Pediatric formulation development - Challenges of today and strategies for tomorrow: Summary report from M-CERSI workshop 2019.

A workshop on "Pediatric Formulation Development: Challenges of Today and Strategies for Tomorrow" was organized jointly by the University of Maryland's Center of Excellence in Regulatory Science and Innovation (M-CERSI), the U.S. Food and Drug Administration (FDA) and the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Drug Product Pediatric Working Group (PWG). This multi-disciplinary, pediatric focused workshop was held over a two-day period (18-19 Jun 2019) and consisted of participants from industry, regulatory agencies, academia and other organizations from both US and Europe. The workshop consisted of sequential sessions on formulation, analytical, clinical, and regulatory and industry lessons learned and future landscape. Each session began with a series of short framing presentations, followed by facilitated breakout sessions and panel discussion. The formulation session was dedicated to three main topics pertaining to drug product acceptability, excipients in pediatrics and oral administration device considerations. The analytical session discussed key considerations for dosing vehicle selection and analytical strategies for testing of different dosage forms, specifically mini-tablets (multiparticulates). The clinical session highlighted the influence of pediatric pharmacokinetics prediction on formulation design, pediatric drug development strategies and clinical considerations to support pediatric formulation design. The regulatory and industry lessons learned and future landscape session explored the regional differences that exist in regulatory expectations, requirements for pediatric formulation development, and key patient-centric factors to consider when developing novel pediatric formulations. This session also discussed potential collaboration opportunities and tools for pediatric formulation development. This manuscript summarizes the key discussions and outcomes of all the sessions in the workshop with a broadened review and discussion of the topics that were covered.

Effects of mutating aromatic surface residues of the heme domain of human sulfite oxidase on its heme midpoint potential, intramolecular electron transfer, and steady-state kinetics.

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

Identity of the exchangeable sulfur-containing ligand at the Mo(V) center of R160Q human sulfite oxidase.

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

Determination of the distance between the Mo(V) and Fe(III) heme centers of wild type human sulfite oxidase by pulsed EPR spectroscopy.

Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, R(MoFe) = 32.3 Å, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date, there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron-electron double resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about R(MoFe) in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with R(MoFe) = 32 Å, similar to that determined by X-ray crystallography for chicken SO, although the orientation of the R(MoFe) radius-vector with respect to the heme center was found to be somewhat different. The implications of these findings for the flexible tether model are discussed.

Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids.

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (k (ET)) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher k (cat) values than their corresponding k (ET), implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.

Structural studies of the molybdenum center of mitochondrial amidoxime reducing component (mARC) by pulsed EPR spectroscopy and 17O-labeling.

Mitochondrial amidoxime reducing components (mARC-1 and mARC-2) represent a novel group of Mo-containing enzymes in eukaryotes. These proteins form the catalytic part of a three-component enzyme complex known to be responsible for the reductive activation of several N-hydroxylated prodrugs. No X-ray crystal structures are available for these enzymes as yet. A previous biochemical investigation [Wahl, B., et al. (2010) J. Biol. Chem., 285, 37847-37859 ] has revealed that two of the Mo coordination positions are occupied by sulfur atoms from a pyranopterindithiolate (molybdopterin, MPT) cofactor. In this work, we have used continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy and density functional theoretical (DFT) calculations to determine the nature of remaining ligands in the Mo(V) state of the active site of mARC-2. Experiments with samples in D(2)O have identified the exchangeable equatorial ligand as a hydroxyl group. Experiments on samples in H(2)(17)O-enriched buffer have shown the presence of a slowly exchangeable axial oxo ligand. Comparison of the experimental (1)H and (17)O hyperfine interactions with those calculated using DFT has shown that the remaining nonexchangeable equatorial ligand is, most likely, protein-derived and that the possibility of an equatorial oxo ligand can be excluded.

Characterization of chloride-depleted human sulfite oxidase by electron paramagnetic resonance spectroscopy: experimental evidence for the role of anions in product release.

The Mo(V) state of the molybdoenzyme sulfite oxidase (SO) is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Vertebrate SO at pH <7 and >9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center termed the low-pH (lpH) and high-pH (hpH) forms, respectively. Both EPR forms have an exchangeable equatorial OH ligand, but its orientation in the two forms is different. It has been hypothesized that the formation of the lpH species is dependent on the presence of chloride. In this work, we have prepared and purified samples of the wild type and various mutants of human SO that are depleted of chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH but rather exhibit spectra that are characteristic of the blocked species that contains an exchangeable equatorial sulfate ligand. Addition of chloride to these samples results in the disappearance of the blocked species and the formation of the lpH species. Similarly, if chloride is added before sulfite, the lpH species is formed instead of the blocked one. Qualitatively similar results were observed for samples of sulfite-oxidizing enzymes from other organisms that were previously reported to form a blocked species at low pH. However, the depletion of chloride has no effect upon the formation of the hpH species.

Effects of interdomain tether length and flexibility on the kinetics of intramolecular electron transfer in human sulfite oxidase.

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO [Kisker, C., et al. (1997) Cell 91, 973-983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816-5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we altered both its length and flexibility in HSO by site-specific mutagenesis, and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernible systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a 3-fold decrease in the IET rate constant. Deletions of nonconserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a 5-fold increase in the sulfite K(m) value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these proline to alanine and deletion mutants are identical to those of wild-type HSO, indicating no significant change in the Mo active site geometry.

Intramolecular electron transfer in sulfite-oxidizing enzymes: elucidating the role of a conserved active site arginine.

All reported sulfite-oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. To improve our understanding of the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for improving our understanding of residue function in IET without the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of the wild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDH(R55Q) and SDH(R55M) suggests that the reduction in the level of IET seen in HSO(R160Q) is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDH(WT), SDH(R55M), and SDH(R55Q) show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (K(eq)) and rate constants (k(et)) of both variants with respect to the SDH(WT) enzyme. SDH(WT) has a k(et) that is independent of pH and a K(eq) that increases as pH decreases; on the other hand, both SDH(R55M) and SDH(R55Q) have a k(et) that increases as pH decreases, and SDH(R55M) has a K(eq) that is pH-independent. IET in the SDH(R55Q) variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDH(WT), but which also occurs in HSO. IET in SDH(R55K) is slower than in SDH(WT). A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.

Insights into the Nature of Mo(V) Species in Solution: Modeling Catalytic Cycles for Molybdenum Enzymes.

The tris(pyrazolyl)borate and related tripodal N-donor ligands originally developed by Trofimenko stabilize mononuclear compounds containing Mo(VI)O(2), Mo(VI)O, Mo(V)O, and Mo(IV)O units and effectively inhibit their polynucleation in organic solvents. Dioxo-Mo(VI) complexes of the type LMoO(2)(SPh), where L = hydrotris(3,5-dimethylpyrazol-1-yl)borate (Tp*), hydrotris(3-isopropylpyrazol-1-yl)borate (Tp(i) (Pr)), and hydrotris(3,5-dimethyl-1,2,4-triazol-1-yl)borate (Tz) and related derivatives are the only model systems that mimic the complete reaction sequence of sulfite oxidase, in which oxygen from water is ultimately incorporated into product. The quasi-reversible, one-electron reduction of Tp*MoO(2)(SPh) in acetonitrile exhibits a positive potential shift upon addition of a hydroxylic proton donor, and the magnitude of the shift correlates with the acidity of the proton donor. These reductions produce two Mo(V) species, [Tp*Mo(V)O(2)(SPh)](-) and Tp*Mo(V)O(OH)(SPh), that are related by protonation. Measurement of the relative amounts of these two Mo(V) species by EPR spectroscopy enabled the pK(a) of the Mo(V)(OH) unit in acetonitrile to be determined and showed it to be several pK(a) units smaller than that for water in acetonitrile. Similar electrochemical-EPR experiments for Tp(i) (Pr)MoO(2)(SPh) indicated that the pK(a) for its Mo(V)(OH) unit was ∼1.7 units smaller than that for Tp*Mo(V)O(OH)(SPh). Density functional theory calculations also predict a smaller pK(a) for (iPr)Mo(V)O(OH)(SPh) compared to Tp*Mo(V)O(OH)(SPh). Analysis of these results indicates that coupled electron-proton transfer (CEPT) is thermodynamically favored over the indirect process of metal reduction followed by protonation. The crystal structure of Tp(i) (Pr)MoO(2)(SPh) is also presented.

Pentadienyls vs cyclopentadienyls and reversal of metal-ligand bonding affinity with metal oxidation state: synthesis, molecular structures, and electronic structures of high-valent zirconium pentadienyl complexes.

Molecules of the form Cp(6,6-dmch)ZrX(2) (Cp = eta(5)-cyclopentadienyl, X = Cl, Br, I; 6,6-dmch = eta(5)-6,6-dimethylcyclohexadienyl) have been synthesized, and the molecular and electronic structures have been investigated. These molecules allow direct comparison of the bonding and properties of pentadienyl and cyclopentadienyl ligands in the same high-oxidation-state metal complexes. Unlike the well-known Cp(2)ZrX(2) analogues, these Cp(6,6-dmch)ZrX(2) molecules are intensely colored, indicating significantly different relative energies of the frontier orbitals. Also unusual, the average Zr-C distances to the 6,6-dmch pentadienyl ligand are about 0.1 A longer than the average Zr-C distances to the cyclopentadienyl ligand for these Zr(IV) complexes, opposite of what is observed for the Zr(II) complex Cp(2,6,6-tmch)Zr(PMe(3))(2) (tmch = eta(5)-2,6,6-trimethylcyclohexadienyl), reflecting a dramatic reversal in the favorability of the bonding depending on the metal oxidation state. The experimental and computational results indicate that the color of the Cp(6,6-dmch)ZrX(2) complexes is due to a 6,6-dmch ligand-to-metal charge-transfer band. Compared to the Cp(2)ZrX(2) analogues, the Cp(6,6-dmch)ZrX(2) molecules have a considerably less stable HOMO that is pentadienyl-based and an essentially unchanged metal-based LUMO. Also, the lowest unoccupied orbital of pentadienyl is stabilized relative to cyclopentadienyl and becomes a better potential delta electron acceptor, thus contributing to the differences in structure and reactivity of the low-valent and high-valent metal complexes.