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.     

Oxidation of Recombinant Human Parathyroid Hormone: Effect of Oxidized Position on the Biological Activity

Purpose. To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H₂O₂, the amino acid residue oxidized, and the biological activity of the oxidation products. Methods. Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. Results. Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met 18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met 18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met 18. Conclusions. The region around Met8 is important for the activity of the parathyroid hormone.     

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.     

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3‘-BROMOINDOLEMINE

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3′-BROMOINDOLEMINE The single tryptophan residue at position 86 in bovine somatotropin has been cleaved by the use of 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolemine. This reaction was carried out with native bovine growth hormone as well as its reduced and alkylated derivative. When performed on the intact hormone, followed by the reduction and alkylation of the disulfides to liberate the two fragments, the cleavage reaction yielded the fragment containing residues 87–191 in a monomeric state. The other fragment, containing residues 1–86, could only be obtained in a highly aggregated form admixed with other material. When the cleavage reaction was carried out on the reduced and alkylated hormone, both the N-terminal fragment of reduced-carbamidomethylated bovine somatotropin as a monomer and the C-terminal fragment as a dimer were obtained in 35–40% yield. Since the cleavage reaction also oxidized methionine to methionine sulfoxide, the sulfoxides were reduced back to the methionine with N-methylmercapto acetamide. In this manner the following new derivatives and fragments of bovine somatotropin have been prepared: completely reduced and alkylated hormone, reduced and alkylated hormone with its methionines and tryptophan oxidized, reduced and alkylated hormone with its tryptophan oxidized, the N-terminal fragment of the hormone containing residues 1–86 with its methionine and tryptophan oxidized, the C-terminal fragment containing residues 87–191, both with its methionines oxidized and in their native state. The chemical and physical properties of these derivatives and fragments have been determined. Biological activity in the rat tibia was found to be negligible for all derivatives and fragments.     

The Pharmacokinetics of Recombinant Human Relaxin in Nonpregnant Women After Intravenous,Intravaginal, and Intracervical Administration

The pharmacokinetics of recombinant human relaxin (rhRlx) after intravenous (iv) bolus administration and the absorption of rhRlx after intracervical or intravaginal administration were determined in nonpregnant women. The study was conducted in two parts. In part I, 25 women received 0.01 mg/kg rhRlx iv. After a minimum 7-day washout period, these women were dosed intracervically (n = 10) or intravaginally (n = 15) with 0.75 or 1.5 mg rhRlx, respectively, in 3% methylcellulose gel. Part II was a double-blind, randomized, three-way crossover study in 26 women. At 1-month intervals, each woman received one of three intravaginal treatments consisting of 0 (placebo), 1, or 6 mg rhRlx in 3% methylcellulose gel. The serum concentrations of relaxin following iv administration were described as the sum of three exponentials. The mean (±SD) initial, intermediate, and terminal half-lives were 0.09 ± 0.04, 0.72 ± 0.11, and 4.6 ± 1.2 hr, respectively. Most of the area under the curve was associated with the intermediate half-life. The weight-normalized clearance was 170 ± 50 mL/hr/kg. The observed peak concentration was 98 ± 29 ng/mL, and the weight-normalized initial volume of distribution was 78 ± 40 mL/kg, which is approximately equivalent to the serum volume. If central compartment elimination was assumed, the volume of distribution at steady state (V _ss/W) was 280 ± 100 mL/kg, which is approximately equivalent to extracellular fluid volume. V _ss/W could be as large as 1300 ± 400 mL/kg without this assumption. After intravaginal administration of the placebo gel, endogenous relaxin concentrations were evident (i.e., 20 pg/mL) in 9 of the 26 women (maximum concentrations, 23–234 pg/mL). A similar proportion of women (approximately 35–40%) exhibited measurable serum concentrations of relaxin following intravaginal rhRlx treatment; this proportion increased to 90% following intracervical rhRlx treatment. For both routes of administration, the maximum serum concentrations of relaxin were usually within the range of values observed for endogenous relaxin, suggesting that the absorption of rhRlx was minimal.     

Oxidation of Human Insulin-like Growth Factor I in Formulation Studies: Kinetics of Methionine Oxidation in Aqueous Solution and in Solid State

Purpose. The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)¹ in aqueous solution and in the solid state by the aid of quantification of oxygen. Methods. The oxidized form of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode. Results. Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M^–1 month^–l and had an activation energy of 95 (+/–4) kJ/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. Conclusions. Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.     

OXIDATION OF METHIONINE RESIDUES IN BOVINE GROWTH HORMONE BY CHLORAMINE-T

The methionine residues in bovine growth hormone were chemically modified with chloramine-T at pH 7.4 using different chloramine-T methionine molar ratios. Methionine 4 was shown to be the most reactive followed in decreasing order by methionines 148, 123 and 178. With a 50-fold molar excess of chloramine-T over methionine almost full oxidation of all the methionine residues was achieved. The oxidation of the four residues of methionine does not change the growth promoting activity of the hormone.     

Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor

No HeadingPurpose. The objective of this study was to elucidate the different mechanisms of action of different excipients on the oxidation of Met¹, Met¹²², Met¹²⁷, and Met¹³⁸ in granulocyte colony-stimulating factor (G-CSF) by using hydrogen peroxide as the oxidant.Methods. The oxidation of Met¹, Met¹²⁷, and Met¹³⁸ was quantified by peptide mapping analysis. The oxidation of Met¹²² has biphasic oxidation kinetics with a faster second phase. Therefore, the oxidation of Met¹²² was quantified by two different methods: peptide mapping analysis for the first phase of oxidation and direct reverse-phase HPLC for the second phase of oxidation.Results. The current work reveals that the preferential excluding excipients sorbitol, sucrose, and trehalose, in the concentration range 0–30% (w/v), and the preferential binding excipients urea and guanidine hydrochloride, in the concentration range 0–0.8 M, do not affect the oxidation of methionine residues in G-CSF at pH 4.5. The chelating agents citrate and EDTA have different effects on the rates of oxidation of methionine residues in G-CSF. At low concentrations, citrate decreases the rates, while at high concentrations, citrate increases the rates. EDTA decreases the rates of oxidation of methionine residues in G-CSF, such that its effect becomes more and more as its concentration is increased from 0 to 200 mM. The efficacy of EDTA on the rates of oxidation of the four methionine residues in G-CSF follows the order Met¹²² > Met¹²⁷ > Met¹³⁸ > Met¹.Conclusions. Our results indicate that EDTA can protect the methionine residues in G-CSF against oxidation induced by hydrogen peroxide. The more exposed the methionine residue is, the more difficult it is to be protected by EDTA. The mechanism may be due to the specific ion binding of EDTA to proteins.     

Chemical Pathways of Peptide Degradation. V. Ascorbic Acid Promotes Rather than Inhibits the Oxidation of Methionine to Methionine Sulfoxide in Small Model Peptides

The effect of primary structure and external conditions on the oxidation of methionine to methionine sulfoxide by the ascorbate/Fe³⁺ system was studied in small model peptides. Degradation kinetics and yield of sulfoxide formation were dependent on the concentration of ascorbate and H⁺, with a maximum rate observed at pH 6–7. Phosphate buffer significantly accelerated the peptide degradation compared to Tris, HEPES, and MOPS buffers; however, the formation of sulfoxide was low. The oxidation could not be inhibited by the addition of EDTA. Other side products besides sulfoxide were observed, indicating the existence of various other pathways. The influence of methionine location at the C terminus, at the N terminus, and in the middle of the sequence was investigated. The presence of histidine in the sequence markedly increased the degradation rate as well as the sulfoxide production. The histidine catalysis of methionine oxidation occurred intramolecularly with a maximum enhancement of the oxidation rate and sulfoxide production when one residue was placed between the histidine and the methionine residue.     

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography–mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G–Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.     

The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity

Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.     

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 Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

No HeadingPurpose. The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH₂), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H₂O₂) were quantified and analyzed.Methods. The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC.Results. At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH₂ promotes the rates of oxidation of methionine residues in G-CSF. The H₂O₂-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M^-1h^-1, respectively, while the oxidation rate constant for Met¹, the most readily oxidizable methionine residue in G-CSF, is 13.95 M^–1h^–1. Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H₂O₂. By using hPTH 13-34, the effect of AscH₂ on the H₂O₂-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH₂ is prone to be a prooxidant, increasing the hydroxyl radical (OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH₂ tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH₂ is increased from 0 to 200 mM, and the reason for this is still not clear.Conclusions. Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH₂ is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H₂O₂ in protein formulation, whether or not EDTA is present.     

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.     

Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress

We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 1.5. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.     

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 Pharmacokinetics and Absorption of Recombinant Human Relaxin in Nonpregnant Rabbits and Rhesus Monkeys After Intravenous and Intravaginal Administration

Recombinant human relaxin (rhRlx) is being developed as a potential cervical ripening agent to be applied intravaginally or intracervically prior to parturition. The pharmacokinetics and absorption of rhRlx were determined in nonpregnant female rabbits and rhesus monkeys after intravenous bolus (iv) and intravaginal administration of 0.1 mg/kg; additionally, rabbits were dosed with 0.5 mg/kg intravaginally. In rabbits (n = 6), mean (±SD) peak concentrations following iv bolus administration were 1554 ± 296 ng/mL. The weight-normalized clearance (CL/W) was 5.9 ± 0.4 mL/min/kg, initial volume of distribution (V ₁/W) was 57 ± 9 mL/kg, and volume of distribution at steady state (V _SS/W), assuming central compartment elimination, was 240 ± 20 mL/kg. V _ss/W could be as large as 2000 ± 400 mL/kg without this assumption. The estimated amounts of rhRlx absorbed in rabbits following intravaginal administration of 0.1 and 0.5 mg/kg (n = 5/dose) were 3.1 ± 1.4 and 0.7 ± 0.3%, respectively; peak concentrations were 600 ± 297 and 1066 ± 584 pg/mL, respectively. In rhesus monkeys (n = 5) after iv administration, peak concentrations were 971 ± 277 ng/mL; CL/W was 4.1 ± 0.6 mL/ min/kg, V ₁/W was 78 ± 25 mL/kg, and V _ss/W, assuming central compartment elimination, was 690 ± 220 mL/kg. The upper limit for V _ss/W was 1600 ± 200 mL/kg when no assumptions were made regarding site (compartment) of elimination. After intravaginal administration (n = 6), two monkeys had undetectable rhRlx concentrations throughout the 48-hr sampling interval; one monkey had only one sample containing measurable rhRlx (51 pg/mL) at 24 hr; and three monkeys absorbed <2% of the 0.1 mg/kg dose. Peak concentrations in these three animals ranged from 64 to 1475 pg/mL. The absorption of rhRlx was low and variable in both species, and similar results have been observed in women.     

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.

     

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.     

The sensitivity of carboxyl-terminal methionines in calmodulin isoforms to oxidation by H(2)O(2) modulates the ability to activate the plasma membrane Ca-ATPase

The oxidative modification of methionines within the primary sequence of calmodulin (CaM) results in an inability to activate the PM-Ca-ATPase fully, and may contribute to alterations in calcium homeostasis under conditions of oxidative stress. To identify differences in the sensitivities of CaM isoforms to oxidative modification, we have compared the function and patterns of oxidative modification resulting from the exposure of CaM isolated from bovine testes and wheat germ to H(2)O(2). In comparison to CaM isolated from wheat germ, vertebrate CaM is functionally resistant to oxidant-induced loss of function. The decreased functional sensitivity of vertebrate CaM correlates with a 75 +/- 3% reduction in the rate of oxidative modification of a methionine near the carboxyl terminus (i.e., Met(144) or Met(145)). The extent of oxidative modification to other methionines in these CaM isoforms is similar. These results suggest that the sensitivity of Met(144) or Met(145) to oxidation modulates the ability of CaM to activate the PM-Ca-ATPase. Consistent with this interpretation, a CaM mutant in which glutamines were substituted for Met(144) and Met(145) fully activates the PM-Ca-ATPase irrespective of the oxidative modification of the other seven methionines to their corresponding methionine sulfoxides. The extent of oxidative modification to individual methionines in vertebrate CaM by H(2)O(2) correlates with the time-averaged surface accessibility of individual sulfurs calculated from molecular dynamics simulations. Thus, the sensitivity of individual methionines to oxidative modification is directly related to the solvent accessibility. These results indicate that sequence differences between vertebrate and plant CaM alter the sensitivity of methionines near the carboxyl terminus to oxidative modification because of alterations in their solvent accessibility. We suggest that these sequence differences between CaM isoforms have a regulatory role in modulating the functional sensitivity of CaM to conditions of oxidative stress.