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.     

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.     

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.     

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.     

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.     

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 Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.     

Theory and Computer Programs for Calculating Solution pH,Buffer Formula,and Buffer Capacity for Multiple Component System at a Given Ionic Strength and Temperature

Purpose. A theory and computer programs running on Microsoft^® Excel for Windows for calculation of solution pH, buffer formula, and buffer capacity at a given ionic strength and temperature were developed. The theory does not limit the category of buffer components, the number of buffer components, or the number of ionizations for each buffer component. The usefulness of the programs was examined. Methods. The formulas for 7 single component buffer solutions and 2 multiple component buffer solutions composed of citrate, phosphate, Tris, borate, and glycine were calculated. The solution pH values were measured at 25, 40, 55 and 70°C for comparison with the calculated pH values. Results. Of the 108 predictions made, 96 were of pH values within ± 0.1 pH unit of the measured values, at temperatures ranging from 25°C to 70°C and at ionic strengths ranging from 0.1 M to 0.5 M. Conclusions. These programs will be useful for identifying appropriate buffer solutions at various temperatures and/or ionic strengths.     

Oxidation of Human Insulin-Like Growth Factor I in Formulation Studies,II. Effects of Oxygen,Visible Light,and Phosphate on Methionine Oxidation in Aqueous Solution and Evaluation of Possible Mechanisms

Purpose. The oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I) in aqueous solution was studied with respect to oxygen, visible light and sodium phosphate. Methods. Aqueous solutions of hIGF-I were prepared with different amounts of phosphate and dissolved oxygen. The solutions were stored either in darkness or exposed to artificial visible light. The oxidized hIGF-I was quantified by RP-HPLC. A two level full factorial experimental design, with two levels of each of the three factors studied, was used. Results. Oxidation was found to be positively correlated with light, oxygen content and, interestingly, phosphate. The increasing effect of phosphate on the oxidation appears not to originate from metal contaminants. The influence of both oxygen and phosphate increased with time. The pH dependence of oxidation indicated the formation of a phosphorylated sulfonium ion as an oxidation intermediate. A significant interaction effect between phosphate and visible light suggested participation of radicals. Conclusions. Factorial experiments provide a valuable tool when studying complex mechanisms with interacting factors. The oxidation of methionine in hIGF-I is significantly affected by light but also by the presence of phosphate buffer.     

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.     

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.     

Effects of Solution Conditions on Methionine Oxidation in Albinterferon Alfa-2b and the Role of Oxidation in its Conformation and Aggregation

Physical and chemical degradation of therapeutic proteins can occur simultaneously. In this study, our first objective was to investigate how solution conditions that impact conformational stability of albinterferon alfa-2b, a recombinant fusion protein, modulate rates of methionine (Met) oxidation. Another objective of this work was to determine whether oxidation affects conformation and rate of aggregation of the protein. The protein was subjected to oxidation in solutions of varying pH, ionic strength, and excipients by the addition of 0.02% tertiary-butyl hydroperoxide (TBHP). The rate of formation of Met-sulfoxide species was monitored by reversed-phase high-performance liquid chromatography and compared across solution conditions. Albinterferon alfa-2b exhibited susceptibility to Met oxidation during exposure to TBHP that was highly dependent on solution parameters, but there was not a clear correlation between oxidation rate and protein conformational stability. Met oxidation resulted in significant perturbation of both secondary and tertiary structure of albinterferon alfa-2b as shown by both far-ultraviolet (UV) and near-UV circular dichroism. Moreover, oxidation of the protein caused a noticeable reduction in the protein's resistance to thermal denaturation. Surprisingly, despite its negative effect on solution structure and conformational stability, oxidation actually reduced the protein's aggregation rate during agitation at room temperature as well as during quiescent incubation at 40°C. Oxidation of the protein resulted in improved colloidal stability of the protein, which is manifested by a more positive B₂₂ value in the oxidized protein. Thus, the reduced aggregation rate after oxidation suggests that increased colloidal stability of oxidized albinterferon alfa-2b counteracted oxidation-induced decreases in conformational stability.     

Xenobiotic metabolism in humans during early pregnancy: peroxidase-mediated oxidation and bioactivation of 2-aminofluorene

1. Arylamines such as 2-aminofluorene (2-AF) are known teratogens and transplacental carcinogens in laboratory animal species. Although exposure of women to arylamines is likely to occur during pregnancy, how these chemicals are metabolized by the enzymes from the human conceptual tissues is currently unknown.

2. Highly purified preparations of peroxidase isolated from human intrauterine conceptual tissues at 8 weeks of gestation were used to study in vitro metabolism of 2-AF. The oxidation of 2-AF was examined spectrophotometrically whereas the bioactivation was assessed from the covalent binding to protein and DNA using [³H] 2-AF.

3. Using guaiacol as a model substrate, the purified preparations of peroxidase used exhibited a specific activity of 15–20 μmol/min/mg protein. 2-AF oxidation was found to be enzymatic in nature. Kinetic data obtained under optimal assay conditions yielded a K_m = 41 μM for 2-AF, 8.33 μM for H₂O₂, and a V_max = 1.2 μmol 2-AF oxidized/min/mg protein.

4. Under optimal assay conditions, the covalent binding of reactive intermediate(s) to protein and DNA (nmol equivalent/min/mg enzyme/mg bovine serum albumin or calf thymus DNA) was observed at the rate of about 3.75 ± 0.39 and 1.90 ± 0.11 respectively.

5. A significant decline in the rate of both oxidation and bioactivation of 2-AF was observed in the presence of classical peroxidase inhibitors, KCN and NaN₃.     

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.     

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.     

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.     

Physiological parameters of the gastrointestinal fluid impact the dissolution behavior of the BCS class IIa drug valsartan

Abstract

The objective of this study was to investigate the effect of the physiological parameters (pH, buffer capacity, and ionic strength) of the gastrointestinal (GI) fluid on the dissolution behavior of the class II weakly acidic (BCS class IIa) drug valsartan. A series of in vitro dissolution studies was carried out on Diovan^® immediate release tablets using media that cover the physiological range of pH (1.2–7.8), buffer capacity (0–0.047?M/ΔpH), and ionic strength (0–0.4?mol/L) of the GI fluid during fasted and fed states using the conventional USP II apparatus. Valsartan exhibited pH- and buffer capacity-dependent dissolution behavior, where valsartan release was slow and incomplete in media simulating gastric fluid with low pH, and fast and complete in media simulating intestinal fluid with high pH. In addition, the rate of valsartan release increased with increasing the buffer capacity of the dissolution medium. In water and NaCl solutions, valsartan release was incomplete and the dissolution profiles were similar regardless of the ionic strength of the medium, indicating an ionic strength-independent dissolution behavior. These results highlight the significant effect of the physiological parameters of the GI fluid on the dissolution behavior of BCS class IIa drugs.     

Response Surface Method: A Novel Strategy to Optimize lontophoretic Transdermal Delivery of Thyrotropin-releasing Hormone

Purpose. To maximize the iontophoretic transdermal delivery rate of thyrotropin-releasing hormone (TRH) facilitated by periodically monophase-pulsed current across excised skin. Methods. The pH of the buffer, the ionic strength in the solution, the frequency of the periodically monophase-pulsed current and the current on/off ratio were chosen as the key variables. A response surface method was applied to optimize the transdermal delivery rate of TRH under different operational conditions. Results. The optimum operating conditions were achieved via experimentation based on the response surface method by systematically adjusting the pH of the buffer, the ionic strength in the solution, the current amplitude, frequency and the active temporal ratio of the pulsed current. The rate of permeation of TRH crossing the skin during iontophoresis varied from two to ten-fold, depending on operating conditions. Conclusions. Only a few steps, two in this work, were needed to reach the optimal. The response surface near the region of the maximal point was thoroughly described with a quadratic function. A maximal transdermal rate of permeation of TRH, 103.2 µg h^–1 cm^–2, was obtained when the donor solution was at pH = 7.0, ionic strength = 0.037, and with a periodically monophase-pulsed current iontophoresis with duty cycle = 75%. The effect of pulse frequency was not statistically significant.     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.     

Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.