Crystalline Preparations Research Articles

Hot Melt Extrusion-Triggered Amorphization as a Continuous Process for Inducing Extended Supersaturable Drug Immediate-Release from saSMSDs Systems.

Hot melt extrusion (HME), a continuous manufacturing process for generating supersaturating amorphous self-micellizing solid dispersion systems (saSMSDs), holds promise for achieving amorphization of many pharmaceutical formulations. For saSMSDs generation, HME-triggered continuous processes offer advantages over traditional non-continuous processes such as fusion/quench cooling (FQC) and co-precipitation (CP). Here we employed HME, FQC, and CP to generate saSMSDs containing the water-insoluble BCS II drug nitrendipine (NIT) and self-micellizing polymer Soluplus®. Scanning electron microscopy, powder X-ray diffraction, and differential scanning calorimetry results revealed that saSMSDs formed when NIT–Soluplus® mixtures were subjected to the abovementioned amorphization methods. All saSMSDs outperformed crystalline NIT preparations and physical mixtures in achieving extended supersaturable immediate release states with superior solubility, “spring-parachute” process characteristics, and dissolution behaviors. Notably, Fourier transform-infrared spectroscopic results obtained for saSMSDs detected hydrogen bonding interactions between the drug and the carrier. Ultimately, our results revealed the advantages of HME-triggered amorphization as a continuous process for significantly improving drug dissolution, increasing solubility, and maintaining supersaturation as compared to traditional amorphization-based techniques.

Penyuluhan Pembuatan Disinfectant kepada Karyawan Laundry di Condongcatur Yogyakarta

Various attempts have been made to combine in one composition to kill bacteria and detergents. So antiseptics such as coal tar, phenolic and crystalline preparations, mercury salts, as compounds of chlorides and iodides, arsenic and silver, and essential oils such as resin oil, terpenes, almond oil, and thyme oil. Has been used with soap, with alkaline salts of Long-chain fatty acids. The effective disinfectant is determined based on several parameters, namely: the phenol coefficient of the disinfectant and the water quality results measured from pH, dissolved oxygen (DO, dissolved oxygen), temperature and disinfectant cost. The antimicrobial effectiveness of the four selected disinfectants (Dettol, Izal, Z-Germicide and Jik) was determined using the agar diffusion method (disc paper). The results show that Dettol is most effective against the test organisms compared to other disinfectants. All noted the least antimicrobial activity. The phenol coefficient value also shows that Dettol is better when compared to other tested disinfectants. Disinfectant for external use is required to avoid infection or related disease caused by this test microorganism. This counselling was successful even though it encountered several obstacles during implementation. The steps that need to be done in making a disinfectant are determining the ingredients that are safest for humans but effective in eliminating various types of bacteria and viruses. The second stage is the implementation of activities. Activities carried out jointly by employees according to what has been planned. In the third stage, monitoring and evaluation of activities are carried out. This process is also carried out jointly with employees. Employees who work directly to random consumers will really need special knowledge on how to make disinfectant fluids. Keywords: Antiseptic, Disinfectant, Virus

Comparative Study of the Antioxidant and Antimicrobial Activities of Compounds Isolated from Solvent Extracts of the Roots of <i>Securinega virosa</i>

There are several lines of indications on the importance of plants as accepted sources of therapeutic agents since most synthetic antioxidants and antibiotics have been associated with cytotoxicity and/or microbial resistance. In this study, the antioxidant and antimicrobial activities of a brown compound recovered from the ethanol extractable fraction (EEF) and white crystals isolated from the chloroform-methanol elution (CME) following column chromatography of the ethanolic extracts of roots of Securinega virosa were examined. Preparations from the white crystalline compound recorded greater antioxidant activity and antimicrobial activities than the crude ethanol extracts of S. virosa. Both the ethanol extract and the crystals showed significant antimicrobial activities against all microbes employed in this study. The crystalline compound demonstrated the highest zone of inhibition against the microbes Enterococcus faecalis with varied inhibitory activities with respect to other microorganisms. The least inhibition was against Pseudomonas fluorescens. The overall range was 8.0 - 22.5 mm. The EEF of the roots of S. virosa recorded its highest activity against Staphylococcus aureus and Escherichia coli. There was varied inhibitory activity against other organisms; the least was against Micrococcus liteus. The zone of inhibition ranged 6.33 - 17.67 mm. Similar to the trends in susceptibility test, the EEF showed appreciable minimum inhibitory concentration (MIC), ranging from 3.13 - 25 mg/mL against test microorganisms while the crystalline preparation had a MIC range of 1.5 - 25 mg/mL except against Salmonella typhii which was 50 mg/mL. Both extracts demonstrated their highest activity against E. faecalis reducing significantly in a dose-dependent manner by each sample against the rest of the test microbes. However, the standard broad-spectrum antibiotic, chloramphenicol, used elicited a zone of inhibition ranging from 0 - 30 mm but with no activity against Streptococcus thermophilus. We conclude that both the EEF and the white crystal compound isolated from the CME chromatographic fraction are potentially excellent sources of antioxidant and antimicrobial compounds.

Biphasic Liquid Crystal and the Simultaneous Measurement of Isotropic and Anisotropic Parameters by Spatially Resolved NMR Spectroscopy.

Residual dipolar couplings and other anisotropic NMR parameters are powerful tools for molecular structure elucidation when conventional techniques do not suffice. With current liquid crystalline preparations it is necessary to prepare two samples to extract isotropic and anisotropic data from spectra and to derive the residual dipolar couplings. Here, we present the preparation, measurement, and interpretation of a novel biphasic liquid crystalline phase where a single sample can be used to generate both isotropic and anisotropic data. First, we introduce the synthesis of the chiral polymer leading to the biphasic liquid crystal. Second, we present two approaches to measure spatially selective CLIP-HSQC spectra. From these spectra, we extracted the couplings, performed an assignment of diastereotopic protons, and achieved the enantiomeric discrimination of isopinocampheol as a well-studied test molecule.

Two-Dimensional Crystallization of the Ca(2+)-ATPase for Electron Crystallography.

Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

The international standard for vitamin D.

Abstract The International Standards for the determination of the different vitamins are part of the indispensable equipment of the laboratories in which this work is carried out. Though the standard materials themselves are so well known, the amount of labour which has been devoted to their creation and adoption and the infinite care which has been taken to ensure their suitability, accessibility and integrity are not so well known or appreciated. The adoption of a crystalline preparation of vitamin D3 to serve as the International Standard for Vitamin D affords a suitable opportunity for outlining the nature of this work, the importance of which to scientific research, medical practice, the problems of nutrition, and the international exchange of ideas, results and materials is now well recognised.

Protocols for the Sequential Solid‐State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET‐s(1‐227)

The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well-dispersed Cbeta resonances. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N-terminal globular domain of the HET-s prion, a 227-residue protein.

Transition States

Transition States

Suppressed or recovered intensities analysis in site-directed 13C NMR: Assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed 13C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1- 13C]Val- and [3- 13C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account. We analyzed the SRI data of [1- 13C]Val-labeled wild-type bR and D85N mutants, as a function of temperature and pH, respectively, based on so-far assigned peaks including newly assigned or revised ones. Global conformational change of the protein backbone, caused by neutralization of the anionic D85 by D85N, can be visualized by characteristic displacement of peaks due to the conformation-dependent 13C chemical shifts. Concomitant dynamics changes if any, with fluctuation frequencies in the order of 10 4 Hz, were evaluated by the decreased peak intensities in the B–C and D–E loops of D85N mutant. The resulting fluctuation frequencies, owing to subsequent, accelerated dynamics changes in the M-like state by deprotonation of the Schiff base at alkaline pH, were successfully evaluated based on the SRI plots as a function of pH, which were varied depending upon the extent of interference of induced fluctuation frequency with frequency of magic angle spinning or escape from such interference. Distinguishing fluctuation frequencies between the higher and lower than 10 4 Hz is now possible, instead of a simple description of the data around 10 4 Hz available from one-point data analysis previously reported.

Evaluation of the State of Active Ingredients in Pharmaceutical Preparations Using Fourier Transform-Raman Difference Spectra

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR

13C NMR spectra of [3- 13C]Ala- and [1- 13C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α II-helices. Surprisingly, the 13C NMR spectra of [3- 13C]Ala-D85N turned out to be very similar to those of [3- 13C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting 13C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10 4–10 5 Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl 13C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1- 13C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, 13C NMR spectral feature of reconstituted [1- 13C]Val and [3- 13C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.

Crystalline monoclonal antibodies for subcutaneous delivery.

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity in vitro. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.

Influence of lid conformation on lipase enantioselectivity

The enantioselectivity of porcine pancreatic lipase (PPL) in the hydrolysis reaction of racemic glycidyl butyrate has been observed to increase substantially upon interfacial activation of the enzyme. The enantioselectivity of Candida antarctica lipase B ( CalB), a lipase that does not display interfacial activation, does not change when the substrate concentration exceeds the solubility limit. A hypothesis, based on a kinetic model, is presented that relates the change of enantioselectivity to the conformational changes that accompany movements of the lid upon interfacial activation. The hypothesis was investigated using various forms of the C. rugosa lipase ( Crl). For several substrates, the enantioselectivities of hydrolysis reactions catalyzed by crude, purified, and crystalline (CLEC®) preparations of Crl in open and closed conformations were measured. As anticipated, the enantioselectivity of open-lid Crl-CLECs in the hydrolysis of racemic ibuprofen methyl ester exceeded that of the closed-lid form. For other esters, however, correlations were less straightforward. It was concluded that apart from affecting the activation barrier leading to the Michaelis–Menten complex, modifications of the lid (open, closed, or modified lid) also induce additional conformational changes in the active site affecting enantioselectivity.

The effect of liquid crystalline structure on chlorhexidine diacetate release

The aim of this study was to examine different liquid crystalline preparations containing chlorhexidine diacetate and to find connection between their structure and the kinetic of drug release. Nonionic surfactant, Synperonic A7 (PEG 7–C 13–15) was selected for the preparation of the examined liquid crystalline systems. Mixtures of different ratios of Synperonic A7 and water were produced. By increasing the water content of the systems, lamellar and hexagonal liquid crystal structures were observed. For the analysis of the prepared liquid crystalline systems polarising microscopy, rheology study, differential scanning calorimetry and dynamic swelling tests were carried out. The chlorhexidine diacetate release was examined by Franz-type vertical diffusion cell apparatus. The chlorhexidine diacetate release from hexagonal liquid crystalline preparations was characterised by zero-order release kinetics, while the drug release from lamellar liquid crystalline systems was described by anomalous (non-Fickian) transport. The results indicate that the drug release kinetic is strongly dependent on the liquid crystalline structure.

X-ray Absorption Spectroscopy of Chicken Sulfite Oxidase Crystals

We have investigated the active site of crystalline sulfite oxidase using X-ray absorption spectroscopy. Our results indicate that the crystalline preparation contains molybdenum in the fully oxidized MoVI form, with a local structure that is identical to that in solution, with two MoO ligands at 1.71 A and three Mo−S at 2.41 A.

Toxicity of Bacillus thuringiensisδ‐ endotoxins toward the potato aphid in an artificial diet bioassay

AbstractRecent investigations indicated that Bacillus thuringiensis delta‐endotoxins (DET) possess aphidicidal activity in an artificial diet bioassay. Crystalline preparations of CryIIA, CryIIIA and CryIVD solubilized in a slightly alkaline sucrose/amino acid diet clearly imparted toxicity toward adults of potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) after 4–5 days of continuous feeding. No obvious feeding deterrence was noted in these assays, as copious honeydew was produced and aphids often died in a feeding position. CryIIIA which was solubilized in aphid diet, but filtered to remove spores or crystalline toxin lacked aphidicidal activity. Spores from an acrystalliferous strain (EG2205) were not toxic by themselves at 7.75 × 105 spores/ml aphid diet, but did restore toxicity to the filtered CryIIIA solution. Therefore, low levels of spores may be very effective in concert with DET for aphicidal activity. Results also clearly demonstrated that a suspension of crystalline CryIIIA alone, without spores, exhibited toxicity. Therefore, DET may be more toxic to the aphids when imbibed as a fine suspension, perhaps indicating the need for slow solubilization into the aphid midgut.

Gastric acid secretion and vitamin B12 absorption after vertical Roux-en-Y gastric bypass for morbid obesity.

This study sought to determine the basal and peak-stimulated acid secretion from the proximal gastric pouch and its relationship to absorption of free and food-bound vitamin B12 after gastric bypass for morbid obesity. Gastric bypass can be performed safely and provides acceptable weight loss, but concerns remain about possible long-term complications such as vitamin B12 malabsorption. The authors hypothesized that by constructing a small pouch of gastric cardia, acid secretion into the pouch would be low, leading to maldigestion of food-bound vitamin B12 with subsequent malabsorption. Basal and pentagastrin-stimulated peak acid outputs from the proximal gastric pouch were measured in ten patients after vertical Roux-en-Y gastric bypass using a perfused orogastric tube technique. Absorption of free and food-bound 57Co-vitamin B12 was evaluated separately using 24-hour urinary excretion. Basal (mEq/hr, mean +/- standard error of the mean [SEM]) and peak-stimulated (mEq/30 min) acid secretions from the proximal gastric pouch were markedly decreased compared to those for age- and sex-matched hospital control subjects (0.01 +/- 0.01 vs. 4.97 +/- 0.66 and 0.08 +/- 0.04 vs. 12.11 +/- 1.34, respectively; p < 0.001 for each). While absorption of free vitamin B12 was not statistically different from that of control subjects (11 +/- 2 vs. 15 +/- 2%; p > 0.05), absorption of food-bound vitamin B12 was decreased (0.8 +/- 0.2 vs. 3.7 +/- 0.5%; p < 0.01). After vertical Roux-en-Y gastric bypass for morbid obesity, acid secretion is virtually absent and food-bound vitamin B12 is maldigested and subsequently malabsorbed. The results of this study suggest that postoperative vitamin B12 supplementation is important and can be achieved with either monthly parenteral vitamin B12 or daily oral crystalline preparations.

Crystalline Preparation of Botulinum Toxin Type a (Botox): Degradation in Potency with Storage

Laryngeal injection of botulinum toxin type A is currently the most effective method of treating spasmodic dysphonia. Botox, a crystalline preparation of botulinum toxin type A, is the only toxin approved for clinical use in the United States and is packaged in vials of 100 mouse units (MU). One MU corresponds to the calculated median lethal intraperitoneal dose (LD50) injected in mice. The logistic problems arising from the need for repeated injections of small amounts of Botox have been addressed by several investigators by refreezing unused Botox for use at a later time. Although FDA labeling recommends that Botox not used within 4 hours of reconstitution be discarded, data regarding degradation in potency after reconstitution and refreezing are not currently available. Using the LD50 Swiss-Webster mouse bioassay and statistical analysis by the Probit procedure, a 69.8% loss in potency was found when Botox was reconstituted, immediately frozen, and then assayed 2 weeks later (p < 0.0001). Statistically significant degradation in potency was seen after refrigerator storage for 12 hours (p = 0.007), but not for 6 hours (p = 0.16). Clinical implications regarding the dilution, use, and storage of Botox are discussed.

HLö 7 dimethanesulfonate, a potent bispyridinium-dioxime against anticholinesterases.

HLö 7 dimethanesulfonate (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis [(hydroxyimino)methyl]pyridinium dimethanesulfonate) is a broad-spectrum reactivator against highly toxic organophosphorus compounds. The compound was synthesized by a new route with the carcinogenic bis(chloromethyl)ether being substituted by the non-mutagenic bis(methylsulfonoxymethyl)ether. The very soluble dimethanesulfonate of obidoxime was also prepared by this way. HLö 7 dimethanesulfonate is the first water-soluble salt of HLö 7 that should be suitable for the wet/dry autoinjector technology, because aqueous solutions of HLö 7 are not very stable (calculated shelf-life 0.2 years when stored at 8 degrees C, 1 M solution, pH 2.5). The crystalline preparation contains 96% of the syn/syn-isomer, less than 2% of the syn/anti-isomer and some minor identified by-products. HLö 7 was very efficient in reactivating acetylcholinesterase (AChE) blocked by organophosphates as long as ageing did not prevent dephosphylation. HLö 7 was superior to HI 6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]pyridinium dichloride) in reactivating soman and sarin-inhibited AChE from erythrocytes, and literature data indicate that HLö 7 exceeds HI 6 by far in reactivating tabun-inhibited AChE. In atropine-protected, soman-poisoned mice HLö 7 was three times more potent than HI 6 (protective ratio 5 versus 2.5), and in sarin-poisoned mice HLö 7 was 10 times more potent than HI 6 (protective ratio 8 for both oximes). In atropine-protected guinea-pigs HLö 7 was less effective than HI 6 (protective ratio: 2.3 versus 5.2 for soman; 5.2 versus 6.8 for sarin; 4.3 versus 3.8 for tabun). The mean survival time of anaesthetized guinea-pigs exposed to 5 LD50 soman (6.3 min) was increased by atropine (27 min) and atropine + HLö (57 min). HLö 7 alone did not prolong the survival. The most impressive effect of HLö 7 was on respiration: 3 min after i.v. injection of HLö 7 and atropine, the depressed respiration increased rapidly to 60% of control and remained at that level during the observation period (60 min). With atropine alone, respiration recovered only slowly. Behavioural and physiologic parameters were determined in atropine-protected mice exposed to a sublethal soman dose. The running performance was significantly improved by HLö 7. Even central symptoms, e.g. hypothermia and convulsions, were decreased markedly by HLö 7 (evaluation 60 min after poisoning). The pharmacokinetic data for HLö 7 in male beagle dogs are similar to those of HI 6.(ABSTRACT TRUNCATED AT 400 WORDS)