The klotho mouse shows multiple phenotypes resembling human aging caused by the mutation of a single gene. This mutation is caused by the insertion of ectopic DNA into the regulatory region of the α-klotho gene. The α-klotho gene encodes a type I membrane protein that is expressed predominantly in the kidney and brain. As a result of a defect in α-klotho gene expression, the klotho mouse exhibits multiple age-associated disorders, such as arteriosclerosis, osteoporosis, pulmonary emphysema and short life span. However, the mechanism by which the α-klotho gene product suppresses the aging phenomena has not been identified. Analysis of the pathophysiology of klotho mice is expected to give clues not only to understanding the mechanisms of individual diseases associated with aging but also the molecular mechanisms during human aging. We previously reported that the aberrant activation of μ-calpain is caused by the α-klotho mutation, and such change leads to degradation of cytoskeletal elements. Similar phenomena were observed in normal aged mice. Such deterioration may trigger tissue abnormalities in klotho mice and aged mice, but klotho protein may suppress these processes. We will summarize the function of α-klotho protein based on our research on the relationship between proteolysis and age-related disorders and the recent advanced researches.
Senescence accelerated mouse (SAM), a murine model of accelerated senescence, was established by Toshio Takeda and colleagues. SAM consists of series of SAMP (prone) and SAMR (resistant) lines. All SAMP lines (from SAMP1 to SAMP11) are characterized by accelerated accumulation of senile features, earlier onset and faster progress of age-associated pathological phenotypes, such as amyloidosis, impaired immune response, senile osteoporosis and deficits in learning and memory. These SAMP lines are useful for evaluation of putative anti-aging therapies. For example, SAMP1 line is used to study the anti-aging effect of the antioxidant containing foods and various anti-oxidants, such as coenzyme Q10, vitamin C, lycopene. SAMP8 line exhibiting an early onset of impaired learning and memory is often used for test strategies for therapeutic intervention of dementia of early onset. SAMP6 is used as an animal model for developing new strategies for the treatment of osteoporosis in humans. Various lines of SAM (P1, P6, P8, P10 and R1) are now commercially available for research. In this review, I will briefly introduce various usages of SAM in anti-aging research.
Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that converts toxic O2- to H2O2. Previous studies have reported that a systemic deficiency in Mn-SOD causes neonatal lethality in mice. Therefore, no mouse model is available for the analysis of the pathological role of O2- injuries in adult tissues. To explore an adult-type mouse model, we generated tissue-specific Mn-SOD conditional knockout mice using a Cre-loxp system. First, we generated liver-specific Mn-SOD-deficient mice by crossbreeding with albumin-Cre transgenic mice. Mn-SOD proteins were significantly downregulated in the liver of liver-specific Mn-SOD knockout mice. Interestingly, the mutant mice showed no obvious morphological abnormalities or biochemical alterations in the liver, suggesting a redundant or less important physiological role for Mn-SOD in the liver than previously thought. Next, we generated heart/muscle-specific Mn-SOD-deficient mice by crossbreeding with muscle creatine kinase-Cre transgenic mice. The mutant mice developed progressive dilated cardiomyopathy with specific molecular defects in mitochondrial respiration. Furthermore, skeletal muscle-specific Mn-SOD-deficient mice that had been generated by crossbreeding with human skeletal actin-Cre transgenic mice developed a severe physical disturbance associated with impaired cellular ATP metabolism. These results imply that the superoxide generated in mitochondria plays a pivotal role in the development and progression of pathologies in the heart and skeletal muscle, but not in the liver. In conclusion, we successfully generated various tissue-specific Mn-SOD conditional knockout mice that provide useful tools for the analysis of various oxidative stress-associated diseases.
Senescence Marker Protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases with aging. Recently, we identified SMP30 as the lactone-hydrolyzing enzyme gluconolactonases (GNL) of animal species. GNL was a key enzyme which involved in vitamin C biosynthesis, and the essential role of SMP30 in this synthetic process was verified by a nutritional study. SMP30 knockout mice developed symptoms of scurvy when fed a vitamin C-deficient diet, verifying the pivotal role of SMP30 in vitamin C biosynthesis. Moreover, SMP30 knockout mice were shorter in life span than the wild type when fed autoclaved mouse chow contained ∼55 mg/kg of vitamin C, which we now know contains too little vitamin C to maintain normal levels of vitamin C in tissues. These results demonstrate that vitamin C deficiency shortens longevity, that is, vitamin C deficiency accelerates aging.
Since Harman proposed the “free-radical theory of aging”, oxidative stress is postulated to be a major causal factor of senescence. Accumulation of oxidative stress-induced oxidatively modified macromolecules including protein, DNA, and lipid, were found in tissues during the aging process. However, it is not necessarily clear which factor is more critical for an increase in endogenous reactive oxygen and/or decrease in antioxidative defense, to the age-related increase in oxidative damage. To clarify the production of reactive oxygen increasing with age, we examined reactive oxygen-dependent chemiluminescent (CL) signals in ex-vivo brain slices prepared from different aged animal brain during hypoxia-reoxygenation treatment using a novel photonic imaging method. CL signal was intensified during reoxygenation. The signals in SAMP10 (short life strain) and SAMR1 (control) brain slices increased with aging. The slope of increase of CL intensity with age in P10 was steeper than those in R1. Age-dependent increase of CL intensity was also observed in C57BL/6 mouse, Wistar rat, and pigeon. However, SOD activity in brain was not changed with age. These results suggest that reactive oxygen production itself increase with aging. The rate of age-related increase of CL intensity was inversely related to the maximum life span of the animals. We speculate that reactive oxygen may be a kind of signal for aging and its levels in tissue may determine the aging process and life span. To decelerate the age-related increases of reactive oxygen production is expected as a potent strategy for anti-aging interventions.
Artificial transcription factors targeting any desired genes are very attractive from the standpoint of regulating biological functions for life science studies and clinical applications. In order to generate such transcription factors, specific DNA binding domains are required to address a single site for each gene promoter. C2H2 type zinc finger motif is one of the best frameworks to create new artificial DNA binding proteins for the following features: the zinc finger motif can recognize three bases DNA, be tandemly repeated by covalent linkage, and work as a monomer. Taking advantage of these features, manifold zinc finger proteins targeting various DNA sequences have been created so far. For application to a target in sequences as complex as the human genome, the significantly strict specificity in DNA binding must be required. Conjugating multiple fingers (multi-zinc fingers) enables to recognize longer sequences which are sufficient for addressing a single site in the human genome, whereas it has become known that as the number of finger motifs increases, the equilibrium time with the target sequence is significantly longer by in vitro experiments. Our recent study showed that the multi-zinc finger type artificial transcription factor could activate the reporter gene promptly. There is much interest in creating gene regulators, and the artificial transcription factors based on multi-zinc finger motifs could be a superior scaffold.
Monoclonal antibodies are being used as therapeutics for a number of cancers, such as leukemia, breast and colon cancers, and a lot of monoclonal antibodies specific for tumor-related antigens have been on clinical trials. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms by which antibodies exert anti-tumor effects. ADCC occurs through interaction between the Fc domains of IgG antibodies bound to target cells and Fcγ receptors on the surface of effector cells. In our study, a chimeric antibody, designated M-Ab, was constructed with the V regions from mouse anti-CD20 mAb 1F5 and the C regions from human IgG1 and κ chain. Two or three Fc domains were tandemly repeated downstream of the C-terminus of the M-Ab to give D0-Ab (Fc dimer Ab without a linker), T0-Ab (Fc trimer Ab without a linker), and T3-Ab (Fc trimer Ab with a (GGGGS)3 linker in front of the second and third hinge regions). Here, we show that Fc tandem repeat antibodies bind to all the low-affinity Fcγ receptors with very potent avidities and have greatly enhanced ADCC activity. T3-Ab is about 100 times more potent than the parental 1F5 chimeric antibody in terms of both Fcγ receptor binding and exerted ADCC activity at a 50-100 times less concentration as compared with the parental antibody. Thus, Fc tandem repeat antibodies are anticipated to be candidates for anti-tumor therapeutics and useful tools to elucidate the biological roles of Fcγ receptors.
A large number of emerging pathogens, such as severe acute respiratory syndrome (SARS), human immunodeficiency virus (HIV), and influenza virus are mucosally transmitted and must cross mucosal barriers to infect the host. Thus, to induce a maximal protective effect, it is desirable to apply vaccines by the mucosal route where virus infections start. Mucosal vaccines administered either orally or nasally have been shown to be effective in inducing antigen-specific immune responses at both systemic and mucosal compartments. However the mucosal antigen-specific immune response is weak because most protein antigens can evoke only a weak immune response when they are applied mucosally. Therefore, one strategy to overcome the weakness of the immune response is a co-administration of mucosal adjuvant with the vaccine antigen. Unfortunately, the development of safe and effective mucosal adjuvant has proved to be challenging. Cytokines are promising adjuvants because they are human-derived safe material and display potent immune-modulating functions. In this regards, we have created a mutant tumor necrosis factor-α (TNF-α), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. In this report, we examined the potential of mTNF-K90R as a mucosal adjuvant and evaluated its effectiveness and safety.
Onset and exacerbation of autoimmune disease, such as rheumatoid arthritis and Crohn's disease would be regulated with hundreds of disease-related proteins changing in quality and quantity. Recently, tumor necrosis factor-α (TNF-α) comes to be known that is the key molecule for development of the autoimmune diseases and recognized as the drug target which should be inhibited to overcome the diseases using neutilizing antibodies. Because the functions of TNF-α are regulated with the manner binding to two receptors, TNFR1 and TNFR2, unexpected side-effects would happen with complete inhibition or activation of the TNF receptor signaling. Thus, it is essential to develop a novel drug developing technology, which regulates the binding pattern of TNF-α definitely for therapeutic purposes. In this regard, we have aimed to create the TNF-α mutant, which has selectivity for binding TNFR1 or TNFR2 and regulates the onset and exacerbation of inflammatory diseases. Recently, we have succeeded in creating several TNF-α mutants by phage display techniques which can substitute aimed amino acids to the other, randomly. In this review, we introduce our unique TNFR1-selective antagonist, which can only inhibit the function via TNFR1 correlating with the onset and exacerbation of autoimmune disease. This TNFR1-selective antagonist does not inhibit the host defense function via TNFR2. This mutant TNF-α did not show the increase of virus infection suggested that it may overcome the risk of infectious disease, which is a major side-effect of anti-TNF-α therapy. These results would provide widely the strategy of regulating protein function in molecular level and would show the attractive approach to create safe and effective medical drug reducing side-effects.
Vancomycin (VCM) is a glycopeptide antibiotic that is generally used to treat methicillin-resistant Staphylococcus aureus (MRSA). Recently, it has been reported that VCM clearance (CL) is significantly higher in elderly patients and infants and children with malignancies, compared with those without malignancies. We have treated many patients with a variety of malignant tumors in whom serum VCM concentrations were found to be moderately lower during therapeutic drug monitoring (TDM). The aim of this study was to determine whether the presence of malignant tumors influences trough concentrations of VCM and VCM pharmacokinetics in elderly patients during treatment for MRSA infection. Comparison of the clinical characteristics and VCM pharmacokinetic parameters between patients with and without malignancies was undertaken in 49 elderly Japanese patients infected with MRSA. The mean trough concentration of VCM in patients with malignancies was significantly lower than that in patients without malignancies. Our results showed significantly higher values of VCM CL and volume of distribution and a shorter elimination half-life in patients with malignancies. Univariate logistic regression analysis of the pharmacokinetic parameters indicated that VCM CL contributed as a significant factor independent of the relation to malignant tumor. In conclusion, it is suggested that the therapeutic effects and side effects of VCM should be actively monitored using TDM, because concentrations may decrease when CL increases in VCM therapy for elderly patients with malignant tumors.
Many orally disintegrating tablets have recently been developed to improve oral ingestion and usability and are widely administered clinically, resulting in improved quality of life for patients. Since orally disintegrating tablets rapidly disintegrate in the mouth, the masking of unpleasant taste is important. We investigated the masking of the taste of furosemide (FU) as a model drug with correctives and prepared orally disintegrating tablets. Using maltitol (MA) as a corrective, granules were prepared employing mixing, coating, and mixing/coating methods using a desktop granulator. Each preparation was subjected to tasting. The taste was masked well when granules were prepared by the mixing and mixing/coating methods. Tablets were prepared from these granules with mannitol and crystalline cellulose added as fillers. Tablets made from granules prepared by the mixing and mixing/coating methods showed appropriate strength and disintegrated rapidly. When the amount of MA was increased in the mixing method, the disintegration time was prolonged, and thus the amount should be determined considering both taste masking and disintegration property. The results showed that orally disintegrating tablets of insoluble drugs with an unpleasant taste such as FU should be prepared with the taste masked employing the methods used in this study.
We investigated several methods of taste masking in the preparation of orally disintegrating tablets (ODTs), using furosemide (FU) as a model drug. Four types of FU preparations were prepared: granules with maltitol (MA), granules with yogurt powder (YO), a physical mixture of FU and MA, and a physical mixture of FU and YO. All taste-masking granules were prepared using the dry granulation method. The taste of each type of preparation was evaluated. All four preparations markedly improved the taste of the FU tablets, but the mixing ratios of the correctives did not affect the masking effect. No difference in masking effect was found between MA and YO in the physical mixtures, but the masking effect in the granules with YO was superior to that of the granules with MA. Taste-masked FU tablets were prepared using the direct compression method; crystalline cellulose (Avicel PH-302) and mannitol were added as excipients at the mixing ratio of 1/1. All four types of tablets displayed sufficient hardness, but MA-containing tablets were harder than YO-containing tablets. The hardness of the tablets prepared from YO granules increased as the YO content increased. The most rapidly disintegrating tablets were those of YO granules prepared at a mixing ratio of FU/YO=1/1, which disintegrated within 20 s, followed by the tablets of MA granules prepared at a mixing ratio of FU/MA=1/1. The disintegration times of the tablets made from physical mixtures, in contrast, were longer than 200 s. Disintegration time lengthened as the mixing ratio of YO or MA increased. The hardness and disintegration time of these tablets could be controlled by varying the compression pressure. We found that YO is more useful than MA in masking unpleasant tastes and confirmed that orally disintegrating tablets with taste-masking function can be prepared using granules of YO prepared using the dry granulation method as a new corrective.
Objectives of the prospective, open-label study were to investigate pharmacokinetics of doripenem and determine appropriate doripenem regimens during continuous hemodiafiltration (CHDF) in critically ill patients with renal failure (creatinine clearance <30 ml/min) in the intensive care unit at a university hospital in Japan. Six patients received intravenous (IV) administration of 250 mg of doripenem every 12 or 24 hours during CHDF (dialysis rate, 500 ml/h; hemofiltration rate, 300 ml/h) via a polysulfone hemofilter. Doripenem concentrations in pre- and post-membrane blood (plasma) samples collected at specified times during one dosing interval were measured in order to calculate pharmacokinetic parameters and clearance via hemodiafiltration. Mean half-life (±standard deviation) of doripenem was 7.9±3.7 hours. Total body clearance of doripenem was 58.0±12.7 ml/min, including clearance of 13.5±1.6 ml/min via CHDF. An IV dose of 250 mg of doripenem every 12 hours during CHDF provided adequate plasma concentrations for critically ill patients with renal failure, without resulting in accumulation upon steady-state. Thus, under the conditions tested, CHDF appeared to have little effect on doripenem clearance. Therefore, the blood level of doripenem can be satisfactorily controlled by adjustment of doripenem dose and dosing interval, in accordance with residual renal function in patients receiving CHDF.
The purpose of this study was to evaluate the incompatibility of ceftriaxone sodium with calcium-containing products using the ionic product of precipitation, and the measurement of insoluble microparticles using a light obscuration particle counter. Appropriate volumes of 2% (w/v) calcium chloride solution were added to 0.4-2 mg/ml ceftriaxone isotonic sodium chloride solution, to make solutions with a final calcium ion concentration of 1.25 mmol/l. The solutions were gently agitated and stored at 37°C for 24 h. The number of insoluble microparticles with a diameter less than 10 μm in the mixed sample solution, determined using a light obscuration particle counter, was increased when the ceftriaxone concentration was >0.8 mg/ml. The Saturation Index (defined as the ratio of the ionic product to the solubility product constant) of the prepared mixed solution was 1.1. A white precipitate could be observed visually when the ceftriaxone concentration of the sample solution was 7 mg/ml; the Saturation Index of the solution was 9.7. The effect of the calcium source on incompatibility with ceftriaxone sodium was also evaluated. The numbers of insoluble microparticles in sample solutions made by adding calcium chloride to the sample were significantly higher than those made by adding calcium gluconate. These results suggest that ceftriaxone should not be co-administered with calcium-containing products even if no precipitation is observed visually. There will still be insoluble microparticles caused by incompatibility in the sample solution when the Saturation Index of the solution is over 1.0.
The aim of this study was to prepare poly (lactide-co-glycolide) (PLGA) microspheres encapsulating highly concentrated glycyrrhizin (GZ), a hydrophilic drug, and to compare the release characteristics of GZ in in vitro experiments and GZ elimination into bile after subcutaneous administration in rats. The preparation was carried out based on water drying using a (w/o)/w emulsion. The encapsulation rate of GZ in microspheres was 76% when the GZ concentration in the outer water phase was equal to that in the inner water phase for the preparation of (w/o)/w emulsion. The release of GZ from the microspheres showed a biphasic zero-order profile, that is, the behavior boundary was approximately 12 h. The release of GZ from the microspheres at the periods of 0.5-8 h and 12-672 h was 0.18 mg/h and 0.0012 mg/h, respectively. On the other hand, 0.25% of GZ administered (5.0 mg) was eliminated into bile by 12 h, and the bile clearance rate (1.13 ml/h) of GZ after the subcutaneous administration of the microspheres was the same as that (1.13 ml/h) after the administration of GZ solution. From the results, it is suggested that the initial controlled release (0.18 mg/h) of GZ from microspheres may be beneficial for the hepatic bioavailability of GZ.
The pharmacokinetics of orally administered tacrolimus were examined in six female lupus nephritis patients (mean age 43 years, range 24-55 years). Tacrolimus (3 mg) was administered after supper, and blood tacrolimus concentrations were measured just prior to dosing and 1, 2, 4, 6, 8, 12 and 24 h after administration. The maximum blood concentration (Cmax) was observed 4-8 h (mean: 6.7 h) after administration. The mean Cmax and area under the tacrolimus concentrationti-me curve (AUC0-24 h) were 12.7 ng/ml and 163.1 ng·h/ml, respectively. Although there was a weak correlation between AUC0-24 h values and tacrolimus concentrations 2, 4, and 6 h after administration, concentrations at 12 h and 24 h were highly correlated with AUC0-24 h values, suggesting that the trough concentration (C24 h) and C12 h are valid markers for therapeutic tacrolimus monitoring. Enzyme-linked immunoabsorbent assay (ELISA) and microparticle enzyme immunoassay (MEIA) measurements of blood tacrolimus concentrations were similar. We recommend that monitoring should be carried out by C12 h in lupus nephritis outpatients.
A semisolid dosage form of clonazepam (CZP), administered to the oral cavity between the lower gum and bottom lip with small volume of saline, was developed to obtain the stable dosage which can replace the injection dosage form. Semisolid dosage forms were prepared using a mixture of CZP/(polyethylene glycol 1500 (PEG))/(oleic acid (OA)) at the ratios of 1/39/0, 1/37/2 and 2/36/2 (w/w), named CZP1-PEG, CZP1-PEG-OA and CZP2-PEG-OA, respectively, and were evaluated in vitro and in vivo. No crystal of CZP was observed in CZP1-PEG-OA for at least 8 days, while CZP crystal appeared before administration for CZP2-PEG-OA. When a small volume of saline was added to CZP1-PEG-OA just before the oral cavity administration, more than 80% (w/w) was found to exist in the soluble form. Each semisolid dosage form (40 mg) was administered to the oral cavity in rats, and CZP 1 mg suspension in 0.5% (w/v) sodium carboxymethylcellulose aqueous solution was administered into rat stomach as a control. CZP1-PEG-OA gave the plasma concentrations of more than 5 ng/ml and 12 ng/ml at 30 min and 1 h after administration, respectively, which might be near the plasma levels effective for the suppression of epileptic seizures in human, while the plasma concentration was less than 5 ng/ml at 30 min or did not reach 10 ng/ml at 1 h for the other formulations. It is proposed that the semisolid dosage form CZP1-PEG-OA should be a possibly useful preparation for the antiepileptic or sedative medication.
ARTCEREB® irrigation and perfusion solution (Artcereb) is typically used as an artificial fluid for applications inside the skull and spinal cavity. This in vitro study was conducted to assess the effects of Artcereb in cultures of rat fetal brain cells. Cell function following exposure to Artcereb was evaluated by measuring 3H-deoxy-D-glucose incorporation activity. Cell function was significantly reduced in primary cultures of rat fetal brain cells at 0 h and 24 h after 1-h or 3-h exposure to normal saline as compared with Artcereb. Cell function was also significantly reduced at 24 h after 3-h exposure to lactated Ringer's solution as compared with Artcereb. Furthermore, cell function was significantly reduced at 24 h after 3-h exposure to a modified Artcereb formulation lacking either HCO3- or Mg2+ as compared with Artcereb, while cell function was unaffected at 24 h after exposure to lactated Ringer's solution with HCO3- or normal saline with HCO3- as compared with Artcereb. These findings suggest the importance of the presence of HCO3- and Mg2+ in the formulation of Artcereb.
Since we do not know much about the efficacy and safety of dietary supplements and functional foods (DS), it is important to collect DS-related events and to share the information among healthcare professionals. Therefore, we aimed to develop an internet-based system which would allow medical doctors and pharmacists to report DS-related events. We conducted a questionnaire survey among pharmacists about their experiences and views of DS-related adverse event reporting. Many pharmacists did not report events because they never had any patient who reported an event. This might have been, in part, owing to lack of awareness of an occurrence, so we collected events using our internet-based system, which periodically offers educational DS information. After educational commentaries and elucidation were appended, collected cases were distributed to the registered members via web pages to encourage them to be more concerned about the safety of DS. Additionally, we constructed a simple posting system for members to easily report similar events, because the questionnaire survey revealed that lack of time and uncertainty of causal relation between an event and DS were sometimes reasons not to report. We obtained several DS-related events both via the normal data collecting form and the simple posting system, and subsequently confirmed reports by e-mail contact. Our interactive system enabled us to obtain more detailed information about posted events. In conclusion, this information system for DS was proved to be useful to facilitate reporting of DS-related events by healthcare professionals and to accumulate events similar to already reported cases.