Composites Containing Nanohydroxyapatites and a Stable TEMPO Radical: Preparation and Characterization Using Spectrophotometry, EPR and 1H MAS NMR.

Hydroxyapatite is the main constituent of mammalian hard tissues. Basic applications of synthetic hydroxyapatites include bone and dental implantology and drug delivery systems. The study of hydroxyapatite surface properties could give greater insight into the processes of bone mineralization and degradation. Nitroxide radicals are stable radicals that exhibit anticancer and antioxidative properties and are often used as spin probes to study the dynamics of complex biological systems. In this work, we attempted to adsorb the stable 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) on two hydroxyapatites (HAs) differing in specific surface area and the degree of hydration. The adsorption was carried out from cyclohexane, 1-chlorobutane and water. The solutions after adsorption were studied spectrophotometrically, while the obtained composites were characterized via NMR and EPR spectroscopy. The results show that it is possible to reproducibly obtain fairly stable composites, where the main factors influencing the adsorbed amount of the radical are solvent polarity and specific surface area of hydroxyapatite. The Langmuir isotherm was determined to be the most suitable adsorption model. The analysis of EPR and NMR spectra allowed us to determine the distribution of the TEMPO molecules on the hydroxyapatite surface, as well as a probable adsorption mechanism. The HA/TEMPO composites could potentially be used to study certain properties of hydroxyapatite surfaces with EPR spectroscopy. They could also be used as fillers after hard tissue surgery, as well as metal-free MRI contrasts.

The solid-state proton NMR study of bone using a dipolar filter: apatite hydroxyl content versus animal age.

The hydroxyl content of bone apatite mineral has been measured using proton solid-state NMR performed with a multiple-pulse dipolar filter under slow magic angle spinning (MAS). This new method succeeded in resolving and relatively enhancing the main hydroxyl peak at ca. 0 ppm from whole bone, making it amenable to rigorous quantitative analysis. The proposed methodology, involving line fitting, the measurement of the apatite concentration in the studied material and adequate calibration, was proved to be convenient and suitable for monitoring bone mineral hydroxylation in different species and over the lifetime of the animal. It was found that the hydroxyl content in the cranial bone mineral of pig and rats remained in the 5-10% range, with reference to stoichiometric hydroxyapatite. In rats, the hydroxyl content showed a non-monotonic increase with age, which was governed by biological processes rather than by chemical, thermodynamically driven apatite maturation.

Synthesis and characterization of MCM-48/hydroxyapatite composites for drug delivery: Ibuprofen incorporation, location and release studies.

MCM-48 is crystalline, mesoporous silicate used in catalysis and for adsorption various molecules to create useful materials for chemistry, pharmacy and medicine. In this work, the MCM-48 silicate has been coated with nanohydroxyapatite (HA) to design a new drug delivery system. The MCM-48/hydroxyapatite composite was synthesized using a new method involving precipitation of HA onto the earlier prepared MCM-48. The method gives an interesting mesoporous material capable of adsorbing drugs on its external surface and on the internal surface in mesopores. This was shown using ibuprofen (Ibu). The MCM-48/HA composites and MCM-48/HA/Ibu conjugates have been thoroughly characterized using various physicochemical methods. It was concluded that the MCM-48/HA composite offers a significant potential for controlled drug delivery systems involving small API molecules.

Pharmaceutical cocrystals, salts and polymorphs: Advanced characterization techniques.

The main goal of a novel drug development is to obtain it with optimal physiochemical, pharmaceutical and biological properties. Pharmaceutical companies and scientists modify active pharmaceutical ingredients (APIs), which often are cocrystals, salts or carefully selected polymorphs, to improve the properties of a parent drug. To find the best form of a drug, various advanced characterization methods should be used. In this review, we have described such analytical methods, dedicated to solid drug forms. Thus, diffraction, spectroscopic, thermal and also pharmaceutical characterization methods are discussed. They all are necessary to study a solid API in its intrinsic complexity from bulk down to the molecular level, gain information on its structure, properties, purity and possible transformations, and make the characterization efficient, comprehensive and complete. Furthermore, these methods can be used to monitor and investigate physical processes, involved in the drug development, in situ and in real time. The main aim of this paper is to gather information on the current advancements in the analytical methods and highlight their pharmaceutical relevance.

Convenient UV-spectrophotometric determination of citrates in aqueous solutions with applications in the pharmaceutical analysis of oral electrolyte formulations.

Herein, we present a convenient method for quantitative spectrophotometric determination of citrate ions in aqueous solutions in the middle-UV range. It involves measuring the absorbance of citric acid at 209 nm under suppressed dissociation at pH < 1.0 in the presence of hydrochloric acid. Validation of the method was performed according to the guidelines of the International Conference on Harmonization. A very good linear dependence of the absorbance on concentration (r2 = 0.9999) was obtained in a citrate concentration range of 0.5-5.0 mmol/L. This method is characterized by excellent precision and accuracy; the coefficient of variation in each case is below the maximal permissible value (%RSD < 2). The proposed analytical procedure has been successfully applied to the determination of citrates in oral electrolyte formulations.

Pharmaceutical properties of two ethenzamide-gentisic acid cocrystal polymorphs: Drug release profiles, spectroscopic studies and theoretical calculations.

The aim of this study was to evaluate the stability and solubility of the polymorphic forms of the ethenzamide (ET) - gentisic acid (GA) cocrystals during standard technological processes leading to tablet formation, such as compression and excipient addition. In this work two polymorphic forms of pharmaceutical cocrystals (ETGA) were characterized by 13C and 15N solid-state nuclear magnetic resonance and Fourier transformed infrared spectroscopy. Spectroscopic studies were supported by gauge including projector augmented wave (GIPAW) calculations of chemical shielding constants.Polymorphs of cocrystals were easily identified and characterized on the basis of solid-state spectroscopic studies. ETGA cocrystals behaviour during direct compressionand tabletting with excipient addition were tested. In order to choose the best tablet composition with suitable properties for the pharmaceutical industry dissolution profile studies of tablets containing polymorphic forms of cocrystals with selected excipients were carried out.

Prazosin-Conjugated Matrices Based on Biodegradable Polymers and α-Amino Acids--Synthesis, Characterization, and in Vitro Release Study.

Novel and promising macromolecular conjugates of the α1-adrenergic blocker prazosin were directly synthesized by covalent incorporation of the drug to matrices composed of biodegradable polymers and α-amino acids for the development of a polymeric implantable drug delivery carrier. The cyto- and genotoxicity of the synthesized matrices were evaluated using a bacterial luminescence test, protozoan assay, and Salmonella typhimurium TA1535. A new urethane bond was formed between the hydroxyl end-groups of the synthesized polymer matrices and an amine group of prazosin, using 1,1'-carbonyldiimidazole (CDI) as a coupling agent. The structure of the polymeric conjugates was characterized by various spectroscopy techniques. A study of hydrogen nuclear magnetic resonance ((1)H-NMR) and differential scanning calorimetry (DSC) thermodiagrams indicated that the presence of prazosin pendant groups in the macromolecule structures increased the polymer's rigidity alongside increasing glass transition temperature. It has been found that the kinetic release of prazosin from the obtained macromolecular conjugates, tested in vitro under different conditions, is strongly dependent on the physicochemical properties of polymeric matrices. Furthermore, the presence of a urethane bond in the macromolecular conjugates allowed for obtaining a relatively controlled release profile of the drug. The obtained results confirm that the pharmacokinetics of prazosin might be improved through the synthesis of polymeric conjugates containing biomedical polymers and α-amino acids in the macromolecule.

Near-Infrared (NIR) Spectroscopy of Synthetic Hydroxyapatites and Human Dental Tissues.

Near-infrared spectroscopy (NIR) was used to analyze synthetic hydroxyapatite calcined at various temperatures, synthetic carbonated hydroxyapatite, and human hard dental tissues (enamel and dentin). The NIR bands of those materials in the combination, first-overtone, and second-overtone spectral regions were assigned and evaluated for structural characterization. They were attributed to adsorbed and structural water, structural hydroxyl (OH) groups and surface P-OH groups. The NIR spectral features were quantitatively discussed in view of proton solid-state magic-angle spinning nuclear magnetic resonance ((1)H MAS NMR) results. We conclude that the NIR spectra of apatites are useful in the structural characterization of synthetic and biogenic apatites.

Crystal structures of tiotropium bromide and its monohydrate in view of combined solid-state nuclear magnetic resonance and gauge-including projector-augmented wave studies.

Tiotropium bromide is an anticholinergic bronchodilator used in the management of chronic obstructive pulmonary disease. The crystal structures of this compound and its monohydrate have been previously solved and published. However, in this paper, we showed that those structures contain some major errors. Our methodology based on combination of the solid-state nuclear magnetic resonance (NMR) spectroscopy and quantum mechanical gauge-including projector-augmented wave (GIPAW) calculations of NMR shielding constants enabled us to correct those errors and obtain reliable structures of the studied compounds. It has been proved that such approach can be used not only to perform the structural analysis of a drug substance and to identify its polymorphs, but also to verify and optimize already existing crystal structures.

Solid-state NMR as an effective method of polymorphic analysis: solid dosage forms of clopidogrel hydrogensulfate.

Clopidogrel hydrogensulfate (HSCL) is an antiplatelet agent, one of top-selling drugs in the world. In this paper, we have described a rapid and convenient method of verification which polymorph of HSCL is present in its final solid dosage form. Our methodology based on solid-state NMR spectroscopy and ab initio gauge-including projector-augmented wave calculations of NMR shielding constants is appropriate for currently available commercial solid dosage forms of HSCL. Furthermore, such structural characterization can assist with the development of new pharmaceutical products containing HSCL and also be useful in the identification of counterfeit drugs.

Solid-state NMR studies of theophylline co-crystals with dicarboxylic acids.

In this work, three polycrystalline materials containing co-crystals of theophylline with malonic, maleic, and glutaric acids were studied using (13)C, (15)N and (1)H solid-state NMR and FT-IR spectroscopy. The NMR assignments were supported by gauge including projector augmented waves (GIPAW) calculations of chemical shielding, performed using X-ray determined geometry. The experimental (13)C cross polarization/magic angle spinning (CP/MAS) NMR results and the calculated isotropic chemical shifts were in excellent agreement. A rapid and convenient method for theophylline co-crystals crystal structure analysis has been proposed for co-crystals, which are potentially new APIs.

Ampicillin-ester bonded branched polymers: characterization, cyto-, genotoxicity and controlled drug-release behaviour.

The development and characterization of novel macromolecular conjugates of ampicillin using branched biodegradable polymers has been described in this study. The conjugates have been prepared coupling the ß-lactam antibiotic with branched polymer matrices based on the natural oligopeptide core. The cyto- and genotoxicity of the synthesized polymers were evaluated with a bacterial luminescence test, two protozoan assays and Salmonella typhimurium TA1535. The presence of a newly formed covalent bond between the drug and the polymer matrices was confirmed by 1H-NMR and FTIR studies. A drug content (15.6 and 10.2 mole %) in the macromolecular conjugates has been determined. The obtained macromolecular products have been subjected to further in vitro release studies. The total percentage of ampicillin released after 21 days of incubation was nearly 60% and 14% and this resulted from the different physicochemical properties of the polymeric matrices. This is the first report on the application of branched biodegradable polymeric matrices for the covalent conjugation of ampicillin. The obtained results showed that the synthesized macromolecular drug-conjugates might slowly release the active drug molecule and improve the pharmacokinetics of ampicillin.

Comparative proton nuclear magnetic resonance studies of amantadine complexes formed in aqueous solutions with three major cyclodextrins.

Host-guest complexes of alpha-, beta-, and gamma-cyclodextrins (α-CD, ß-CD, and γ-CD, respectively) with amantadine (1-aminoadamantane, AMA; an antiviral agent) were characterized in aqueous solutions using proton nuclear magnetic resonance (NMR) spectroscopy. Host-guest molecular interactions were manifested by changes in the chemical shifts of AMA protons. NMR Job's plots showed that the stoichiometry of all the studied complexes was 1:1. Two-dimensional T-ROESY experiments demonstrated that the complexes were formed by different degrees of incorporation of the adamantyl group of AMA into the CD cavity. The mode of AMA binding was proposed. The AMA molecule came into the α-CD cavity (the smallest size) or ß-CD cavity (the intermediate size) through its wide entrance to become shallowly or deeply accommodated, respectively. In the complex of AMA with γ-CD (the largest cavity size), the adamantyl group was also quite deeply inserted into the CD cavity, but it arrived there through the narrow cavity entrance. It was found that the adamantyl group of AMA was best accommodated by the ß-CD cavity. The binding constants Kaa of the studied complexes (in M(-1) ), determined from DOSY NMR, were fairly high; their values in an ascending order were: α-CD (183) < γ-CD (306) ≪ ß-CD (5150).

Solid-state MAS NMR, TEM, and TGA studies of structural hydroxyl groups and water in nanocrystalline apatites prepared by dry milling.

A series of nanocrystalline calcium hydroxyapatites was prepared by dry milling and characterized using proton and 31P MAS NMR, TEM, and TGA methods. The samples contained stubby rod-shaped crystals, which length and width varied in the 130-30 and 95-20 nm ranges, respectively. It was confirmed that concentration of structural hydroxyl groups in nanocrystalline apatites decreases with the decreasing crystal size. In the series of the studied apatites, the decrease was from 86 to ca. 50 % in reference to stoichiometric apatite. Water was found in the surface hydrated layer and in the c-axis channels, in which compartments existed as adsorbed and structural, respectively. Molecules of the adsorbed water were capable of moving from the crystal surface into the lattice c-axis channels of apatite. This process introduced considerable structural disorder within and around those channels and reduced the content of the structural hydroxyl groups, particularly in the region underneath the apatite crystal surface.

Solid-state NMR and IR characterization of commercial xenogeneic biomaterials used as bone substitutes.

Three commercial xenogeneic biomaterials (Gen-Os, Apatos Spongiosa and Apatos Cortical; all from Tecnoss Dental, Torino, Italy) originated from porcine bone were characterized by various analytical methods, such as powder X-ray diffraction (XRD), thermogravimetry (TGA), high-resolution solid-state nuclear magnetic resonance (ssNMR) and infrared spectroscopy (FT-IR). The studies were focused on structural properties and chemical compositions of the samples. It was found that the main constituents of the analyzed biomaterials were nanocrystalline apatite mineral, organic collagenous matrix and water. For comparison, synthetic carbonated hydroxyapatite and natural collagen type I from bovine tendon were used. Differences in various physicochemical parameters such as crystal size, specific surface area, concentration of structural hydroxyl groups, contents of CO(3)(2-) and HPO(4)(2-) ions and their location were discussed. It was shown that various techniques of ssNMR and elaborate analysis of the FT-IR spectra, applied together, provide valuable information on xenogeneic biomaterials.

Synthesis and properties of a star-shaped poly(ϵ-caprolactone)-ibuprofen conjugate.

A series of novel star-shaped poly(ϵ-caprolactone) (PCL) biodegradable polyesters were synthesized through ring-opening polymerization of ϵ-caprolactone in the presence of a poly(amidoamine) (PAMAM) dendrimer initiator. The polymers (PAMAM/PCLs) were obtained with a high yield (92%) and a number-average molecular weight of up to 14 000 g/mol. The nonlinear structure of PAMAM/PCLs was confirmed by nuclear magnetic resonance, gel-permeation chromatography, thermogravimetric analysis and differential scanning calorimetry. Thermal analysis indicated that the star-shaped PAMAM/PCLs had a melting point, degree of crystallinity, glass transition temperature and maximum decomposition temperature all lower than those of linear PCL. Ibuprofen (IBU), a popular non-steroidal anti-inflammatory drug, was co-valently (ester) bonded to the PAMAM/PCL molecules using the DCC/DMAP coupling method (DCC, N,N'-dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine). A high drug content (33.1 mol%) in the conjugate was obtained. The in-vitro drug-release profiles of IBU from the PAMAM/PCL/IBU conjugate were examined and found adequate for drug-carrier applications of our star-shaped polymers.

Preparation and characterization of polyester- and poly(ester-carbonate)-paclitaxel conjugates.

The polyester- and poly(ester-carbonate)-paclitaxel conjugates with low molecular weight were synthesized using dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) as catalysts. Polymeric matrices were obtained by ring-opening polymerization of ɛ-caprolactone (CL), rac-lactide (rac-LA), l-lactide (LLA) and trimethylene carbonate (TMC). The macromolecular conjugates were characterized by using spectroscopic techniques, such as (1)H, (13)C NMR and FTIR. The degree of degradation of polyester- and poly(ester-carbonate)-paclitaxel conjugates was tested in vitro under different conditions. The preliminary results of drug release were discussed.

Use of aliphatic poly(amide urethane)s for the controlled release of 5-fluorouracil.

The controlled release of 5-fluorouracil (5FU) from aliphatic poly(amide urethane)s (PURs) was studied in vitro. Linear PUR conjugates were prepared by the reaction between oligo(ɛ-caprolactone) (PCL), oligolactide (PLA), copolymers of ɛ-caprolactone (CL) and rac-lactide (rac-LA), dihydroxy(polyethylene adipate) (OEDA) with 1,6-diisocyanatohexane (HDI). Release of 5FU from PURs was found to depend on the nature of oligoester units and consist of soft and hard segments.

Kinetics of 1H→13C NMR cross-polarization in polymorphs and solvates of the antipsychotic drug olanzapine.

The (1)H→(13)C NMR cross-polarization (CP) was studied under magic-angle spinning at 7.5 kHz in various crystal forms of the antipsychotic drug olanzapine: two polymorphs (metastable I and stable II) and eight solvates containing organic solvent and water molecules. The CP kinetics followed the non-classical I-I(*)-S model, in which CP begins in a spin cluster of proximate abundant spins I(*) and rare spins S, then is controlled by spin diffusion of the abundant spins I from bulk to the I(*) spins of the spin cluster and finally is governed by spin-lattice relaxation of the abundant spins in the rotating frame. The corresponding CP kinetics parameters were determined and analyzed. It was demonstrated that the, λ and T(df) values (the CP time constant, the cluster composition parameter and the (1)H spin-diffusion constant, respectively) were very useful to discriminate the functional groups, especially in the 3D parameter space. In order to conveniently analyze the large amount (175) of the collected CP parameters, the number of the observed variables was reduced using the principal component (PC) analysis. The 2D plot of PC2 vs. PC1 showed adequate separation of the CH(3), CH(2), CH and C cases (C stands for carbons without adjacent hydrogens). It was demonstrated that those cases were located along the PC1 axis in the order of increasing (1)H-(13)C dipolar couplings: C

Synthesis and study of controlled release of ofloxacin from polyester conjugates.

New polymeric conjugates were prepared coupling ofloxacin to two-, three-, four and six-arm, star-shaped poly(ɛ-caprolactone) and polylactide. The homopolymers were synthesized via ring-opening polymerization of ɛ-caprolactone, l-lactide and rac-lactide in the presence of glycerol, penthaerythritol, dipentaerythritol and poly(ethylene glycol) as initiators and stannous octoate as a catalyst. The conjugates were characterized by GPC, MALDI-TOF MS, NMR, IR and viscosity methods. Content of Sn has been investigated in polymers by electrothermal atomic absorption spectrometry. Toxicity of monomers, initiators and polymers were evaluated with bacterial luminescence test and two protozoan assays. The in vitro release of ofloxacin from obtained conjugates in pH 7 was investigated.