Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Study of the impact of tissue density heterogeneities on 3-dimensional abdominal dosimetry: comparison between dose kernel convolution and direct monte carlo methods.

Dose kernel convolution (DK) methods have been proposed to speed up absorbed dose calculations in molecular radionuclide therapy. Our aim was to evaluate the impact of tissue density heterogeneities (TDH) on dosimetry when using a DK method and to propose a simple density-correction method. METHODS: This study has been conducted on 3 clinical cases: case 1, non-Hodgkin lymphoma treated with (131)I-tositumomab; case 2, a neuroendocrine tumor treatment simulated with (177)Lu-peptides; and case 3, hepatocellular carcinoma treated with (90)Y-microspheres. Absorbed dose calculations were performed using a direct Monte Carlo approach accounting for TDH (3D-RD), and a DK approach (VoxelDose, or VD). For each individual voxel, the VD absorbed dose, D(VD), calculated assuming uniform density, was corrected for density, giving D(VDd). The average 3D-RD absorbed dose values, D(3DRD), were compared with D(VD) and D(VDd), using the relative difference Δ(VD/3DRD). At the voxel level, density-binned Δ(VD/3DRD) and Δ(VDd/3DRD) were plotted against ρ and fitted with a linear regression. RESULTS: The D(VD) calculations showed a good agreement with D(3DRD). Δ(VD/3DRD) was less than 3.5%, except for the tumor of case 1 (5.9%) and the renal cortex of case 2 (5.6%). At the voxel level, the Δ(VD/3DRD) range was 0%-14% for cases 1 and 2, and -3% to 7% for case 3. All 3 cases showed a linear relationship between voxel bin-averaged Δ(VD/3DRD) and density, ρ: case 1 (Δ = -0.56ρ + 0.62, R(2) = 0.93), case 2 (Δ = -0.91ρ + 0.96, R(2) = 0.99), and case 3 (Δ = -0.69ρ + 0.72, R(2) = 0.91). The density correction improved the agreement of the DK method with the Monte Carlo approach (Δ(VDd/3DRD) < 1.1%), but with a lesser extent for the tumor of case 1 (3.1%). At the voxel level, the Δ(VDd/3DRD) range decreased for the 3 clinical cases (case 1, -1% to 4%; case 2, -0.5% to 1.5%, and -1.5% to 2%). No more linear regression existed for cases 2 and 3, contrary to case 1 (Δ = 0.41ρ - 0.38, R(2) = 0.88) although the slope in case 1 was less pronounced. CONCLUSION: This study shows a small influence of TDH in the abdominal region for 3 representative clinical cases. A simple density-correction method was proposed and improved the comparison in the absorbed dose calculations when using our voxel S value implementation.

Dinâmica de nutrientes na solução do solo em pomar fertirrigado de citros

O objetivo deste trabalho foi avaliar a dinâmica de nutrientes na solução do solo após a aplicação, via fertirrigação, de nitrogênio, fósforo e potássio a laranjeiras. O experimento foi realizado entre setembro de 2007 e outubro de 2009, em pomares de laranjeiras 'Valência' e 'Hamlin', enxertadas sobre citrumeleiro 'Swingle'. Foram avaliadas cinco doses de N, P2O5 e K2O (0, 25, 50, 100 e 200% da dose recomendada). A solução do solo foi extraída a 30 e 60 cm de profundidade, com o auxílio de extratores com cápsulas porosas. Foram realizadas 11 avaliações durante o período experimental, com as extrações iniciadas após 12 horas das fertirrigações. O aumento das doses reduziu o pH (pH~3,5, na maior dose), e aumentou a condutividade elétrica (CE~1,5 dS m-1, na maior dose) e os teores de NH4, NO3, P, K, Mn e Zn na solução do solo, nas duas profundidades amostradas. Nos meses com maior precipitação pluvial, houve perda potencial de nutrientes por lixiviação, pois maiores concentrações de NO3, K e B foram observadas à profundidade de 60 cm. A análise da solução do solo, obtida por extratores com cápsula de cerâmica porosa, pode ser considerada ferramenta auxiliar para monitorar e avaliar a disponibilidade de nutrientes às plantas.

Teor de óleo e produtividade da mamoneira de acordo com a adubação nitrogenada e irrigação com água salina

O objetivo deste trabalho foi avaliar o teor de óleo e a produção de sementes da mamoneira 'BRS Energia', submetida a doses de adubação nitrogenada e irrigação com água com diferentes índices de salinidade. O experimento foi realizado em lisímetros, em condições de campo, em delineamento de blocos ao acaso, com três repetições. Os tratamentos consistiram dos índices salinos da água de 0,4 (controle), 1,4, 2,4, 3,4 e 4,4 dS m-1, associados a 50, 75, 100, 125 e 150% das doses de N recomendadas para ensaio. A interação entre salinidade da água de irrigação e doses de N não foi significativa para nenhuma variável estudada. A salinidade da água até 1,4 dS m-1 propiciou teor de óleo das sementes de 47%. Doses de N acima de 64% da recomendada promoveram teor de óleo inferior a 48%. A maior produção de matéria seca da parte aérea foi obtida com 150% da dose de N recomendada. O incremento salino a partir de 0,4 dS m-1 aumenta o tempo para emissão do racemo primário e reduz os valores dos componentes de produção, dos quais a massa de matéria seca da parte aérea e o número de frutos são os mais afetados. Condutividade elétrica da água de até 1,9 dS m-1 e doses de N a partir de 134% da recomendada propiciam maior número de racemos.

Plantas de cobertura e calagem na implantação do sistema plantio direto

O objetivo deste trabalho foi avaliar o efeito de plantas de cobertura e da calagem sobre a produtividade da soja e as características químicas do perfil do solo, após implantação do plantio direto. O experimento foi realizado em campo por dois anos, em Latossolo Vermelho‑Amarelo. A área era explorada como pastagem há cinco anos. Avaliaram-se três sistemas de cultivo: soja/pousio/soja, soja/Pennisetum glaucum/soja e soja/Urochloa ruziziensis/soja, com aplicação de calcário à dosagem de 0, 0,5, 1,0 e 2,0 vezes a quantidade necessária para elevar a saturação de bases (V) a 50%, na camada de 0-20 cm. O calcário foi incorporado ao solo, a 20 cm de profundidade, previamente à implantação dos sistemas de cultivo. Aos 490 dias após a calagem, foram coletadas amostras de solo das profundidades de 0-­10, 10-­20, 20-­40 e 40­-60 cm. Avaliaram-se o estado nutricional das plantas, as alterações das características químicas do solo e a produtividade da soja. As plantas de cobertura associadas à calagem promovem melhorias nos parâmetros de acidez do solo - sobretudo quando a dose aplicada é igual ou mais elevada do que a recomendada para V 50% -, e aumentam o teor foliar de P e a produtividade da soja, independentemente da calagem. Quanto maior a dose de calcário, maior é o avanço da frente alcalina no solo e a produtividade de grãos.

Amlodipine and valsartan as components of a rational and effective fixed-dose combination.

Pharmacological treatment of hypertension is effective in preventing cardiovascular and renal complications. Calcium antagonists and blockers of the renin-angiotensin system are widely used today to initiate antihypertensive therapy but, when given as monotherapy, do not suffice in most patients to normalize blood pressure. Combining the two types of agents considerably increases the antihypertensive efficacy, but not at the expense of a deterioration of tolerability. This is exemplified by the experience accumulated with the recently developed fixed dose combination containing the AT(1)-receptor blocker valsartan (160 mg) and the dihydropyridine amlodipine (5 or 10 mg). In a randomized trial, an 8-week treatment normalized blood pressure (<140/90 mmHg) within 8 weeks in a large fraction of hypertensive patients (78.4% and 85.2% using the 5/160 [n = 371] and 10/160 mg [n = 377] dosage, respectively). Like all AT(1)-receptor blockers valsartan has a placebo-like tolerability. Valsartan prevents to a large extent the occurrence amlodipine-induced peripheral edema. Both amlodipine and valsartan have beneficial effects on cardiovascular morbidity and mortality, as well as protective effects on renal function. The co-administration of these two agents is therefore very attractive, as it enables a rapid and sustained blood pressure control in hypertensive patients. The availability of a fixed-dose combination based on amlodipine and valsartan is expected therefore to facilitate the management of hypertension, to improve long-term adherence with antihypertensive therapy and, ultimately, to have a positive impact on cardiovascular and renal outcomes.

Association of urethral toxicity with dose exposure in combined high-dose-rate brachytherapy and intensity-modulated radiation therapy in intermediate- and high-risk prostate cancer.

INTRODUCTION: To report acute and late toxicities in patients with intermediate- and high-risk prostate cancer treated with combined high-dose-rate brachytherapy (HDR-B) and intensity-modulated radiation therapy (IMRT). MATERIALS AND METHODS: From March 2003 to September 2005, 64 men were treated with a single implant HDR-B with 21 Gy given in three fractions, followed by 50 Gy IMRT along with organ tracking. Median age was 66.1 years, and risk of recurrence was intermediate in 47% of the patients or high in 53% of the patients. Androgen deprivation therapy was received by 69% of the patients. Toxicity was scored according to the CTCAE version 3.0. Median follow-up was 3.1 years. RESULTS: Acute grade 3 genitourinary (GU) toxicity was observed in 7.8% of the patients, and late grades 3 and 4 GU toxicity was observed in 10.9% and 1.6% of the patients. Acute grade 3 gastrointestinal (GI) toxicity was experienced by 1.6% of the patients, and late grade 3 GI toxicity was absent. The urethral V(120) (urethral volume receiving &gt; or =120% of the prescribed HDR-B dose) was associated with acute (P=.047) and late &gt; or = grade 2 GU toxicities (P=.049). CONCLUSIONS: Late grades 3 and 4GU toxicity occurred in 10.9% and 1.6% of the patients after HDR-B followed by IMRT in association with the irradiated urethral volume. The impact of V(120) on GU toxicity should be validated in further studies.

Étude des biais sur la dose parentérale d'un nouveau médicament lors d'un essai clinique avec profil cinétique

Introduction et objectif: Lors d'essais cliniques, le pharmacien est responsable de la préparation et de la dispensation des médicaments à évaluer. Un article récent a toutefois montré que les aspects pharmaceutiques liés au contrôle de la dose administrée in fine étaient souvent mal contrôlés. Il peut exister une différence entre la dose nominale fournie par le certificat d'analyse du fabricant et la dose réellement administrée au sujet, biais qui se reporte en cascade sur l'estimation des paramètres pharmacocinétiques (PK), comme la clairance ou le volume de distribution. Ce travail visait à évaluer les biais entachant la quantité de médicament réellement injectée (iv/sc) aux volontaires d'un essai clinique étudiant la PK et la relation dose-réponse d'un nouveau produit biotechnologique. Méthode: La dose de médicament administrée lors de l'essai clinique (D) a été calculée de la manière suivante: D = C * V - pertes. La concentration du produit (C; titre nominal du fabricant) a été vérifiée par immuno-essai. Le volume de médicament injecté (V) a été déterminé pour chaque injection par pesée (n=72), en utilisant la masse de la seringue avant et après injection et la densité du produit. Enfin, une analyse in vitro a permis d'évaluer les pertes liées à l'adsorption du produit dans les lignes de perfusion et de choisir le dispositif adéquat in vivo. Résultats: La concentration du médicament s'est révélée proche du titre nominal (96 ± 7%), et a été utilisée comme référence. Le volume injecté était quant à lui entaché d'un biais systématique par rapport à la valeur théorique correspondant à 0.03 mL pour la dose minimale (i.e. 75% du volume à injecter à cette dose). Une analyse complémentaire a montré que cela s'expliquait par une réaspiration partielle de la solution médica-menteuse avant le retrait de la seringue après injection sc, due à l'élasticité du piston. En iv, le biais était par contre provoqué par une réaspiration du soluté de perfusion co-administré. Enfin, la mesure des quantités de médicament récupérées après injection dans le dispositif de perfusion a démontré des pertes minimales par adsorption. Discussion-conclusion: Cette étude confirme l'existence de biais inversement corrélés au volume et à la concentration du médicament administré, pouvant provoquer des erreurs importantes sur les paramètres PK. Ce problème est négligé ou insuffisamment considéré dans les protocoles de Phase I et nécessiterait une planification rigoureuse. Les procédures opératoires devraient attirer l'attention sur ce point crucial.

Intravenous immunoglobulin for treatment of mild-to-moderate Alzheimer's disease: a phase 2, randomised, double-blind, placebo-controlled, dose-finding trial.

BACKGROUND: Three small trials suggest that intravenous immunoglobulin can affect biomarkers and symptoms of mild-to-moderate Alzheimer's disease. We tested the safety, effective dose, and infusion interval of intravenous immunoglobulin in such patients. METHODS: We did a multicentre, placebo-controlled phase 2 trial at seven sites in the USA and five in Germany. Participants with probable Alzheimer's disease aged 50-85 years were randomly assigned (by a computer-generated randomisation sequence, with block sizes of eight) to infusions every 4 weeks (0·2, 0·5, or 0·8 g intravenous immunoglobulin per kg bodyweight, or placebo) or infusions every 2 weeks (0·1, 0·25, or 0·4 g/kg, or placebo). Patients, caregivers, investigators assessing outcomes, and staff at imaging facilities and the clinical research organisation were masked to treatment allocation, but dispensing pharmacists, the statistician, and the person responsible for final PET analyses were not. Treatment was masked with opaque pouches and infusion lines. The primary endpoint was median area under the curve (AUC) of plasma amyloid β (Aβ)(1-40) between the last infusion and the final visit (2 weeks or 4 weeks depending on infusion interval) in the intention-to-treat population. The trial is registered at ClinicalTrials.gov (NCT00812565) and controlled-trials.com (ISRCTN64846759). FINDINGS: 89 patients were assessed for eligibility, of whom 58 were enrolled and 55 included in the primary analysis. Median AUC of plasma Aβ(1-40) was not significantly different for intravenous immunoglobulin compared with placebo for five of the six intervention groups (-18·0 [range -1347·0 to 1068·5] for 0·2 g/kg, -364·3 [-5834·5 to 1953·5] for 0·5 g/kg, and -351·8 [-1084·0 to 936·5] for 0·8 g/kg every 4 weeks vs -116·3 [-1379·0 to 5266·0] for placebo; and -13·8 [-1729·0 to 307·0] for 0·1 g/kg, and -32·5 [-1102·5 to 451·5] for 0·25 g/kg every 2 weeks vs 159·5 [51·5 to 303·0] for placebo; p>0·05 for all). The difference in median AUC of plasma Aβ(1-40) between the 0·4 g/kg every 2 weeks group (47·0 [range -341·0 to 72·5]) and the placebo group was significant (p=0·0216). 25 of 42 (60%) patients in the intervention group versus nine of 14 (64%) receiving placebo had an adverse event. Four of 42 (10%) patients in the intravenous immunoglobulin group versus four of 14 (29%) receiving placebo had a serious adverse event, including one stroke in the intervention group. INTERPRETATION: Intravenous immunoglobulin may have an acceptable safety profile. Our results did not accord with those from previous studies. Longer trials with greater power are needed to assess the cognitive and functional effects of intravenous immunoglobulin in patients with Alzheimer's disease.

Canakinumab Relieves Symptoms of Acute Flares and Improves Health-Related Quality Of Life (HRQoL) in Difficult-To-Treat Gouty Arthritis Patients by Suppressing Inflammation: Results of a Randomized, Dose-Ranging Study

RATIONALE:We investigated the impact of canakinumab, a fully human anti-interleukin-1b monoclonal antibody on inflammation and HRQoL in gouty arthritis patients.METHODS: In this 8-week, single-blind, dose-ranging study, patients with acute gouty arthritis flares, unresponsive/intolerant or contraindicated to NSAIDs and/or colchicine were randomized to single subcutaneous canakinumab (10, 25, 50, 90, or 150mg, N5143) or single intramuscular triamcinolone acetonide (TA, 40mg, N557). Patients assessed pain (Likert scale), physicians assessed clinical signs of joint inflammation, and HRQoL was recorded using SF-36.RESULTS: At baseline, 98% patients had moderate-to-extreme pain, 85% had moderate/severe joint swelling, 64-79% had elevated inflammatory markers and HRQoL scores indicated impaired physical function. Percentage of patients with no/mild pain was numerically greater in most canakinumab groups vs. TA, 24-72h post-dose; difference significant for 150mg group at these time-points (P<0.05). Canakinumab 150mg was associated with significantly lower Likert scores for tenderness [OR, 3.2; 95% CI, 1.27-7.89; P50.014] and swelling (OR, 2.7; 95% CI, 1.09-6.50, P50.032) at 72h vs. TA; erythema was not different. Median CRP and SAA levels normalized by 7 days post-dose in most canakinumab groups, but remained elevated in TA. Physical function improved at 7 days postdose in all groups, highest improvement for canakinumab 150mg. SF-36 scores for physical functioning and bodily pain with canakinumab 150mg approached US general population scores by 7 days post-dose and exceeded normal values by 8 weeks post-dose.CONCLUSION: Canakinumab 150mg produced significantly greater and rapid pain-relief and improvements in HRQoL vs. TAin acute gouty arthritis patients.

Disponibilidade e mineralização do nitrogênio após aplicações sucessivas de lodo de esgoto no solo, estimadas por meio de incubação anaeróbica

O objetivo deste trabalho foi avaliar, por meio de incubação anaeróbica, a mineralização e a disponibilidade de N em solos tratados com aplicações sucessivas de lodo de esgoto, e determinar o efeito residual das aplicações anteriores na taxa de mineralização do nitrogênio (TMN). Dois experimentos de longa duração foram realizados com doses anuais de lodo de esgoto, para o cultivo de milho: um com dose recomendada de lodo, correspondente a 120 kg ha-1 de N, e 2, 4 e 8 vezes essa dose, em área da Embrapa Meio Ambiente, em Jaguariúna; e outro com 80 kg ha-1 de N aplicado via adubo mineral, e 1 e 2 vezes a dose recomendada de lodo de esgoto, em área do Instituto Agronômico, em Campinas; além de testemunha, sem aplicação de lodo. A disponibilidade de N e a TMN foram estimadas em condições de campo e por incubações anaeróbicas em laboratório. O N total absorvido pelas plantas também foi determinado. O lodo de esgoto é mais efetivo em fornecer nitrogênio ao milho, em longo prazo, do que a adubação mineral. O efeito residual do lodo de esgoto não influencia a TMN do lodo recém-adicionado. O método de incubação anaeróbica é eficiente em estimar a mineralização do N proveniente de lodo de esgoto recém-adicionado.

EFEITOS DA SIMULAÇÃO DE DERIVA DE CLOMAZONE EM PLANTAS DE LARANJEIRA 'HAMLIN'

Em trabalho realizado em 1998, no município de Taiúva-SP, objetivou-se avaliar o efeito da simulação da deriva de doses crescentes, até atingir a recomendada comercialmente, de clomazone, em duas formulações, e de clomazone em mistura com ametryne, em laranjeira-'Hamlin' com frutos com 2 a 4 cm de diâmetro. O delineamento experimental utilizado foi o de blocos casualizados, com 15 tratamentos, em 3 repetições. As avaliações basearam-se em possíveis alterações morfofisiológicas das plantas, com determinações do teor de clorofila total nas folhas, porcentagem de abortamento de frutos, além de análise tecnológica dos frutos. Concluiu-se que a dose comercial de clomazone isolado e em mistura com ametryne a 50 e 100% da dose resultou na formação de manchas cloróticas e/ou necróticas na casca do fruto e causou mortalidade de ramos que se encontravam em crescimento vegetativo, não acarretando qualquer alteração qualitativa do suco.