments Related to ...Follow‐On Proteins
Scientific Issues Related to Follow‐On Proteins (Data Requirements)
Proposed Regulatory Paradigm: Case Studies
Summary and Conclusions
References
•Weight of evidence (WoE) method to evaluate key events in adverse outcome pathways.•Biological plausibility, empirical evidence and essentiality are WoE determinants.•Application to human health and ...ecological AOPs is illustrated with six examples.•A prototype quantitative decision analysis (MCDA) WoE model for AOPs is described.
Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users’ Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.
Rabbit articular chondrocytes maintained in monolayer, synthesized and secreted a 46 kDa protein into the culture medium. N-terminal sequence analysis and immunoprecipitation of the radiolabeled ...material revealed this protein to be osteonectin (ON)/SPARC, a protein previously shown to be present in bone. When chondrocytes were exposed to interleukin-1, a cytokine with matrix degradative properties, ON synthesis and secretion was greatly inhibited. However, this was specific to IL-1 since two other pro-inflammatory cytokines (tumor-necrosis factor-α and interleukin-6) with properties similar to IL-1, failed to cause any discernible effect on ON synthesis. Several growth factors (TGF-β, PDGF, and IGF-1), that have been shown to stimulate other cartilage matrix macromolecular synthesis, also stimulated ON synthesis and were also able to reverse the inhibitory effect of IL-1 on ON synthesis. These observations were also demonstrated in explant cultures of cartilage. Our studies suggest that ON is a biosynthetic product of articular cartilage and could play a role in cartilage structure and/or function.
This chapter contains sections titled:
Introduction
Formulation Assessment
Analytical Method Development
Formulation Development
Drug Product Stability
Quality by Design and Scale‐Up
Concluding ...Remarks
References
An efficient secretion vector containing a gene coding for an
E. coli signal peptide fused to human growth hormone (hGH) was cloned into
E. coli. The recombinant fusion protein was expressed and ...correctly processed hGH was secreted into the periplasmic space at a yield of 10–15 μg hGH/
A
600. Purification of hGH from the periplasmic fraction by anion exchange and size exclusion gave hGH of greater than 90% purity. Characterization by SDS-PAGE, amino terminal analysis, trypsin mapping, and circular dichroism demonstrated that the fusion protein was correctly processed to authentic hGH and that the
E. coli periplasm provided an appropriate environment for proper folding of hGH and disulfide bond formation.
The present studies were designed to provide structural characterization of peptide metabolites of biosynthetic human growth hormone (hGH) formed by rat thyroid gland proteases in vitro. Electrospray ...ionization mass/spectrometry (ESI-MS) and N-terminal sequencing were used to characterize the peptide metabolites. The predominant enzyme in the thyroid gland preparations was a chymotrypsin-like serine protease which was biochemically similar to rat mast cell protease-I. Metabolic intermediates were formed by cleavage of hGH exclusively at Tyr/Phe/Leu-Xaa bonds. After a 5- or 45-min incubation of hGH with thyroid gland S9 pellet fraction, the majority of metabolites formed were two-chain variants of hGH having masses ranging from 16,002 to 22,143 Da. These metabolites were formed as a result of proteolysis in the large disulfide loop region of hGH in combination with processing at Tyr42-Ser43. Based upon the time-related appearance and structural characterization of these intermediates, a sequence of metabolic events is proposed. The initial event appears to be cleavage by the chymotrypsin-like protease between Tyr143-Ser144 to form a two-chain hGH. This intermediate is then cleaved between Tyr42-Ser43, liberating the N-terminal peptide, Phe1-Tyr42. Two other metabolites were generated as a result of the deletion of the peptides Lys140-Tyr143 and Ser144-Phe146 from the large loop region. The identification of similar metabolites truncated by a single amino acid at the carboxyl terminus indicated the action of a carboxypeptidase on these metabolic products. After a 4.5-hr incubation, the protease isolated from the S9 pellet fraction degraded hGH to > 20 small peptides, having masses < or = 2300 Da.
The presence of several endogenous molecular forms of human GH (hGH), including proteolytically cleaved two-chain forms, has been proposed to be related to the diverse biological activity of hGH. The ...present study characterized hGH degradation in the rat to determine how peripheral metabolism may influence the kinetics and pharmacology of exogenously administered hGH. In vitro studies indicated that hGH was proteolytically degraded by thyroid gland and skeletal muscle, but not liver and kidney homogenates. The proteolytic activity, localized to the 9000 x g pellet fraction, was characterized as a chymotrypsin-like serine protease using class-specific inhibitors. N-Terminal sequencing of hGH peptides formed by the thyroid gland and skeletal muscle indicated that cleavage sites were almost exclusively at Tyr/Phe-Xaa bonds, with similar points of cleavage observed in the two tissues. Immunoreactive two-chain forms of hGH were also formed. The two-chain molecules had similar cleavage sites, but differed in apparent mol wt when analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To understand the potential significance of two-chain product formation, we compared the kinetics and degradation of hGH with those of a synthetic two-chain derivative of hGH (Des-1-8,135-145; 2-CAP). The in vitro tissue distribution of 2-CAP proteolysis was the same as that for hGH. The fragmentation pattern of 2-CAP was less complex when analyzed by reverse phase HPLC. The major peptide fragments formed from 2-CAP were chromatographically similar to those formed from hGH. The plasma kinetics of 2-CAP were compared to those of hGH with a RIA using polyclonal antiserum to hGH. After im and sc administration of 2-CAP (125 micrograms/kg), the area under the plasma concentration curve was 3.2- and 4.5-fold greater, respectively, than after administration of hGH (125 micrograms/kg). Both compounds had a greater area under the curve by the im than the sc route. 2-CAP had 2- to 3-fold greater bioavailability than hGH by the im and sc routes. Plasma from rats treated 30 min earlier with hGH im was immunoextracted and analyzed by Western blotting. A circulating immunoreactive fragment was detected which had similar electrophoretic mobility as a two-chain hGH product formed during the in vitro incubations of hGH with skeletal muscle and thyroid gland homogenates. The results indicate that hGH is proteolytically processed in peripheral tissue homogenates, with the formation of two-chain products. The greater bioavailability of 2-CAP suggests that metabolism of hGH to two-chain forms may influence the in vivo kinetics of hGH.
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