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1.
Lancet Oncol ; 25(4): 488-500, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38547893

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma is characterised by low immunogenicity and an immunosuppressive tumour microenvironment. LOAd703, an oncolytic adenovirus with transgenes encoding TMZ-CD40L and 4-1BBL, lyses cancer cells selectively, activates cytotoxic T cells, and induces tumour regression in preclinical models. The aim of this study was to evaluate the safety and feasibility of combining LOAd703 with chemotherapy for advanced pancreatic ductal adenocarcinoma. METHODS: LOKON001 was a non-randomised, phase 1/2 study conducted at the Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA, and consisted of two arms conducted sequentially; the results of arm 1 are presented here. In arm 1, patients 18 years or older with previously treated or treatment-naive unresectable or metastatic pancreatic ductal adenocarcinoma were treated with standard 28-day cycles of intravenous nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 (up to 12 cycles) and intratumoural injections of LOAd703 every 2 weeks. Patients were assigned using Bayesian optimal interval design to receive 500 µL of LOAd703 at 5 × 1010 (dose 1), 1 × 1011 (dose 2), or 5 × 1011 (dose 3) viral particles per injection, injected endoscopically or percutaneously into the pancreatic tumour or a metastasis for six injections. The primary endpoints were safety and treatment-emergent immune response in patients who received at least one dose of LOAd703, and antitumour activity was a secondary endpoint. This study was registered with ClinicalTrials.gov, NCT02705196, arm 2 is ongoing and open to new participants. FINDINGS: Between Dec 2, 2016, and Oct 17, 2019, 23 patients were assessed for eligibility, leading to 22 patients being enrolled. One patient withdrew consent, resulting in 21 patients (13 [62%] men and eight [38%] women) assigned to a dose group (three to dose 1, four to dose 2, and 14 to dose 3). 21 patients were evaluable for safety. Median follow-up time was 6 months (IQR 4-10), and data cutoff was Jan 5, 2023. The most common treatment-emergent adverse events overall were anaemia (96 [8%] of 1237 events), lymphopenia (86 [7%] events), hyperglycaemia (70 [6%] events), leukopenia (63 [5%] events), hypertension (62 [5%] events), and hypoalbuminaemia (61 [5%] events). The most common adverse events attributed to LOAd703 were fever (14 [67%] of 21 patients), fatigue (eight [38%]), chills (seven [33%]), and elevated liver enzymes (alanine aminotransferase in five [24%], alkaline phosphatase in four [19%], and aspartate aminotransferase in four [19%]), all of which were grade 1-2, except for a transient grade 3 aminotransferase elevation occurring at dose 3. A maximum tolerated dose was not reached, thereby establishing dose 3 as the highest-evaluated safe dose when combined with nab-paclitaxel plus gemcitabine. Proportions of CD8+ effector memory cells and adenovirus-specific T cells increased after LOAd703 injections in 15 (94%) of 16 patients for whom T-cell assays could be performed. Eight (44%, 95% CI 25-66) of 18 patients evaluable for activity had an objective response. INTERPRETATION: Combining LOAd703 with nab-paclitaxel plus gemcitabine in patients with advanced pancreatic ductal adenocarcinoma was feasible and safe. To build upon this novel chemoimmunotherapeutic approach, arm 2 of LOKON001, which combines LOAd703, nab-paclitaxel plus gemcitabine, and atezolizumab, is ongoing. FUNDING: Lokon Pharma, the Swedish Cancer Society, and the Swedish Research Council.


Assuntos
Adenocarcinoma , Anemia , Vírus Oncolíticos , Neoplasias Pancreáticas , Trombocitopenia , Masculino , Humanos , Feminino , Gencitabina , Vírus Oncolíticos/genética , Teorema de Bayes , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/tratamento farmacológico , Paclitaxel , Anemia/induzido quimicamente , Trombocitopenia/induzido quimicamente , Adenocarcinoma/terapia , Adenocarcinoma/tratamento farmacológico , Albuminas , Terapia Genética/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Microambiente Tumoral
2.
J Cell Mol Med ; 28(7): e18162, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38494863

RESUMO

Immunostimulatory gene therapy using oncolytic viruses is currently evaluated as a promising therapy for cancer aiming to induce anti-tumour immunity. Here, we investigate the capacity of oncolytic adenoviruses (LOAd) and their transgenes to induce immunogenicity in the infected tumour cells. Oncolysis and death-related markers were assessed after infection of eight human solid cancer cell lines with different LOAd viruses expressing a trimerized, membrane-bound (TMZ)-CD40L, TMZ-CD40L and 41BBL, or no transgenes. The viruses induced transgene expression post infection before they were killed by oncolysis. Death receptors TRAIL-R1, TRAIL-R2 and Fas as well as immunogenic cell death marker calreticulin were upregulated in cell lines post infection. Similarly, caspase 3/7 activity was increased in most cell lines. Interestingly, in CD40+ cell lines there was a significant effect of the TMZ-CD40L-encoding viruses indicating activation of the CD40-mediated apoptosis pathway. Further, these cell lines showed a significant increase of calreticulin, and TRAIL receptor 1 and 2 post infection. However, LOAd viruses induced PD-L1 upregulation which may hamper anti-tumour immune responses. In conclusion, LOAd infection increased the immunogenicity of infected tumour cells and this was potentiated by CD40 stimulation. Due to the simultaneous PD-L1 increase, LOAd viruses may benefit from combination with antibodies blocking PD1/PD-L1.


Assuntos
Ligante de CD40 , Neoplasias , Humanos , Ligante de CD40/genética , Adenoviridae/genética , Antígeno B7-H1/genética , Calreticulina/genética , Antígenos CD40
3.
J Transl Med ; 21(1): 506, 2023 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-37501121

RESUMO

BACKGROUND: The activation of dendritic cells (DCs) is pivotal for generating antigen-specific T-cell responses to eradicate tumor cells. Hence, immunotherapies targeting this interplay are especially intriguing. Moreover, it is of interest to modulate the tumor microenvironment (TME), as this harsh milieu often impairs adaptive immune responses. Oncolytic viral therapy presents an opportunity to overcome the immunosuppression in tumors by destroying tumor cells and thereby releasing antigens and immunostimulatory factors. These effects can be further amplified by the introduction of transgenes expressed by the virus. METHODS: Lokon oncolytic adenoviruses (LOAd) belong to a platform of chimeric serotype Ad5/35 viruses that have their replication restricted to tumor cells, but the expression of transgenes is permitted in all infected cells. LOAd732 is a novel oncolytic adenovirus that expresses three essential immunostimulatory transgenes: trimerized membrane-bound CD40L, 4-1BBL and IL-2. Transgene expression was determined with flow cytometry and ELISA and the oncolytic function was evaluated with viability assays and xenograft models. The activation profiles of DCs were investigated in co-cultures with tumor cells or in an autologous antigen-specific T cell model by flow cytometry and multiplex proteomic analysis. Statistical differences were analyzed with Kruskal-Wallis test followed by Dunn's multiple comparison test. RESULTS: All three transgenes were expressed in infected melanoma cells and DCs and transgene expression did not impair the oncolytic activity in tumor cells. DCs were matured post LOAd732 infection and expressed a multitude of co-stimulatory molecules and pro-inflammatory cytokines crucial for T-cell responses. Furthermore, these DCs were capable of expanding and stimulating antigen-specific T cells in addition to natural killer (NK) cells. Strikingly, the addition of immunosuppressive cytokines TGF-ß1 and IL-10 did not affect the ability of LOAd732-matured DCs to expand antigen-specific T cells and these cells retained an enhanced activation profile. CONCLUSIONS: LOAd732 is a novel immunostimulatory gene therapy based on an oncolytic adenovirus that expresses three transgenes, which are essential for mediating an anti-tumor immune response by activating DCs and stimulating T and NK cells even under imunosuppressive conditions commonly present in the TME. These qualities make LOAd732 an appealing new immunotherapy approach.


Assuntos
Melanoma , Linfócitos T , Humanos , Proteômica , Melanoma/genética , Melanoma/terapia , Células Matadoras Naturais , Citocinas/metabolismo , Terapia Genética , Células Dendríticas , Microambiente Tumoral
4.
Cancer Immunol Immunother ; 70(10): 2851-2865, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33666760

RESUMO

Pretreatment of B-cell lymphoma patients with immunostimulatory gene therapy using armed oncolytic viruses may prime tumor lesions for subsequent chimeric antigen receptor (CAR) T-cell therapy, thereby enhancing CAR T-cell functionality and possibly increasing response rates in patients. LOAd703 (delolimogene mupadenorepvec) is an oncolytic adenovirus (serotype 5/35) that encodes for the transgenes CD40L and 4-1BBL, which activate both antigen-presenting cells and T cells. Many adenoviruses failed to demonstrate efficacy in B-cell malignancies, but LOAd703 infect cells via CD46, which enables B cell infection. Herein, we investigated the therapeutic potential of LOAd703 in human B-cell lymphoma models, alone or in combination with CAR T-cell therapy. LOAd703 could infect and replicate in B-cell lymphoma cell lines (BC-3, Karpas422, Daudi, DG-75, U-698) and induced an overall enhanced immunogenic profile with upregulation of co-stimulatory molecules CD80, CD86, CD70, MHC molecules, death receptor Fas and adhesion molecule ICAM-1. Further, CAR T-cell functionality was boosted by stimulation with lymphoma cells infected with LOAd703. This was demonstrated by an augmented release of IFN-γ and granzyme B, increased expression of the degranulation marker CD107a, fewer PD-1 + TIM-3+ CAR T cells in vitro and enhanced lymphoma cell killing both in in vitro and in vivo xenograft models. In addition, LOAd703-infected lymphoma cells upregulated the secretion of several chemokines (CXCL10, CCL17, CCL22, CCL3, CCL4) essential for immune cell homing, leading to enhanced CAR T-cell migration. In conclusion, immunostimulatory LOAd703 therapy is an intriguing approach to induce anti-lymphoma immune responses and to improve CAR T-cell therapy in B-cell lymphoma.


Assuntos
Ligante de CD40/imunologia , Linfoma/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Receptores de Antígenos Quiméricos/imunologia , Animais , Feminino , Humanos , Linfoma/patologia , Camundongos , Camundongos Nus
5.
J Immunol ; 202(3): 787-798, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30617223

RESUMO

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-ß. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mechanisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-ß-1 and collagen type I. Virus infection prevented IL-6/GM-CSF-mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infection of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-γ. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer.


Assuntos
Ligante de CD40/metabolismo , Ativação Linfocitária , Terapia Viral Oncolítica , Receptores de Interleucina-6/antagonistas & inibidores , Transdução de Sinais , Adenoviridae , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Ligante de CD40/genética , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Células Dendríticas/citologia , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , Interleucina-6/imunologia , Zíper de Leucina , Melanoma Experimental , Camundongos , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/citologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Interleucina-6/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/imunologia
6.
Biochim Biophys Acta Biomembr ; 1860(5): 1205-1215, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29470946

RESUMO

Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).


Assuntos
Bicamadas Lipídicas/química , Membranas Mitocondriais/química , Membranas Mitocondriais/efeitos dos fármacos , Ubiquinona/farmacologia , Adsorção , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacocinética , Terpenos/química , Terpenos/farmacologia , Ubiquinona/farmacocinética
7.
Biochim Biophys Acta ; 1848(10 Pt A): 2233-43, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25986530

RESUMO

Ubiquinone-10 is mostly known for its role as an electron and proton carrier in aerobic cellular respiration and its function as a powerful antioxidant. Accumulating evidence suggest, however, that this well studied membrane component could have several other important functions in living cells. The current study reports on a previously undocumented ability of ubiquinone-10 to modulate the mechanical strength and permeability of lipid membranes. Investigations of DPH fluorescence anisotropy, spontaneous and surfactant induced leakage of carboxyfluorescein, and interactions with hydrophobic and hydrophilic surfaces were used to probe the effects caused by inclusion of ubiquinone-10 in the membrane of phospholipid liposomes. The results show that ubiquinone in concentrations as low as 2 mol% increases the lipid packing order and condenses the membrane. The altered physicochemical properties result in a slower rate of release of hydrophilic components, and render the membrane more resistant towards rupture. As judged from comparative experiments using the polyisoprenoid alcohol solanesol, the quinone moiety is essential for the membrane stabilizing effects to occur. Our findings imply that the influence of ubiquinone-10 on the permeability and mechanical properties of phospholipid membranes is similar to that of cholesterol. The reported data indicate, however, that the molecular mechanisms are different in the two cases.


Assuntos
Lipossomos/química , Fluidez de Membrana , Fosfatidilcolinas/química , Ubiquinona/química , Interações Hidrofóbicas e Hidrofílicas , Porosidade , Propriedades de Superfície , Resistência à Tração , Viscosidade
8.
J Transl Med ; 14(1): 282, 2016 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-27687804

RESUMO

BACKGROUND: Cancer immunotherapy can be potentiated by conditioning regimens such as cyclophosphamide, which reduces the level of regulatory T cells (tregs). However, myeloid suppressive cells are still remaining. Accordingly to previous reports, gemcitabine improves immune status of cancer patients. In this study, the role of gemcitabine was further explored to map its immunological target cells and molecules in patients with pancreatic cancer. METHODS: Patient blood was investigated by flow cytometry and cytokine arrays at different time points during gemcitabine treatment. RESULTS: The patients had elevated myeloid-derived suppressor cells (MDSCs), and Tregs at diagnosis. Myeloid cells were in general decreased by gemcitabine. The granulocytic MDSCs were significantly reduced while monocytic MDSCs were not affected. In vitro, monocytes responding to IL-6 by STAT3 phosphorylation were prevented to respond in gemcitabine medium. However, gemcitabine could not prevent STAT3 phosphorylation in IL-6-treated tumor cell lines. TGFß-1 was significantly reduced after only one treatment and continued to decrease. At the same time, the effector T cell:Treg ratio was increased and the effector T cells had full proliferative capacity during the gemcitabine cycle. However, after a resting period, the level of suppressor cells and TGFß-1 had been restored showing the importance of continuous conditioning. CONCLUSIONS: Gemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGFß-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy.

9.
Mol Cancer ; 13: 155, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24950741

RESUMO

BACKGROUND: With increased long-term survivors of childhood cancer patients, therapy-associated infertility has become one of the most common late side-effects and significantly affects their life-quality. Therefore, evaluation of anti-cancer agents on male reproduction and infertility prevention are urgently demanding. The proteasome inhibitor bortezomib has been launched in clinical trials for childhood cancers, however, its potential side effects on reproduction have so far been neither investigated experimentally nor reported in treated children. Thus the present study is designed to explore the impact of bortezomib on male reproductive function and to gain insights into how bortezomib exerts its adverse effects on man gonad, thereby providing pediatric oncologists relevant information. METHODS: 35 day-old male mice were treated with one 11-day cycle of bortezomib and then sacrificed 2 days, 45 days, or 6 months later. A mating study was performed in the group followed for 6 months, and their pups were analyzed on postnatal day 50. Serum follicle-stimulating hormone (FSH) and testicular testosterone levels were measured. Testicular morphology was evaluated by light- and electron microscopy, and the underlying mechanisms and pathways of testis damage were investigated. RESULTS: Testicular damage was visible already 2 days after stopping bortezomib and increased in severity by day 45. Then 80% of seminiferous tubules exhibited hypospermatogenesis with arrest at the levels of spermatogonia, spermatocytes and round spermatids. Germ cells were specifically targeted by bortezomib as evidenced by increased apoptosis mediated through activation of p53 and caspases. Even six months after the bortezomib treatment, testis weight, sperm concentration and seminiferous tubule length remained at a decreased level, indicating that spermatogenesis and tubular outgrowth could not fully recover. Combined with persistently increased serum levels of FSH in these mice, our results demonstrate that bortezomib can have long-term effects on testicular function, although fertility of bortezomib-exposed males remained and their offspring looked healthy. CONCLUSION: Bortezomib treatment causes long-term gonadal dysfunction in male mice. Careful monitoring of gonadal function in male childhood cancer patients treated with bortezomib is thus strongly recommended.


Assuntos
Ácidos Borônicos/efeitos adversos , Pirazinas/efeitos adversos , Espermatogênese/efeitos dos fármacos , Doenças Testiculares/patologia , Testosterona/biossíntese , Animais , Ácidos Borônicos/administração & dosagem , Bortezomib , Hormônio Foliculoestimulante/sangue , Humanos , Masculino , Camundongos , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/administração & dosagem , Pirazinas/administração & dosagem , Doenças Testiculares/induzido quimicamente , Testículo/efeitos dos fármacos , Testículo/patologia
10.
Genes (Basel) ; 15(3)2024 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-38540435

RESUMO

Considering the growing importance of microbiome analyses in forensics for identifying individuals, this study explores the transfer of the skin microbiome onto clothing, its persistence on fabrics over time, and its transferability from the environment and between different garments. Furthermore, this project compares three specific QIAGEN microbiome extraction kits to test their extraction efficiency on fabric samples. Additionally, this study aims to check if these extracts contain human DNA, providing a chance to obtain more information from the same evidence for personal identification. The results obtained show: (1) variations in the skin microbiome between the volunteers, potentially due to their different sex; (2) differences in microbial composition between worn and unworn clothing; (3) the influence of the environment on the microbial signature of unworn clothing; (4) the potential use of certain phyla as biomarkers to differentiate between worn and unworn garments, even over extended periods; (5) a tendency towards extraction biases in the QIAampMP® DNA microbiome kit among the three tested ones; and (6) none of the extraction kits allow for the typing of human genetic profiles suitable for comparison. In conclusion, our study offers supplementary insights into the potential utility of time-transferred microbiome analysis on garments for forensic applications.


Assuntos
Vestuário , Microbiota , Humanos , Pele , DNA Ribossômico , Microbiota/genética
11.
Vaccines (Basel) ; 11(2)2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36851086

RESUMO

BACKGROUND: COVID-19 vaccine uptake in Kenya is still low compared to other countries, especially in Europe and North America. In most parts of the country, a large percentage of the Kenyan population remains unvaccinated. As of October 2022, the Ministry of Health (Kenya) estimates that only 36.2% of the adult population had been fully vaccinated. METHODS: We conducted an experimental study in April 2022 targeting unvaccinated adults who had a history of hypertension and/or diabetes and those in the 60+ age group. We tested various messaging approaches using two different intervention channels. RESULTS: Although the overall rate of vaccinated individuals according to national records is low, responses from the study group collected through phone call conversations show that higher-risk adults such as those older than 60 or those with chronic illnesses have a remarkably high vaccination rate of 89%. After the study, four participants received a COVID-19 vaccine within 1 month of the intervention. These four participants all received a loss-messaging intervention approach during the study. CONCLUSION: This study supports a national approach to increasing COVID-19 vaccination rates using loss-messaging directed at unvaccinated, high-risk individuals.

12.
J Pharm Sci ; 112(3): 844-858, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36372229

RESUMO

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.


Assuntos
Antineoplásicos , Dendrímeros , Dendrímeros/química , Lisina , Antineoplásicos/química , Polietilenoglicóis/química
13.
Phys Chem Chem Phys ; 14(36): 12637-46, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22892582

RESUMO

The exact cellular target for the potent anti-cancer agent hypericin has not yet been determined; this thus encourages the application of computational chemistry tools to be employed in order to provide insights that can be employed in further drug development studies. In the present study computational docking and molecular dynamics simulations are applied to investigate possible interactions between hypericin and the Ca(2+) pump SERCA as proposed in the literature. Hypericin was found to bind strongly both in pockets within the transmembrane region and in the cytosolic region of the protein, although the two studied isoforms of SERCA differ slightly in their preferred binding sites. The calculated binding energies for hypericin in the four investigated sites were of the same magnitude as for thapsigargin (TG), the most potent SERCA inhibitor, or in the range between TG and di-tert-butylhydroquinone (BHQ), which is also known to possess inhibitory activity. The hydrophobic character of hypericin indicates that the molecule initially binds in the ER membrane from which it diffuses into the transmembrane region of the protein and to binding pockets therein. The transmembrane TG and BHQ binding pockets provide suitable locations for hypericin as they allow for favourable interactions with the lipid tails that surround these. High binding energies were noted for hypericin in these pockets and are expected to constitute highly possible binding sites due to their accessibility from the ER membrane. Hypericin most likely binds to both isoforms of SERCA and acts as an inhibitor or, under light irradiation, as a singlet oxygen generator that in turn degrades the protein or induces lipid peroxidation.


Assuntos
Antineoplásicos/química , Simulação de Dinâmica Molecular , Perileno/análogos & derivados , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimologia , Antracenos , Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidroquinonas/química , Hidroquinonas/farmacologia , Modelos Moleculares , Estrutura Molecular , Perileno/química , Perileno/farmacologia , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Relação Estrutura-Atividade , Tapsigargina/química , Tapsigargina/farmacologia
14.
Arch Toxicol ; 86(10): 1613-25, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22669514

RESUMO

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.


Assuntos
Antineoplásicos/farmacologia , Oxidiazóis/farmacologia , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Peróxido de Hidrogênio/metabolismo , Dose Letal Mediana , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Oxidiazóis/administração & dosagem , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
15.
Mol Ther Oncolytics ; 24: 429-442, 2022 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-35141399

RESUMO

Immune checkpoint inhibitors have revolutionized the treatment of metastatic melanoma, but most tumors show resistance. Resistance is connected to a non-T cell inflamed phenotype partially caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Herein, we investigated whether the adenoviral gene vehicle mLOAd703 carrying both DC- and T cell-activating genes can lead to inflammation in a B16-CD46 model and thereby overcome resistance to checkpoint inhibition therapy. B16-CD46 cells were injected subcutaneously in one or both flanks of immunocompetent C57BL/6J mice. mLOAd703 treatments were given intratumorally alone or in combination with intraperitoneal checkpoint inhibition therapy (anti-PD-1, anti-PD-L1, or anti-TIM-3). Tumor, lymph node, spleen, and serum samples were analyzed for the presence of immune cells and cytokines/chemokines. B16-CD46 tumors were non-inflamed and resistant to checkpoint blockade. In contrast, mLOAd703 treatment led to infiltration of the tumor by CD8+ T cells, natural killer (NK) cells, and CD103+ DCs, accompanied by a systemic increase of pro-inflammatory cytokines interferon γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-27 (IL-27). This response was even more pronounced after combining the virus with checkpoint therapy, in particular with anti-PD-L1 and anti-TIM-3, leading to further reduced tumor growth in injected lesions. Moreover, anti-PD-L1 combination also facilitated abscopal responses in non-injected lesions.

16.
J Anal Toxicol ; 46(7): 765-775, 2022 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34746960

RESUMO

We have identified a clinical need for a sensitive, specific, flexible, comprehensive and affordable analytical technology to efficiently detect polydrug use. In addition, the current standard practice of surveilled urine sampling is uncomfortable for the patient; hence, more patient-friendly sample collection methods are requested. To fill these needs, we have developed and validated a high-throughput liquid chromatography-high-resolution mass spectrometry (LC--HRMS) method for the analysis of drugs of abuse (DoA) in oral fluid (OF). The method covers a panel of 71 substances including traditional DoA, prescription narcotics and new psychoactive substances (NPS), with a guaranteed limit of identification of <3 µg/L for 87% of the analytes. Method validation showed high accuracy (>99.7%), sensitivity (>99.7%) and specificity (100%). Most analytes had a high process efficiency during the salting-out liquid-liquid extraction sample preparation and no or only a minor matrix effect during the analysis. We have implemented this method in clinical routine and present data from 18,579 OF samples collected during routine patient treatment in mainly psychiatric and addiction clinics in West Sweden between September 2020 and June 2021. Seventy-one percent of the samples were positive and a total of 41,472 DoA findings were detected. Amphetamine (27%), buprenorphine (25%), nordiazepam (18%) and alprazolam (16%) were most prevalent. New psychoactive substances were detected in 189 samples (1.0%). The occurrence of polydrug use was common; 34% of the positive samples contained three analytes or more and 12% six or more. To the best of our knowledge, this is the first method for comprehensive analysis of DoA in OF using LC--HRMS and the largest dataset published on the detection of DoA in OF. With the current complex and variable drug use pattern, this broad, cost-effective and reliable method has largely replaced immunoassay screening in urine in our laboratory.


Assuntos
Detecção do Abuso de Substâncias , Transtornos Relacionados ao Uso de Substâncias , Cromatografia Líquida/métodos , Atenção à Saúde , Humanos , Espectrometria de Massas/métodos , Psicotrópicos/análise , Detecção do Abuso de Substâncias/métodos , Transtornos Relacionados ao Uso de Substâncias/diagnóstico
17.
Phys Chem Chem Phys ; 13(24): 11590-6, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21594267

RESUMO

The porphyrin and chlorin parent compounds constitute the base of many potent photosensitizers aimed to be utilized in photodynamic therapy (PDT). However, the photosensitizers available on the market today are not ideal for use in PDT; many of them suffering from drawbacks such as long-lasting photosensitization or absorption at wavelengths below the optimal tissue penetration. This has emphasized the need of new photosensitizers with improved photodynamic properties. In the present study we have used density functional theory based methods to design new chlorin compounds with conjugated substituents such as vinyl groups and carboxylic acids, aiming for strong absorption in the therapeutic window of PDT. The specific substituent positions were found to have a significant effect on the spectra. A chlorin with four propenoic acids was able to red-shift the absorption the most compared with non-substituted chlorin, generating the red-most absorption at 755 nm, and with significantly enhanced oscillator strengths. The results from the present study constitute a useful starting point for further design of tetrapyrrole derivatives as improved photosensitizers.


Assuntos
Fármacos Fotossensibilizantes/química , Porfirinas/química , Absorção , Clorofilídeos , Mesoporfirinas/química
18.
Phys Chem Chem Phys ; 13(15): 7207-17, 2011 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-21409255

RESUMO

Porphyrin and chlorin based compounds possess promising properties to be utilized as photosensitizers in photodynamic therapy (PDT). However, the photosensitizers available on the market today are not ideal for use in PDT, which has emphasized the need for new photosensitizers with improved photodynamic properties to be developed. Computational drug-design can be utilized in the search for improved pharmaceutical compounds, provided that the methods used are able to reproduce experimental data. In the present study we investigated, by the use of time-dependent density functional theory (TD-DFT), the performance of the long-range corrected functionals ωB97, ωB97X and ωB97XD on their ability to predict low-lying singlet excitations (>600 nm) of a set of well-known photosensitizing compounds. It was found that ωB97X reproduced the experimental red-most absorption band most satisfactorily. The use of either B3LYP, ωB97XD or M06 in geometry optimizations has a minor effect on the spectra in most cases. Calculated energy differences between the optimized singlet ground states and optimized first excited triplet states show consistent and overall higher triplet state energies for B3LYP, M06, and PBE0 compared with ωB97, ωB97X, and ωB97XD. The calculated triplet state energies are, however, sufficient to generate singlet oxygen in most cases.


Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Análise Espectral , Tetrapirróis/química , Absorção , Porfirinas/química , Teoria Quântica
19.
Eur J Oncol Nurs ; 51: 101923, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33618229

RESUMO

PURPOSE: Information can help parents of children with cancer by reducing uncertainty and giving them a sense of control in a chaotic situation. Although providing information to parents is a core activity of paediatric oncology nursing, few studies focus on interventions for informing parents. Thus, the aim of this study is to evaluate parents' experiences after participating in a person-centred information intervention for parents of children with cancer. METHOD: This study is part of a process evaluation of a person-centred informational intervention in paediatric oncology for patients' parents. Qualitative semi-structured interviews with 13 parents who had taken part in the intervention were analysed using qualitative content analysis. RESULTS: An opening for healing emerged as the overarching theme, consisting of three categories. Gaining a deeper understanding of the entire situation describes how parents benefitted from processing current topics and moving forward by learning. Caring reflections in a safe space describes how parents appreciated having a moment just for themselves and feeling better by venting their feelings. Meeting a competent and compassionate nurse describes how parents experienced trust and being listened to. CONCLUSION: Having individual information meetings integrated as a primary nursing responsibility, mediated by competent and compassionate nurses also responsible for the care of the child, could enhance person-centred care and individualise parental education.


Assuntos
Comunicação , Neoplasias/enfermagem , Enfermagem Oncológica/métodos , Pais/psicologia , Enfermagem Pediátrica/métodos , Relações Profissional-Família , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pesquisa em Avaliação de Enfermagem , Satisfação do Paciente , Pesquisa Qualitativa
20.
Mol Ther Oncolytics ; 20: 508-518, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33738337

RESUMO

Immunostimulatory gene therapy utilizing oncolytic viruses (OVs) as gene vehicles is a promising immunotherapy for cancer. Since viruses are immunogenic, systemic delivery can be troublesome due to neutralizing antibodies. Nevertheless, local delivery by intratumoral injection seems to induce systemic immune reactions. In this study, we demonstrate a novel mechanism of action of armed OV therapy suggesting that exosomes released by tumor cells infected with armed OV may participate to activate the immune system and this may also support systemic immunity. Tumor cell-derived exosomes commonly exert immunosuppressive functions. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. Human melanoma cells were infected by OVs armed with costimulatory molecules CD40 ligand (CD40L) and 4-1BB ligand (4-1BBL). Exosomes were purified and investigated for the presence of CD40L/4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results show that the exosomes cargo transgenes. The exosomes from CD40L/4-1BBL-expressing tumor cells, or the viruses themselves, could stimulate robust dendritic cell (DC) activation with an enhanced level of major histocompatibility complex (MHC) and costimulatory molecules. Hence, exosomes after OV infection can locally activate immune responses at the tumor site and encounter immune cells such as DCs.

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