Effects of solute miscibility on the micro- and macroscopic structural integrity of freeze-dried solids.

The purpose of this study was to elucidate the effect of solute miscibility in frozen solutions on their micro- and macroscopic structural integrity during freeze-drying. Thermal analysis of frozen solutions containing poly(vinylpyrrolidone) (PVP) and dextran showed single or multiple thermal transitions (T'g: glass transition temperature of maximally freeze-concentrated solutes) depending on their composition, which indicated varied miscibility of the concentrated noncrystalline polymers. Freeze-drying of the miscible solute systems (e.g., PVP 10,000 and dextran 1060, single T'g induced physical collapse during primary drying above the transition temperatures T'g). Phase-separating PVP 29,000 and dextran 35,000 mixtures (two T'g s) maintained their cylindrical structure following freeze-drying below both of the T'g s (<-24 °C). Primary drying of the dextran-rich systems at temperatures between the two T'g s (-20 to -14 °C) resulted in microscopically disordered "microcollapsed" cake-structure solids. Freeze-drying microscopy (FDM) analysis of the microcollapsing polymer system showed locally disordered solid region at temperatures between the collapse onset (T(c1)) and severe structural change (T(c2)). The rigid dextran-rich matrix phase should allow microscopic structural change of the higher fluidity PVP-rich phase without loss of the macroscopic cake structure at the temperature range. The results indicated the relevance of physical characterization and process control for appropriate freeze-drying of multicomponent formulations.

Ammonium ion level in serum affects doxorubicin release from liposomes.

In this study, we measured the release of drug from liposome-encapsulated doxorubicin (DXR) in human and mouse serum. While human serum did not induce DXR-release, mouse serum significantly induced DXR-release in a temperature- and time-dependent manner. Release of DXR was clearly observed in ultrafiltrated mouse serum, indicating that low-molecular substances affect DXR-release. Therefore, the level of Na+, Cl(-), NH4+, and urea nitrogen in each type of serum was measured. Only the concentration of NH4+ in mouse serum was significantly higher than that in human serum. Furthermore, addition of ammonium acetate to human serum induced DXR release at the same level observed in mouse serum. These results indicate that the NH4+ concentration in serum might greatly affect the release of DXR from liposomes.

Development and release characterization of hyaluronan-doxycycline gels based on metal coordination.

A simple mixing with hyaluronan (HA), doxycycline (DC) and divalent metal cation in an aqueous solution enabled a thermoreversible water-soluble gel to form. For the cross-linking, two kinds of interactions were supposed. One was an electrostatic interaction between a positively charged group in DC and a negatively charged carboxyl function of HA, and the other was a chelation at the phenolic diketone moiety in DC. Since the gel was formed physically, the critical polymer concentration for gelation was present, and it was about 0.05% for HA with a molecular weight of 1.6x10(6). The hydrogel would be formed holding water in the HA entanglement network when DCs on HA chains made coordinate bonds through metal chelation. By changing the mixing ratio, two types of gels with different characteristics in drug release could be prepared. One was a gel with zero-order release prepared by mixing the same amount of HA and DC in equivalent. The other was a gel indicating Fickian diffusion-type release by mixing more DC than HA. Further, by controlling the absolute concentration of HA and DC, or the molecular weight of HA, some gels with desired release profiles could be prepared.

Recyclable characteristics of hyaluronate-polyhydroxyethyl acrylate blend hydrogel for controlled releases.

Hyaluronate-hydroxyethyl acrylate blend hydrogels which have a wide range of composition and characteristics were investigated. Glycidyl methacrylate derivatized hyaluronate (GMA-HA) were synthesized by coupling GMA to hyaluronate (HA) in the presence of a photoinduced initiator for polymerization. By copolymerizing radically GMA-HA and hydroxyethyl acrylate (HEA) under various compositions (weight ratios of HEA and GMA-HA: 1-20), GMA-HA hydrogels could be prepared in wide ranges of characteristics. These HA-PHEA gels possessed the feature that the dried ones recovered completely to the original swelling states on repeated runs, i.e. recyclable gels. The water contents of these hydrogels in equilibrium swellings in water (W(w)) were 0.99-0.86, and their viscoelastic properties were measured by a creep. The spontaneous elastic moduli were 1.05x10(5)-1.94x10(5) N m(-2), and they were mechanically tough. Their effective charge densities were estimated from the partition coefficients of sodium benzoate (NaBA) and decreased from -0.033 to -0.044 mol dm(-3) with increasing contents of HEA. Release of NaBA was studied, and the diffusion coefficients were found to be from 6.95x10(-10) to 0.12x10(-10) m(2) s(-1) with increasing the ratio of HEA. Their diffusion coefficients were found to be much less than the values estimated from the lattice model.

Hyaluronate depolymerization following thermal decomposition of oxytetracycline.

Depolymerization of sodium hyaluronate (HA) by tetracyclines was investigated. Reduction in HA molecular weight was followed by size exclusion chromatography with a low angle laser light scattering detector. On mixing with oxytetracycline hydrochloride (OTC) solution and incubating at 37 degrees C, HA was gradually depolymerized. OTC, a representative antibiotic, is known as a photosensitizer, and phototoxic side effects relevant to radicals have been reported. However, HA depolymerization required no irradiation. As time passed, OTC solution incubated at 37 degrees C got colored reddish brown, even in the dark. With reversed-phase HPLC separation, several peaks derived from decomposed OTC appeared. One of the peaks had an absorbance in the visible range. A quantitative correlation between the discoloration and the HA depolymerization rate was obtained. On the other hand, when samples were incubated below 25 degrees C, change of color was slight, and practically no HA depolymerization was observed after up to 4 h. Oxygen depletion by nitrogen saturation or addition of mannitol also prevented the depolymerization. Under anaerobic conditions, the color of the solution did not change, whereas it turned red under aerobic conditions in the presence of mannitol. The mannitol did not inhibit the OTC decomposition, but it preserved HA from damage. On the basis of the known decomposition of OTC and the results of HPLC separation, anhydrooxytetracycline can be proposed as the derivative causing HA depolymerization.

Statistical and probabilistic approaches to confidence intervals of linear calibration in liquid chromatography.

The aim of this paper is to compare the reliability of two approaches to estimate the 95% confidence intervals of linear calibration in real situations. One is the statistical approach, which is well known in statistics, and the other is the probabilistic approach, which is based on a theory to predict the precision of instrumental analyses mainly from signal and noise, called FUMI (Function of Mutual Information) theory. The high-performance liquid chromatographic determination of quisalofop and maltose is taken as an example. Calibration lines obtained under the same experimental conditions are superimposed on the 95% confidence intervals to investigate whether the resulting confidence intervals can include all the calibration lines reasonably. A pair of 95% confidence intervals (upper and lower limits) can be calculated from each calibration line, but varies from calibration line to calibration line, although obtained under the same experimental conditions. The variability and reliability of the 95% intervals are also examined.

Determination of enantiomeric purity of hyoscyamine from scopolia extract using HPLC-CD system without chiral separation.

Enantiomeric ratio of hyoscyamine from Scopolia extract was determined by chiral HPLC-CD analysis. It was found that circular dichroism (CD) detection allowed the analysis of the sample without any special pretreatment whereas UV detection required an ammonia-ether extraction. To obtain a shorter analysis time for the determination, reversed-phase HPLC-CD analysis was applied by using a g-factor calibration curve (EE% vs. CD/UV). The analysis time was shortened from 35 to 18 min. EE% values obtained were consistent with those by chiral HPLC analysis.

Preparation and characterization of hyaluronate-hydroxyethyl acrylate blend hydrogel for controlled release device.

Hyaluronate-hydroxyethyl acrylate blend hydrogels were investigated as matrices for controlled release devices. Glycidyl methacrylate (GMA) derivatized HA (GMA-HA) was synthesized by coupling of GMA to HA in the presence of a suitable catalyst. These hydrogels were prepared by a free radical copolymerization of GMA-HA and hydroxyethyl acrylate. The water content of these hydrogels at equilibrium swelling in water (Ww) was 0.978+/-0.0073 (n=18); however, these hydrogel was mechanically tough and could be used as disk shape. The hydrogels swelling were found to depend on ionic strength and pH. The dried hydrogels quickly regained their original condition in water, and they swelled to more than 90% of its initial water contents after 30 min. This swelling-deswelling behavior was reproducible. The release of chlorpromazine HCl as a model cationic drug from the gels was suppressed significantly in water. The release increased with increasing the ionic strength and decreasing pH of bulk solutions.

[Evaluation of molecular weight of hyaluronate preparations by size-exclusion chromatography].

Hyaluronate (HA), a glycosaminoglycan polysaccharide, has been used as a biomedical polymer to treat osteoarthritis by intra-articular injections and in ophthalmic surgery, such as anterior segment surgery. In this study, the molecular weight (MW) of HA preparations was estimated by size-exclusion chromatography (SEC), using HA and pullulan fractions as molecular weight standards, and the MW values obtained were compared to those obtained with a low angle laser light scattering detector (LALLS). The results showed that the universal calibration with pullulan as the standard is useful for HA preparations. The conditions of SEC for HA were also investigated, and the results suggested that a high ionic strength and low flow rate of the eluent were preferable for high molecular weight HA preparations.

[Analysis of B-3 desamido insulin in human insulin preparations by HPLC].

The content of A-21 desamido insulin (A-21 DI) and B-3 desamido insulin (B-3 DI) in human insulin preparations was measured by new RP-HPLC method using a neutral eluent (pH 6.5). Sometimes the content of B-3 DI in human insulin preparations was about two times larger than that of A-21 DI. In particular, in the case of neutral insulin injection, the content of B-3 DI has increased remarkably as the increase of total desamido content. The content of B-3 DI obtained by the new RP-HPLC method will open the new aspect of the impurity test for insulin preparations.

[Studies on the molecular weight standards for size-exclusion chromatography--standardization of a molecular weight standard for water-soluble polymers].

The possibility of the standardization of molecular weight standards for size-exclusion chromatography (SEC) was investigated in the case of water-soluble polysaccharides such as dextran, hyaluronate and chitosan. In the case of dextran, comparing the method adopted for dextran in EP and BP, it was shown that pullulan, one of commercially available molecular weight standards, can be used in usual SEC method. For hyaluronate and chitosan, applying the Mark-Houwink correction to the calibration curve obtained by pullulan, the acceptable molecular weights could be estimated.

Discrimination limit for purity test of human insulin by capillary electrophoresis.

Because of the inevitable noise in instrumental analysis, a purity test can overlook an illegitimate drug that contains an undesired substance in a higher amount than the prescribed limit. The lowest (average) amount of undesired substance which leads to the right results of the purity test with 95% probability is referred to here as 95% discrimination limit. This paper presents a method for predicting the discrimination limit for the purity test of human insulin in capillary electrophoresis (CE). The theory and experiments show that if the legitimate limit of a degradation product (desamido insulin) is 3.0% of the total amount of the insulin formulation, the 95% discrimination limit in the CE system used in this study is 3.24% desamido insulin. Since the statistical aspects of the purity test are provided by the interpretation of the baseline fluctuation in the instrument, the usual strategy to repeat the instrumental analysis on the same samples is unnecessary in the present study.

Separation of B-3 monodesamidoinsulin from human insulin by high-performance liquid chromatography under alkaline conditions.

The separation of human insulin (HI) and related compounds such as A-21 monodesamido HI (A-21DHI) and B-3 monodesamido HI (B-3DHI) was investigated using reversed-phase HPLC and capillary zone electrophoresis (CZE). In the case of HPLC under the usual acidic conditions, whereas A-21DHI was separately eluted, B-3DHI was included in the HI peak. Then it was found that by applying a novel ODS column resistant to alkaline conditions, a good separation of B-3DHI from HI could be achieved using an alkaline eluent, and two peaks corresponding to B-3DHI were eluted in front of the HI peak.

[The Somatropin Reference Standard (Control 951) of the National Institute of Health Sciences].

Somatropin material was examined for preparation of the "Somatropin Reference Standard". The candidate material was evaluated by a domestic collaborative study in which eight laboratories participated. The protein content was determined to be 4.5 mg/Vial based on amino acid analysis. Because of the possibility of application as a chemical reference standard for assay by the HPLC method, a physico-chemical evaluation of the candidate material was also performed. By SE-HPLC, the content of polymer, dimer were determined to be 0.54%, 0.98%, respectively. By RP-HPLC, the early peak area ascribed to desamido and sulfoxide form was 1.07% of the total peak area. And for informational data, the potency of the candidate material, being estimated by three different biological methods, weight gain assay, tibia test and adiposeconversion assay is 14.8 IU/vial. Based on the above results, the candidate was authorized as the Somatropin Reference Standard of the National Institute of Health Sciences.

[Interaction between sodium hyaluronate and tetracyclines].

The interaction between sodium hyaluronate (HA) and 6 kinds of tetracycline (TCs) was investigated by dissolution tests, conductivity, viscosity and turbidity measurements. The 6 TCs were tetracycline (TEC), oxytetracycline (OTC), doxycycline (DOC), methacycline (METC), minocycline (MINC) and chlortetracycline (CTC). In dissolution tests, the release of TCs from the HA solution contained in the cellulose tube was measured and it was found that the 50% release time (T50) of TCs was prolonged with the increase of HA concentration. And the dependence of T50 on HA concentration was different in TCs: the release of METC was most delayed and that of OTC was least affected. By measuring the conductivity of TCs solutions, the binding of TCs to HA was suggested. In viscosity measurements of the HA solution at various pHs, the increase of viscosity was observed by the addition of METC. However in the case of OTC such an increase was not observed. The dissolution tests at various pHs were further tried and the release rate of METC depended on the pH, while that of OTC showed no dependency. Finaly, it was found that T50 of TCs from the HA solution correlates well with the literature values of apparent partition coefficients of TCs between n-octanol and aqueous buffers. Comparing T50 of 6 TCs from carboxymethylchitin (DS = 0.56) solution, it was clarified that the lipophilicity of TCs are more effective to retard the release in the case of the HA solution. From the above results, an electrostatic interaction between HA and TCs can be assumed to be almost similar among 6 TCs.(ABSTRACT TRUNCATED AT 250 WORDS)

[Sustained-release effect of the direct compressed tablet based on chitosan and Na alginate].

In the dissolution test of tablets prepared by the direct compression of chitosan with 10% sodium alginate (NaAlg), the release of theophylline was extended independently of the medium pH. The drug release mechanism of the tablet was investigated by measuring the dissolution behavior of such excipients as chitosan and NaAlg. It was shown that at the lower pH the drug release was accompanied with the dissolution of chitosan, while at the neutral pH it was controlled by the gelation and dissolution of NaAlg. Further more by the oral administration of the tablet to beagle dogs, the sustained release of theophylline was also confirmed by some bioavailability parameters. Thus the chitosan tablet with 10% NaAlg was expected to be a pH-independent sustained release dosage form.

[The Human Insulin Reference Standard (Control 921) of the National Institute of Health Sciences].

Raw human insulin material was examined for preparation of the "Human Insulin Reference Standard". The candidate material was evaluated by a domestic collaborative study in which four laboratories participated. The biological activity was determined to be 26.0 Unit/mg against the International Human Insulin Reference Standard (Code 83/500), based on the rabbit blood-glucose method specified in the JP XII (1991). Because of the possibility of application as a chemical reference standard for assay by the HPLC method, a physico-chemical evaluation of the candidate material was also performed. The total desamide form, dimer and oligomer impurities were estimated to be about 1% by HPLC. Based on the above findings, this raw material was authorized as the Human Insulin Reference Standard of the National Institute of Health Sciences.

[Elcatonin Reference Standard (Control 921) of National Institute of Health Sciences].

The raw material of elcatonin was examined for preparation of the "Elcatonin Reference Standard". The candidate material was evaluated by a domestic collaborative study in which five laboratories participated. The biological activity was determined to be 11.3 Unit/Amp. against the International Elcatonin Reference Standard (Code 84/614), based on one hour hypocalcaemia rat bioassay. In spite of the differences in rat strain, sex, administration method, dosage and assay method for serum calcium etc., the separately obtained biological activities for the candidate agreed closely with each other (95% confidence limits 11.08-11.53 Unit/Amp.). The physico-chemical evaluation of the candidate material was also performed, by using HPLC and amino acid chromatography. Based on the above results, this raw material was authorized to be the Elcatonin Reference Standard of the National Institute of Health Sciences.

[Partition behavior of tropane alkaloids between organic solvents and water].

The partition behavior of tropane alkaloids (TrA) between organic solvent and water was studied under various conditions. The apparent partition coefficient, log P', increases with pH with a slope of +1 in the acidic to neutral pH range, then tends to approach to a constant value in the alkaline region. Thus the partition of TrA to the organic phase is based on the neutral form of TrA. From the pH dependency of the partition and pH titration of TrA, the intrinsic partition coefficients log P0 and pKa values for each TrA were estimated. Values of pKa and log P0 for scopolamine were smaller than those for atropine or homatropine. It was considered that the presence of the epoxide ring in scopolamine molecule reduces the log P0 and pKa because of its polar and electro-inductive effect. It was also expected from the results of pH-dependent partition behavior that in the low pH region some portion of TrA cations is transferred to the organic phase by forming an ion-pair complex with an anion, in the aqueous phase. The log P' of TrA was measured in the presence of various kinds of anions (halides and alkyl sulfonates, C5-C8) at about pH 2, where almost all TrA are present as a protonated form. The relations between the log P' and the logarithmic concentration of anions is linear at low concentrations of anions as expected from the ion-pair partition equation.(ABSTRACT TRUNCATED AT 250 WORDS)

[High Molecular Weight Urokinase Reference Standard (Control 901) of National Institute of Health Sciences].

A candidate for "High Molecular Weight Urokinase Reference Standard (HMW-UK RS, Control 901)" of the National Institute of Health Sciences (NIHS) was examined for evaluating the fibrinolytic activity. The urokinase activity of the candidate HMW-UK RS was determined by "two-stage method" as 800 Unit/Amp. against the present Urokinase Reference Standard (Control 881), through the collaborative study involving five laboratories. According to the gel-permeation chromatography of the candidate for the molecular determination, it was confirmed that more than 97% of the total urokinase activity was attributed to the higher molecular fraction, named HMW-UK. Thus it was authorized as the High Molecular Weight Urokinase Reference Standard of the NIHS.