Synthesis of BACE1 inhibitors against Alzheimer's disease

Alzheimer’s disease (AD) is a fast growing disease with a high socioeconomic impact in industrial countries making it urgent to find a drug capable of blocking or delaying the disease progression.
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Focusing on the mechanistic cause, the amyloid cascade is a well known process for the formation of toxic oligomers of amyloid-beta (Abeta42) peptide that deposit in amyloid plaques, an AD characteristic. Beta-secretase (BACE-1) is one of two enzymes in this cascade: beta-secretase and gamma-secretase, needed for the production of Abeta42 peptide and its inhibition has become attractive as a therapeutic target.
BACE-1 [Hong et al., 2000]
Unfortunately, the treatment of AD through brain inhibition of BACE-1 is a very hard task, mainly due to the limitation of the inhibitors to cross the blood-brain barrier (BBB).
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Following this, we are focused on overcoming the BBB low permeability of BACE-1 inhibitors in order to turn them effective drugs against AD. The strategy to accomplish this consists on obtaining peptidomimetic BACE-1 inhibitors containing a targeting sequence for a BBB receptor that will promote its transcytosis across the BBB. This is a clearly breakthrough approach, contrasting with the current approach that consists on reducing the size and peptidic character of the inhibitors, which are necessary conditions to allow passive diffusion, but that till now showed inefficiency to cross the BBB.


The results of our work will provide the basis for future development of a truly AD modifying drug.

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Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.; Zhang, X. C.; Tang, J., Structure of the protease domain of memapsin 2 (β-secretase) complexed with inhibitor. Science 2000, 290, (5489), 150-153.
In vitro models for prediction of drug absorption and metabolism
“Over the past years a shift has been observed towards the regulatory acceptance of scientifically valid in vitro methods as well as formally validated in vitro methods as part of an integrated testing strategy. Moreover, focus has broadened to the application of all 3 R’s, replacement, reduction and refinement, whilst historically much emphasis has been placed only on replacement of animal studies by one or more in vitro or in silico approaches. “
17 March 2011, EMA/CHMP/SWP/169839/2011, Concept paper on the Need for Revision of the Position on the Replacement of Animal Studies by in vitro Models (CPMP/SWP/728/95))

In vitro models are now routinely used by academia and industry for prediction of drug absorption and metabolism, but increased attention is being devoted to the usage of these models in the simultaneous evaluation of metabolism and bioavailability.

In vitro assays are more prone to automation and consequently high throughput screening, but also allow reduction of the use of animals in preliminary tests of drug development. These are in fact the stages during drug research and development where usually most compounds are discarded. Being usually less complex systems, in comparison with animal models, they are easier to interpret and correlate with in vivo observations. Additionally, they allow the usage of human cells, minimising the inter-species variation that may pose severe constraints to the in vitro-in vivo correlations.

Among the in vitro systems used for the study of drug intestinal absorption, there are two models which are widely used by the industry: Caco-2 and PAMPA (parallel artificial membrane permeability assay).
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Caco-2
PAMPA
In the Caco-2 model, permeability is tested across a differentiated monolayer of cells of the human colon adenocarcinoma, while in the PAMPA method an artificial phospholipidic membrane is used with the same purpose.
Caco-2 and the PAMPA models are routinely used in our laboratory.
They have been applied on the evaluation of the permeability of different compounds such as: flavonoids, prodrugs designed for colon delivery and some natural products, such as wine extracts or dietary supplements. We are now also using the Caco-2 model for evaluating the pathogenic potential of some environmental bacteria in a project we share with the Microbiology of Man-made Environments group.
Caco-2 model provides significant information in terms of active transport and efflux prediction. Nonetheless, this cell line fails to express some relevant enzymes at the same relative level that they can be encountered in the intestine such as CYP3A4 and other phase I metabolising enzymes.
In our group we are willing to contribute to the development of more physiologically relevant models, thus we are putting significant effort in the refinement strategy and therefore we are now focused in developing an improved Caco-2 model which will more closely resemble human enterocytes. This work is done in collaboration with the Animal Cell Technology Unit.
Although specially interested in in vitro models mimicking human intestinal processes, we have also experience in the usage of hepatic models, such as the Hep-G2 cells for predicting drug metabolism and citotoxicity in the liver.
Protein production and characterization
Protein production and protein characterization are other fields that can greatly benefit from new in-house analytical development.

Our work in cooperation with Animal Cell Technology UNIT, iBET’s Pilot Plant and ITQB Crystallography groups are examples of the mutual benefits of joining multiple disciplinary fields. Together we have recently produced a recombinant human enzyme and through its characterization we have contributed:
- To increase the fundamental knowledge of the enzyme, in terms of its stability and activity, potentiating the success of its structure determination through crystallographic studies;
- To increase the relevance of in vitro models in comparison to what happens in vivo, meaning, in the organism. The rationale behind it is that, through a better understanding of the parts, a more relevant interpretation and applicability of the system may be achieved;
- To increase the awareness of species differences.
Analytical development
In the field of analytical development we are particularly interested in the distinction and quantification of specific enzymes in complex biological samples such as cellular extracts and animal sera.
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Our expertise in Capillary Electrophoresis ranges from off-line to in-capillary reactions.
Electro-mediated micro analysis has been frequently used to follow enzymatic reactions; however, correlation with off-line methodologies is difficult because the fluid dynamics inside the capillary is not thoroughly characterized.
We have previously successfully engaged in the evaluation of in-capillary reactions for the study of enzyme kinetics, taking into consideration stacking and/or diffusion effects.
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Currently, we are focused in increasing the physiological relevance of the analytical assays. Our goal is to develop improved tools allowing detecting and quantifying specific enzymes in their naturally occurring environment, such as the intracellular milieu.
We have also been contributing, with new analytical methods, to the characterization of potentially new biological drugs such as rotavirus like particles.
Drug-drug interactions
The issue of drug-drug interactions has generated significant concern within the pharmaceutical industry and among US and European regulatory authorities in recent years.


The co-administration of different drugs or dietary supplements can affect the therapeutic outcome of a drug. These interactions may include alterations in the pharmacokinetics of the drug, such as alterations in the absorption, distribution, metabolism and excretion (ADME) of the drug. Alternatively, the interactions can also result in alterations in the pharmacodynamic properties of the drug, e.g. the co-administration of a receptor antagonist and an agonist for the same receptor.
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Many drug interactions are due to alterations in drug metabolism, by enzime inhibition/induction. In the same way, drug interactions can also affect the absorption and consequently the bioavailabilty of a drug, by inhibition/induction of transporter proteins, such as the p-glycoprotein.

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In our group, we are currently studying the effect that some flavonoid formulations can have in the metabolism of drugs wich are substrates for esterases. We are also studying their effect on the bioavailability of the same drugs and for that purpose we are using the Caco-2 cell model, rat hepatocytes and pure enzymes.
Physicochemical characterization of small drug molecules by capillary electrophoresis
The physicochemical properties of pharmaceuticals such as acid dissociation constant (pKa), octanol-water partition coefficiente (log Pow), solubility or protein binding constant are very important in drug design, candidate selection, drug delivery and pharmacokinetic studies.
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Capillary electrophoresis (CE) is a simple, versatile, automated, and powerful separation technique and widely applied in physicochemical profiling for pharmaceuticals. It has advantages over other methodologies, such as high separation efficiency, short analysis time and low sample and electrolyte (buffer) consumption.

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In our laboratory capillary electrophoresis is routinely used for the establishment of physicochemical properties of pharmaceuticals and have been applied on the determination of the ionization profiles of flavonoids, amines prodrugs and others small drug molecules.
PHARMACOKINETICS AND BIOPHARMACEUTICAL ANALYSIS