Studies on quality and safety control of drugs for human use from transgenic animals/clone animals

Recently the pharmaceuticals, which were produced using transgenic animals, have been developed, and will be submitted for registration in nearly future in Japan as well as in USA and EU. In addition, clone animals are also thought to be useful for the productions of the pharmaceuticals. This study has been, therefore, undertaken to establish the technical requirement for registration of the pharmaceuticals. They should be evaluated from the following standpoints: 1) Transgene construct and its characterization; 2) Creation and characterization of the transgenic founder animal; 3) Establishment of a reliable and continuous source of transgenic founder animals; 4) Generation and selection of the production animals; 5) Maintenance of transgenic animals; 6) Recovery and purification of products from transgenic animals; 7) Characterization of products; 8) Process validation/evaluation and in-process test; 9) Specification of products; 10) Stability of products; 11) Preclinical safety evaluation and clinical evaluation. Cloning technology by nuclear transfer of a transformed somatic cell has been already applied to the creation of the transgenic founder animal for the production of pharmaceuticals. The pharmaceuticals produced using the clone animals could be evaluated from almost the same standpoints. However, the flexible evaluation will be also needed depending on the development of the technology.     

Factors affecting pulsatile ocular blood flow in normal subjects

BACKGROUND: The factors that influence pulsatile ocular blood flow (POBF) were evaluated in normal subjects. METHODS: POBF was measured in 80 normal subjects using Langham OBF computerised tonometry. The effect of age, systolic and diastolic blood pressure, refractive error, intraocular pressure, and axial length on POBF was evaluated using multiple regression analysis. RESULTS: The mean (SD) POBF value was 593.3 (203.6) microl/min (range 290.7-1201.6). Of all the independent variables in the model, only the axial length was statistically significant (p = 0.008). The regression coefficient was negative, indicating that the axial length decreased with increasing POBF. CONCLUSIONS: These data suggest that, in normal subjects, the POBF decreases as axial length increases. Choroidal blood flow may decrease as the axial length increases. The axial length may therefore be a major factor affecting POBF.     

Multi-level disruption of the spinal nerve root sleeves in spontaneous spinal cerebrospinal fluid leakage--two case reports

A 37-year-old male and an 18-year-old male presented with spontaneous spinal cerebrospinal fluid (CSF) leakage from multiple nerve root sleeves. Both patients suffered abrupt onset of intense headache followed by nausea, dizziness, and one patient with and one without positional headache. Radioisotope spinal cisternography of both patients revealed that the CSF leaks were not localized in a special zone but distributed to multiple spinal nerve root sleeves. Magnetic resonance (MR) myelography suggested that the spinal CSF column was fully expanded to the root sleeves. The extraspinal nerve bundles demonstrated numerous high intensity spots. Both patients were treated conservatively, and their symptoms resolved within one month. Repeat radioisotope cisternography and MR myelography confirmed the spine was normal after recovery. We suggest that spreading disruption of the arachnoid membrane occurs at the nerve root sleeves due to CSF overflow into the spinal canal.     

Prednisolone inhibits an IgE-mediated late-phase allergic cutaneous reactionby interfering with the activation of mast cells in mice

Epicutaneous antigen challenge in passively sensitized mice with IgE produces a biphasic cutaneous response which peaks 1 h (immediate-phase reaction) and 24 h (late-phase reaction; LPR) after the antigen challenge. In this model, anaphylactic degranulation and interleukin 6 (IL-6) expression between 4 and 8 h are observed in resident mast cells as the preceding stage of LPR. Prednisolone at a dose of 3 mg kg(-1) clearly inhibited the LPR when administered 2 h before and 4 h after antigen challenge. Slight or no inhibition of LPR was observed by prednisolone administered 6-12 h after challenge. Histologically, prednisolone treatment 2 h before antigen challenge completely inhibited edema and inflammatory cell infiltration, while treatment at 6 h did not at all. In order to investigate the relationship between inhibition of LPR by prednisolone and mast cell activation, the effects of prednisolone on degranulation of mast cells and IL-6 expression in mast cells were investigated. 8 h after antigen challenge, prednisolone clearly inhibited the increase in the number of anaphylactic degranulated and IL-6-positive mast cells by administration 2 h before challenge, but did not affect it by administration 6 h after challenge. These data indicate that the inhibitory mechanism of prednisolone on LPR, at least, involves the inhibition of mast cell activation before LPR.     

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.     

Determination of serotonin in microdialysis samples from rat brain by microbore column liquid chromatography with post-column derivatization and fluorescence detection

The present paper describes a new method for on-line determination of 5-HT in brain microdialysates from awake rats by microbore column liquid chromatography with post-column derivatization and fluorescence detection. The derivatization reagent contained 1 mM benzylamine and 0.5 mM potassium hexacyanoferrate (III), both dissolved in a mixture of acetonitrile and 25 mM borate buffer (pH 11.0) (1:1, v/v). The limit of detection (S/N=3) for 5-HT was 0.5 fmol/20 microl. The samples were injected every 20 min onto a microbore column packed with C18 silica gel. The method exhibits an excellent stability over the periods of at least 12-24 h. The basal levels of 5-HT from 25 awake rats were 7.10+/-1.06 fmol/20 microl in the dorsal hippocampus and 4.64+/-0.91 fmol/20 microl (mean+/-SD) in the striatum. The 5-HT release increased to about 1500% during the perfusion with 100 mM K(+) containing Ringer solution or it was reduced to 60 or 40% during the perfusion with 1 microM tetrodotoxin or calcium free Ringer, respectively. The new method can be used to monitor extracellular 5-HT following acute systemic drug administration.     

Depressive symptoms and associated factors in a cognitively normal elderly population: the Tajiri Project

BACKGROUND AND OBJECTIVE: Since depression is one of the main problems of elderly subjects, it is important to examine the prevalence of this condition and to identify associated factors. METHODS: A total of 1525 cognitively normal subjects aged 65 years and over in the town of Tajiri, a typical agricultural town in Japan, were analysed. Their MMSE (mini-mental state examination) scores were 24 or over. Depressive state was assessed by Zung's SDS (self-rating depression scale) with a comprehensive interview to examine ADL, demographics and symptoms associated with illness, etc. The prevalence of depression was calculated. To determine the factors associated with depression, the t-test and the Chi-square test were used. To examine the relative strength of each factor, logistic regression analysis was performed. RESULTS: The ratio of the depressive subjects was 6.4%, lower than those of previous reports, probably due to the effect of excluding dementia subjects. The ratio for older females aged 80 years and over was 14.3%, which was significantly higher than that of the males. Among socio-demographic factors, sex, age, number of children and perception of economic status, were significantly related. For health status and ADL, such factors as perception of health and medical history of heart disease and rheumatism were related. For familial and social status, factors such as daily activity and several conversation abilities were related. The logistic regression analysis indicated that perception of health and daily activity were associated. CONCLUSIONS: In this study, we isolated some factors related to depression in a cognitively normal population. Knowledge of such factors is important for appropriate mental care of aged subjects.