Does route of administration affect the outcome of TNF antagonist therapy?

The tumor necrosis factor (TNF) antagonists are parenterally administered biologic response modifiers indicated for the management of rheumatoid arthritis. Although infliximab, etanercept, and adalimumab are all members of this class, they differ in route of administration and dosing regimen. In the USA and in Europe, infliximab, in combination with oral methotrexate, is administered intravenously, initially at a dose of 3 mg/kg at weeks 0, 2, and 6, then every 8 weeks thereafter. The US Food and Drug Administration (FDA) has further approved that the dosage can be increased to 10 mg/kg and the doses can be given as often as every 4 weeks to optimize patient outcome (information based on the US package insert dated June 2002). Etanercept and adalimumab are given subcutaneously and can be self-injected. The FDA-approved dose of etanercept is 25 mg twice weekly, and of adalimumab is 40 mg every 2 weeks with methotrexate, or 40 mg alone. Medication adherence, possibly the most important factor in maintaining the benefits of anti-TNF therapy, is influenced by the interaction between the patient and his or her healthcare team, the patient's attitude toward the disease and medication regimen, and the choice of therapy.     

Absence of α-methyldopamine in rat striatum after chronic administration of d-amphetamine

Direct measurement by gas chromatography methane chemical ionization mass spectrometry of α-methyldopamine and α-methylnorepinephrine in rat striatum has shown the failure of these compounds to be accumulated in vivo after chronic administration of d-amphetamine despite the accumulation of α-methyltyramine, an immediate in vitro precursor. Further, both α-methyldopamine and α-methyltyrosine. These data suggest striatum after administration of α-methyltyrosine. These data suggest that, after administration of α-methyltyrosine, α-methyldopamine is formed via decarboxylation of α-methyldopa and not from hydroxylation of α-methyltyramine. Finally, our results indicate that α-methyldopamine does not play a role in the development of tolerance to d-amphetamine.     

Influences of β-HCG administration on carbon isotope ratios of endogenous urinary steroids

Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body - the testes and the adrenal glands. By administration of β-HCG it is possible to strongly stimulate the steroid production within the testes without influencing the production at the adrenal glands. Therefore, this treatment should result in changed CIR of urinary androgens in contrast to the undisturbed pre-treatment values. Four male volunteers received three injections of β-HCG over a time course of 5 days and collected their urine samples at defined intervals after the last administration. Those samples showing the largest response in contrast to the pre-administration urines were identified by steroid profile measurements and subsequent analysed by GC/C/IRMS. CIR of androsterone, etiocholanolone, testosterone, 5α- and 5β-androstanediol and pregnanediol were compared. While pregnanediol was not influenced, most of the investigated androgens showed depleted values after treatment. The majority of differences were found to be statistically significant and nearly all showed the expected trend towards more depleted δ(13)C-values. These results support the hypothesis of different CIR at different production sites inside the human body. The impact of these findings on doping control analysis will be discussed.     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Amelioration of diabetic nephropathy by oral administration of d-α-tocopherol and its mechanisms

Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKC?activity was involved in this amelioration.     

Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ～70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.     

Studies of the mechanisms of toxicity of the administration of recombinant tumor necrosis factor α in normal and tumor-bearing mice

Summary Tumor-bearing mice have a greater sensitivity to the acute lethal effects of the administration of high-dose recombinant human tumor necrosis factor (rhTNF-) compared to normal, non-tumor-bearing mice. We studied whether or not the presence of tumor per se was responsible for the enhanced rhTNF- toxicity. Tumor-bearing mice underwent tumor excision or sham operation before the systemic administration of rhTNF at staged times (0.5–24 h) following surgery. There was little survival difference between sham-operated tumor-bearing mice and tumor-bearing mice undergoing tumor excision (at 24 h, treatment with 12 µg rhTNF-, survival:sham-operated tumor bearers = 0/12, excised tumor-bearers = 0/12;P ² <0.01 compared to non-tumor-bearers). Mice without tumors receiving sham operation, had minimal toxicity (10 of 12 mice surviving). The injection of 3 ml Ringer's lactate i.p. before i.v. rhTNF- therapy increased survival in tumor-bearing animals; following pretreatment with Ringer's lactate 30/42 mice survived 12 µg rhTNF- compared to 6/42 surviving a similar rhTNF- dose without hydration (P ² <0.001). Since the production of oxygen free-radical metabolites has been postulated to play a role in the acute toxicity of rhTNF-, bismuth subnitrate was used to induce the enzyme metallothionein to act as a natural scavenger for these metabolites. Daily oral bismuth subnitrate treatments improved survival of mice with MCA-106 or MCA-102 sarcoma and of mice without tumors, with higher rhTNF- doses (12–20 µg), without reducing the therapeutic effect of rhTNF- against the weakly immunogenic MCA-106 sarcoma. These studies suggest methods for reducing the toxicity of rhTNF- administration in clinical trials.     

Dissolution of pre-existing platelet thrombus by synergistic administration of low concentrations of bifunctional antibodies against β3 integrin

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a ~2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 μM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T(50%)) by APAC (95 ± 6.1 min) or SLK (145 ± 7.1 min) was much longer than that by combined APAC + SLK (65 ± 7.6 min) at the final concentration of 0.025 μM (APAC + SLK vs APAC, p<0.05; APAC + SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.     

Peripheral administration of α-MSH reduces fever in older and younger rabbits

In these experiments IV, ICV and intra-gastric administration of α-MSH reduced fever caused by injections of leukocytic pyrogen (LP). 2.5 μg α-MSH injected IV reduced fever caused by IV LP, more so in rabbits over 3 yrs old than in those under 2 yrs of age; 5 mg of acetaminophen given IV had no antipyretic effect in either age group. ICV administration of 25 ng α-MSH reduced fever caused by IV LP injection in the older but not in the younger rabbits. α-MSH given IV (2.5 μg) also lowered fever induced by ICV injection of LP in older but not in younger animals. Both older and younger rabbits showed reductions in fever evoked by IV LP after 2.5 mg α-MSH was given by gastric tube. The results indicate that this peptide which occurs naturally within the brain has potent antipyretic properties when given systemically, presumably as a result of a central antipyretic action. Greater sensitivity to central α-MSH in the older rabbits may account for the reduced febrile response seen in the aged. The findings support previous data which suggest that central α-MSH has a physiological role in the limitation of fever.     

Effects of Δ9-tetrahydrocannabinol administration on marker proteins of rat testicular cells

The effects of chronic administration of 2 mg Δ⁹-tetrahydrocannabinol per kg body weight upon rat testicular cell function was examined by use of selected testicular cell marker proteins. γ-Glutamyl transpeptidase was used as a marker of Sertoli cell plasma membranes; sorbitol dehydrogenase was used as a marker of pachytene spermatocytes. The interstitial cells were marked by cytochrome P-450, a microsomal component, and β-glucuronidase, a lysosomal component. The results of this study show a rapid reduction in microsomal P-450 content following 2 days of tetrahydrocannabinol administration. In addition, γ-glutamyl transpeptidase was significantly reduced at 2 days and continued to decline to day 9. β-Glucuronidase and sorbitol dehydrogenase exhibited no significant change over the course of the experiment. It is suggested that the reduction of testosterone synthesis in testes of tetrahydrocannabinol treated rats may be the result of a reduction in P-450 content.     

Intravesical administration of γδ T cells successfully prevents the growth of bladder cancer in the murine model

Background  Superficial bladder cancers are usually managed with transurethral resection followed by the intravesical administration of Bacillus Calmette-Guerin which requires major histocompatibility complex (MHC) class I expression on cancer cells. Since cancer cells often loose MHC expression, a novel immunotherapy such as MHC-unrestricted γδ T cell therapy is desired. Objective  To clarify the relationship between the expression of MHC class I and clinicopathological features in bladder cancer patients, and investigate the effects of the administration of intravesical γδ T cells on bladder cancer. Methods  Samples from 123 patients who had undergone either transurethral resection or radical cystectomies were examined for MHC expression and the relationship between this and the clinicopathological features was analyzed statistically. The in vitro and in vivo effects of γδ T cells expanded by zoledronic acid (ZOL) against several types of cancer cell line and an orthotopic bladder cancer murine model which was pretreated with ZOL were investigated. Results  MHC-diminished superficial bladder cancer was significantly more progressive than MHC-conservative bladder cancer (P = 0.047). In addition, there was a significant association between diminished MHC expression and poor disease free survival (P = 0.041) and overall survival (P = 0.018) after radical cystectomy. In vitro, all of the cell lines pretreated with 5-μM ZOL showed a marked increase in sensitivity to lysis by γδ T cells. Moreover, intravesical administration of γδ T cells with 5-μM ZOL significantly demonstrated antitumor activity against bladder cancer cells in the orthotopic murine model (P < 0.001), resulting in prolonged survival. Conclusion  The present murine model provides a potentially interesting option to develop immunotherapy using γδ T cells for bladder cancer in human. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Yuasa and K. Sato contributed equally to the study.     

Systemic administration of neuregulin-1β1 protects dopaminergic neurons in a mouse model of Parkinson's disease

Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1β(1) (Nrg1β(1)-ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1β(1)-ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1β(1)-ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1β(1)-ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for Parkinson's disease patients.     

Altered expression of γ-secretase components in animal model of major depressive disorder induced by reserpine administration

Altered expression of neurotrophic factors as well as neuroinflammation is commonly associated with Major depressive disorder (MDD) and Alzheimer's disease (AD). To investigate whether or not reserpine-induced MDD affects the expression of AD-related proteins, the expression of γ-secretase components and substrate were measured in brains of ICR mice following reserpine treatment for 15 days. In active avoidance test, total response time and peak slightly increased in the 2 mg/kg reserpine (RSP2)-treated group compared to vehicle-treated group (P<0.05). Expression and phosphorylation of MKP-1, which is a key factor in MDD pathology, were both higher in the RSP2-treated group than the vehicle- and 1 mg/kg reserpine (RSP1)-treated groups (P<0.02). Furthermore, full-length expression of amyloid precursor protein (APP) was enhanced in the RSP1 and RSP2-treated groups compared to the vehicle-treated group, whereas expression of γ-secretase components decreased (P<0.03). Among the three components of the γ-secretase complex, nicastrin protein underwent the largest decrease in expression, as detected by Western blotting (P<0.03). Therefore, the data presented here provide additional evidence about the pathological correlation between MDD and AD.     

Effects of nicotine administration in a mouse model of familial hypertrophic cardiomyopathy, α-tropomyosin D175N

The effects of nicotine (NIC) on normal hearts are fairly well established, yet its effects on hearts displaying familial hypertrophic cardiomyopathy have not been tested. We studied both the acute and chronic effects of NIC on a transgenic (TG) mouse model of FHC caused by a mutation in α-tropomyosin (Tm; i.e., α-Tm D175N TG, or Tm175). For acute effects, intravenously injected NIC increased heart rate, left ventricular (LV) pressure, and the maximal rate of LV pressure increase (+dP/dt) in non-TG (NTG) and Tm175 mice; however, Tm175 showed a significantly smaller increase in the maximal rate of LV pressure decrease (-dP/dt) compared with NTGs. Western blots revealed phosphorylation of phospholamban Ser16 and Thr17 residue increased in NTG mice following NIC injection but not in Tm175 mice. In contrast, phosphorylation of troponin I at serine residues 23 and 24 increased equally in both NTG and Tm175. Thus the attenuated increase in relaxation in Tm175 mice following acute NIC appears to result primarily from attenuated phospholamban phosphorylation. Chronic NIC administration (equivalent to smoking 2 packs of cigarettes/day for 4 mo) also increased +dP/dt in NTG and Tm175 mice compared with chronic saline. However, chronic NIC had little effect on heart rate, LV pressure, -dP/dt, LV wall and chamber dimensions, or collagen content for either group of mice.     

Effect of prolonged administration of bovine lactoferrin in neuropathic pain: Involvement of opioid receptors,nitric oxide and TNF-α

AimsThis study aimed to investigate the effect of prolonged administration of bovine milk lactoferrin (bLF) on hyperalgesia and allodynia in a rat model of neuropathic pain and to determine the involvement of c-Fos, TNF-α, nitric oxide and opioidergic systems in this effect.Main methodsNeuropathic pain was induced in rats by loose ligation of the right sciatic nerve and evaluated by tests measuring the mechanical and thermal hyperalgesia and allodynia. bLF (50, 100, and 200 mg/kg) alone or in combination with opioidergic antagonists were administered intraperitoneally to the rats with neuropathic pain. c-Fos and NADPH-d immunocytochemistry and Western blotting for TNF-α, iNOS and nNOS were performed in the lumbar spinal cord of rats. Plasma TNF-α levels were determined with ELISA.Key findingsProlonged, but not single, administration of bLF produced antihyperalgesic and antiallodynic effects in neuropathic rats. Pretreatment with opioidergic antagonists significantly decreased this effect. Prolonged administration of bLF decreased c-Fos and NADPH-d immunoreactivity and TNF-α and iNOS expressions at 50 and 100 mg/kg and nNOS expression at 100 mg/kg in the lumbar spinal cord of neuropathic rats. Plasma TNF-α levels remained unchanged after bLF treatment.SignificanceProlonged administration of bLF exerts antihyperalgesic and antiallodynic effect in neuropathic rats; down-regulation of both TNF-α and iNOS expressions and potentiation of opioidergic system in the lumbar spinal cord can contribute to this effect.