The gut microbiome influences the bioavailability of olanzapine in rats.

BACKGROUND: The role of the gut microbiome in the biotransformation of drugs has recently come under scrutiny. It remains unclear whether the gut microbiome directly influences the extent of drug absorbed after oral administration and thus potentially alters clinical pharmacokinetics. METHODS: In this study, we evaluated whether changes in the gut microbiota of male Sprague Dawley rats, as a result of either antibiotic or probiotic administration, influenced the oral bioavailability of two commonly prescribed antipsychotics, olanzapine and risperidone. FINDINGS: The bioavailability of olanzapine, was significantly increased (1.8-fold) in rats that had undergone antibiotic-induced depletion of gut microbiota, whereas the bioavailability of risperidone was unchanged. There was no direct effect of microbiota depletion on the expression of major CYP450 enzymes involved in the metabolism of either drug. However, the expression of UGT1A3 in the duodenum was significantly downregulated. The reduction in faecal enzymatic activity, observed during and after antibiotic administration, did not alter the ex vivo metabolism of olanzapine or risperidone. The relative abundance of Alistipes significantly correlated with the AUC of olanzapine but not risperidone. INTERPRETATION: Alistipes may play a role in the observed alterations in olanzapine pharmacokinetics. The gut microbiome might be an important variable determining the systemic bioavailability of orally administered olanzapine. Additional research exploring the potential implication of the gut microbiota on the clinical pharmacokinetics of olanzapine in humans is warranted. FUNDING: This research is supported by APC Microbiome Ireland, a research centre funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan (grant no. 12/RC/2273 P2) and by Nature Research-Yakult (The Global Grants for Gut Health; Ref No. 626891).

A Simultaneous Differential Scanning Calorimetry-X-ray Diffraction Study of Olanzapine Crystallization from Amorphous Solid Dispersions.

Amorphous solid dispersions (ASDs) of class II and IV biopharmaceutics classification system drugs in water-miscible polymers are a well-recognized means of enhancing dissolution, while such dispersions in hydrophobic polymers form the basis of micro- and nanoparticulate technologies. However, drug recrystallization presents significant problems for product development, and the mechanisms and pathways involved are poorly understood. Here, we outline the use of combined differential scanning calorimetry (DSC)-synchrotron X-ray diffraction to monitor the sequential appearance of polymorphs of olanzapine (OLZ) when dispersed in a range of polymers. In a recent study (Cryst. Growth Des.2019,19, 2751-2757), we reported a new polymorph (form IV) of OLZ which crystallized from a spray-dried dispersion of OLZ in polyvinylpyrrolidone. Here, we extend our earlier study to explore OLZ dispersions in poly(lactide-co-glycolide) (PLGA), polylactide (PLA), and hydroxypropyl methyl cellulose acetate succinate (HPMCAS), with a view to identifying the sequence of form generation on heating each dispersion. While spray-dried OLZ results in the formation of crystalline form I, the spray-dried material with HPMCAS comprises an ASD, and forms I and IV are generated upon heating. PLGA and PLA result in a product which contains both amorphous OLZ and the dichloromethane solvate; upon heating, the amorphous material converts to forms I, II, and IV and the solvate to forms I and II. Our data show that it is possible to quantitatively assess not only the polymorph generation sequence but also the relative proportions as a function of temperature. Of particular note is that the sequence of form generation is significantly more complex than may be indicated by DSC data alone, with coincident generation of different polymorphs and complex interconversions as the material is heated. We argue that this may have implications not only for the mechanistic understanding of polymorph generation but also as an aid to identifying the range of polymorphic forms that may be produced by a single-drug molecule.

Olanzapine crystal symmetry originates in preformed centrosymmetric solute dimers.

The symmetries of a crystal are notoriously uncorrelated to those of its constituent molecules. This symmetry breaking is typically thought to occur during crystallization. Here we demonstrate that one of the two symmetry elements of olanzapine crystals, an inversion centre, emerges in solute dimers extant in solution prior to crystallization. We combine time-resolved in situ scanning probe microscopy to monitor the crystal growth processes with all-atom molecular dynamics simulations. We show that crystals grow non-classically, predominantly by incorporation of centrosymmetric dimers. The growth rate of crystal layers exhibits a quadratic dependence on the solute concentration, characteristic of the second-order kinetics of the incorporation of dimers, which exist in equilibrium with a majority of monomers. We show that growth by dimers is preferred due to overwhelming accumulation of adsorbed dimers on the crystal surface, where it is complemented by dimerization and expedites dimer incorporation into growth sites.

Investigation of Clozapine and Olanzapine Reactive Metabolite Formation and Protein Binding by Liquid Chromatography-Tandem Mass Spectrometry.

Drug-induced toxicity has, in many cases, been linked to oxidative metabolism resulting in the formation of reactive metabolites and subsequent covalent binding to biomolecules. Two structurally related antipsychotic drugs, clozapine (CLZ) and olanzapine (OLZ), are known to form similar nitrenium ion reactive metabolites. CLZ-derived reactive metabolites have been linked to agranulocytosis and hepatotoxicity. We have studied the oxidative metabolism of CLZ and OLZ as well as two known metabolites of CLZ, desmethyl-CLZ (DCLZ), and CLZ-N-oxide (CLZ-NO), using in vitro rat liver microsomal (RLM) incubations with glutathione (GSH) trapping of reactive metabolites and liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Reactive metabolite binding to selected standard peptides and recombinant purified human proteins was also evaluated. Bottom-up proteomics was performed using two complementary proteases, prefractionation of peptides followed by LC-HRMS/MS for elucidating modifications of target proteins. Induced RLM was selected to form reactive metabolites enzymatically to assess the complex profile of reactive metabolite structures and their binding potential to standard human proteins. Multiple oxidative metabolites and several different GSH adducts were found for CLZ and OLZ. Modification sites were characterized on human glutathione S-transferase (hGST) alpha 1 (OLZ-modified at Cys112), hGST mu 2 (OLZ at Cys115), and hGST pi (CLZ, DCLZ, CLZ-NO and OLZ at Cys170), human microsomal GST 1 (hMGST1, CLZ and OLZ at Cys50), and human serum albumin (hSA, CLZ at Cys34). Furthermore, two modified rat proteins, microsomal GST 1 (CLZ and OLZ at Cys50) and one CYP (OLZ-modified, multiple possible isoforms), from RLM background were also characterized. In addition, direct effects of the reactive metabolite modifications on proteins were observed, including differences in protease cleavage specificity, chromatographic behavior, and charge-state distributions.

Measurement of the amorphous fraction of olanzapine incorporated in a co-amorphous formulation.

Amorphous and co-amorphous formulations have been used to enhance the solubility and bioavailability of poorly water-soluble drugs. However, during handling and/or storage amorphous solids present inherent instability and overtime recrystallize back into their crystalline counterpart. The development of tools capable of quantifying and monitoring the recrystallization of amorphous materials is required to ensure the delivery of solid dosage forms with improved performance. This work describes the development and validation of a computational model for simple measurement of amorphous and co-amorphous olanzapine (OLZ) fractions in tablets. Amorphous OLZ produced by quench cooling and co-amorphous OLZ by solvent evaporation using saccharin (SAC) as a co-former were characterized by calorimetry (DSC), diffractometry (XRPD) and spectroscopy (FTIR and NIR). Spectral differences were used to predict the fraction of amorphous OLZ in samples containing different fractions of powdered amorphous and co-amorphous OLZ:SAC. The models were shown to be linear, accurate and reproducible. Blends of (co)amorphous OLZ and excipients were directly compacted at different pressures and dwell times to impose physical stress on the systems. Data collected from the analysis of the tablets was used in the model to monitor the stability of amorphous and co-amorphous OLZ demonstrating the applicability and validity of the model.

Poloxamer-407-Co-Poly (2-Acrylamido-2-Methylpropane Sulfonic Acid) Cross-linked Nanogels for Solubility Enhancement of Olanzapine: Synthesis, Characterization, and Toxicity Evaluation.

Current study is focused to enhance the solubility of poorly soluble drug olanzapine (OLZ) by nanogels drug delivery system, as improved solubility is one of the most important applications of nanosystems. Poor solubility is a major issue, and 40% of marketed and about 75% of new active pharmaceutical ingredients are poorly water soluble which significantly affect the bioavailability and therapeutic effects of these drugs. In this study, nanogels, a promising system for solubility enhancement, were developed by free-radical polymerization technique. Different formulations were synthesized in which poloxamer-407 was cross-linked with 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with the help of cross-linker methylene bisacrylamide (MBA). The chemically cross-linked nanogels were characterized by Fourier transform infrared spectroscopy (FT-IR), thermos gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), zeta size, swelling, sol-gel analysis, drug loading, solubility, and in vitro drug release studies. In order to determine the biocompatibility and cytotoxicity of nanogels to biological system, toxicity study on rabbits was also carried out. It was confirmed that the developed nanogels was thermally stable, safe, effective, and compatible to biological system, and the solubility of olanzapine (OLZ) was enhanced up to 38 folds as compared with reference product.

Ecofriendly chromatographic methods for determination of co-prescribed drugs, olanzapine and metformin, in rat plasma.

Background: Olanzapine (OLZ) is one of most recommended drugs for the treatment of schizophrenia while metformin (MET) is the most commonly used hypoglycemic agent. Aim: Development and validation of two green, sensitive and accurate chromatographic methods for the simultaneous determination of OLZ along with the co-prescribed, MET. Materials & methods: TLC-densitometric method with a developing system consisting of methylene chloride:methanol:ethyl acetate:triethylamine (4:4:5:0.1, by volume) and a reversed-phase (RP)-HPLC method where the chromatographic separation was performed using ethanol:water mixture (50: 50, v/v) as a mobile phase. Results: TLC-densitometric method had linearity over concentration ranges of 160-4000 ng/band for OLZ and 150-4500 ng/band for MET, while RP-HPLC method was linear and validated over concentration range of 300-20000 ng/ml for OLZ and MET. Conclusion: Pharmacokinetic study was successfully performed and suggested the possibility of co-administration of MET with OLZ and their further formulation in one pharmaceutical preparation to enhance patient's compliance.

Brain-targeting by optimized 99mTc-olanzapine: in vivo and in silico studies.

Purpose: Olanzapine (OLZ) is an atypical antipsychotic agent that is characterized by low brain porousness. The present work aimed to develop radiolabeled olanzapine (OLZ) without colloidal impurities and evaluate its biodistribution following intravenous (I.V.) and intranasal (I.N.) administration as a potential agent for brain diagnosis. Materials and methods: OLZ was radiolabeled with technetium-99m by using sodium dithionite as the reducing agent. Biodistribution of 99mTc-OLZ complex in mice following I.V. and I.N. administrations was examined. Furthermore, a molecular docking study was performed.Results: Sodium dithionite labeling procedure resulted in highest radiochemical yield (96.30 ± 0.09%) and in vitro stability in serum up to 8 h. Biodistribution study of 99mTc-OLZ complex showed high brain uptake following I.N. (6.2 ± 0.12% ID/g) and I.V. (5.5 ± 0.09% ID/g) at 0.5 and 1 h post administration (P.I.), respectively. Docking into two brain targets predicts higher affinity of 99mTc-OLZ than free OLZ. Additionally, docking to P-glycoproteins shows less affinity for the radiolabelled OLZ and hence it is expected to be associated with better brain exposure than free OLZ.Conclusion: These chemical and preliminary biological merits strongly suggest that the 99mTc-OLZ complex with new reducing agent could be used as a potential diagnostic agent for brain.

Spray-Drying, Solvent-Casting and Freeze-Drying Techniques: a Comparative Study on their Suitability for the Enhancement of Drug Dissolution Rates.

PURPOSE: Solid dispersions (SDs) represent the most common formulation technique used to increase the dissolution rate of a drug. In this work, the three most common methods used to prepare SDs, namely spray-drying, solvent-casting and freeze-drying, have been compared in order to investigate their effect on increasing drug dissolution rate. METHODS: Three formulation strategies were used to prepare a polymer mixture of polyvinyl-alcohol (PVA) and maltodextrin (MDX) as SDs loaded with the following three model drugs, all of which possess a poor solubility: Olanzapine, Dexamethasone, and Triamcinolone acetonide. The SDs obtained were analysed and compared in terms of drug particle size, drug-loading capacity, surface homogeneity, and dissolution profile enhancement. Physical-chemical characterisation was conducted on pure drugs, as well as the formulations made, by way of thermal analysis and infrared spectroscopy. RESULT: The polymers used were able to increase drug saturation solubility. The formulation strategies affected the drug particle size, with the solvent-casting method resulting in more homogenous particle size and distribution when compared to the other methods. The greatest enhancement in the drug dissolution rate was seen for all the samples prepared using the solvent-casting method. CONCLUSION: All of the methods used were able to increase the dissolution rate of the pure drugs alone, however, the solvent-casting method produced SDs with a higher surface homogeneity, drug incorporation capability, and faster dissolution profile than the other techniques.

Surface modified niosomes of olanzapine for brain targeting via nasal route; preparation, optimization, and in vivo evaluation.

Olanzapine (OL) is an atypical antipsychotic drug which suffers from an extensive hepatic metabolism and poor bioavailability. In addition, it has low brain permeability due to efflux by P-glycoproteins. In the current investigation, surface modified niosomes containing OL were prepared for brain targeting of the drug through nasal route. Spans were mixed with cholesterol at ratios of 1:1, 1:2, 1:3, and 1:4 of cholesterol to surfactant, respectively to prepare niosomes. Chitosan (CS) coated vesicles were prepared by mixing optimum niosomal formula with CS solution (0.6%). Physicochemical and stability parameters and confocal laser scanning microscopy (CLSM) of developed vesicles were determined. Also, the brain targeting properties of the optimized formula were measured in rats. Niosomes had entrapment efficiency more than 90% and particle size ranging from 201.3 ± 2.4 nm to 1446 ± 9 nm. TEM photomicrographs of developed vesicles showed a clear shell surrounding the coated vesicles. The produced vesicles exhibited 2.46 folds increase in the amount of drug that permeated nasal mucosa and prolonged OL release compared to drug solution. Coated niosomes further improved drug permeation. CLSM of coated optimum formula showed high permeation across the nasal mucosa. Stability studies revealed non-significant changes in the physicochemical parameters of optimum formula over the storage period. The optimized nasal CS-coated niosomes showed a three-fold increase in OL concentration in the brain compared to the intranasal solution of the drug. In conclusion, the developed vesicles were efficient in nasal delivery of OL into the brain.

Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics.

Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors not only are a powerful tool to test causality in basic neuroscience but also are potentially amenable to clinical translation. A major obstacle, however, is that the widely used agonist clozapine N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations but is not approved for use in humans by the Food and Drug Administration or the European Medicines Agency, limiting its translational potential. Here, we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a potent agonist of the human M4 muscarinic receptor-based DREADD, facilitating clinical translation of chemogenetics to treat central nervous system diseases.

Agranulocytosis-Protective Olanzapine-Loaded Nanostructured Lipid Carriers Engineered for CNS Delivery: Optimization and Hematological Toxicity Studies.

Potential risk of agranulocytosis is one of the drug-induced adverse effects of the second-generation antipsychotic agents. The present investigation aimed to formulate and investigate olanzapine (OLZ)-loaded nanostructured lipid carriers (OLZ-NLCs) via intranasal (i.n.) route. The NLC was prepared by melt emulsification method and optimized by Box-Behnken design. Mucoadhesive NLC was prepared by using 0.4% Carbopol 974P (OLZ-MNLC (C)) and the combination of 17% poloxamer 407 and 0.3% of HPMC K4M (OLZ-MNLC (P+H)). The particle size, zeta potential, and entrapment efficiency were found to be 88.95 nm ± 1.7 nm, - 22.62 mV ± 1.9 mV, and 88.94% ± 3.9%, respectively. Ex vivo permeation of OLZ-NLC, OLZ-MNLC (P+H), and OLZ-MNLC (C) was found to be 545.12 µg/cm2 ± 12.8 µg/cm2, 940.02 µg/cm2 ± 15.5 µg/cm2, and 820.10 µg/cm2 ± 11.3 µg/cm2, respectively, whereas the OLZ-MNLC (P+H) formulation showed rapid drug permeation than the OLZ-NLC and OLZ-MNLC (C) formulations. The OLZ-MNLC (P+H) formulation was shown to have 13.57- and 27.64-fold more Jss than the OLZ-MNLC (C) and OLZ-NLC formulations. The OLZ nanoformulations showed sustained release of up to 8 h. Finally, the brain Cmax of technetium-99m (99mTc)-OLZ-MNLC (i.n.) and 99mTc-OLZ-NLC (i.v.) was found to be 936 ng and 235 ng, respectively, whereas the Cmax of i.n. administration was increased 3.98-fold more than the Cmax of i.v. administration. The in vivo hematological study of OLZ-MNLC (P+H) confirmed that the i.n. formulation did not reflect any variation in leukocyte, RBC and platelet counts. Hence, it can be concluded that the nose-to-brain delivery of OLZ-MNLC (P+H) can be considered as an effective and safe delivery for CNS disorders.

Determination of olanzapine for therapeutic drug monitoring in schizophrenia patients by LC/MS method.

Olanzapine is an atypical antipsychotic drug from the thienobenzodiazepine family which displays efficacy in patients with schizophrenia and related psychoses. A novel LC/MS method was developed and validated for determination of olanzapine in schizophrenia patients' plasma. A liquid-liquid extraction procedure was carried out using 5 mL diethyl ether-diisopropyl ether mixture (1:1, v/v). Average recovery of the extraction procedure was 94.8%. Chromatographic separation was performed on reversed-phase C18 column (250 × 2.0 mm, 5 µm) using mixture of deionized water (trifluoro acetic acid 0.1%)-acetonitrile (20:80, v/v) as mobile phase at a flow rate of 1 mL/min. Irbesartan was used as internal standart and total run time was 2.5 min. Mass spectrometric analysis were carried out in selective-ion montoring mode, and detected olanzapine at m/z 313.1 and IS at m/z 429.4 in all forms of the ions. The calibration curve of olanzapine was linear in the range 2-300 ng/mL (r2 > 0.9993). The interday and intraday precisions (RSD) were <7.55%, and accuracy was >7.59% (n = 6). The proposed study was successfully validated with respect to the US Food and Drug Administration guidelines.

Challenging identification of polymorphic mixture: Polymorphs I, II and III in olanzapine raw materials.

Olanzapine (OLZ), a drug for the treatment of schizophrenia, presents in more than 60 crystal forms. Polymorphs I, II and III were reported, however, the preparation conditions for pure II and III have not been reported. Polymorph IV was reported but this form is actually polymorph II described at different temperature. The diversity of solid forms of OLZ, the change in the nomenclature found in the literature and the presence of polymorphic mixture in samples, increase the difficulty for a correct solid state characterization. Therefore, the goal was the polymorphic identification of three OLZ raw materials, highlighting the limitation of conventional techniques (typically used in analytical control) and the necessity to use a combination of advanced ones to solve this challenge. The samples were studied by conventional techniques such as powder X-ray diffraction, thermoanalytical techniques, infrared spectroscopy. In apart from that, synchrotron powder X-ray diffraction (SPXRD) and solid state nuclear magnetic resonance (ss-NMR) were used. All samples were in accordance with the pharmacopoeia criteria. However, the conventional techniques were not specific for the complete polymorphic identification. Therefore, a combination of advanced techniques (SPXRD and ss-NMR) was necessary to identify the mixture of polymorphs (I, II and III) in all samples.

The Development of Quasi-isothermal Calorimetry for the Measurement of Drug-Polymer Miscibility and Crystallization Kinetics: Olanzapine-Loaded PLGA Microparticles.

The assessment of drug-polymer equilibrium solubility is of critical importance for predicting suitable loading and physical stability of solid dispersion formulations. However, quantitative measurement of this parameter is nontrivial due to the difficulties associated with ascertaining equilibrium values in systems that are prone to supersaturation and are simultaneously highly viscous, thereby slowing the equilibration process considerably; no standard methodology has yet been agreed for such measurements. In this study, we propose a new approach involving quasi-isothermal modulated temperature DSC (QiMTDSC), whereby unsaturated and supersaturated samples are held at defined temperatures and subject to a sinusoidal heating signal at a zero underpinning heating rate, thereby allowing the heat capacity of the sample to be measured as a function of time and temperature. We are not only able to ascertain whether equilibrium has been reached by monitoring the time-dependent heat capacity signal, but we can also measure solubility as a function of temperature via the absolute heat capacity values of the components. We are also able to measure the kinetics of recrystallization from the supersaturated systems. Dispersions of olanzapine in PLGA at concentrations up to 50% w/w, prepared by spray drying, were prepared and characterized using conventional and QiMTDSC as well as hot stage microscopy. The new QiMTDSC protocol was successfully able to determine olanzapine solubility in PLGA at 90 °C to be 23.1 ± 6.1% w/w, which was comparable to the values calculated using other established methods at this temperature, while a temperature/solubility profile was obtained using the method at a range of temperatures. Drug crystallization kinetics from the solid dispersions could also be modeled directly from the QiMTDSC data using the Avrami approach, thereby allowing the effect of drug loading on the rate of crystallization and the effective completion of crystallization to be investigated. Overall, an alternative protocol for measuring drug-polymer solubility has been developed and validated via comparison to established methods, the approach allowing solubility as a function of temperature, identification of equilibrium following demixing, and kinetic analysis of crystallization to be performed within one set of experiments.