Pulmonary Administration of Soluble Antigen Arrays Is Superior to Antigen in Treatment of Experimental Autoimmune Encephalomyelitis.

Antigen-specific immunotherapy has been used to hyposensitize patients to allergens and offers an enticing approach for attenuating autoimmune diseases. Applying antigen-specific immunotherapy to mucosal surfaces such as the lungs may engage unique immune response pathways to improve efficacy. Pulmonary delivery of soluble antigen arrays (SAgAs) was explored in mice with experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. SAgAs were designed to impede immune response to autoantigen epitopes and are composed of a hyaluronan backbone with peptides PLP139-151 (proteolipid protein) and LABL, a disease-causing proteolipid peptide epitope and an intracellular cell-adhesion molecule-1 ligand, respectively. Pulmonary instillation of SAgAs decreased disease score, improved weight gain, and decreased incidence of disease in EAE mice compared to pulmonary delivery of hyaluronic acid polymer, LABL, or PLP. Interestingly, treating with PLP alone also showed some improvement. Splenocytes from SAgA-treated animals showed increased interferon-gamma levels, and interleukin-6 (IL-6) and IL-17 were elevated in SAgA-treated animals compared to PLP treatments. IL-10, IL-2, and tumor necrosis factor-alpha levels showed no significant difference, yet trends across all cytokines suggested SAgAs induced a very different immune response compared to treatment with PLP alone. This work suggests that codelivery of peptide components is essential when treating EAE via pulmonary instillation, and the immune response may have shifted toward immune tolerance.

Hyaluronic Acid Molecular Weight Determines Lung Clearance and Biodistribution after Instillation.

Hyaluronic acid (HA) has emerged as a versatile polymer for drug delivery. Multiple commercial products utilize HA, it can be obtained in a variety of molecular weights, and it offers chemical handles for cross-linkers, drugs, or imaging agents. Previous studies have investigated multiple administration routes, but the absorption, biodistribution, and pharmacokinetics of HA after delivery to the lung is relatively unknown. Here, pharmacokinetic parameters were investigated by delivering different molecular weights of HA (between 7 and 741 kDa) to the lungs of mice. HA was labeled with either a near-infrared dye or with iodine-125 conjugated to HA using a tyrosine linker. In initial studies, dye-labeled HA was instilled into the lungs and fluorescent images of organs were collected at 1, 8, and 24 h post administration. Data suggested longer lung persistence of higher molecular weight HA, but signal diminished for all molecular weights at 8 h. To better quantitate pharmacokinetic parameters, different molecular weights of iodine-125 labeled HA were instilled and organ radioactivity was determined after 1, 2, 4, 6, and 8 h. The data showed that, after instillation, the lungs contained the highest levels of HA, as expected, followed by the gastrointestinal tract. Smaller molecular weights of HA showed more rapid systemic distribution, while 67 and 215 kDa HA showed longer persistence in the lungs. Lung exposure appeared to be optimum in this size range due to the rapid absorption of <67 kDa HA and the poor lung penetration and mucociliary clearance of viscous solutions of HA > 215 kDa. The versatility of HA molecular weight and conjugation chemistries may, therefore, provide new opportunities to extend pulmonary drug exposure and potentially facilitate access to lymph nodes draining the pulmonary bed.

Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies.

UNLABELLED: When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H2O2) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H2O2, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H2O2 was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H2O2. The consumption of H2O2 by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H2O2. However, mAb formulation excipients may affect the rate of H2O2 consumption. mAb formulations containing trehalose or sucrose had faster H2O2 consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H2O2 spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H2O2 level. Our kinetic modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study to support the use of VPHP isolator for antibody drug product manufacture. LAY ABSTRACT: Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H2O2) spiking studies may be performed to determine the sensitivity of monoclonal antibody (mAb) drug products to residual peroxide in the isolator. In this study, mAbs were spiked with H2O2; an increase in methionine (Met) oxidation of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Met and H2O2 was 1:1, and its rate was dependent on mAb and H2O2 concentrations. Consumption of H2O2 by Met followed pseudo first-order kinetics; the rate was proportional to mAb concentration. Formulations containing trehalose or sucrose had faster consumption rates than formulations without the sugars, which could be due to excipient impurities. Based on H2O2 spiking study results, we can predict the amount of Met oxidation for a given mAb concentration and H2O2 level. Our modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study that supports using VPHP isolators during manufacture of mAb products.

Pulmonary Delivery of Vancomycin Dry Powder Aerosol to Intubated Rabbits.

Antibiotic multiresistant pneumonia is a risk associated with long-term mechanical ventilation. Vancomycin is commonly prescribed for methicillin-resistant Staphylococcus aureus infections; however, current formulations of vancomycin are only given intravenously. High doses of vancomycin have been associated with severe renal toxicity. In this study, we characterized dry powder vancomyin as a potential inhaled therapeutic aerosol and compared pharmacokinetic profiles of iv and pulmonary administered vancomycin in intubated rabbits through an endotracheal tube system. Cascade impaction studies indicated that using an endotracheal tube, which bypasses deposition in the mouth and throat, increased the amount of drug entering the lung. Bypassing the endotracheal tube with a catheter further enhanced drug deposition in the lung. Interestingly, intubated rabbits administered 1 mg/kg vancomycin via inhalation had similar AUC to rabbits that were administered 1 mg/kg vancomycin via a single bolus iv infusion; however, inhalation of vancomycin reduced Cmax and increased Tmax, indicating that inhaled vancomycin resulted in more sustained pulmonary levels of vancomycin. Collectively, these results suggested that dry powder vancomycin can successfully be delivered by pulmonary inhalation in intubated patients. Furthermore, as inhaled vancomycin is delivered locally to the site of pulmonary infection, this delivery route could reduce the total dose required for therapeutic efficacy and simultaneously reduce the risk of renal toxicity by eliminating the high levels of systemic drug exposure required to push the pulmonary dose to therapeutic thresholds during iv administration.

Routes of administration and dose optimization of soluble antigen arrays in mice with experimental autoimmune encephalomyelitis.

Soluble antigen arrays (SAgAs) were developed for treating mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. SAgAs are composed of hyaluronan with grafted EAE antigen and LABL peptide (a ligand of ICAM-1). SAgA dose was tested by varying injection volume, SAgA concentration, and administration schedule. Routes of administration were explored to determine the efficacy of SAgAs when injected intramuscularly, subcutaneously, intraperitoneally, intravenously, or instilled into lungs. Injections proximal to the central nervous system (CNS) were compared with distal injection sites. Intravenous dosing was included to determine if SAgA efficiency results from systemic exposure. Pulmonary instillation (p.i.) was included as reports suggest T cells are licensed in the lungs before moving to the CNS. Decreasing the volume of injection or SAgA dose reduced treatment efficacy. Treating mice with a single injection on day 4, 7, and 10 also reduced efficacy compared with injecting on all three days. Surprisingly, changing the injection site did not lead to a significant difference in efficacy. Intravenous administration showed efficacy similar to other routes, suggesting SAgAs act systemically. When SAgAs were delivered via p.i., however, EAE mice failed to develop any symptoms, suggesting a unique lung mechanism to ameliorate EAE in mice.

NanoClusters surface area allows nanoparticle dissolution with microparticle properties.

Poorly water-soluble drugs comprise the majority of new drug molecules. Nanoparticle agglomerates, called NanoClusters, can increase the dissolution rate of poorly soluble compounds by increasing particle surface area. Budesonide and danazol, two poorly soluble steroids, were studied as model compounds. NanoCluster suspensions were made using a Netzsch MiniCer media mill with samples collected between 5 and 15 h and lyophilized. Differential scanning calorimetry (DSC) and powder X-ray Diffraction were used to evaluate the physicochemical properties of the powders, and Brunauer, Emmett and Teller (BET) analysis was used to determine surface area. Scanning electron microscopy confirmed NanoClusters were between 1 and 5 µm. NanoCluster samples showed an increase in dissolution rate compared with the micronized stock and similar to a dried nanoparticle suspension. BET analysis determined an increase in surface area of eight times for budesonide NanoClusters and 10-15 times for danazol NanoClusters compared with the micronized stock. Melting temperatures decreased with increased mill time of NanoClusters by DSC. The increased surface area of NanoClusters provides a potential micron-sized alternative to nanoparticles to increase dissolution rate of poorly water-soluble drugs.

Ytterbocene charge-transfer molecular wire complexes.

A systematic study of the novel charge-transfer [(f)14-(pi)0-(f)14 --> (f)13-(pi)2-(f)13] electronic state found in 2:1 metal-to-ligand adducts of the type [(Cp)2Yb](BL)[Yb(Cp)2] [BL = tetra(2-pyridyl)pyrazine (tppz) (1), 6',6' '-bis(2-pyridyl)-2,2':4',4'':2'',2'''-quaterpyridine (qtp) (2), 1,4-di(terpyridyl)-benzene (dtb) (3), Cp = (C5Me5)] has been conducted with the aim of determining the effects of increased Yb-Yb separation on the magnetic and electronic properties of these materials. The neutral [(f)13-(pi)2-(f)13], cationic [(f)13-(pi)1-(f)13] and dicationic [(f)13-(pi)0-(f)13] states of these complexes were studied by cyclic voltammetry, UV-vis-NIR electronic absorption spectroscopy, NMR, X-ray crystallography, and magnetic susceptibility measurements. The spectroscopic and magnetic data for the neutral bimetallic complexes is consistent with an [(f)13(pi)2(f)13] ground-state electronic configuration in which each ytterbocene fragment donates one electron to give a singlet dianionic bridging ligand with two paramagnetic Yb(III) centers. The voltammetric data demonstrate that the electronic interaction in the neutral molecular wires 1-3, as manifested in the separation between successive metal reduction waves, is large compared to analogous transition metal systems. Electronic spectra for the neutral and monocationic bimetallic species are dominated by pi-pi and pi-pi transitions, masking the f-f bands that are expected to best reflect the electronic metal-metal interactions. However, these metal-localized transitions are observed when the electrons are removed from the bridging ligand via chemical oxidation to yield the dicationic species, and they suggest very little electronic interaction between metal centers in the absence of pi electrons on the bridging ligands. Analysis of the magnetic data reveals that the qtp complex displays antiferromagnetic coupling of the type Yb(alpha)(alphabeta)Yb(beta) at approximately 13 K.

Ligand substituent effect observed for ytterbocene 4'-cyano-2,2':6',2' '-terpyridine.

A new N-heterocyclic complex of ytterbocene (Cp(2)Yb(II), Cp = C(5)Me(5)) has been prepared by the addition of 4'-cyano-2,2':6',2' '-terpyridine (tpyCN) to Cp(2)Yb(II)(OEt(2)) in toluene to give a dark blue species designated as Cp(2)Yb(tpyCN). The effect of the electron-withdrawing group (-CN) on the redox potentials of the charge-transfer form of this complex [in which an electron is transferred from the f(14) metal center to the lowest unoccupied (pi) molecular orbital of the tpyCN ligand to give a 4f(13)-pi(1) electronic configuration] has been quantified by cyclic voltammetry. The tpyCN ligand stabilizes this configuration by 60 mV more than that in the unsubstituted tpy ligand complex and by 110 mV more than that in the unsubstituted bpy ligand complex. Magnetic susceptibility measurements corroborate the enhanced stabilization of the 4f(13)-pi(1) configuration by the substituted terpyridyl ligand complex. Furthermore, the temperature dependence of the magnetic data is most consistent with a thermally induced valence tautomeric equilibrium between this paramagnetic 4f(13)-pi(1) form that dominates near room temperature and the diamagnetic 4f(14)-pi(0) form that dominates at low temperature. Differing coordination modes for the tpyCN ligand to the ytterbocene center have also been confirmed by isolation and X-ray crystallographic characterization of complexes binding through either the cyano nitrogen of tpyCN or the three terpyridyl nitrogen atoms of tpyCN.

Toward new paradigms in mixed-valency: ytterbocene-terpyridine charge-transfer complexes.

(C5Me5)2Yb x OEt2 reacts with terpyridine and tetrapyridinylpyrazine to afford new mixed-valent systems.

Electrochemical and spectroscopic characterization of the novel charge-transfer ground state in diimine complexes of ytterbocene.

The novel charge-transfer ground state found in alpha,alpha'-diimine adducts of ytterbocene (C(5)Me(5))(2)Yb(L) [L = 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen)] in which an electron is spontaneously transferred from the f(14) metal center into the lowest unoccupied (pi*) molecular orbital (LUMO) of the diimine ligand to give an f(13)-L(*)(-) ground-state electronic configuration has been characterized by cyclic voltammetry, UV-vis-near-IR electronic absorption, and resonance Raman spectroscopies. The voltammetric data demonstrate that the diimine ligand LUMO is stabilized and the metal f orbital is destabilized by approximately 1.0 V each upon complexation for both bpy and phen adducts. The separation between the ligand-based oxidation wave (L(0/-)) and the metal-based reduction wave (Yb(3+/2+)) in the ytterbocene adducts is 0.79 V for both bpy and phen complexes. The UV-vis-near-IR absorption spectroscopic data for both the neutral adducts and the one-electron-oxidized complexes are consistent with those reported recently, but previously unreported bands in the near-IR have been recorded and assigned to ligand (pi*)-to-metal (f orbital) charge-transfer (LMCT) transitions. These optical electronic excited states are the converse of the ground-state charge-transfer process (e.g., f(13)-L(*-) <--> f(14)-L(0)). These new bands occur at approximately 5000 cm(-1) in both adducts, consistent with predictions from electrochemical data, and the spacings of the resolved vibronic bands in these transitions are consistent with the removal of an electron from the ligand pi* orbital. The unusually large intensity observed in the f --> f intraconfiguration transitions for the neutral phenanthroline adduct is discussed in terms of an intensity-borrowing mechanism involving the low-energy LMCT states. Raman vibrational data clearly reveal resonance enhancement for excitation into the low-lying pi* --> pi* ligand-localized excited states, and comparison of the vibrational energies with those reported for alkali-metal-reduced diimine ligands confirms that the ligands in the adducts are reduced radical anions. Differences in the resonance enhancement pattern for the modes in the bipyridine adduct with excitation into different pi* --> pi* levels illustrate the different nodal structures that exist in the various low-lying pi* orbitals.

Metalla-supramolecular rectangles as electron reservoirs for multielectron reduction and oxidation.

The electron-transfer capacity of molecular rectangle ions [Pt(II)(4)(PEt(3))(8)(mu-anth(2-))(2)(mu-L)(2)](4+) with anth = anthracene-1,8-diyl and L = 4,4'-bipyridine (bp) or 1,2-bis(4-pyridyl)ethene (bpe) was investigated in acetonitrile and dichloromethane using cyclic voltammetry, EPR, and UV-vis-near-IR spectroelectrochemistry. The compounds can be reversibly reduced, first in a two-electron process and then via two closely separated one-electron steps. Oxidation was also possible at rather low potentials in a reversible two-electron step, followed by an electrochemically irreversible process. The spectroscopic results indicate reduction at the neutral acceptor ligands L and oxidation at the formally dianionic anthracene "clips". In contrast, the prototypical molecular square ([Pt(triphos)(mu-bp)](4))(8+) undergoes only irreversible reduction.

Coordination-driven self-assembly: solids with bidirectional porosity.

Coordination-driven self-assembly reactions have been used in the preparation of a variety of discrete supramolecular species, some of which have shown promise as synthetic receptors. Many highly ordered coordination polymers and porous networks have been prepared in a similar fashion. While a few of these solids are capable of the uptake of small organic molecules in the resultant molecular channels, the formation of truly porous structures has frequently been thwarted by lattice interpenetration. A strategy for the formation of porous solids that may circumvent this problem is based on the covalent construction of nanoscale macrocycles which, when eclipsed in the solid state, may lead to porous, tubular assemblies. We have incorporated these concepts toward the realization of a bidirectionally porous solid. The metal-directed, self-assembly of a conjugated, macrocyclic ligand provides a discrete, supramolecular entity in solution and the solid state. X-ray crystallographic analysis establishes that this assembly packs such that bidirectional channels are realized, and the incorporation of only ClCH2CH2Cl into the crystal lattice demonstrates that these channels are potentially suitable for the selective uptake of small organic guests.

Self-assembly of molecular prisms via an organometallic "clip".

[reaction: see text] Under the appropriate conditions, the combination of two tritopic pyridyl ligands with three metal-containing molecular "clips" spontaneously generates supramolecular coordination cages with trigonal prismatic frameworks.

Self-assembly of nanoscopic coordination cages of D(3h) symmetry.

A family of nanoscale-sized supramolecular cage compounds with a trigonal prismatic framework was prepared by means of spontaneous self-assembly from the combination of a predesigned molecular "clip" with tritopic pyridyl subunits. As confirmed by x-ray crystallography, the smallest structure of the reported series is approximately 1 x 2 nm and possesses a nitrate anion incarcerated inside its molecular cavity. The largest structure has dimensions of approximately 1 x 4 nm.