Crystallization of Amorphous Nifedipine Under Isothermal Conditions: Inter-laboratory Reproducibility and Investigation of the Factors Affecting Reproducibility.

High inter-laboratory reproducibility is required for conducting collaborative experiments among several laboratories. The primary aim of our evaluation of the physical stability of amorphous drugs, conducted in co-operation with eight laboratories, was to establish a protocol for isothermal storage tests to obtain data of the same quality from all the participating laboratories. Sharing a protocol that contained the same level of detail as the experimental section of general papers was insufficient for high inter-laboratory reproducibility. We investigated the causes of variations in the data from the various laboratories and restricted the protocol step-by-step to achieve high inter-laboratory reproducibility. The various experimentalists had very different levels of awareness regarding how to control the temperature of a sample as the samples were transferred into and out of thermostatic chambers. Specific instructions on how to conduct this operation, such as regarding the time required for the transfer and thermal protection of the container during the transfer, helped to reduce variation. Improved inter-laboratory reproducibility revealed that the physical stabilities of amorphous drugs differed when samples were prepared in differently shaped aluminum pans designed for various differential scanning calorimeters.

Influence of the crystallization tendencies of pharmaceutical glasses on the applicability of the Adam-Gibbs-Vogel and Vogel-Tammann-Fulcher equations in the prediction of their long-term physical stability.

Amorphization is a powerful approach for improving the aqueous solubility and bioavailability of poorly water-soluble compounds. However, it can cause chemical and physical instability, the latter of which can lead to crystallization during storage, diminishing the solubility advantage of the amorphous state. As there is no standard method for predicting the physical stability of amorphous materials, a long-term stability study is needed in drug development. This study investigated the correlation between the physical stability of amorphous compounds and molecular mobility based on the assumption that physical stability is governed by the diffusional motion of a molecule. Model compounds were evaluated for crystallization onset time, structural relaxation time, fragility, and fictive temperature. The crystallization onset time of acetaminophen glass correlated with its relaxation time calculated from the Adam-Gibbs-Vogel equation; however, that of felodipine glass correlated with the relaxation time calculated from the Vogel-Tammann-Fulcher equation. The different crystallization tendencies of these compounds can be explained by the differences in the rate limiting steps in their crystallization processes, indicating the importance of distinguishing the critical process associated with crystallization. These findings will be useful for more accurate prediction of long-term physical stability of amorphous materials.

Application of Co-Amorphous Technology for Improving the Physicochemical Properties of Amorphous Formulations.

Co-amorphous technology was recently introduced to stabilize drugs in the amorphous state for drug development. We examined the predictability of the formation of co-amorphous systems and identified two reliable indicators of successful formation: (1) a negative Δ Hmix value and (2) small Δlog P between components. Moreover, we found that the stability of co-amorphous systems was improved when (1) Δ Hmix was negative and (2) amorphous forms of the constituent compounds were stable. Furthermore, we concluded that co-amorphous systems with small (negatively large) Δ Hmix values had lower hygroscopicity. Typically, amorphous solid dispersions exhibit hygroscopicity because polymers exhibit large hygroscopicity. We proved the superiority of co-amorphous technology over amorphous solid dispersion in this respect. Our results provide methods for (1) establishing a screening method and (2) improving hygroscopicity, which may make co-amorphous technology more useful than amorphous solid dispersion technology.

Selective activation of two sites in RNA by acridine-bearing oligonucleotides for clipping of designated RNA fragments.

Artificial enzymes for selective scission of RNA at two designated sites, which are valuable for advanced RNA science, have been prepared by combining lanthanide(III) ion with an oligonucleotide bearing two acridine groups. When these modified oligonucleotides form heteroduplexes with substrate RNA, the two phosphodiester linkages in front of the acridines are selectively activated and preferentially hydrolyzed by lanthanide ion. This two-site RNA scission does not require any specific RNA sequence at the scission sites, and the length of clipped RNA fragment is easily and precisely controllable by changing the distance between two acridine groups in the modified oligonucleotide. The two-site scission is also successful even when the substrate RNA has higher-order structures. By using these two-site RNA cutters, RNA fragments of predetermined length were obtained from long RNA substrates and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single nucleotide polymorphisms in homozygous and heterozygous samples were accurately and easily detected in terms of the difference in mass number. Multiplex analyses of in vitro transcripts from human genome were also successful.

Tandem site-selective RNA scission utilizing acridine-DNA conjugates.

Useful technique to clip designated short RNA fragments from long substrates has been prepared by combining oligonucleotides bearing two acridine groups and lanthanide(III) ions. The substrate RNA is site-selectively activated at two designated phosphodiester linkages by complementary bis-acridine-modified DNA, and is promptly cleaved by lanthanide(III) ions to produce short RNA fragment between the two scission sites. By applying this technique, efficient genotyping method for single nucleotide polymorphism (SNP) have been developed.

Site-selective RNA scission at two sites for precise genotyping of SNPs by mass spectrometry.

Short RNA fragments containing single nucleotide polymorphism (SNP) sites have been selectively clipped out of substrate RNA by using complementary DNA having two acridine residues and Lu(III), and the genotype of the substrate is accurately and easily determined by mass analysis of these fragments.

Metal ion-induced site-selective RNA hydrolysis by use of acridine-bearing oligonucleotide as cofactor.

New types of noncovalent ribozyme-mimics for site-selective RNA scission are prepared by combining metal ions with oligonucleotides bearing an acridine. Lanthanide(III) ions and various divalent metal ions (Zn(II), Mn(II), Cu(II), Ni(II), Co(II), Mg(II), and Ca(II)) are employed without being bound to any sequence-recognizing moiety. The modified oligonucleotide forms a heteroduplex with the substrate RNA, and selectively activates the phosphodiester linkages in front of the acridine. As a result, these linkages are preferentially hydrolyzed over the others, even though the metal ions are not fixed anywhere. The scission is efficient under physiological conditions, irrespective of the sequence at the target site. Site-selective RNA scission is also successful with the combination of an oligonucleotide bearing an acridine at its terminus, another unmodified oligonucleotide, and the metal ion. In a proposed mechanism, the acridine pushes the unpaired ribonucleotide out of the heteroduplex and changes the conformation of RNA at the target site for the sequence-selective activation.

Conjugation of various acridines to DNA for site-selective RNA scission by lanthanide ion.

Three types of DNA conjugates having 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro-2-methoxyacridine at the 5'-ends were synthesized and used for site-selective RNA scission together with another unmodified DNA and Lu(III) ion. The target phosphodiester linkages in the substrate RNA were selectively and efficiently activated and were hydrolyzed by free Lu(III) ion. The conjugate bearing 9-amino-6-chloro-2-methoxyacridine was the most active. However, its duplex with the substrate RNA was almost as stable as that of the 9-aminoacridine-bearing conjugate, which was much less active for the RNA activation. The 9-acridinecarboxamide-bearing conjugate was only marginally active. The substituents on the acridine groups in these conjugates positively participate in the present RNA activation, probably by fixing the orientation of the acridine rings.

Novel approach for SNP genotyping based on site-selective RNA scission.

Novel genotyping method for single nucleotide polymorphisms (SNPs), based on site-selective RNA scission, has been developed. A substrate RNA is activated at two sites by complementary acridine-modified DNA having two acridine residues, and is site-selectively cleaved by metal ion catalyst to produce short RNA fragment containing the SNP site. Genotype of the substrate is accurately and easily determined by mass analysis of the fragment.