BAP Certificate in Non-Clinical Psychopharmacology


At the beginning of the month, I was lucky enough to attend a residential course held by the British Association for Psychopharacology (BAP) in Cambridge. The training, which was held over four days, provided an overview of many major techniques used in this area of scientific research, as well as advances within the field.

We heard about cutting-edge research, from experts in academia, industry and the heath care sector. Our first lecture started with the basic concepts of genomics, and went on to the difficulties involved in interpreting genome-wide association studies (GWAS), an approach which is sometimes used to identify candidate genes for genetically complex neurological disorders. Another talk covered techniques including optogenetics and designer G-protein-coupled receptors (DREADDs). We went over the advantages of both these methods, which are used to precisely control neural activity, but also touched upon some of the limitations that still exist with these technologies. Other talks covered application of imaging methods and behavioural models.

Lectures were broken up by workshops on statistics and experimental design, as well as a group project. In a workshop focusing on PK/PD calculations, I was introduced to the concept of counter clockwise hysteresis plots. This is when you see two different response levels at a given drug concentration, the result of a delayed effect of the drug at the target. During this session, we spoke about the importance of considering these factors when designing a study as to avoid producing misleading data.

For our group project, we were given the task to form a Drug Discovery leadership team, where we had to choose a drug target for a neurodegenerative disorder, which we deemed to be the strongest candidate. With this target in mind, we put together a plan outlining why it is a worthy target and how we would go about identifying a molecule to take to clinic. Our conclusions were pitched later in the week in a “Dragons’ Den” like situation to see if our case was strong enough to get funding.

As part of the course, we took a trip to the Addenbrook’s hospital where we had the opportunity to take a tour around The Wolfson Brain Imaging Centre. Here we were able to see their clinical and pre-clinical imaging facilities, which included Positron Emission Tomography and Magnetic Resonance Imaging and heard about some of the ongoing research in the department.

After the intensive days, we all had the chance to sit down as a group for dinner and talk to the individuals who had presented throughout the day and had after dinner talks including one from the president of BAP. On the final evening, we headed down to Queens’ College, where we were presented with our certificates in Non-clinical psychopharmacology, which was a perfect way to finish off the course.

As someone who is relatively new to the area of research, I took away a lot from the course. I would definitely recommend this programme and believe that it could be beneficial for individuals at any stage of their career. The programme provided a fantastic platform to network and interact with others from many different area of psychopharmacology. I am excited to attend the BAP summer meeting 2018 to hear more from world leading scientist in both clinical and non-clinical psychopharmacology, and attend the evening disco, which has even been described as legendary!

Blog written by Olivia Simmonds

 

Advertisements

Development of Ultra-Rapid Insulins


The goal of insulin therapy for diabetic patients is to mimic closely the physiologic pattern of insulin release by the pancreas in order to maintain normoglycaemia.

Available as the beef/pig pancreas derived hormone since 1922, the first human recombinant insulin was developed by Genentech and marketed by Eli Lilly in 1982.

Standard 2 Zinc-insulin (which is hexameric) must be injected ~30 minutes before a meal to allow for disassembly in the subcutaneous depot into dimers and monomers (the active species).

At the turn of the millenium, to facilitate more accurate dosing, the principles of protein engineering were applied to destabilize the dimer and hexamer interfaces and produce rapid-acting insulin analogs (Fig. 1). Raj 1

Figure 1. Amino acid compositions of the rapid-acting insulin analogues (1).

Lispro/Humalog (Eli Lilly), Aspart/Novalog (Novo Nordisk) and Glulisine/Apidra (Sanofi-Aventis) can all be injected 5 to 15 minutes before a meal (Fig. 2).

Raj 2

Figure 2. 4 hour physiological plasma insulin profiles plotted together with pharmacokinetic profiles for insulin lispro and human insulin in type 1 diabetes (2).

Attention has more recently focused on the development of ultra-rapid insulins for dosing at (or even after) the time of the meal, for the benefit of children, insulin pump users and for highly insulin-resistant type 2 diabetics. Current approaches to speeding up the onset of absorption include modification of excipients and enabling of tissue diffusion.

Affreza

Powdered insulin delivered by a nebulizer into the lungs is absorbed more rapidly than subcutaneous insulin and absorption is of short duration. Exubera, developed by Inhale Therapeutics (then Nektar) was the first hexameric inhaled insulin product to be marketed by Pfizer in 2006. Unfortunately a 2007 study concluded that Exubera “appears to be as effective, but no better than injected short-acting insulin”. Exubera was dispensed using a bulky device (Fig. 3) with little dosing flexibility and poor sales led to its withdrawal in 2007. More recently, Afrezza, a monomeric inhaled insulin developed by Mannkind was approved by the FDA in 2014. Afrezza is delivered using a small device about the size of an asthma inhaler (Fig. 3), peaks at ~15-20 minutes and is eliminated from the body within ~2-3 hours. Raj 3

Figure 3.

The rapid absorption and decreased duration of Afrezza closely resembles physiological insulin release (Figs. 2 & 4).

Raj 4

 Figure 4. Pharmacokinetic profiles for inhaled Afrezza and SC insulin lispro (in type 1 DM patients) and for inhaled Exubera and SC human insulin (in type 2 DM patients)(4).

Biochaperone Lispro

An alternative to engineering the insulin aggregation interfaces is to introduce biotechnological enhancers. Eli Lilly has complexed Lispro insulin with French biotech company Adocia’s proprietary BioChaperone (BC) to accelerate absorption (licensed in 2014) (Fig. 5).

Raj 5

Figure 5. Adocia’s BioChaperone technology is based on polymers, oligomers and organic compounds. The BC-insulin complex forms spontaneously in water, protecting it from enzymatic degradation and enhancing absorption after injection (5).

BC Lispro promoted a statistically significant 63% increase in metabolic effect over the first hour in comparison with Novolog, having previously been demonstrated to outperform Eli Lilly’s Humalog (Fig. 6).

Raj 6

Figure 6. Comparison of mean blood glucose profiles after subcutaneous injection of lispro and BC lispro (6).

Despite results from 6 clinical studies indicating that BC Lispro performs better than Humalog, Eli Lilly decided to terminate its collaboration with Adocia in 2017 (possibly because of the costly failure of its Alzheimer’s drug solanezumab). Lilly is now developing its own ultra-rapid Lispro in house (LY900014 currently in phase 3), formulated with two new excipients, treprostinil (a vasodilator) and citrate (a vascular permeabilizer). The rights to BC Lispro reverted back to Adocia from Eli Lilly at no cost and the company is currently seeking a new partner to shoulder the costs of phase 3 clinical trials, regulatory and marketing hurdles.

Fiasp

Novo’s Faster-acting insulin aspart (FIASP), the first ultra-rapid insulin to be approved and marketed (Fig. 7), is an innovative formulation containing Vitamin B3 (niacinamide) to increase the speed of absorption, and the naturally occurring amino acid (L-Arginine) for stability.

FIASP was sidelined by the FDA in October 2016 but approved in September 2017 following clarification of immunogenicity and clinical pharmacology data.

Raj 7

Figure 7. Fiasp FlexTouch Prefilled Pens -100 units/mL (7).

 FIASP can be injected from 2 minutes before to up to 20 minutes into a meal and acts twice as fast as Novolog/Aspart (Fig. 8).

rAJ 8

Figure 8. Blood insulin Aspart concentration after subcutaneous injection of Fiasp and Novolog in patients with type 1 diabetes (8).

This was achieved without a significant difference in the overall rate of severe or confirmed hypoglycemia. Clinical trial data showed that FIASP gave a lower post-meal spike and that patients also lowered their A1C levels.

Novo Nordisk is very keen to expand the use of ultra-rapid acting FIASP in an artificial pancreas setting and it is already approved for use in insulin pumps in Europe.

Given that Novolog was the third best selling diabetes medication in 2015 with $3.03 billion in global sales (9), Fiasp is well poised to become the leader in this enormous market segment.

Raj 9

http://www.doctablet.com

References

  1. https://www.diapedia.org/management/8104096115/short-acting-insulin-analogues
  2. Home, P.D. (2015) Plasma insulin profiles after subcutaneous injection: how close can we get to physiology in people with diabetes? Diabet. Obes. Metab. 17, 1011-1020.
  3. http://pharmamkting.blogspot.co.uk/2015/02/look-ma-no-bong-afrezza-inhaled-insulin.htmlAl-Tabakha, M.M. (2015) Future prospect of insulin inhalation for diabetic patients: The case of Afrezza versus Exubera. J. Control. Release 215, 25-38.
  4. https://www.adocia.com/technology/biochaperone-technology-2/
  5. http://www.diyabetimben.com/ultra-hizli-etkili-insulin-biochaperone-lispro/
  6. https://online.pharmacy/product/fiasp-insulin-3/
  7. https://www.fiasppro.com/the-fiasp-story/onset-of-appearance.html
  8. pharmaceutical-technology.com

Blog written by Raj Gill

Reducing attrition in drug discovery: the AstraZeneca 5R-framework


The high attrition in drug discovery is responsible for the extremely high cost of developing a new medicine. Some suggestions aimed at reducing the attrition have been put forward such as: improving efficacy and safety profiles, reducing toxicity, improving preclinical models, better understanding of mechanism (Nat Rev Drug Discov. 2004, 3, 711-716) and shifting the attrition to earlier phases (Nat. Rev. Drug Discov. 2010, 9, 203–214).

Based on this, scientists at AstraZeneca have established a five-dimensional framework (5R framework) (Nat. Rev. Drug Discov. in 2014) aimed at improving the low success rates in the process. The framework includes five determinants: right target, right tissue, right safety, right patient and right commercial potential, identified as key features in the drug discovery process (summary depicted in Figure 1).

Marco 1

Figure 1. The 5R framework (from Nat. Rev. Drug Discov. 2018).

In addition to the “5R framework”, AstraZeneca scientists decided to reduce their diseases portfolio and to potentiate their capabilities for target selection and validation (better understanding of biology and mechanism of the disease, stronger target rational).

Furthermore they improved their lead generation strategy (expansion of their compounds library including library sharing, and integration with other screening approaches), and their pharmacokinetic/pharmacodynamics modelling, patient stratification and biomarkers. In a more recent paper published in Nat. Rev. Drug Discov. in 2018, they show how the application of these guidelines have led to an increase in project success rates (Fig. 2) and to a reduction of cycle time and cost (Fig. 3).

Marco 2

Figure 2. Project success rates for the AstraZeneca (AZ) portfolio.

The overall project success rates have increased from 4% (2005-2010) to 19% (2012-2016); the cost to reach clinical proof of concept has decreased by 31% when comparing the two time cohorts, and by 42% compared to the industry average.

Marco 3

 

Figure 3. Metrics for projects costs (a. first good laboratory practice dose; b. clinical proof of concept) and cycle times.

With regard to cycle times, they observed a considerable reduction of the length for phase II (50% shorter than industry average) although this may return in line to the average in the future.

To further support how the introduction of the 5R framework has influenced AstraZeneca pipelines, Table 1 reports the new molecular entities and new biologics that entered phase III in 2012-2016, highlighting how their progression was influenced by these guidelines. The 5R framework can be used at any stage of the process, as it has been done for Olaparib (selected as drug candidate before 2011) resulting in the initiation of novel clinical studies. (Readers are encourage to check Box 1 in the paper for an example of the 5R framework applied to Osimertinib).

Marco 4

Table 1. Influence of 5R framework for 15 projects new molecular entities and new biologics entering phase III.

Although these guidelines have clearly improved productivity, 81% of the projects still failed at some stage of the process, but it is clear they are moving the process in the right direction and it will be interesting to see how these guidelines may affect the R&D strategy of other companies.

Blog written by Marco Derudas

Serine Racemase Showdown: Clash of the Crystal Structures


Having been sitting on an X-ray crystal structure and half-finished manuscript for quite some time now, imagine my chagrin when during a casual perusal of the PDB, I found an extremely similar structure had just been deposited to the one I was aiming to publish.

The deposited structure (5X2L) is for wild-type human serine racemase (SR) and the associated paper focuses on in silico screening and medicinal chemistry, while my structure contains two cysteine-to-aspartate point mutations (C2D, C6D) with a (speculative) paper that explores the crystallography and biophysical side. As well as briefly summarising the findings of the study, I thought it would be fun to compare our two crystal structures in what I have dubbed…

Clash of the Crystals

Chloe 1

But firstly, some background on the contestant/s: 5X2L (published) vs. CRK1 (mine, unpublished).

In the blue corner…(and the red corner)…weighing in at 37.4 kDa, we have a pyridoxial-5’-phosphate (PLP)-dependent enzyme that catalyses the racemisation of L-serine to D-serine, as well as the α,β-elimination of water from L-serine. This forebrain-localised enzyme produces about 90% of D-serine in the brain, and because D-serine is a co-agonist of N-methyl-D-aspartate glutamate receptors (NMDARs), SR inhibition has been touted as an up-and-coming approach to indirectly modulate NMDAR activity. This is a potential game-changer for treating disorders underpinned by NMDAR overactivation, such as neuropathic pain, neurodegenerative disorders, and epileptic states.

So let’s hear it for…serine racemase! [Thunderous applause]

 Round 1: Paper summary

Anyone well versed in SR literature will know the paper in question, ‘Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase’ by Takahara et al. (2018)1 is an additional chapter to an ongoing story. Several groups have previously tried to identify new SR inhibitors that are potent, selective, and structurally distinct from the countless amino acid analogue inhibitors that are already well-described2, and for many this has proved to be a challenging endeavour.

The status quo shifted when a series of dipeptide-like inhibitors with a clear structural motif and slow-binding kinetics was identified by Dixon et al. (2006)3, which later provided the query molecule for an in silico screen performed by the same group behind 5X2L4. The resulting inhibitors contained an essential central amide structure with a phenoxy substituent, and substitution of parts of the structure for heavier halogen atoms such as bromine and iodine produced derivatives with improved inhibitory activity (comparable to classical SR inhibitors), binding affinity, and ligand efficiency. The Mori group took their explorations even further by testing the most potent derivative in vivo to demonstrate the SR inhibitor suppressed neuronal activity-dependent Arc expression to regulate NMDAR overactivation5.

The current paper expands on these studies by firstly, solving the crystal structure of wild-type SR for molecular docking and in silico screening; secondly, using these methods to identify new SR inhibitors related to their previously described peptide compounds; and thirdly, testing these inhibitors in an in vitro assay. The team synthesised 15 derivatives, of which one showed relatively high inhibitory activity, making a nice addition to their growing rolodex of peptide SR inhibitors.
Chloe 2

Figure 1. Structure and binding pocket of the novel peptide SR inhibitor derivative identified by Takahara et al.

Round 2: Clash of the Crystals!

Both contestants were crystallised using the sitting-drop vapour diffusion method in very similar experimental conditions (Table 1). Both structures were determined to a highly respectable resolution of 1.8 Å, and organised into a large domain and small domain connected by a flexible loop region (Fig. 2). The PLP cofactor (Fig. 2; yellow sticks), on which SR is dependent for its catalytic activity, can be seen covalently linked to Lys56.

Table 1. Summary of key features of 5X2L and CRK1.

Feature 5X2L CRK1
Crystallisation conditions 10% PEG 8000, 5 mM MgCl2, 0.1 M Bis-Tris pH 6.0, 10% ethylene glycol, 20 °C 15% PEG 3350, 250 mM MgCl2, 0.1 M Bis-Tris pH 6.5, 20 °C
Resolution 1.8 Å 1.8 Å
Space group P212121 P21
a, b, c (Å)

α, β, γ (°)

80.1   112.6   88.0

90.0   90.0   90.0

69.0   53.8  79.4

90.0   106.1   90.0

Crystal system Orthorhombic Monoclinic
No. residues resolved 305/340 321/340
Ligands PLP, Mg2+ PLP, Mg2+

 

SR belongs to the fold-type II family of PLP-dependent enzymes, meaning it contains a β-sheet core surrounded by α-helices, with the active site located in a cleft between the two domains. Accordingly, both domains of 5X2L and CRK1 contain a parallel-stranded β-sheet surrounded by nine α-helices in the large domain and three in the small domain. A magnesium ion (pink sphere) that helps to stabilise protein folding and increase maximal activity6 is octahedrally coordinated by three water molecules, the acid groups of Glu210 and Asp216, and the carbonyl oxygen of Ala214.

Chloe 3

Figure 2. Overall X-ray crystal structure of the human SR holoenzyme CRK1. Residues are coloured from red to violet, N-terminus to C-terminus, and all helices are numbered 1–12 based on the order they occur in the polypeptide sequence. Each SR monomer comprises a large domain (helices 1–3 and 7–12) and small domain (helices 4–6) connected by a flexible loop region.

 

CRK1 boasts good ordering of residues, with only a few not well-defined: 1–3, 132–135, and 339–340. Aside from those at the C- and N-terminus, which are often poorly resolved during structure solution anyway, the only other undefined residues (132–135) were localised to the top of helix 5 in the highly-mobile small domain. 5X2L shows similarly undefined residues at the termini (1–2, 318–340) although in addition it is also missing residues of the flexible loop region (67–76) that connects the two domains.

Solvent-exposed loops are notorious for being tricky to model due to their high occupancy. The loop may be visible in CRK1 but not 5X2L because CRK1 has the help of its (symmetry) mates. By viewing the symmetry partners in the crystal lattice, the loop residues 69–73 are seen to be stabilised by a water-mediated interaction between Leu72 in one monomer and Lys221 in an adjacent monomer.

These favourable contacts may not occur for both structures because 5X2L crystallised in the orthorhombic space group P212121 while CRK1 crystallised in the monoclinic space group P21. Both possess 2-fold symmetry, but differences in molecular packing would have influenced whether the loop region would be suitably positioned to receive stabilising crystal lattice contacts.

Round 3: Best [Super]Pose!

A superposition of 5X2L and CRK1 (Fig. 3) revealed that, unsurprisingly, the two structures were well aligned with a Cα RMSD of 0.55 Å. Any remaining conformational differences are likely to result from the unresolved loop region, the missing helix and polypeptide strand that make up residues 318–340, and random structural variations. So hardly a ‘clash’ but at least it makes for a nice picture.

Chloe 4

Figure 3. Superposition of the X-ray crystal structures of 5X2L (blue) and CRK1 (red).

By now there is no doubt you are wondering who the champion is of the undeniably riveting Clash of the Crystals.

The answer is both, and neither, because any discovery that contributes to scientific advancement is a champion in my book J

Yes, even when said discovery beats me to the punch.

Blog written by Chloe Koulouris

References

  1. Takahara S, Nakagawa K, Uchiyama T, Yoshida T, Matsumoto K, Kawasumi Y et al. Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase. Bioorg Med Chem Lett. 2017 Dec 13.
  2. Jirásková-Vaníčková J, Ettrich R, Vorlová B, Hoffman HE, Lepšík M, Jansa P et al. Inhibition of human serine racemase, an emerging target for medicinal chemistry. Curr Drug Targets. 2011 Jun; 12(7):1037-55.
  3. Dixon SM, Li P, Liu R, Wolosker H, Lam KS, Kurth MJ et al. Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries. J Med Chem. 2006 Apr; 49(8):2388-97.
  4. Mori H, Wada R, Li J, Ishimoto T, Mizuguchi M, Obita T et al. In silico and pharmacological screenings identify novel serine racemase inhibitors. Bioorg Med Chem Lett. 2014 Aug; 24(16):3732-5.
  5. Mori H, Wada R, Takahara S, Horino Y, Izumi H, Ishimoto T et al. A novel serine racemase inhibitor suppresses neuronal over-activation in vivo. Bioorg Med Chem. 2017 Jul 15; 25(14):3736-45.
  6. De Miranda J, Panizzutti R, Foltyn VN, Wolosker H. Cofactors of serine racemase that physiologically stimulate the synthesis of the n-methyl-d-aspartate (nmda) receptor coagonist d-serine. Proc Natl Acad Sci U S A. 2002 Oct; 99(22):14542-7.