The World Health Organization has reported that when morphine is used to control pain in cancer patients, psychological dependence is not a major concern. Our studies were undertaken to ascertain the modulation of psychological dependence on morphine under a chronic pain-like state in rats. Morphine induced a dose-dependent place preference. We found that inflammatory and neuropathic pain-like states significantly suppressed the morphine-induced rewarding effect. In an inflammatory pain-like state, the suppressive effect was significantly recovered by treatment with a κ-opioid receptor antagonist. In addition, in vivo microdialysis studies clearly showed that the morphine-induced increase in the extracellular levels of dopamine (DA) in the nucleus accumbens (N.Acc.) was significantly decreased in rats pretreated with formalin. This effect was in turn reversed by the microinjection of a specific dynorphin A antibody into the N.Acc. These findings suggest that the inflammatory pain-like state may have caused the sustained activation of the κ-opioidergic system within the N.Acc., resulting in suppression of the morphine-induced rewarding effect in rats. On the other hand, we found that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with a reduction in the μ-opioid receptor-mediated G-protein activation in the ventral tegmental area (VTA). Moreover, nerve injury results in the continuous release of endogenous β-endorphin to cause the dysfunction of μ-opioid receptors in the VTA. This paper also provides a review to clarify misunderstandings of opioid analgesic use to control pain in cancer patients.
Adenosine and its precursors, ATP and ADP, exert various physiological effects via binding to purinergic receptors. We previously used co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and immunoelectron microscopy to demonstrate the hetero-oligomerization of purinergic receptor subtypes. Furthermore, pharmacological studies found significant changes in receptor-mediated signaling in human embryonic kidney (HEK) 293T cells co-transfected with these receptors. These findings suggest that heterodimers of purinergic receptors may have distinct pharmacological profiles, possibly due to dimerization-induced conformational changes, further suggesting that hetero-dimerization may be employed to “fine-tune” purinergic receptor signaling. Adenosine A2A receptor (A2AR), P2Y1 receptor (P2Y1R) and P2Y12 receptor (P2Y12R) are predominantly expressed on human platelets. ADP activates human platelets by stimulating both P2Y1R and P2Y12R, which act sequentially and in concert to achieve complete platelet aggregation. In contrast, adenosine stimulates Gs-coupled A2AR, followed by activativation of adenylate cyclase, leading to an increase in intracellular cAMP levels, which potently inhibits platelet activation. We examined the hetero-oligomerization and functional interactions of A2AR, P2Y1R, and P2Y12R. In HEK293T cells triply expressing all three receptors, hetero-oligomerization was observed among the three receptors. Additionally, P2Y1R agonist-evoked Ca2+ signaling was significantly inhibited by co-treatment with an A2AR antagonist in HEK293T cells. In human platelets, we identified endogenous A2AR/P2Y1R and A2AR/P2Y12R heterodimers. We also observed functional Ca2+-signaling-related cross-talk similar to those found in HEK293T cells, and found that they appeared to affect platelet shape. These results collectively suggest that intermolecular signal transduction and specific conformational changes occur among components of the hetero-oligomers formed by these three receptors.
Sialidase removes sialic acid residues from sialoglycoconjugates such as glycoproteins and glycolipids. Since sialic acid plays crucial roles in synaptic plasticity and memory in the hippocampus, the regulation of sialyl signaling by sialidase is also necessary for neural functions. However, since mammalian sialidase activity is remarkably weak, it has been difficult to detect sialidase activity in mammalian tissues. Determination of the distribution of sialidase activity in living mammalian tissues would provide much valuable information for understanding the roles of sialidase in physiological functions. Therefore, we synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. After cleavage of BTP-Neu5Ac, which is water soluble and shows little fluorescence, with sialidase, the water-insoluble fluorophore benzothiazolylphenol (BTP) released from BTP-Neu5Ac stains tissue and shows bright fluorescence. BTP-Neu5Ac can visualize sialidase activity in brain tissue with high levels of sensitivity and specificity. The sialidase expression level is markedly high in various human cancers such as colon, renal, prostate, and ovarian cancers. BTP-Neu5Ac can detect human colon cancers sensitively. Thus, BTP-Neu5Ac is useful not only for physiological research but also as a cancer probe. BTP-Neu5Ac is now being used in virology research. In this review, methods for histochemical imaging of sialidase activity and the role of sialidase in hippocampal memory are described based on the author's study of multidimensional analysis of hippocampal excitatory neurotransmission and development of analytical tools for glycans, which was awarded a prize by the Tokai branch of the Pharmaceutical Society of Japan.
Replication-incompetent adenovirus (Ad) vectors have gained attention as gene delivery vehicles. Theoretically, no Ad genes should be expressed following transduction; however, Ad genes are expressed from the vector genome, leading to induction of cellular immunity against Ad proteins and Ad protein-induced toxicity. To suppress the leaky expression of Ad genes, a microRNA (miRNA)-regulated gene expression system was utilized. We developed novel Ad vectors by incorporating targeted sequences of miR-122a or miR-142-3p, which exhibit liver- or spleen-specific expression, respectively, in the 3′-untranslated region (UTR) of the E2A, E4, or pIX genes. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional human embryonic kidney 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold in an miRNA-dependent manner, compared with a conventional Ad vector, by the insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3′-UTR of the E4 gene (Ad-E4-122aT) expressed 1.5- to 34-fold higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver compared with a conventional Ad vector. miR-122a-mediated suppression of E4 gene expression in the liver significantly reduced the hepatotoxicity that an Ad vector causes via both adaptive and non-adaptive immune responses. Ad-E4-122aT would be a promising framework for efficient gene delivery due to its ability to mediate higher and longer-term transgene expression and lower hepatotoxicity than a conventional Ad vector.
In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.
Many pharmacists have requested optimization of aluminum packaging of medicinal products in terms of usability. To improve operational efficiency of aluminum packaging, we used Universal Design (UD)-based approach, which enables products to be used properly and consistently regardless of users. The UD-pack used in this research is composed of a film that can be easily opened and torn linear. Here, we compared the UD-pack to conventional aluminum packaging by evaluating the practical use of each under the cooperation of 24 pharmacists. Following opening and removal of contents of one sample for both types of packaging, monitors were asked which type was easier to use in each case. Also, monitors were to repeat the opening and removal of contents of five samples in a row, and were asked the same question. Monitors were recorded by digital camera to measure the time required to finish the procedure for five samples in a row. After opening one sample, approximately 83% of monitors preferred the UD-pack, and after opening five samples, all (100%) preferred the UD-pack. Regarding the time required for opening five samples and removing the contents measured by analyzing the recorded video, the UD-pack significantly reduced the time required for all monitors. The average time ratio of the UD-pack to conventional aluminum packaging was approximately 59%, and no significant difference was observed between male and female pharmacists. Our results indicate the UD-pack improves ease of opening and removal of contents and increases efficiency of dispensing in a clinical setting compared with conventional aluminum packaging.
White petrolatum is frequently used as an oleaginous base, but has a drawback of poor usability. In this trial, white petrolatum was prepared at a lower melting point to improve its usability. Characteristic pharmaceutical values such as melting point, yield, and consistency were compared between a conventional product and ophthalmic white petrolatum. Usability was compared by administering a survey questionnaire and evaluating the comparable moisturizing effect by conductivity in humans. The melting point and yield value of the improved product were significantly lower compared with other white petrolatum products. In the survey, the improved product was rated excellent in five criteria. On a scale of 1 to 5, the average values for the five criteria for the improved product were 4.7, while the conventional product and ophthalmic white petrolatum were rated 3.0 and 3.5, respectively. No difference in moisturizing effect was observed among all petrolatums after application, from day 1 to day 14. In conclusion, the improved white petrolatum demonstrated better usability, and the moisturizing effect was equivalent to conventional product, suggesting that the use of this improved product may lead to improved adherence.
Since the Great East Japan Earthquake, the Ministry of Health, Labour, and Welfare, municipalities, and medical organizations have made various revisions to medical systems employed at the time of a disaster. To educate pharmacists who can contribute to medical teams conducting healthcare activities at the time of disasters, there is a need to develop disaster medical instructions in pharmaceutical education. However, the “Model Core Curriculum for Pharmaceutical Education”, a new curriculum, contains little disaster medical care education. In the present study, in cooperation with the Ehime Society of Hospital Pharmacists and Ehime Pharmaceutical Association, we surveyed pharmacists living in Ehime Prefecture in order to investigate their views regarding the necessity of disaster medical instructions in pharmaceutical education, and what they considered essential to be taught. Our subjects considered that there is a strong need for teaching disaster medical instruction in college. In addition, they regarded all of the 16 investigated items concerning disaster medical instruction as highly necessary. Factor analysis led to the classification of these items into “disaster medical activities performed by pharmacists” and “responses to secondary issues in affected areas”. On the basis of this classification, we established specific goals. Disaster medical instructions should be taught as a part of pharmaceutical education in college. However, to develop these instructions, it is important for universities to cooperate with local pharmaceutical and hospital pharmacist associations, as it is difficult for universities to teach such instructions independently.
We performed a survey of cancer patients' needs for drug treatment and support from pharmacists during treatment and evaluated the support that cancer patients can expect from community pharmacists in the future. The patients consisted of 16 members of the Cancer Patient Association in Aichi prefecture who underwent chemotherapy. The results of a semistructured group interview were qualitatively analyzed using the grounded theory method. Patients undergoing chemotherapy had high hopes for its effectiveness but were worried about side effects and medical costs. To overcome these problems, they hoped for a decrease in the economic burden, compassionate-use system, and development of novel drugs. The patients had anxiety because the side effects of chemotherapy often caused physical and psychological damage. Despite patients' confusion, pharmacists sometimes did not give adequate explanations to them. The patients expected more from pharmacists regarding medication support and hoped for a system allowing continuous side effect monitoring and consultation without hesitation. For patients undergoing cancer chemotherapy who are confused regarding side effects, pharmacists should understand the patient explanatory model and become more involved with patients as partners in treatment.
Stomatitis frequently occurs during chemotherapy and radiotherapy for cancer. Because of its pharmacological properties including anti-inflammatory activity and stimulatory effects on endogenous prostaglandin synthesis, rebamipide has been suggested as a potentially effective treatment against stomatitis. In the present study we tested the stability of oral rebamipide solutions prepared in our hospital pharmacy using sodium alginate as a thickener to increase retention of this agent in the oral cavity, and the addition of different flavoring mixtures intended for use in enteral diets to reduce the bitterness of rebamipide and sodium alginate. Samples of oral rebamipide solution prepared with 13 kinds of flavoring and sodium alginate were evaluated in terms of their appearance, redispersibility, pH, viscosity, and rebamipide content immediately after preparation and 1, 3, 7, and 10 days after storage at room temperature under ambient light or in a cool, dark place. After 10 days of storage, favorable stability was observed in four sample solutions supplemented with green apple, pineapple, yogurt, and tomato flavoring mixtures intended for use in Elental® diets. These oral solutions may have potential clinical application.
Cetuximab was approved in Japan as the only clinically available molecular targeted drug for the treatment of head and neck cancer. Hypomagnesemia associated with cetuximab is considered one of the most serious adverse events. However, the factors influencing the development of hypomagnesemia are not clear, although the drug was previously approved for the treatment of patients with colorectal cancer. Thus, we studied the factors involved in the development of hypomagnesemia in patients receiving cetuximab therapy for head and neck cancer. Patients' background data and laboratory values before starting cetuximab therapy did not affect the development of hypomagnesemia. Among patients who had never been treated with cisplatin (NT group), 36.4% developed hypomagnesemia. In contrast, all patients who had previously been treated with cisplatin (T group) developed hypomagnesemia (p=0.034). Magnesium is reabsorbed by transient receptor potential subfamily melastatin 6 (TRPM6) in the distal convoluted tubule. The expression level of TRPM6 is controlled by the epidermal growth factor (EGF) pathway. Cetuximab is an EGF receptor inhibitor and reduces the expression of TRPM6. Additionally, recent studies have shown that the expression of TRPM6 is reduced by cisplatin. Therefore, we considered that the serum magnesium level was cumulatively reduced by cetuximab and cisplatin. In conclusion, the T group was more likely to develop hypomagnesemia than the NT group, and therefore the serum magnesium level in the T group requires careful monitoring so that magnesium supplementation can be provided to patients when the level decreases.