Importance of the Direct Contact of Amorphous Solid Particles with the Surface of Monolayers for the Transepithelial Permeation of Curcumin.

The amorphization has been generally known to improve the absorption and permeation of poorly water-soluble drugs through the enhancement of the solubility. The present study focused on the direct contact of amorphous solid particles with the surface of the membrane using curcumin as a model for water-insoluble drugs. Amorphous nanoparticles of curcumin (ANC) were prepared with antisolvent crystallization method using a microreactor. The solubility of curcumin from ANC was two orders of magnitude higher than that of crystalline curcumin (CC). However, the permeation of curcumin from the saturated solution of ANC was negligible. The transepithelial permeation of curcumin from ANC suspension was significantly increased as compared to CC suspension, while the permeation was unlikely correlated with the solubility, and the increase in the permeation was dependent on the total concentration of curcumin in ANC suspension. The absorptive transport of curcumin (from apical to basal, A to B) from ANC suspension was much higher than the secretory transport (from basal to apical, B to A). In vitro transport of curcumin through air-interface monolayers is large from ANC but negligible from CC particles. These findings suggest that the direct contact of ANC with the absorptive membrane can play an important role in the transport of curcumin from ANC suspension. The results of the study suggest that amorphous particles may be directly involved in the transepithlial permeation of curcumin.

Risk factors for clozapine-induced central nervous system abnormalities in Japanese patients with treatment-resistant schizophrenia.

The purpose of this study was to assess the risk factors for clozapine-induced central nervous system (CNS) abnormalities (i.e., electroencephalogram [EEG] abnormalities, myoclonus, and seizures). We retrospectively analyzed data from 106 patients with schizophrenia who received clozapine treatment through our hospital. A review of the EEG recordings showed that 71 of these patients (67.0 %) developed CNS abnormalities after initiating clozapine treatment. EEG abnormalities, myoclonus, and seizures occurred in 53.8 %, 38.7 %, and 8.5 % of the patients, respectively. Multivariate logistic regression analysis showed that the risk factors for clozapine-induced CNS abnormalities were concomitant lithium usage (odds ratio, 4.560; 95 % confidence interval, 1.750-11.900) and shorter illness durations before clozapine initiation (odds ratio, 0.796; 95 % confidence interval, 0.649-0.976). However, plasma clozapine levels and the usage of antiepileptics did not exhibit associations with the risks of CNS abnormalities. Clinicians should monitor their patients for incident CNS abnormalities when administering lithium in combination with clozapine regardless of plasma clozapine levels or the usage of antiepileptics. This is especially true for patients with short illness durations.

Effects of introduction of hydrophobic group on ribavirin base on mutation induction and anti-RNA viral activity.

One of the possible mechanisms of antiviral action of ribavirin (1-beta- d-ribofuranosyl-1,2,4-triazole-3-carboxamide, 1) is the accumulation of mutations in viral genomic RNA. The ambiguous incorporation of 5'-triphosphate of ribavirin (RTP, 8) by a viral RNA-dependent RNA polymerase (RdRp) is a key step of the mutation induction. We synthesized three ribavirin analogues that possess hydrophobic groups, 4-iodo-1-beta- d-ribofuranosylpyrazole-3-carboxamide ( 7a), 4-propynyl-1-beta- d-ribofuranosylpyrazole-3-carboxamide ( 7b), and 4-phenylethynyl-1-beta-D-ribofuranosylpyrazole-3-carboxamide ( 7c), and the corresponding triphosphates ( 9a, 9b, and 9c, respectively). Steady-state kinetics analysis of the incorporation of these triphosphate analogues by a poliovirus RdRp, 3D (pol), revealed that while the incorporation efficiency of 9a was comparable to RTP, 9b and 9c showed lower efficiency than RTP. Antipolioviral activity of 7a and 7b was much more moderate than ribavirin, and 7c showed no antipolioviral activity. Effects of substituting groups on the incorporation efficiency by 3D (pol) and a strategy for a rational design of more active ribavirin analogues are discussed.

Template properties of mutagenic cytosine analogues in reverse transcription.

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome.

Fluorescence-Based High-Throughput Salt Screening.

The present study reports a high-throughput screening method for the salt formation of amine-containing active pharmaceutical ingredients (APIs) based on fluorescence measurements. A free form amine API was alkynylated by a solid-vapor reaction using propargyl bromide, and a fluorescent compound was produced by a subsequent reaction using 9-azidomethylanthracene. In contrast, salts were inert to propargyl bromide; thus, no fluorescence was observed. Samples for salt screening were prepared by grinding haloperidol with various counter acids, and these mixtures were derivatized in a 96-well microplate to determine whether the salt formation had occurred between haloperidol and the counter acids. Samples that turned into fluorescent and nonfluorescent were confirmed to be free form and salt form, respectively, using powder X-ray diffraction and Raman spectroscopy. In conclusion, our method adequately functions as an indicator of the salt formation of amine APIs. Further, this method allows for the rapid evaluation of the salt formation of APIs using 96-well microplates without the need for special reagents or techniques; thus, it is valuable for the discovery of an optimal salt form of newly developed amine APIs in the pharmaceutical industry.

[Applications of Raman Spectroscopy on Quality Control of Hospital Formulations].

Hospital formulation has several advantages, including the flexibility of customization as per the disease state or the patients' precise requirements. However, compared with commercial formulations, hospital formulations are usually not under the same level of quality check. In the present study, we tested mixed powder formulations prepared in a hospital pharmacy using Raman spectroscopy to investigate the feasibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. For this purpose, we first established a numerical evaluation method to determine the uniformity of a powder mixture using Raman chemical imaging data with atropine sulfate/lactose mixture samples and revealed that the mixing uniformity correlated to the experience level of the pharmacist. Next, we developed a content quantification method in a one-dose packaged powder formulation by measuring the Raman spectra from the outside of the package. Because this method allows for quantification of the components inside the package in a non-destructive and non-contact manner, it can be applied for content confirmation after one-dose packaging. Using this method, the content uniformity of the mixed powder formulation in the one-dose package was compared between the formulations prepared by the pharmacists and those prepared by a pharmacy robot. Our study indicates the possibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. Studies on further applications of Raman spectroscopy in the field of clinical pharmacology are expected.

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs.

Direct site-specific biotinylation of RNA molecules was achieved by specific transcription mediated by unnatural base pairs. Unnatural base pairs between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y), or 2-amino-6-(2-thiazolyl)purine (denoted as v) and y specifically function in T7 transcription. Using these unnatural base pairs, the substrate of biotinylated-y (Bio-yTP) was selectively incorporated into RNA, opposite s or v in the DNA templates, by T7 RNA polymerase. This method was applied to the immobilization of an RNA aptamer on sensor chips, and the aptamer accurately recognized its target protein. This direct site-specific biotinylation will provide a tool for RNA-based biotechnologies.

Visualization of Protonation/Deprotonation of Active Pharmaceutical Ingredient in Solid State by Vapor Phase Amine-Selective Alkyne Tagging and Raman Imaging.

Here, we report a simple and direct method to visualize the protonation/deprotonation of an amine active pharmaceutical ingredient (API) in the solid state using a solid-vapor reaction with propargyl bromide and Raman imaging for the assessment of the API during the manufacturing process of solid formulations. An alkyne tagging occurred on the free form of solid haloperidol by the vapor phase reaction, and a distinct Raman signal of alkyne was detected. Alkyne signal monitoring by Raman imaging enabled us to visualize the distribution of the free-form haloperidol in a solid formulation. On the other hand, haloperidol hydrochloride did not react with propargyl bromide in the solid-vapor reaction, and the alkyne signal was not observed. Using the difference in reactivity, the protonation/deprotonation of the amine API in the solid state could be visualized. As an example of application, we tried to visually assess the protonation/deprotonation state when the free-form haloperidol was ground with acids using the solid-vapor reaction and Raman imaging and found that haloperidol was partially protonated when ground with 2 equivalents of hydrogen chloride. Furthermore, we demonstrated the relationship between the degree of protonation and the amount of water added as a medium for grinding haloperidol with succinic acid.

Advanced Applications of Raman Imaging for Deeper Understanding and Better Quality Control of Formulations.

The importance of using the Raman imaging technique is increasing in pharmaceutical sciences, particularly in the quality control of active pharmaceutical ingredients, formulation design, and manufacturing development. Formulation design based on Raman imaging data is important for achieving quality by design. Recently, several novel Raman imaging measurement and analytical techniques have been reported. It is undoubtedly essential for pharmaceutical researchers and manufacturing engineers to use modern Raman imaging technology to produce the best quality pharmaceutical products. This short review seeks to inform researchers and engineers about recent developments in Raman imaging techniques applicable to formulation design and manufacturing.

Assessment of drug content uniformity of atropine sulfate triturate by liquid chromatography-tandem mass spectrometry, X-ray powder diffraction, and Raman chemical imaging.

BACKGROUND: Atropine sulfate is an anticholinergic agent for treatment of hypertrophic pyloric stenosis and is orally administrated as a triturate with lactose hydrate. Because of the low safety margin of atropine sulfate, triturate uniformity is a key safety factor. In this study, we assessed the uniformity of atropine sulfate in 1000-fold triturates prepared by wet mixing and dry mixing methods and discussed the cause of the difference in uniformity between two preparation methods. METHODS: A 1000-fold triturate of atropine sulfate with lactose hydrate was prepared by two different methods: wet mixing and dry mixing. The wet mixing was performed according to Kurashiki Central Hospital protocol and the dry mixing was a simple physical mixing by a rocking mixer. The uniformity of atropine sulfate content in aliquots of a 1000-fold triturate with lactate hydrate was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification. Solid-state analyses of the triturates by Raman chemical imaging and X-ray powder diffraction (XRPD) were performed to investigate the difference in uniformity. RESULTS: The LC-MS/MS quantification showed that the uniformity of atropine sulfate in the 1000-fold triturate was excellent for wet mixing but was significantly variable for dry mixing. On the basis of the Raman chemical imaging and XRPD analyses, it was indicated that an amorphous thin film of atropine sulfate coated the surfaces of the lactose hydrate particles during wet mixing and contributed to the uniformity of the triturate. In contrast, clusters of the crystalline atropine sulfate were found in the dry mixing samples. CONCLUSION: The results showed that better atropine sulfate triturate uniformity was achieved using the wet mixing method rather than the dry method and the cause of the uniformity difference between two mixing methods was indicated by the multilateral assessment.

Vapor Phase Alkyne Coating of Pharmaceutical Excipients: Discrimination Enhancement of Raman Chemical Imaging for Tablets.

Raman chemical imaging has become a powerful analytical tool to investigate the crystallographic characteristics of pharmaceutical ingredients in tablet. However, it is often difficult to discriminate some pharmaceutical excipients from each other by Raman spectrum because of broad and overlapping signals, limiting their detailed assessments. To overcome this difficulty, we developed a vapor phase coating method of excipients by an alkyne, which exhibits a distinctive Raman signal in the range of 2100-2300 cm(-1) . We found that the combination of two volatile reagents, propargyl bromide and triethylamine, formed a thin and nonvolatile coating on the excipient and observed the Raman signal of the alkyne at the surface. We prepared alkyne-coated cellulose by this method and formed a tablet. The Raman chemical imaging of the tablet cross-section using the alkyne peak area intensity of 2120 cm(-1) as the index showed a much clearer particle image of cellulose than using the peak area intensity of 1370 cm(-1) , which originated from the cellulose itself. Our method provides an innovative technique to analyze the solid-state characteristics of pharmaceutical excipients in tablets.

Crystal face identification by Raman microscopy for assessment of crystal habit of a drug.

Crystal habit is one of the key crystallographic characteristics of active pharmaceutical ingredients (APIs), especially those that are poorly soluble. X-ray powder diffraction has commonly been used to assess crystal habit; however, it can only provide macro-information regarding crystal habit for a whole powder sample, not for individual crystals. We describe an approach that uses Raman microscopy for the identification of crystal faces to assess crystal habit at the individual particle level. An antiepileptic agent, phenytoin, was used as the model substance. Phenytoin crystals form a primitive orthorhombic cell. Raman microscopy was used to identify three different patterns of Raman spectra, corresponding to the crystallographic axis that was parallel to the polarization direction of the excitation laser. Thus, a combination of Raman spectra, in which the polarization direction was horizontal and vertical to the morphologically long axis of the crystal, characterized the crystal face. Phenytoin crystals were prepared under various conditions, and the horizontal/vertical combinations of Raman spectra were recorded for individual crystals. The dominantly exposed crystal faces for each condition were identified. This analytical method enables micro-view assessments of crystal habit, which are helpful for identifying the habits of APIs alone and in formulations.

Prevention of the exposure by cyclophosphamide oral tablet.

BACKGROUND: Unintended exposure to antitumor agents from an oral medicine may place healthcare workers and patients taking medicine at risk. In this study, the exposure to blister pack by CP (cyclophosphamide) and appropriate preventive procedures were examined. FINDINGS: CP detected inside the blister pack of the tested seven lots by LC-MS/MS ranged from 8.2 to 199.6 ng. Raman imaging clearly showed that CP ingredient was completely covered by the tablet coating layer and had not leached out of the tablet. In addition, the amount of CP detected inside the vials was suppressed under the lower detection limit until day 28, and only 6.0 ng was detected only at day 56. CONCLUSIONS: Various amounts of CP were contaminated to not only the inside of the blister pack but also the outside. This contamination may be caused not only by the manufacturing environment but also by the CP oral tablets themselves through volatilization of CP. Refrigerated storage of CP oral tablets may protect healthcare workers and patients from contact with CP.

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo.

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.

Cytostatic evaluations of nucleoside analogs related to unnatural base pairs for a genetic expansion system.

The introduction of an unnatural base pair into DNA enables the expansion of genetic information. To apply unnatural base pairs to in vivo systems, we evaluated the cytostatic toxicity of several nucleoside analogs by an MTT assay. Several nucleoside analogs based on two types of unnatural base pairs were tested. One is a hydrogen-bonded base pair between 2-amino-6-(2-thienyl)purine (s) and pyridin-2-one (y), and the other is a hydrophobic base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa). Among the nucleoside analogs, the ribonucleoside of 6-(2-thienyl)purine possessed the highest cytostatic activity against CCRF-CEM and especially HT-1080, as well as the normal fibroblast cell line, WI-38. The other analogs, including its 2'-deoxy, 2-amino, and 1-deazapurine nucleoside derivatives, were less active against CCRF-CEM and HT-1080, and the toxicity of these nucleosides toward WI-38 was low. The nucleosides of y and Pa were inactive against CCRF-CEM, HT-1080, and WI-38. In addition, no cytostatic synergism was observed with the combination of the pairing nucleosides of s and y or Ds and Pa.

Chemistry of bisulfite genomic sequencing; advances and issues.

Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3-5 M sodium bisulfite at pH 5 and at 50-55 degrees for 4-20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases.

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.

Simple preparation of biotinylated RNA by transcription via an unnatural base pair.

To develop a convenient method for RNA fragment biotinylation at a specific position, we synthesized ribonucleoside 5'-triphosphates of biotinylated unnatural bases. These unnatural bases were composed of 2-oxo(1H)pyridine (denoted as y), specifically pairing with 2-amino-6-(2-thienyl)purine (denoted as s) in transcription. The y base was linked with biotin through an allylamine or propynylamine linker at position 5 of y. These triphosphate derivatives were specifically incorporated by T7 RNA polymerase into RNA opposite s in templates. The specific biotinylation and immobilization of an RNA aptamer were demonstrated by this system.