Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Local delivery of minocycline-loaded PLGA nanoparticles from gelatin-coated neural implants attenuates acute brain tissue responses in mice.

BACKGROUND: Neural interfaces often elicit inflammatory responses and neuronal loss in the surrounding tissue which adversely affect the function and longevity of the implanted device. Minocycline, an anti-inflammatory pharmaceutics with neuroprotective properties, may be used for reducing the acute brain tissue responses after implantation. However, conventional administration routes require high doses which can cause adverse systemic side effects. Therefore, the aim of this study was to develop and evaluate a new drug-delivery-system for local and sustained administration of minocycline in the brain. METHODS: Stainless steel needles insulated with Parylene-C were dip-coated with non-crosslinked gelatin and minocycline-loaded PLGA nanoparticles (MC-NPs) were incorporated into the gelatin-coatings by an absorption method and subsequently trapped by drying the gelatin. Parylene-C insulated needles coated only with gelatin were used as controls. The expression of markers for activated microglia (CD68), all microglia (CX3CR1-GFP), reactive astrocytes (GFAP), neurons (NeuN) and all cell nuclei (DAPI) surrounding the implantation sites were quantified at 3 and 7 days after implantation in mice. RESULTS: MC-NPs were successfully incorporated into gelatin-coatings of neural implants by an absorption method suitable for thermosensitive drug-loads. Immunohistochemical analysis of the in vivo brain tissue responses, showed that MC-NPs significantly attenuate the activation of microglial cells without effecting the overall population of microglial cells around the implantation sites. A delayed but significant reduction of the astrocytic response was also found in comparison to control implants. No effect on neurons or total cell count was found which may suggest that the MC-NPs are non-toxic to the central nervous system. CONCLUSIONS: A novel drug-nanoparticle-delivery-system was developed for neural interfaces and thermosensitive drug-loads. The local delivery of MC-NPs was shown to attenuate the acute brain tissue responses nearby an implant and therefore may be useful for improving biocompatibility of implanted neuro-electronic interfaces. The developed drug-delivery-system may potentially also be used for other pharmaceutics to provide highly localized and therefore more specific effects as compared to systemic administration.

The allosteric modulation effects of doxycycline, minocycline, and their derivatives on the neuropeptide receptor PAC1-R.

Class B G-protein coupled receptors (GPCR) PAC1-R is a neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptor that mediates the effective neuroprotective activity. Based on our previous data showing that doxycycline and minocycline work as the positive allosteric modulator (PAM) of PAC1-R, we used computer molecular docking and isothermal titration calorimetry assay to further determine the bindings of doxycycline/minocycline's derivatives including tetracycline/tigecycline with the N-terminal extracellular domain of PAC1-R (PAC1-EC1). Then the cAMP assay combined with the PAC1-R natural agonist PACAP27 was used to confirm the possible PAM roles of the small-molecule antibiotics. The results showed that tetracycline/tigecycline had significant lower affinity to PAC1-EC1 than doxycycline/minocycline, which was consistent with their non-positive allosteric modulation activity on PAC1-R. Furthermore, by comparing the key residues contributing to the PAM binding with the predicted allosteric site in PAC1-EC1, we characterized four motifs contributing to PAM binding in PAC1-EC1. The site-directed mutation results showed that ASN60 played the most important role in the PAM binding of the small-molecule antibiotics, while ASP116 played a sensitive marginal role in the PAM binding. These results not only help to explain the clinical and experimental neuroprotective effects of doxycycline/minocycline, but also help to characterize the PAM binding site in PAC1-EC1, which will promote the screening and characterization of novel small-molecule PAMs targeting PAC1-EC1 with drug development potency in nerve system disease.

Preparation and Properties of Minocycline-Loaded Carboxymethyl Chitosan Gel/Alginate Nonwovens Composite Wound Dressings.

As derivatives from marine natural biomaterials, alginate-based and chitosan-based biomaterials are commonly used in wound dressings. Calcium alginate fiber (CAF) dressings possess excellent absorption and unique gel forming performance, but the low bioactivity limits its application in wound healing. Carboxymethyl chitosan (CM-Chit) has excellent antibacterial activity, but the gel structure with weak mechanical properties restricts its application. In this study, minocycline (Mino)/CM-Chit solution was coated on the surface of plasma treated CAF needle-punched nonwovens, and then Mino loaded CM-Chit gel/CAF nonwovens composite dressings were fabricated by EDC/NHS (1-3-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide) crosslinking. The dressings had a porous composite structure, which allowed them to quickly absorb and store a large number of wound exudates. Skin-like tensile performance allowed the dressings to provide a better healing environment. Antibacterial assay against Escherichia coli and Staphylococcus aureus indicated that the addition of Mino significantly improved the antibacterial activity of the wound dressings. The tight structure of CM-Chit gel prevented the burst release of Mino so that the dressings had antibacterial activity in a certain period of release time. Cell culture assay showed that the dressings had excellent cell biocompatibility. As new functional dressings, the prepared composite dressings had excellent potential in the clinical healing of wounds.

Kinetic analysis of the inhibition of the drug efflux protein AcrB using surface plasmon resonance.

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl ß-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Surface functionalization of TiO2 nanotubes with minocycline and its in vitro biological effects on Schwann cells.

BACKGROUND: Minocycline has been widely used in central nervous system disease. However, the effect of minocycline on the repairing of nerve fibers around dental implants had not been previously investigated. The aim of the present study was to evaluate the possibility of using minocycline for the repairing of nerve fibers around dental implants by investigating the effect of minocycline on the proliferation of Schwann cells and secretion of neurotrophic factors nerve growth factor and glial cell line-derived neurotrophic factor in vitro. METHODS: TiO2 nanotubes were fabricated on the surface of pure titanium via anodization at the voltage of 20, 30, 40 and 50 V. The nanotubes structure were characterized by scanning electron microscopy and examined with an optical contact angle. Then drug loading capability and release behavior were detected in vitro. The TiO2 nanotubes loaded with different concentration of minocycline were used to produce conditioned media with which to treat the Schwann cells. A cell counting kit-8 assay and cell viability were both selected to study the proliferative effect of the specimens on Schwann cell. Reverse transcription-quantitative PCR and western blot analyses were used to detect the related gene/protein expression of Schwann cells. RESULTS: The results showed that the diameter of TiO2 nanotubes at different voltage varied from 100 to 200 nm. The results of optical contact angle and releasing profile showed the nanotubes fabricated at the voltage of 30 V met the needs of the carrier of minocycline. In addition, the TiO2 nanotubes loaded with the concentration of 20 µg/mL minocycline increased Schwann cells proliferation and secretion of neurotrophic factors in vitro. CONCLUSIONS: The results suggested that the surface functionalization of TiO2 nanotubes with minocycline was a promising candidate biomaterial for the peripheral nerve regeneration around dental implants and has potential to be applied in improving the osseoperception of dental implant.

Determination of tigecycline in human plasma by LC-MS/MS and its application to population pharmacokinetics study in Chinese patients with hospital-acquired pneumonia.

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of tigecycline (TGC) in human plasma, using tigecycline-d9 as an internal standard (IS). Analytical samples were prepared using a protein precipitation method coupled with a concentration process. The analyte and IS were separated on a reversed-phase Waters Acquity UPLC® BEH-C18 column (2.1 × 50 mm i.d., 1.7 µm) with a flow rate of 0.25 mL/min. The mobile phase consisted of water, containing 0.2% formic acid (v/v) with 10 mm ammonium formate (A) and acetonitrile (B). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 586.2 → 513.1 and m/z 595.1 → 514.0 for TGC and IS, respectively. The linearity of the method was in the range of 10-5000 ng/mL. Intra- and inter-batch precision (CV) for TGC was <9.27%, and the accuracy ranged from 90.06 to 107.13%. This method was successfully applied to the analysis of samples from hospital-acquired pneumonia patients treated with TGC, and a validated population pharmacokinetic model was established. This developed method could be useful to predict pharmacokinetics parameters and valuable for further pharmacokinetics/pharmacodynamics studies.

Design of minocycline-containing starch nanocapsules for topical delivery.

Pharmaceutical research has been focussed on developing improved delivery systems while exploring new ways of using approved excipients. The present work investigated the potential of starch nanocapsules (StNC) as a topical delivery platform for hydrophilic antimicrobial drugs using minocycline hydrochloride (MH) as a model drug. Thus, a quality by design approach was used to assess the role of different factors that affect the main pharmaceutical properties of StNC prepared using an emulsification-solvent evaporation method. Full characterisation was performed in terms of particle size, encapsulation efficiency, morphology and physical stability at 5 ± 3 °C. Results show the surfactant and lipid contents play a major role in StNC particle size distribution. The MH loading only promoted minor changes upon StNC properties. Formulations were stable without variations on physicochemical properties. All tested formulations presented a zeta-potential of +33.6 ± 6.7 mV, indicating a good physical stability and evidencing that StNC are suitable nanocarriers for topical use.

Development and Evaluation of Minocycline Hydrochloride-Loaded In Situ Cubic Liquid Crystal for Intra-Periodontal Pocket Administration.

In the present study, an injectable in situ liquid crystal formulation was developed for local delivery of minocycline hydrochloride (MH) for chronic periodontitis treatment. The physicochemical properties, phase structures, in vitro drug release and pharmacodynamics of in situ liquid crystals were investigated. The optimal formulation (phytantriol (PT)/propylene glycol (PG)/water, 63/27/10, w/w/w) loaded with 20 mg/g MH was proved to be injectable. The precursor formulation can form a cubic phase gel in excess water in 6.97 ± 0.10 s. The results of in vitro drug release suggested the MH presented a sustained release for 4 days. Liquid crystal precursor formulation significantly reduced gingival index, probing depth and alveolar bone loss compared to the model group (p < 0.01). Besides, the pathological characteristics of model rats were improved. The results suggested that MH-loaded in situ cubic liquid crystal possessed of sustained release ability and periodontal clinical symptoms improvement. The developed in situ cubic liquid crystal may be a potentially carrier in the local delivery of MH for periodontal diseases.

The complexity of minocycline serum protein binding.

Objectives: Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Methods: Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. Results: The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. Conclusions: The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines.

Activated Microglia Targeting Dendrimer-Minocycline Conjugate as Therapeutics for Neuroinflammation.

Brain-related disorders have outmatched cancer and cardiovascular diseases worldwide as the leading cause of morbidity and mortality. The lack of effective therapies and the relatively dry central nervous system (CNS) drug pipeline pose formidable challenge. Superior, targeted delivery of current clinically approved drugs may offer significant potential. Minocycline has shown promise for the treatment of neurological diseases owing to its ability to penetrate the blood-brain barrier (BBB) and potency. Despite its potential in the clinic and in preclinical models, the high doses needed to affect a positive therapeutic response have led to side effects. Targeted delivery of minocycline to the injured site and injured cells in the brain can be highly beneficial. Systemically administered hydroxyl poly(amidoamine) (PAMAM) generation-6 (G6) dendrimers have a longer blood circulation time and have been shown to cross the impaired BBB. We have successfully prepared and characterized the in vitro efficacy and in vivo targeting ability of hydroxyl-G6 PAMAM dendrimer-9-amino-minocycline conjugate (D-mino). Minocycline is a challenging drug to carry out chemical transformations due to its inherent instability. We used a combination of a highly efficient and mild copper catalyzed azide-alkyne click reaction (CuAAC) along with microwave energy to conjugate 9-amino-minocycline (mino) to the dendrimer surface via enzyme responsive linkages. D-mino was further evaluated for anti-inflammatory and antioxidant activity in lipopolysaccharides-activated murine microglial cells. D-mino conjugates enhanced the intracellular availability of the drug due to their rapid uptake, suppressed inflammatory cytokine tumor necrosis factor α (TNF-α) production, and reduced oxidative stress by suppressing nitric oxide production, all significantly better than the free drug. Fluorescently labeled dendrimer conjugate (Cy5-D-mino) was systematically administered (intravenous, 55 mg/kg) on postnatal day 1 to rabbit kits with a clinically relevant phenotype of cerebral palsy. The in vivo imaging study indicates that Cy5-D-mino crossed the impaired blood-brain barrier and co-localized with activated microglia at the periventricular white matter areas, including the corpus callosum and the angle of the lateral ventricle, with significant implications for positive therapeutic outcomes. The enhanced efficacy of D-mino, when combined with the inherent neuroinflammation-targeting capability of the PAMAM dendrimers, may provide new opportunities for targeted drug delivery to treat neurological disorders.

Fungal Biotransformation of Tetracycline Antibiotics.

The commercial antibiotics tetracycline (3), minocycline (4), chlortetracycline (5), oxytetracycline (6), and doxycycline (7) were biotransformed by a marine-derived fungus Paecilomyces sp. to yield seco-cyclines A-H (9-14, 18 and 19) and hemi-cyclines A-E (20-24). Structures were assigned by detailed spectroscopic analysis, and in the case of 10 X-ray crystallography. Parallel mechanisms account for substrate-product specificity, where 3-5 yield seco-cyclines and 6 and 7 yield hemi-cyclines. The susceptibility of 3-7 to fungal biotransformation is indicative of an unexpected potential for tetracycline "degradation" (i.e., antibiotic resistance) in fungal genomes. Significantly, the fungal-derived tetracycline-like viridicatumtoxins are resistant to fungal biotransformation, providing chemical insights that could inform the development of new tetracycline antibiotics resistant to enzymatic degradation.

The Role of pH in Modulating the Electronic State Properties of Minocycline Drug and Its Inclusion within Micellar Carriers.

A detailed investigation of the spectral and photophysical properties of minocycline (MC) in water at different pHs, solvents of different polarity, and micellar surfactant solutions was carried out in this study. An unusual behavior was highlighted with respect to other tetracyclines due to the presence of an additional dimethylamino group in the MC molecular structure. In particular, four equilibrium constants associated with mono-deprotonation reactions were characterized by steady-state spectroscopy. Femtosecond time-resolved pump-probe and fluorescence up-conversion measurements allowed the dynamics of the lowest excited singlet state of the five different acid-base species of MC to be characterized in terms of lifetimes and transient spectra. Two emissive species associated with keto-enol tautomerism resulting from excited-state intramolecular proton transfer (ESIPT) were revealed with time constants of a few and tens of picoseconds. TD-DFT quantum mechanical calculations were also performed to define the state order and nature of the differently protonated species, together with their absorption spectra. The role of pH proved to be fundamental in modulating the drug charge and therefore the interaction with cationic micelles where the neutral form of MC, that is the biologically active one, resulted efficiently included.

Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis.

Here we present an X-ray crystallography structure of the clinically relevant tigecycline antibiotic bound to the 70S ribosome. Our structural and biochemical analysis indicate that the enhanced potency of tigecycline results from a stacking interaction with nucleobase C1054 within the decoding site of the ribosome. Single-molecule fluorescence resonance energy transfer studies reveal that, during decoding, tigecycline inhibits the initial codon recognition step of tRNA accommodation and prevents rescue by the tetracycline-resistance protein TetM.

Global in vitro activity of tigecycline and comparator agents: Tigecycline Evaluation and Surveillance Trial 2004-2013.

BACKGROUND: The Tigecycline Evaluation and Surveillance Trial (TEST) is a global antimicrobial susceptibility surveillance study which has been ongoing since 2004. This report examines the in vitro activity of tigecycline and comparators against clinically important pathogens collected globally between 2004 and 2013. METHODS: Antimicrobial susceptibility was determined using guidelines published by the Clinical and Laboratory Standards Institute. The Cochran Armitage Trend Test was used to identify statistically significant changes in susceptibility between 2004 and 2013. RESULTS: Among the Enterobacteriaceae susceptibility was highest to the carbapenems [imipenem 97.1% (24,655/25,381), meropenem 97.0% (90,714/93,518)], tigecycline (97.0%, 115,361/118,899) and amikacin (96.9%, 115,200/118,899). Against Acinetobacter baumannii the highest rates of susceptibility were for minocycline (84.5%, 14,178/16,778) and imipenem (80.0%, 3,037/3,795). The MIC90 for tigecycline was 2 mg/L. 40% (6,743/16,778) of A. baumannii isolates were multidrug-resistant. Enterococci were highly susceptible to tigecycline and linezolid (>99%); vancomycin resistance was observed among 2% of Enterococcus faecalis (325/14,615) and 35% of Enterococcus faecium (2,136/6,167) globally. 40% (14,647/36,448) of Staphylococcus aureus were methicillin-resistant while 15% (2,152/14,562) of Streptococcus pneumoniae were penicillin-resistant. Against S. aureus and S. pneumoniae susceptibility to linezolid, vancomycin, and tigecycline was ≥99.9%. Globally, 81% (331/410) of statistically significant susceptibility changes during the study period were decreases in susceptibility. CONCLUSIONS: Amikacin, the carbapenems, and tigecycline were active against most gram-negative pathogens while linezolid, tigecycline, and vancomycin retained activity against most gram-positive pathogens collected in TEST during 2004-2013.

Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop.

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.

Comparison of bond strength of self-etch adhesive to pulp chamber dentin after placement of calcium hydroxide and various antibiotic pastes.

OBJECTIVE: The aim of this study was to evaluate the effect of calcium hydroxide, double antibiotic paste (DAP) and triple antibiotic paste (TAP) with minocycline, cefaclor and amoxicillin on the micro tensile bond strength (µTBS) of self-etch adhesive to pulp chamber dentin. MATERIALS AND METHODS: Sixty mandibular first molars were cut horizontally and randomly divided into a control group and five experimental groups, which received an intra-canal dressing, as follows: calcium hydroxide, DAP, TAP with minocycline, TAP with cefaclor and TAP with amoxicillin. After storing the specimens for 4 weeks, the medicaments were removed by irrigation with 10 mL each of the following solution: 2.5% NaOCl, 17% EDTA and distilled water. A self-etch adhesive (Clearfil S3 Bond, Okayama, Japan) was applied and composite resin (Clearfil Majesty Posterior, Kuraray Medical Inc., Japan) was placed into the cavity. A µTBS test was performed on each specimen using a universal test machine. RESULTS: The DAP reduced the µTBS of self-etch adhesive compared to the control group, calcium hydroxide and TAP with minocycline and with cefaclor (p < 0.05). However, the other medicaments did not result in a decreased µTBS of self-etch adhesive to pulp chamber dentin as compared to the control group (p > 0.05). CONCLUSIONS: The use of DAP resulted in a reduced µTBS of self-etch adhesive to pulp chamber dentin.

Switch-loop flexibility affects transport of large drugs by the promiscuous AcrB multidrug efflux transporter.

Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å(2) are less well transported than other substrates.

New thermoresistant polymorph from CO2 recrystallization of minocycline hydrochloride.

PURPOSE: To prepare and thoroughly characterize a new polymorph of the broad-spectrum antibiotic minocycline from its hydrochloride dehydrate salts. METHODS: The new minocycline hydrochloride polymorph was prepared by means of the antisolvent effect caused by carbon dioxide. Minocycline recrystallized as a red crystalline hydrochloride salt, starting from solutions or suspensions containing CO2 and ethanol under defined conditions of temperature, pressure and composition. RESULTS: This novel polymorph (ß-minocycline) revealed characteristic PXRD and FTIR patterns and a high melting point (of 247 ºC) compared to the initial minocycline hydrochloride hydrates (α-minocycline). Upon dissolution the new polymorph showed full anti-microbial activity. Solid-state NMR and DSC studies evidenced the higher chemical stability and crystalline homogeneity of ß-minocycline compared to the commercial chlorohydrate powders. Molecular structures of both minocyclines present relevant differences as shown by multinuclear solid-state NMR. CONCLUSIONS: This work describes a new crystalline structure of minocycline and evidences the ability of ethanol-CO2 system in removing water molecules from the crystalline structure of this API, at modest pressure, temperature and relatively short time (2 h), while controlling the crystal habit. This process has therefore the potential to become a consistent alternative towards the control of the solid form of APIs.

Comparative study to investigate the effect of meloxicam or minocycline HCl in situ gel system on local treatment of periodontal pockets.

In situ gelling formulations allow easy application to the target area. Gelation is induced by physiological stimuli at the site of application where the formula attains semisolid properties and exerts sustained drug release. In situ gelling formulations containing either 3% meloxicam (Mx) or 2% minocycline HCl (MH) were prepared for local application into the periodontal pockets. Gel formulations were based on the thermosensitive Pluronic(®) (Pl) and the pH-sensitive Carbopol(®) (C) polymers. C gels were prepared in combination with HPMC (H) to decrease its acidity. The total percent drug released from Pl formulae was 21.72% after 1 week for Mx and 85% after 3 days for MH. Their release kinetics data indicated anomalous non-Fickian behavior that could be controlled by both diffusion and chain relaxation. Addition of MH to C/H gels (1:2.5) resulted in liquefaction, followed by drug precipitation. Regarding C/H gel containing Mx, it showed a prolonged release rate up to 7 days with an initial burst effect; the kinetics data revealed Fickian-diffusion mechanism. The in vitro antibacterial activity studies for MH gel in Pl revealed that the drug released exceeded the minimum inhibitory concentration (MIC) of MH against Staphylococcus aureus ATCC 6538; placebo gel showed no effect on the microorganism. Clinical evaluation of Pl gels containing either Mx or MH showed significant improvement in chronic periodontitis patients, manifested by decrease in pocket depth and gingival index and increase in bone density.