The Stability and Degradation Pathway of Recombinant Human Parathyroid Hormone: Deamidation of Asparaginyl Residue and Peptide Bond Cleavage at Aspartyl and Asparaginyl Residues

Purpose. The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. Methods. Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60°C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. Results. At pH2, eight degradation products were found: l-30rhPTH, l-74rhPTH, l-71rhPTH, l-56rhPTH, l-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Aspl6-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. Conclusions. Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent. rhPTH is most stable at pH5.     

Comparison Between Light Induced and Chemically Induced Oxidation of rhVEGF

Purpose. The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. Methods. A liquid formulation of rhVEGF was exposed to fluorescent light (2 × 10⁴ lux for up to 4 weeks), hydrogen peroxide (H₂O₂), or t-butylhydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. Results. Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78,81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%, possibly due to the role of Met18 in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H₂O₂, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H₂O₂ and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. Conclusions. Methionine residues with exposed surface areas greater than 65 Ų and sulfur surface areas greater than 16 Ų were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H₂O₂ to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 Ų, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.     

Oxidative Degradation of Antiflammin 2

Purpose. To study the oxidation of the methionine residue of antiflammin 2 (HDMNKVLDL, AF2) as a function of pH, buffer concentration, ionic strength, and temperature using different concentrations of hydrogen peroxide and to determine the accessibility of methionine residue to oxidation. Methods. Reversed-phase high-performance liquid chromatography (RPHPLC) was used as the main analytical method in determining the oxidation rates of AF2. Calibration curves for AF2 and the oxidation product, methionine sulfoxide of AF2 (Met(O)-3-AF2), were constructed for each measurement using standard materials. Fast Atom Bombardment Mass Spectroscopy (FABMS) was used to characterize the product. Results. Met(O)-3-AF2 was the only oxidation product detected at pH 3.0 to 8.0. The oxidation rates were independent of buffer concentrations, ionic strength, and pH from 3.0 to 7.0. However, there was an acceleration of the rates at basic pHs, and small amounts of degradation products other than Met(O)-3-AF2 were observed in this alkaline region. Conclusions. Oxidation of methionine in AF2 does not cause the biological inactivation reported by other laboratories since this drug is relatively stable under neutral conditions in the absence of oxiding agent.     

Effects of Polyaminocarboxylate Metal Chelators on Iron-thiolate Induced Oxidation of Methionine- and Histidine-Containing Peptides

Purpose. Site-specific protein oxidation induced by prooxidant/metal/ oxygen has been recognized as one of the major degradation pathways of protein pharmaceuticals. Polyaminocarboxylate (PAC) metal chelators are commonly employed to prevent metal-catalyzed oxidation, for they sequester metals. However, studies have indicated that iron chelates may still be catalytically active due to their specific coordination geometry. The purpose of this study was to investigate how PAC chelators affect prooxidant/metal/oxygen-catalyzed oxidation of peptides containing histidine (His) and methionine (Met). Methods. PACs were applied to a model oxidizing system, dithiothreitol/iron/oxygen, which was shown to promote the oxidation of Met to Met sulfoxide in the two model peptides, GGGMGGG and GHGMGGG. Results. PAC chelators did not suppress the peptide oxidation but significantly changed the product pattern. In particular, the yield of Met sulfoxide dropped significantly, while a number of other products emerged, including oxidation products from the N-terminus and His (if present). Overall, the oxidation became rather non-selective in the presence of PACs. The oxidation kinetics were significantly accelerated by nitrilotriacetate (NTA), ethylenediaminediacetate (HDDA), and ethylenediaminetetraacetate (EDTA), but they were slowed down by ethyl-enebis(oxyethylenenitrilo)tetraacetate (EGTA) and diethylenetriaminepentaacetate (DTPA). Meanwhile the PAC chelators were also observed to undergo degradation. Scavengers of hydrogen peroxide or hydroxyl radicals exerted only partial inhibition on the peptide oxidation. Conclusions. The results of this study are rationalized by the abilities of PAC chelators (i) to extract iron from potential binding sites of the peptides to impair site-specific oxidation, and (ii) to promote the formation of ROS different from the species formed at the peptide metal-binding sites.     

Combined application of high resolution and tandem mass spectrometers to characterize methionine oxidation in a parathyroid hormone formulation

Identification and monitoring of degradation products is a critical aspect of drug product stability programs. This process can present unique challenges when working with complex biopharmaceutical formulations that do not readily lend themselves to straightforward HPLC analysis. The therapeutic 34 amino acid parathyroid hormone fragment (PTH1–34) contains methionine (Met) residues at positions 8 and 18. Oxidation of these Met residues results in reduced biological activity and thus efficacy of the potential drug product. Here, we present an effective approach for the identification of PTH1–34 oxidation products in a drug product formulation in which the stability indicating method used non‐MS compatible HPLC conditions to separate excipients, drug substance and degradation products. High resolution and tandem mass spectrometers were used in conjunction with cyanogen bromide (CNBr) mediated digestion to accurately identify the oxidation products observed in an alternative MS compatible HPLC method used for drug substance analysis. All anticipated CNBr digested peptide fragments, including both oxidized and nonoxidized peptide fragments, were positively identified using TOF MS without the need for additional enzymatic digestion. Once identified, the oxidation products generated were injected onto the original non‐MS compatible HPLC drug product stability indicating method and the respective retention times were confirmed. This allowed the oxidative stability of different formulations to be effectively monitored during the solid state stability program and during variant selection. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1169–1179, 2010     

Stability of human growth hormone: influence of methionine oxidation on thermal folding

Oxidation, particularly of methionine residues, is one of the major chemical degradations of proteins. In a previous publication we studied the conformation of recombinant human growth hormone (r-hGH) selectively oxidized at Met14 and Met125. Conformation of oxidized r-hGH was found not different from that of nonoxidized r-hGH. In this paper, the effect of methionine oxidation on the thermal stability of r-hGH folding was investigated. The thermally induced unfolding process of the oxidized and nonoxidized protein was measured by monitoring the circular dichroism signal at 220 nm. The melting temperatures (T(m)) of the oxidized and nonoxidized r-hGH forms were determined at different pHs and in the presence of salts often used in pharmaceutical formulations. The effect of the location of the oxidized Met residue in the protein and the percentage of oxidation were investigated. Our findings indicate that the monoxidation of the two most accessible methionine residues of r-hGH-Met14 and Met125 - has no effect on the protein conformation. However, oxidation of these residues to form sulfoxides does influence the thermal stability of the protein folding. The presence of the polar oxygen atom on the methionine sulfoxide group thermally destabilizes r-hGH folding. The effect (T(m)) depends upon pH, ionic strength, and the location of the oxidized methionine residues in the protein. The thermal melting of r-hGH and its oxidized products is a highly cooperative process. Methionine oxidation leads to a thermal destabilization of the whole protein folding and is not just a local destabilization.     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

Isolation and Characterization of a Monomethioninesulfoxide Variant of Interferon α-2b

Purpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon -2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN -2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN -2b molecule. The far-UV CD spectra showed a slight loss of -helical content and an increase in the -sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon -2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN -2b variant was found to be present in the rHuIFN -2b bulk drug substance in solution. The Met¹¹¹ residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met¹¹¹ residue did not seem to have a detectable effect on the biological activity of the molecule.     

Safety,Tolerability, Pharmacokinetics and Pharmacodynamics of Recombinant Human Parathyroid Hormone after Single‐ and Multiple‐Dose Subcutaneous Administration in Healthy Chinese Volunteers

Abstract: Recombinant human parathyroid hormone [rhPTH(1–84)] represents a new class of anabolic agents for the treatment of osteoporosis. The present study was designed to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of rhPTH(1–84) after single‐ and multiple‐dose subcutaneous administration in healthy Chinese volunteers. Six cohorts of 32 volunteers received a single dose of rhPTH(1–84) at 0.5–5.0 μg/kg, and two cohorts of 12 volunteers received 2.0 and 3.0 μg/kg of rhPTH(1–84) once daily for 7 consecutive days to assess its safety and tolerability. The results indicated that rhPTH(1–84) appeared to be safe and well tolerated. Additionally, pharmacokinetics of rhPTH(1–84) and its active N‐terminal fragment rhPTH(1–34) were investigated after administration of single 1.0, 2.0 and 4.0 μg/kg doses of rhPTH(1–84) in 30 other volunteers and after multiple doses of 2.0 μg/kg once daily for 7 consecutive days. The pharmacokinetic parameters for rhPTH(1–84) and rhPTH(1–34) after subcutaneous administration of a single dose of 1.0, 2.0 and 4.0 μg/kg were as follows: C_max = (110.54 ± 59.18), (149.70 ± 50.61) and (372.52 ± 94.96) pg/mL; (53.93 ± 6.27), (61.12 ± 11.28) and (89.04 ± 7.08) pg/mL, respectively. AUC_0–10 = (268.87 ± 47.72), (538.93 ± 146.89) and (1364.11 ± 176.82) pg hr/mL; (197.20 ± 50.78), (207.15 ± 72.08) and (344.05 ± 77.06) pg hr/mL, respectively. t_1/2 = (2.34 ± 1.93), (2.58 ± 1.18) and (2.74 ± 1.31) hr; (3.37 ± 1.82), (4.39 ± 3.79), and (3.99 ± 1.85) hr, respectively. Plasma C_max and AUC values of rhPTH(1–84) and rhPTH(1–34) were found to be dose proportional. The pharmacokinetic parameters for rhPTH(1–84) and rhPTH(1–34) after administration of multiple doses of 2.0 μg/kg were as follows: C_{ss_max} = (164.96 ± 52.61) and (75.05 ± 7.31) pg/mL; C_{ss_min} = (6.99 ± 7.73) and (2.05 ± 2.82) pg/mL; AUC_ss = (567.26 ± 118.41) and (306.02 ± 77.55) pg hr/mL; t_1/2 = (1.81 ± 0.89) and (2.27 ± 1.11) hr; DF = (6.93 ± 2.64) and (6.00 ± 1.37), respectively. After multiple doses, the pharmacokinetic parameters for rhPTH(1–84) were consistent with those after single dose. However, the mean C_max and AUC_0–10 of rhPTH(1–34) after multiple dosing were significantly higher than the corresponding values obtained after single‐dose administration. Serum total calcium and phosphate concentrations increased and decreased significantly at 4 hr post‐dosing, respectively.     

Methionine,tryptophan, and histidine oxidation in a model protein,PTH: Mechanisms and stabilization

Recent oxidation events on monoclonal antibody candidates prompted us to investigate the mechanism of oxidation of Met, Trp, and His residues and to search for suitable stabilizers. By using parathyroid hormone (1–34), PTH, as a model protein and various oxidants, aided by liquid chromatography, peptide mapping, and mass spectrometry, we identified and quantified the oxidation of these vulnerable residues. Whereas H₂O₂ and t-butyl hydroperoxide (t-BHP) primarily oxidized the two Met residues, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), and H₂O₂ + Fe(II) oxidized Met and Trp residues, with AAPH more capable of generating oxidized Trp species than the latter. H₂O₂ + Fe(III) generated results comparable to those with H₂O₂ + Fe(II), except that there was a lesser amount of hydroxylated Phe. Oxidation of the His residue in PTH occurred when copper was used instead of iron. AAPH, a free-radical generator, produced alkylperoxides, which simulated the oxidizing species from degraded polysorbate, commonly found in protein formulations. It is prudent to screen stabilizers by using H₂O₂, H₂O₂ + Fe(II), and AAPH because these agents represent potential assaults from the H₂O₂ commonly present in degraded polysorbate, the residue of aseptic agents and the metal from stainless steel surfaces, and alkylperoxides from degraded polysorbate, respectively. Free Met protected the Met residues in PTH from oxidation by H₂O₂ and H₂O₂ + Fe(II). Mannitol and EDTA were effective against H₂O₂ + Fe(II). Free Trp protected only the Trp residue in PTH from oxidation by AAPH, the combination of Trp and Met was effective against all three oxidant conditions. By using AAPH to generate oxidant, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and pyridoxine were also found to exhibit good free-radical scavenging activity and thus protected Trp in PTH against oxidation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4485–4500, 2009     

Protective activity of Hertia cheirifolia extracts against DNA damage,lipid peroxidation and protein oxidation

Context: Hertia cheirifolia L. (Asteraceae), a perennial shrub widely distributed in Northern Africa, is traditionally used to treat inflammatory disorders.

Objective: The protective effect of methanol (Met E) and aqueous (Aq E) extracts of Hertia cheirifolia against DNA, lipid and protein oxidation was investigated.

Materials and methods: Different concentrations (50–1000?μg/mL) of Hertia cheirifolia aerial part extracts were examined against DNA, lipid and protein oxidation induced by H₂O_2?+?UV, FeSO₄, and Fe³⁺/H₂O₂-ascorbic acid, respectively. The DPPH^?, metal ion chelating, reducing power and β-carotene bleaching tests were conducted.

Results: Both extracts were rich in polyphenols, flavonoids and tannins, and were able to scavenge DPPH^? with IC₅₀ values of 138 and 197?μg/mL, respectively. At 300?μg/mL, Aq E exerted stronger chelating effect (99%) than Met E (69%). However, Met E reducing power (IC_50?=_?61?μg/mL) was more than that of Aq E (IC_50?=_?193?μg/mL). Both extracts protected from β-carotene bleaching by 74% and 94%, respectively, and inhibited linoleic acid peroxidation. The inhibitory activity of Aq E extract (64%) was twice more than that of Met E (32%). Interestingly, both extracts protected DNA against the cleavage by about 96–98%. At 1?mg/mL, Met E and Aq E restored protein band intensity by 94–99%.

Conclusions: Hertia cheirifolia exhibits potent antioxidant activity and protects biomolecules against oxidative damage; hence, it may serve as potential source of natural antioxidant for pharmaceutical applications and food preservation. This is the first report on the protective activity of this plant against biomolecule oxidation.     

Validation of the Chloramine-T Induced Oxidation of Human Serum Albumin as a Model for Oxidative Damage in Vivo

Purpose. The validity of using chloramine-T as a model compound for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine-T and by mutating ³⁴Cys for a serine (C34S). Methods. High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5-dithiobis(2-nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques. Results. From the HPLC analysis of human serum, the validity of using chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT_0.1-HSA, CT₁-HSA), ³⁴Cys and Met residues were oxidized, at medium concentrations (CT₁₀-HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration (CT₅₀-HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA (CT₁₀-HSA, CT₅₀-HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half-life and an increased liver clearance. Conclusions. We succeeded in imitating oxidative damage to HSA using chloramine-T and the findings show that Site II is more affected than Site I and ³⁴Cys, when HSA is exposed to oxidative stress.     

An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization

Purpose

The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation.

Methods

Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins.

Results

The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence.

Conclusions

Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

     

Intramolecular cross-linking of insulin. Preparation and properties of oxalyl-and malonyl-bis(methionyl) insulin

The bifunctional reagents, oxalyl-(Met-ONp)₂ and malonyl-(Met-ONp)₂ have been prepared and investigated as reversible cross-linking reagents for insulin and model compounds. The removal of the cross-linking residues was demonstrated by the cyanogen bromide cleavage of oxalyl-(Met-Phe-OMe)₂ and malonyl-(Met-Phe-OMe)₂. Zinc-insulin reacted with a molar equivalent of oxalyl-(Met-ONp)₂ or malonyl-(Met—ONp)₂ in presence of excess triethylamine to yield oxalyl-(Met)₂-insulin and malonyl-(Met)₂-insulin, respectively. In these derivatives the N -terminal phenylalanine (B1 residue) was free. Thus the cross-link was between A1 and B29 residues in insulin. All three disulfide bonds of these insulin derivatives undergo reduction with tributylphosphine to give six sulfhydryls. Airoxidation of reduced oxalyl-(Met)₂-insulin and malonyl-(Met)₂-insulin in 0.05 m disodium phosphate, pH 9.5, yielded products which were indistinguishable from oxalyl-(Met)₂-insulin and malonyl-(Met)₂-insulin respectively, as measured by physicochemical and biological methods. Cyanogen bromide cleavage of reduced and reoxidized malonyl-(Met)₂-insulin in 70% formic acid regenerated insulin quantitatively, but only 40% of insulin was determined from similar treatment of oxalyl-(Met)₂-insulin. The regenerated insulins exhibited the biological activity of native insulin. These studies strongly suggest that disulfide bonds formed during oxidation of reduced oxalyl-(Met)₂-insulin and malonyl-(Met)₂-insulin are identical to those found in insulin.     

Drug safety evaluation of parathyroid hormone for hypocalcemia in patients with hypoparathyroidism

Introduction: Hypoparathyroidism is a rare disorder characterized by low serum calcium levels and high serum phosphate levels, and low or inappropriately normal levels of parathyroid hormone (PTH). This disease is commonly treated with calcium supplements and active vitamin D metabolites or analogues, but large doses of these supplements are often utilized to relieve the symptoms caused by hypocalcemia, without guarantee of a physiological normalization of calcium-phosphate homeostasis.

Areas covered: Several studies have investigated replacement therapy with recombinant human PTH [rhPTH (1-84) and rhPTH (1-34)] for subjects with hypoparathyroidism. In 2015, The Food and Drug Administration (FDA) approved, in the United States, rhPTH (1–84), named Natpara®, a bioenginerred rhPTH, for the management of chronic hypoparathyroidism not well controlled with conventional therapy. This article evaluates the safety and tolerability of rhPTH (1–84) in patients with chronic hypoparathyroidism, and also describes the studies conducted so far on rhPTH (1-34) used for chronic hypoparathyroidism.

Expert opinion: The research done in this field has shown that replacement treatment with rhPTH is an attractive option for subjects with hypoparathyroidism who are unable to maintain stable and safe serum and urinary calcium levels. However, since therapy with rhPTH is a long-term management option in hypoparathyroidism, more long-term safety data are needed.     

Antiinflammatory activity of methionine, methionine sulfoxide and methionine sulfone

The oxidation of methionine in peptides is often associated with the loss of biological activity. Since methionine showed good antiinflammatory activity, its oxidized products methionine sulfoxide and methionine sulfone were tested. The sulfone was more active than the sulfoxide although methionine was most active indicating that the antiinflammatory activity is not correlated with the oxidation state of sulphur. Their hydroxyl radical scavenging activity was measured. Methionine was most active and sulfone was least active. Here also no correlation with antiinflammatory activity was found.     

Metal-Catalyzed Oxidation of Brain-Derived Neurotrophic Factor (BDNF): Analytical Challenges for the Identification of Modified Sites

Purpose. We examined the metal-catalyzed oxidation of brain-derivedneurotrophic factor (BDNF) using the Cu(II)/ascorbate/O₂ modeloxidative system. Methods. Electrospray ionization mass spectrometry, peptide mappingand amino acid analysis were utilized to determine the nature of thecovalent modification induced by the metal-catalyzed oxidative system.Additionally, analytical ultracentrifugation, the Bradford assay, circulardichroism and ANSA dye-binding were used to determine the natureof any conformational changes induced by the oxidation. Results. Exposure of BDNF to the Cu(II)/ascorbate/O₂ system led tothe modification of ca. 35% of Met⁹² to its sulfoxide, and to subsequentconformational changes. The proteolytic digestion procedure wassensitive to this conformational change, and was unable to detect themodification. Chemical digestion with CNBr, however, was not sensitive tothis change, and allowed for the identification of the site ofmodification. Conclusions. The modification of Met⁹² to its sulfoxide rendered theoxidized BDNF inaccessible to proteolytic digestion, due toconformational changes associated with the oxidation.     

In Vitro Methionine Oxidation of Recombinant Human Leptin

Purpose. To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity. Methods. Oxidized leptin derivatives were prepared in the presence of H₂O₂ and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied. Results. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met¹ and Met⁶⁹ proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k ₁ and k ₆₉, were 1.5 × 10^–3 and 2.3 × 10^–4 min^–1. No change in the secondary or tertiary structure was detected for Met¹ mono-oxidized and Met¹, Met⁶⁹ di-oxidized leptins. The Met¹ mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met¹, Met⁶⁹ di-oxidized leptin. Both Met⁵⁵ and Met¹³⁷ were not oxidized in t-butylhydroperoxide but only in H₂O₂. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule. Conclusions. The oxidation of leptin occurred in the order of Met¹ > Met⁶⁹ >> Met⁵⁵ Met¹³⁷, and the importance for maintaining leptin structural integrity was Met⁵⁵ Met¹³⁷ >> Met⁶⁹ Met¹. Met⁶⁹, but not Met¹, plays a critical role in the protein stability and activity.     

Oxidation of Recombinant Human Interleukin-2 by Potassium Peroxodisulfate

Purpose. The oxidation of recombinant human interleukin-2 (rhIL-2) by potassium peroxodisulfate (KPS) with or without N,N,N,N-tetramethylethylenediamine (TEMED), which are used for the preparation of dextran-based hydrogels, was investigated. Methods. The oxidation of (derivatives of) methionine, tryptophan, histidine and tyrosine, as well as rhIL-2 was investigated. Both the oxidation kinetics (RP-HPLC) and the nature of the oxidation products (mass spectrometry) were studied as a function of the KPS and TEMED concentration, and the presence of a competitive antioxidant, methionine. Results. Under conditions relevant for the preparation of rhIL-2 loaded hydrogels, only methionine and tryptophan derivatives were susceptible to oxidation by KPS. The oxidation of these compounds was inhibited once TEMED was present, suggesting that the peroxodisulfate anion, rather than the radicals formed in the presence of TEMED, is the oxidative species. KPS only induced oxidation of the four methionines present in rhIL-2, whereas the tryptophan residue remained unaffected. The radicals, formed after KPS decomposition by TEMED, induced some dimerization of rhIL-2. The oxidation of rhIL-2 could be substantially reduced by the addition of methionine, or by pre-incubation of KPS with TEMED. Conclusions. Only the methionine residues in rhIL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions.     

The Solid State Oxidation of Methionine Containing Peptide: A Preliminary Study Using Time of Flight Secondary Ion Mass Spectrometry

Purpose. A surface sensitive mass spectrometric technique: Time ofFlight Secondary Ion Mass Spectrometry (ToF-SIMS) was introducedto study the solid state instability of a methionine containing peptidecaused by the oxidation of the methionine residue. Methods. The oxidation of a neuropeptide Methinonine-Enkephalin(ME) in air and under UV acceleration was studied by ToF-SIMS. Results. The apparent oxidation rate is defined by the peak ratio ofoxidized molecular ion over unoxidized molecular ion. ME is oxidizedat a faster rate to its sulfoxide derivative in the UV accelerated oxidationenvironment than in lab air. The calibration curve for evaluating theionization probability ratio of the oxidized deprotonated molecular iondivided by the unoxidized deprotonated molecular ion was obtained.This could be used to extract the real oxidation rate of ME in thesolid state. Conclusions. The preliminary results showed that ToF-SIMS with simplesample handling, fast data acquisition, together with excellentsurface sensitivity and detection limit could be an applicable and convenienttool to study peptide reactions in the solid state such as oxidationand deamidation process.