In situ nondestructive sediment characterization and resuspendability evaluation of concentrated aqueous paliperidone palmitate suspensions in prefilled syringes by low-field one-dimensional pulsed-field gradient NMR profilometry

This work aims to demonstrate the usefulness of a low-field one-dimensional pulsed-field gradient NMR (1D pfg NMR) profilometry technique to enable in situ nondestructive sediment characterization and resuspendability quantification of concentrated prefilled injectable suspensions. Aqueous paliperidone palmitate suspensions were used as model samples and low-intensity centrifugation was evaluated as a long-term gravity simulation approach. The low-field 1D pfg NMR technique allowed a detection zone of 2.5 cm in height for water content measurement of syringe samples using a Teflon syringe holder. Thus, the sediment compactness could be deduced from its water content. Quantitative evaluation of resuspendability was realized by front tracking of the NMR profile signals, which yielded the exponential sediment volume decay constant as a resuspendability quantification parameter. The study shows that both active ingredient particle size distribution and storage temperature had significant effects on the sedimentation rate and the resuspendability of the suspensions. The centrifugation method proved to be useful as a long-term gravity simulation and screening method, although the results should be interpreted with caution due to its higher acceleration and compression force imposed on the active ingredient particles.     

Quantification of water content in biological tissues by proton nuclear magnetic resonance.

We have made quantitative measurements of water content in biological tissue using proton nuclear magnetic resonance (NMR). We evaluated the factors which affect the NMR signal intensity measurements in order to quantify the absolute water content of the tissue. It can be said that there is an optimum sample length to achieve the absolute value of water content using NMR. The integrated NMR water signal intensity was determined in the frequency domain. The absolute water content was measured gravimetrically. The NMR detectable water in brain and lung tissue was determined using the signal intensity of the analyzed tissue compared with the signal intensity for the same weight of distilled water. The NMR detectable water was 99% by weight for brain and 60% by weight for peripheral lung tissue. The NMR detectability of water in biological tissues varied for different tissues.     

Quantification of cynaropicrin in artichoke leaf extracts by ¹H NMR spectroscopy

The quantification of cynaropicrin in artichoke leaf extracts (ALE) by means of quantitative 1H NMR was performed. The method relies on solid phase extraction for sample preparation and spectra acquisition in DMSO- D? containing anthracene as the internal standard. The obtained 1H NMR spectra revealed excellent signal purity for H-13(b) of cynaropicrin, which was thus chosen for quantification. Validation was performed in terms of selectivity, linearity, precision, accuracy, and robustness. The content of cynaropicrin in the analyzed samples showed considerable variation, ranging from non detectable to 1.6?%. The NMR approach does not rely on external calibration, which represents the major advantage compared to traditional approaches based on HPLC.     

Determination of tacrine metabolites in microsomal incubate by high performance liquid chromatography-nuclear magnetic resonance/mass spectrometry with a column trapping system

A column trapping system has been incorporated into high performance liquid chromatography-nuclear magnetic resonance-mass spectrometry (HPLC-NMR-MS) to reduce data acquisition time of NMR experiments. The system uses a trapping column to capture analytes after the HPLC column and back flush trapped analyte to the flow cell of the NMR probe for detection. A dilution solvent is mixed with eluent from HPLC column to reduce the influence of the organic content in the mobile phase before column trapping. The trapping column is also coupled with a mass spectrometer (MS) to get complementary MS data on the same peak. Studies on 1-hydroxylated 9-amino-1,2,3,4-tetrahydro-acridine (1-OH tacrine), indomethacin and testosterone with the column trapping system showed good recovery of analytes and over 3-fold mean increase in UV-VIS signal intensity. The time saving on NMR experiments with the column trapping system was demonstrated by the analysis of dog microsomal incubate with tacrine.     

19F NMR spectroscopic study on the binding of triflupromazine to bovine and human serum albumins

The 19F NMR spectrum of triflupromazine hydrochloride (TFZ) in a buffer solution (pH 6.8) showed a single sharp signal of the TFZ CF3 group at 13.5 ppm from the external trifluoroacetic acid. The addition of 1 mM HSA or BSA to the sample solution caused a split of the CF3 signal into two broadened signals shifted to slightly lower (0.2 ppm) and higher (0.7 ppm) fields, respectively, from the original position. Denaturation of the albumins by guanidine hydrochloride (3M) restored the two broadened signals to a slightly broadened single signal, indicating that TFZ has at least two binding sites on HSA and BSA, respectively. From the competitive binding 19F NMR experiments using Warfarin (Site-I ligand), l-tryptophan (Site-II ligand), NaCl, and oleate, the signal at high field was assigned to the TFZ bound to Site II. Comparison of the signal intensity revealed that the affinity of TFZ for Site II on HSA was considerably higher than that on BSA. The low-field signal could be identified as a weight-averaged signal between nonspecifically bound TFZ to HSA (BSA) and free TFZ in the water phase. In the presence of physiological concentrations of NaCl, major binding of TFZ to HSA and BSA was considered to be nonspecific. The present work indicates that 19F NMR is very useful for obtaining important detailed information regarding the binding of fluorinated drugs to serum albumins.     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.     

Validation of a sensitive ion chromatography method for determination of monoethylsulfate in Indinavir sulfate drug substance

The present study relates to the optimization of an ion chromatography method to determine the content of monoethylsulfate at very low levels in Indinavir sulfate drug substance, and subsequent validation of the method to prove its suitability, reliability and sensitivity. Monoethylsulfate is a potential impurity of Indinavir sulfate, and may forms during the preparation as well as during storage. The ion chromatography method was developed in such a way that to enhance the detection level by introducing suppressor, and minimizing acquisition time by using suitable buffer of 3.2 mmole of sodium carbonate and 1 mmole of sodium hydrogen carbonate in water as eluent. The retention time of monoethylsulfate was about 9.5 min and the total acquisition time was 25 min. The optimized method was validated to prove its performance characteristics by demonstrating selectivity, sensitivity (limit of detection and quantification), linearity, precision and accuracy. The established limit of detection and quantification of monoethylsulfate in Indinavir sulfate by this method was found to be 24 ng/ml and 74 ng/ml respectively, and the overall percent accuracy (recovery) of samples evaluated at different concentration levels was found to be 97.1, indicating the sensitivity and accuracy of this optimized ion chromatography method.     

A Solid-State NMR Study of Protein Hydration and Stability

Purpose. The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity. Methods. Magic angle spinning ¹³C, ¹⁵N, ¹H, ²H, and ¹⁷O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (₀, T₁) and low frequency (₁, T_1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. Results. There was little change in T_1p observed with increasing humidity. The results show, however, that there is a decrease in T₁, for DNase, insulin and lysozyme at relative humidities ranging from 0–98%, and we propose that the reduction in T₁, is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using ¹⁷O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data. Conclusions. Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.     

膝关节周围神经源性肿瘤的低场强MRI

目的评价膝关节周围软组织内神经鞘瘤的低场强MRI表现。方法回顾性分析8例经手术病理证实的膝关节周围神经鞘瘤的低场强MRI资料,其中,6例伟神经鞘瘤,2例神经纤维瘤,均进行常规MRI扫描,包括T1WI和T2WI矢状位,冠状位STIR序列。结果6例在T1WI上神经鞘瘤呈低等信号,T2WI上病灶呈不均质高信号,其内可见有低信号,4例可见明显的靶征,表现为瘤体中央低信号,其周围为高信号;2例神经纤维瘤病灶内呈混杂信号,并且是4个患者15个病灶。结论MRI可以明确肿瘤的形态、位置和范围,有利于手术的方案的确定,靶征、梭形肿块、与神经的密切关系是神经源性肿瘤的特征性表现。     

采用核磁共振氟谱定性与定量分析盐酸氟西汀

采用核磁共振氟谱检测盐酸氟西汀,在δ14.15出现信号峰,随着浓度减小(2.00～0.05 mmol.L1),化学位移略往高场移动(δ14.158到14.145),定性分析的进一步确定可以采用盯梢重叠实验。氟谱灵敏度可以达到17 mg.L1,符合药典要求,但需要扫描500次与使用合适的参数。定量分析上制作出浓度氟谱积分的标准工作曲线,藉由内插或公式法进行快速定量分析。氟标准曲线的制作中,本文提出使用毛细管内标法,利用毛细管中的三氟乙酸作为积分的标定参考。此法操作简便并获得极佳的线性拟合度;使用三氟乙醇直接添加法制作的标准曲线的线性拟合度不如预期。     

Effects of typical and atypical antipsychotic drugs on two-way active avoidance. Relationship to DA receptor blocking profile

The dose-dependent and time-dependent effects of the novel antipsychotic compound remoxipride, as well as the reference compounds chlorpromazine, clozapine, haloperidol, pimozide and sulpiride upon the retention of two-way active avoidance (conditioned avoidance responses, CARs) were studied in male rats. The dose-dependent effects of remoxipride as well as haloperidol and chlorpromazine on the acquisition of CARs were also studied. The acquisition and retention of CARs were tested in shuttleboxes using a 1.0-mA shock intensity and a 10-stone signal (1000 Hz). All the compounds studied, including remoxipride, caused a dose-dependent impairment of acquisition and retention of CARs. The effect of remoxipride on CAR acquisition correlated with remoxipride's effectiveness to block the hyperactivity induced by the dopamine (DA) agonist apomorphine. Unlike chlorpromazine and haloperidol, the potency of remoxipride and clozapine for antagonising CAR retention was found at dose levels much lower than those producing cataleptic effects or blocking apomorphine-induced stereotypies. Based on the DA receptor blocking profile and the relative effectiveness to block CAR it is concluded that the mechanism(s) by which clozapine and remoxipride affect CAR differ from typical neuroleptic drugs. This difference may reflect an action upon different subtypes of functionally coupled DA D₂ receptors.     

Usefulness of Applying Partial Least Squares Regression to T2 Relaxation Curves for Predicting the Solid form Content in Binary Physical Mixtures

This study applied partial least squares (PLS) regression to nuclear magnetic resonance (NMR) relaxation curves to quantify the free base of an active pharmaceutical ingredient powder. We measured the T₂ relaxation of intact and moisture-absorbed physical mixtures of tetracaine free base (TC) and its hydrochloride salt (TC·HCl). The obtained T₂ relaxation curves were analyzed by two methods, one using a previously reported T₂ relaxation time (T₂), and the other using PLS regression. The accuracy of estimating TC was inadequate when using previous T₂ values because the moisture-absorbed physical mixtures showed biphasic T₂ relaxation curves. By contrast, the entire measured whole of the T₂ relaxation curves was used as input variables and analyzed by PLS regression to quantify the content of TC in the moisture-absorbed TC/TC·HCl. Based on scatterplots of theoretical versus predicted TC, the obtained PLS model exhibited acceptable coefficients of determination and relatively low root mean squared error values for calibration and validation data. The statistical values confirmed that an accurate and reliable PLS model was created to quantify TC in even moisture-absorbed TC/TC·HCl. The bench-top low-field NMR instrument used to apply PLS regression to the T₂ relaxation curve may be a promising tool in process analytical technology.     

Determination of the structure of [Nle7]-endothelin by 1H NMR

[Nle⁷]-endothelin was synthesized and studied by ¹H NMR and distance geometry calculations. The NMR study was performed first in DMSO-d₆ and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two-dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three-dimensional structure of [Nle⁷]-endothelin is proposed and discussed.     

Optimization of Surface-Enhanced Raman Spectroscopy Detection Conditions for Interaction between Gonyautoxin and Its Aptamer

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO₄ concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm⁻¹ confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm⁻¹ was selected for the GTX1/4–GO18 complex (I₁₆₅₆/I₁₀₉₉) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO₄), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R² = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.     

Blockade of spatial learning by the M1 muscarinic antagonist pirenzepine

Two experiments were conducted to determine the effects of the M₁ muscarinic receptor antagonist pirenzepine on place navigation in a water maze. In the first experiment rats were required to learn the location of a hidden platform following intracerebroventricular injections of equimolar doses of pirenzepine or scopolamine methylbromide. Both drugs dose-dependently impaired spatial learning according to both escape latency data and transfer test analysis. Pirenzepine was approximately 3 times less potent than scopolamine, a potency ratio which suggests M₁ receptor mediation of the impairment. In the second experiment pirenzepine (192.3 g/rat ICV) was injected prior to training on a simultaneous place dicrimination task in the water maze. Impairments of choice accuracy were found with a dose of 20 g/rat in the absence of any marked increases in either errors of omission or choice latency. These data suggest that M₁ receptor blockade impairs processes which are involved in spatial learning.     

Behavioral assessments of learning and attention in rats exposed perinatally to 3,3',4,4',5-pentachlorobiphenyl (PCB 126)

Evidence from humans suggests that cognitive dysfunction may result from perinatal exposure to polychlorinated biphenyls (PCBs), and the results of some animal research with PCBs have been interpreted in terms of possible impairment of attention. Long-Evans rats were fed 3,3',4,4',5-pentachlorobiphenyl (PCB 126), a coplanar congener, at doses of 0.25 or 1 microgram/kg/day [corrected] throughout gestation and nursing. Male offspring of these rats were trained as adults to perform 2 tests of attention for food reward. First, a cued target-detection task, modeled after Posner's covert orienting method for humans, was used to assess visuospatial attention. In this task, a visual target stimulus was presented in 1 visual hemifield on each trial, preceded either by a valid cue, an invalid cue, or no cue. A valid cue appeared in the same hemifield as the target, and an invalid cue appeared in the opposite hemifield. As expected, valid cues increased accuracy and speed of target detection and invalid cues decreased accuracy and speed; moreover, these effects were systematically related to changes in cue intensity and target duration. However, perinatal exposure to PCB 126 did not affect acquisition or performance of this task. The second task assessed sustained attention by means of a signal detection method in which a brief, spatially-constant but temporally unpredictable, visual signal indicated which of 2 responses would yield food. Varying the intensity of the signal greatly affected the probability of correctly reporting the signal. Perinatal exposure to PCB 126 did not affect acquisition of the response rule or performance of the task. Finally, all rats were challenged with chlordiazepoxide (CDP) at doses of 0, 3, 5, 8, or 12 mg/kg SC, 20 min before testing in the sustained attention task. In control rats, low doses (3, 5, and 8 mg/kg) of CDP reduced accuracy at low signal intensities only, suggesting an increase in visual threshold. The high dose of CDP reduced accuracy at all signal intensities and increased the false-alarm rate as well, suggesting an impairment of attention. The rats exposed perinatally to PCB 126 at 0.25 micrograms/kg [corrected] were unaffected by CDP, and those exposed to PCB 126 at 1 microgram/kg [corrected] showed a smaller decrement in performance after CDP than did the controls. Taken together, these data provide little support for the possibility that perinatal exposure to PCB 126 causes deficits in attention, but suggest that PCB 126 may alter GABA-mediated pathways in the CNS during development.     

Probing the Distribution of Water in a Multi-Component System by Solid-State NMR Spectroscopy

Purpose

To characterize the distribution of water among various components in a powder blend using solid-state NMR spectroscopy.

Methods

Water sorption behavior of theophylline anhydrate and excipients was determined by dynamic vapor sorption (DVS) and Karl Fischer Titration (KFT) after storing them in humidity chambers for 1 week at room temperature (RT) and calibration curves were generated for water content vs. ¹H T ₁ relaxation times. Powder blends (either with microcrystalline cellulose or lactose as diluent) were stored at different relative humidity (RH) conditions and analyzed periodically using solid-state NMR, powder X-ray diffraction, and KFT.

Results

Anhydrous theophylline converted to the hydrate at?≥?84% RH. Based on the calibration curves of water content vs. relaxation times, the distribution of water in the powder blends was estimated. The total water content calculated using ssNMR was in good agreement with values measured using KFT. In blends stored at 90% RH, theophylline anhydrate-to-hydrate conversion did not occur in 1 week.

Conclusions

The distribution of water in multi-component powder blends was successfully determined using correlation between ¹H T ₁ relaxation times and total water content. Excipient water sorption inhibited hydrate formation in theophylline at 90% RH. Water distribution was affected by excipient type. The extent of water sorbed by excipients in blends was found to be different than their standalone equilibrium water content.

     

Effect of nonionic surfactants on aqueous primidone suspensions

The following interactions between the soluble surfactant, octoxynol 9, and the very slightly soluble, finely powdered drug, primidone, in aqueous suspension were investigated: adsorption/desorption of the surfactant, micellar solubilization of the drug, and deflocculation of its particles. The last effect, measured by the sedimentation volume of the suspensions, was also investigated for other octoxynols. The adsorption of octoxynol 9 on solid primidone was proportional to the equilibrium surfactant concentration up to the critical micelle concentration. It leveled off at higher concentrations, reaching saturation at completion of a close-packed surfactant monolayer. The adsorption was essentially completely reversible. The solubility of primidone in water was very slight; its micellar solubilization was even less extensive. The sedimentation volume of primidone suspensions decreased with increasing equilibrium concentration of octoxynol 9 and began to level off at the critical micelle concentration of 0.018%. At about twice that concentration, the sedimentation volume became almost constant, but reached its lowest value only at less than or equal to 0.5%. Slow rotation of suspensions prior to sedimentation promoted flocculation and higher sedimentation volumes between 0.01 and 0.03% octoxynol 9. Octoxynols with higher hydrophilic-lipophilic balance than octoxynol 9 produced considerably larger sedimentation volumes at comparable concentrations, due to a lower surface activity and a lesser tendency to adsorb on primidone.     

Determination of Molecular Mobility of Lyophilized Bovine Serum Albumin and γ-Globulin by Solid-State 1H NMR and Relation to Aggregation-Susceptibility

Purpose. Feasibility of solid-state ¹H NMR for determining the mobility of protein molecules in lyophilized cakes was considered. The mobility in cakes with various levels of water content was studied in relation to aggregation-susceptibility. Methods. Spin-spin relaxation time (T₂) of protons in lyophilized bovine serum albumin (BSA) and -globulin (BGG) was measured as a function of hydration level by solid state ¹H NMR using a solid-echo pulse sequence. Moisture-induced aggregation of the lyophilized proteins was also determined by high performance size exclusion chromatography. Results. Lyophilized BSA and BGG became susceptive to aggregation when water content exceeded about 0.2 g/g of protein. T₂ of protein protons in the lyophilized cakes started to increase at lower water contents. The increase in aggregation susceptibility observed with increasing water content appears to follow the increase in T₂ of protein protons. For lyophilized BGG, both aggregation and T₂ of protein protons decreased at water contents above 0.5 g/g protein. Conclusions. Mobility of protein molecules in lyophilized cakes was successfully detemined by solid-state ¹H NMR. The aggregation susceptibility of proteins was strongly related to their molecular mobility as indicated by T₂.     

Partition of N-monodemethylated phenothiazine drugs to phosphatidylcholine bilayer vesicles studied by second-derivative spectrophotometry

The partition coefficients (Kps) of three N-monodemethylated metabolites of phenothiazines (chlorpromazine (CPZ), triflupromazine, and promazine) between phosphatidylcholine (PC) vesicles (SUV) and water were determined to evaluate their affinity to biomembranes by second-derivative spectrophotometry without any separation procedures. The second derivative spectra showed distinct derivative isosbestic points confirming the entire elimination of the residual background signal effects of the SUV, which were observed in the absorption spectra. From the relationship between PC SUV concentration and the derivative intensity change (DeltaD) of each metabolite induced by its interaction with the PC bilayer, the Kp values were calculated and could be obtained with the R.S.D. of below 10% (n = 5) proving an accuracy of the derivative method. The obtained Kp values were similar to those of the parent drugs (the relative differences were within 15%), although the partition coefficient of N-monodemethylated CPZ measured in an octanol/buffer system was reported to be about 1/18 of CPZ. The results obtained from the PC liposome/buffer system do not contradict with psychoactivity and brain accumulation of N-monodemethylated metabolites of phenothiazines, in contrast to the results of the octanol/buffer system.