Estudos de medicamentos biosimilares

In Brazil, the registration of new drugs is carried out only when the regulatory agency (Anvisa, acronym in Portuguese) is fully satisfied with the evidence of their quality, efficacy and safety, presented by a pharmaceutical industry that strive for this registration. With the patent expiration, pharmaceutical companies are attracted to produce biological medicines called biosimilar or biogenerics or simply generics, whose approval may result in reduced treatment costs. But it is necessary that the biosimilar be, at least, equally efective and safe and without contaminants in relation to the original. Recent consensus guidelines aim to establish criteria for efcacy and safety of these medicines. Preclinical studies in vitro and in vivo, the origin of raw materials and clinical studies phase I, II and III are recommended for biosimilar medicine registration in the international market. Low molecular weight heparins are found in this situation. In this review we specifcally addressed this type of medicine, which could serve as a benchmark for other biosimilar medicines.

An HPLC-UV method for the quantification of zidovudine in rat plasma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coleta, processamento, caracterização fenótipa das células-tronco mesenquimais e sua viabilidade de aplicação por via intratecal em equinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pharmacokinetics of hydroxymethylnitrofurazone and its parent drug nitrofurazone in rabbits

The prodrug hydroximethylnitrofurazone (NFOH) presents antichagasic activity with greatly reduced toxicity compared to its drug matrix nitrofurazone (NF). Besides these new characteristics, the prodrug was more active against the parasite T. cruzi amastigotes. These advantages make the prodrug a possible therapeutic alternative for the treatment of both acute and the chronic phase of Chagas disease. However, the knowledge of pharmacokinetic profile is crucial to evaluate the feasibility of a new drug. In this study, our objective was to evaluate the in vivo formation of NF from the NFOH single administration and to evaluate its pharmacokinetic profile and compared it to NF administration. A bioanalytical method to determine the NF and NFOH by LCMS/MS was developed and validated to perform these investigations. Male albino rabbits (n=15) received NF intravenously and orally in doses of 6.35 and 63.5 mg / kg respectively, and NFOH, 80.5 mg / kg orally. The serial blood samples were processed and analyzed by mass spectrometry. The system operated in positive and negative modes for the analites determination, under elution of the mobile phase 50:50 water: methanol. The administration of NFOH allowed the calculation of pharmacokinetic parameters for the prodrug, and the NF obtained from NFOH administration. Using the pharmacokinetic profile obtained from the NF i.v. administration, the oral bioavailability of NF from the administered prodrug was obtained (60.1%) and, as a key parameter in a prodrug administration, should be considered in future studies. The i.v. and oral administrations of NF differ in the constant of elimination (0.04 vs 0.002) and elimination half-life (17.32 min vs 276.09 min) due to the low solubility of the drug that hinders the formation of molecular dispersions in the digestory tract. Still, there was observed no statistical differences were observed between the pharmacokinetic parameters of orally administered NF and NF obtained from NFOH. The calculated area under the curve (AUC 0-∞) showed that the exposure to the parental drug was fairly the same (844.79 vs 566.44) for NF and NF obtained from the prodrug administration. The tendency to higher NF's mean residence time (MRT) as observed in the prodrug administration (956.1 min vs 496.3 min) guarantees longer time for the action of the drug and it allows the expansion of the administration intervals. These findings, added with the beneficial characteristics of the prodrug encourage new efficacy tests towards the clinical use of NFOH.

Memory-rescuing effects of cannabidiol in an animal model of cognitive impairment relevant to neurodegenerative disorders

Rationale Cannabidiol, the main nonpsychotropic constituent of Cannabis sativa, possesses a large number of pharmacological effects including anticonvulsive, sedative, hypnotic, anxiolytic, antipsychotic, anti-inflammatory, and neuroprotective, as demonstrated in clinical and preclinical studies. Many neurodegenerative disorders involve cognitive deficits, and this has led to interest in whether cannabidiol could be useful in the treatment of memory impairment associated to these diseases. Objectives We used an animal model of cognitive impairment induced by iron overload in order to test the effects of cannabidiol in memory-impaired rats. Methods Rats received vehicle or iron at postnatal days 12-14. At the age of 2 months, they received an acute intraperitoneal injection of vehicle or cannabidiol (5.0 or 10.0 mg/kg) immediately after the training session of the novel object recognition task. In order to investigate the effects of chronic cannabidiol, iron-treated rats received daily intraperitoneal injections of cannabidiol for 14 days. Twenty-four hours after the last injection, they were submitted to object recognition training. Retention tests were performed 24 h after training. Results A single acute injection of cannabidiol at the highest dose was able to recover memory in iron-treated rats. Chronic cannabidiol improved recognition memory in iron-treated rats. Acute or chronic cannabidiol does not affect memory in control rats. Conclusions The present findings provide evidence suggesting the potential use of cannabidiol for the treatment of cognitive decline associated with neurodegenerative disorders. Further studies, including clinical trials, are warranted to determine the usefulness of cannabidiol in humans suffering from neurodegenerative disorders.

Human Adipose-Derived Mesenchymal Stromal Cells Injected Systemically Into GRMD Dogs Without Immunosuppression Are Able to Reach the Host Muscle and Express Human Dystrophin

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting I in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

The neonatal immune system: immunomodulation of infections in early life

The innate and adaptive immune responses in neonates are usually functionally impaired when compared with their adult counterparts. The qualitative and quantitative differences in the neonatal immune response put them at risk for the development of bacterial and viral infections, resulting in increased mortality. Newborns often exhibit decreased production of Th1-polarizing cytokines and are biased toward Th2-type responses. Studies aimed at understanding the plasticity of the immune response in the neonatal and early infant periods or that seek to improve neonatal innate immune function with adjuvants or special formulations are crucial for preventing the infectious disease burden in this susceptible group. Considerable studies focused on identifying potential immunomodulatory therapies have been performed in murine models. This article highlights the strategies used in the emerging field of immunomodulation in bacterial and viral pathogens, focusing on preclinical studies carried out in animal models with particular emphasis on neonatal-specific immune deficits.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy (NFLE).

Nocturnal Frontal Lobe Epilepsy (NFLE) is characterized by onset during infancy or childhood with persistence in adulthood, family history of similar nocturnal episodes simulating non-REM parasomnias (sleep terrors or sleepwalking), general absence of morphological substrates, often by normal interictal electroencephalographical recordings (EEGs) during wakefulness. A family history of epilepsy may be present with Mendelian autosomal dominant inheritance has been described in some families. Recent studies indicate the involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in the molecular mechanisms of NFLE. Mutations in the genes encoding for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nAChR induce changes in the biophysical properties of nAChR, resulting generally in a “gain of function”. Preclinical studies report that activation of a nuclear receptor called type peroxisome proliferator-activated receptor (PPAR-α) by endogenous molecules or by medications (e.g. fenofibrate) reduces the activity of the nAChR and, therefore, may decrease the frequency of seizures. Thus, we hypothesize that negative modulation of nAChRs might represent a therapeutic strategy to be explored for pharmacological treatment of this form of epilepsy, which only partially responds to conventional antiepileptic drugs. In fact, carbamazepine, the current medication for NFLE, abolishes the seizures only in one third of the patients. The aim of the project is: 1)_to verify the clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant NFLE and ADNFLE patients; focousing on the analysis of the polysomnographic action of the PPAR- agonist (fenofibrate). 2)_to demonstrate the subtended mechanism of efficacy by means of electrophysiological and behavioral experiments in an animal model of the disease: particularly, transgenic mice carrying the mutation in the nAChR 4 subunit (Chrna4S252F) homologous to that found in the humans. Given that a PPAR-α agonist, FENOFIBRATE, already clinically utilized for lipid metabolism disorders, provides a promising therapeutic avenue in the treatment of NFLE\ADNFLE.

Molekulare Grundlagen der Immunkontrolle des murinen Cytomegalovirus in Gegenwart viruskodierter Immunevasine

Die Kontrolle der Infektion mit dem humanen Cytomegalovirus (HCMV) wird primär durch antivirale CD8 T-Zellen vermittelt. Während der Koevolution zwischen Virus und Wirt wurden Immunevasionsmechanismen entwickelt, die direkt die Expression der Peptid-MHC-Klasse-I-Komplexe an der Zelloberfläche beeinflussen und es dem Virus ermöglichen, der Immunkontrolle des Wirtes zu entkommen. Da HCMV und das murine CMV (mCMV) zum Teil analoge Strategien zur Modulation des MHC-Klasse-I-Antigen-Präsentationswegs entwickelt haben, wurde in der vorliegenden Arbeit auf das experimentelle Modell mit mCMV zurückgegriffen. Die für die Immunevasion verantwortlichen Genprodukte m04/gp34, m06/gp48 und m152/gp40 werden aufgrund ihres regulatorischen Einflusses auf die Antigenpräsentation als vRAPs (viral regulators of antigen presentation) bezeichnet. Diese interferieren mit dem Transport Peptid-beladener MHC-Klasse-I-Moleküle und reduzieren in ihrer konzertierten Wirkung die Präsentation viraler Peptide an der Zelloberfläche.rnDie Transplantation hämatopoietischer Zellen nach Immunoablation stellt eine etablierte Therapieform bei malignen hämatologischen Erkrankungen dar. Zwischen Immunoablation und der Rekonstitution des Immunsystems sind die Empfänger der transferierten Zellen stark immunsupprimiert und anfällig für eine CMV-Erkrankung bei Reaktivierung des Virus. Neben der Gabe antiviraler Medikamente ist der adoptive Transfer antiviraler CD8 T-Zellen eine vielversprechende Therapiemöglichkeit, um reaktivierende CMV zu kontrollieren, bis das körpereigene Immunsystem wieder funktionsfähig ist. Obwohl im murinen Modell sehr wohl etabliert, stellen im humanen System die eingeschränkte Wirkung und die Notwendigkeit der konsequenten Gabe hoher Zellzahlen gewisse logistische Schwierigkeiten dar, welche die Methode bisher von der klinischen Routine ausschließen.rnDas murine Modell sagte eine Rolle von IFN-γ voraus, da Depletion dieses Zytokins zu einer verminderten Schutzwirkung gegen die mCMV-Infektion führt.rnIm ersten Teil dieser Arbeit sollte ein möglicher inhibitorischer Effekt von m04 auf m152 untersucht werden, der bei der Rekombinanten Δm06W beobachtet wurde. Mit neu generierten Viren (Δm06L1+2) konnte dieser Effekt allerdings nicht bestätigt werden. Bei Δm06W fehlte jedoch eine höher N-glykosylierte Isoform des m152-Proteins. Um zu untersuchen, ob die N-Glykosylierung von m152 für seine Funktion notwendig ist, wurde ein rekombinantes Virus generiert, das in Folge einer Deletion aller 3 N-Glykosylierungssequenzen nur eine nicht-glykosylierte Isoform des m152-Proteins bilden kann. In Übereinstimmung mit der zwischenzeitlich publizierten Kristallstruktur das Komplexes von m152 und dem Liganden RAE-1 des aktivierenden NK-Zellrezeptors NKG2D konnte erstmals gezeigt werden, dass die Funktionen von m152 in der adaptiven und in der angeborenen Immunität auch von der nicht N-glykosylierten Isoform wahrgenommen werden können.rnIm zweiten Teil der Arbeit sollte mit Hilfe eines Sets an vRAP Deletionsmutanten der Einfluss von IFN γ auf die einzeln oder in Kombination exprimierten vRAPs untersucht werden. Es zeigte sich, dass Vorbehandlung der Zellen mit IFN-γ die Antigenprozessierung nach Infektion stark erhöht und die vRAPs dann nicht mehr in der Lage sind, die Präsentation aller Peptid-beladener MHC-Klasse-I-Komplexe zu verhindern. Des Weiteren konnte gezeigt werden, dass vorher nicht-schützende CD8 T-Zellen Schutz vermitteln können, wenn das Gewebe der Rezipienten konstitutiv mit IFN-γ versorgt wird. Die zusätzliche Gabe von IFN-γ stellt daher eine vielversprechende Möglichkeit dar, den adoptiven Transfer als Therapie in der klinischen Routine einzusetzen.

Effect of Acute Psychological Stress on Prefrontal GABA Concentration Determined by Proton Magnetic Resonance Spectroscopy

Objective Impaired function of the central gamma-aminobutyric acid (GABA) system, which provides the brain’s major inhibitory pathways, is thought to play an important role in the pathophysiology of anxiety disorders. The effect of acute psychological stress on the human GABA-ergic system is still unknown, however. The purpose of this study was to determine the effect of acute stress on prefrontal GABA levels. Method A recently developed noninvasive magnetic resonance spectroscopy method was used to measure changes in the GABA concentration of the prefrontal cortex in 10 healthy human subjects during a threat-of-shock condition and during a safe condition (two sessions on different days). The main outcome measure was the mean GABA concentration within a 3×3×2-cm3 voxel selected from the medial prefrontal cortex. Results Prefrontal GABA decreased by approximately 18% in the threat-of-shock condition relative to the safe condition. This reduction was specific to GABA, since the concentrations of N-acetyl-aspartate, choline-containing compounds, and glutamate/glutamine levels obtained in the same spectra did not change significantly. Conclusions This result appeared compatible with evidence from preclinical studies in rodents, which showed rapid presynaptic down-regulation of GABA-ergic neurotransmission in response to acute psychological stress. The molecular mechanism and functional significance of this reduced inhibitory effect of acute psychological stress in relation to impaired GABA-ergic function in anxiety disorders merit further investigation.

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.

First clinical evidence that imaging with somatostatin receptor antagonists is feasible

Preclinical studies have indicated that somatostatin receptor (sst)-expressing tumors demonstrate higher uptake of radiolabeled sst antagonists than of sst agonists. In this study, we evaluated whether imaging with sst antagonists was feasible in patients.

Phase I clinical and pharmacokinetic evaluation of the vascular-disrupting agent OXi4503 in patients with advanced solid tumors

Preclinical studies show that OXi4503 (combretastatin A1 diphosphate, CA1P) is more potent than other clinically evaluated vascular-disrupting agents.

A 5-year prospective multicenter study of early loaded titanium implants with a sandblasted and acid-etched surface

For dental implants to be successful, osseointegration must occur, but it is unknown how much time must pass for osseointegration to be established. Preclinical studies suggested that titanium implants with a sandblasted and acid-etched (SLA) surface were more osteoconductive and allowed more rapid osseointegration than machined or turned implant surfaces. The hypothesis of this study was that implants with an SLA surface could be loaded in half the conventional healing time of machined-surface implants and that, after loading, the implants would be successful for 5 years.