Drugs of Abuse - Amphetamines, Ecstasy and Cathinones.

BACKGROUND

People have been using psychoactive drugs for pleasure for thousands of years. From the Areca nut used as a mild stimulant in Timor over 13,000 years ago, to the coca leave cultivated in South America 5,000 years ago it seems the human brain craves stimulation, and consequently, new and interesting stimulants. Modern day humans are no exception. We're all used to hearing news stories about heroin, cocaine, ecstasy and LSD, but lately there has been an explosion in both the number and types of new drugs available.

WHAT'S THE PROBLEM?

Regulatory authorities are struggling to keep up with the number of new "illegal" drugs on the market. I say "illegal" simply because it's not clear how these compounds should be classified. The complexity in the law governing the use of drugs arises from the subtleties of the chemistry at the atom level. For instance, for any given compound, legal or not, a difference of a single atom anywhere in the structure results in a compound which is completely unique. There may not be anything known about this novel compound, and consequently, there is no legislation governing it's use.

For example, lets examine the chemical composition of two drugs, one legal decongestant called pseudoephedrine, and one illegal drug called methamphetamine, (crystal meth). Pseudoephedrine is available from pharmacies as an over the counter medication. It's the active ingredient in Sudafed for example. Structurally, pseudoephedrine looks very similar to methamphetamine, see the picture below. In fact there are just two atoms in the difference.

If nothing was known about pseudoephedrine a toxicologist looking at it for the first time might expect it to have a similar effect to methamphetamine, that is, stimulatory, promoting alertness, and increasing reaction time. But it's very difficult to judge the effect of those two extra atoms. The additional (-OH) present on pseudoephedrine could make it more or less potent as a stimulant. Perhaps it's metabolised more quickly by the body? Perhaps it's more soluble in the blood now? Perhaps it has a harder time getting into the brain? A good toxicologist can make good predictions, but they are just that. Until detailed studies are performed it's really not known how a particular drug will behave.

This is the root of the difficultly for legislators. If there was a decision to make pseudoephedrine illegal this might go some way to curbing it's use. However, a single atom change to it's structure results in an entirely new compound. It's perfectly possible that nothing is known about the physiological effects of this compound, beyond it's ability to produce a chemical high. This is exactly what happened with ecstasy (MDMA) production recently. One of the precursor ingredients for MDMA was made illegal. The idea was to make it more difficult to manufacture MDMA and ultimately make people safer.

However, far from making people safer this change in legislation resulted in the rise of a new ecstasy like compound called PMMA. Both MDMA and PMMA have similar effects, but the effects of PMMA come on much more slowly. Thinking the drugs weren't working, or that they had taken a really low dose, regular MDMA users would take more and more PMMA waiting for the effects to kick in. Needless to say, when the effects did kick in things got serious. Very quickly after the emergence of PMMA there were media reports of overdoses across Ireland and the UK. Sadly, this is a stark example of legislation that was introduced to prevent harm actually causing more harm than good.

Fuck it, lets just make everything legal!

MEDIA ATTENTION ON DRUGS

2014 saw the introduction of over 100 new synthetic drugs of abuse in Europe alone. With such a wide array of new street drugs it's no longer surprising to encounter a news story including the name of a drugs most of us have never heard before. For example, this was a recent headline in America. 

The drug being referred to here is called alpha-pyrrolidinopentiophenone, or alpha-PVP for short, street name Flakka, or Gravel. But what is this drug, and where did it come from?

ALPHA-PVP

Alph-PVP is a synthetic stimulant which belongs to a class of drugs called cathinones. Cathinone itself is naturally occurring, being found in the plant called Khat. The leaves of this plant can be chewed to produce mild stimulation. The structure of cathinone is shown below, along with the khat plant it's found in. Interestingly you can see it looks a little bit like methamphetamine, so it's not too surprising to learn that this drug is a stimulant. It also induces paranoid delusions, with users reportedly fearing for their lives after hallucinating gangs of people chasing them down!

The chemical structure of cathinone (left) and the plant which makes it Catha edulis, (right).

At this point it's important to point out here that whether or not a compound is synthetic has no bearing on its safety or toxicity. There are many synthetic compounds which are safe, and many natural compounds that are dangerous. In the case of PVP, the naturally occurring cathinone molecule was modified by a chemist to produce a novel synthetic compound. This is the case with all synthetic cathinones which include mephedrone (M-CAT, Meow, Meow), and MDPV, (Bath Salts), both of which get sporadic media attention as a result of fatal or near fatal overdoses. The structures of these compounds are shown below. If you know a little chemistry you can see they look a little like the structure of cathinone, shown above. Variants of cathinone are continously made to circumvent the laws which ban them. Thus, it is often not illegal to manufacture or sell these compounds.

You could argue that all that's required for legislators to get a handle on this is an outright ban of all cathinone compounds. The problem with that idea is that some cathinones are actually medicinal, and are used as antidepressants for example. Making all cathinones illegal immediately makes research into these drugs more difficult. Any research laboratory wanting to investigate these compounds would now need to apply for a licence to have them on the premises. This is a bureaucratic nightmare, enough to put off many research scientists who simply don't have the knowledge or the resources to work in such a regulated environment. Regardless of whether or not you think this is a poor attitude for a scientist to take, the reality is more barriers to research means less research is done.

Those using synthetic cathinones recreationally can be often experience far more intense "highs" than expected. Part of the problem with illegal or unregulated drug manufacture is that the contents are not tightly controlled, so the potency between batches is extremely variable. By contrast, any drug manufactured legally by a pharmaceutical company must undergo a multitude of quality control (QC) checks to ensure purity before being distributed.

SUMMARY

New drugs are being manufactured all the time. Legislation designed to protect the population from the risks and harms of drug use have actually compounded the problem resulting in deaths from the distribution of PMMA marketed as Ecstasy (MDMA). At the same time, there is increase in the number of synthetic cathinones, producing powerful psychoactive stimulants. Almost nothing is known about the physiological effects and safety of these stimulants. By contrast, the physiological effects of compounds such as amphetamine, methamphetamine and cathinone itself, are known and documented in detail. I hope this segment has given some insight into the science behind the news headlines, and the difficulties faced by regulators and scientists working in this area.

As always, comments are welcome. 

USEFUL INFORMATION

• Database on new psychoactive drugs in Europe
• Guardian Science Podcast on PMMA
• FOX news article on Alpha-PVP
• Information on Synthetic Cathinones.
• Irish Times News Story on PMMA Deaths
• Review of Flakka

Universal Blood Cells.

BACKGROUND

Some time ago, while making my way home from college, I was listening to an episode of a podcast called Science Friday. It was dark and cold outside and I was keen to get home, eat and be warm, so it's safe to say my mind was not on the material at hand. Even so, what I heard blew my mind, it seemed too simple to be true, and yet it made perfect sense. I was immediately annoyed I hadn't come up with the idea myself. The idea? Universal blood cells. I'm going to keep you in suspense as to what that means while I explain more about blood in general.

BLOOD

Blood is composed of red blood cells (also referred to as RBCs, or erythrocytes), white blood cells (also referred to as WBCs, or leukocytes), and platelets. This varied composition allows it to perform a myriad of essential functions. For example, RBCs are responsible for the transport of oxygen, (O₂), from the lungs to every other organ and cell in the body. In addition, they also exchange this oxygen for a waste product of glucose metabolism, carbon dioxide (CO₂), and return it to the lungs to be expelled. Therefore there is a constant exchange of CO₂ for O₂ going on in the lungs, with blood being continually circulated around the body to remove/supply both. WBCs are a much more complicated group. There are five different subtypes of WBCs, but together they form part of our immune system, allowing us to fend of biological attacks from viruses and bacteria. Finally, plateletes are responsible for some housekeeping activities such as blood clotting, maintenance of blood vessel lining, and digestion of harmful bacteria.

The composition of blood, image taken at http://www.myvmc.com/anatomy/blood-function-and-composition/

In addition to all of the above functions blood also has the added function of regulating pH, regulating body temperature, and generally acting as a delivery mechanism throughout the body for the supply of nutrients such as glucose, amino acids, lipids, vitamins and salts. So, blood is important, and it's able to perform all of these functions because of the complex composition of RBCs, WBCs, and platelets. 

CELL BIOLOGY 101

Cells are the basic unit of life, by which I mean all living things are composed of either a single cell, or many cells, termed unicellular or multicellular respectively. But cells themselves are incredibly complex, and in the same way that we have specific organs performing specific tasks for us, our cells have tiny structures inside them called organelles performing specific tasks for them. This could be the production of protein, or conversion of sugars into cellular energy. But the complexity of a cell continues to the outside. All animal cells have a fatty cell membrane called a phospholipid bilayer separating it from the outside world. This lipid bilayer is protective, but also functional. It's scattered with protein and sugar molecules that allow it to regulate transport in/out of the cell, and generally communicate with other cells around it. Sugar and proteins sometimes combine to form what is called a glycoproteins, and these are important molecules.

BLOOD GROUPS

Most people will have heard the term blood group. But what does it actually mean? Well, there are four different blood groups called group A, B, AB, and O. It turns out that the glycoprotein surface is what determines your blood group. There are many different types of sugars, and they have names like glucose, fructose, and sucrose. In the image below "GAL", "GAL-Nac" and "FUC" are sugars called galactose, N-acetylgalatosamine and Fucose respectively. You can see how all three blood groups share the same GAL-GALNAc-GAL backbone, but differ in their terminal regions. For example, blood group A has an additional Gal-Nac sugar molecule compared to blood group O, while blood group B has an additional GAL sugar molecule compared to blood group O. Blood group A is further subdivided with blood group A₁ and A₂ being the most common.

Schematic of the A, B, and O blood groups. The red oval represents the red blood cell. The coloured hexagons represent the different sugars that attach to the blood cells to make its blood group.

MEDICAL RELEVANCE

Blood groups are extremely important for blood transfusions. The blood groups between donor and recipient must match otherwise the body will reject it. All blood groups can donate to themselves, so group A is compatible with A etc. However, blood group A is not compatible with blood group B, and the reverse is also true, group B is not compatible with group A. Group AB cannot be given to group A, or B. Group O however can be given to every blood type, which makes it extremely valuable. The compatibility of different blood groups can be summarised in the diagram below. Red arrows indicate blood groups which cannot be exchanged.

The compatibility of each of the blood groups, red arrows indicate blood groups which cannot be exchanged.

Invasive surgery requires blood transfusion while the operation is being performed, so if there's no time to test the blood type of your patient then you know it's safe transfuse blood O without resulting in any additional complications. Transfusing the wrong blood will lead to activation of the immune system, destruction of the new blood cells which ultimately can result in death.

UNIVERSAL BLOOD CELLS

The idea I ended up stumbling across that night while listening to my podcast was to generate blood group O cells from bags of group A, B, and AB blood. That is, the ability to take any blood type and modify it so that it's capable of being transfused without any immune response in the patient. This idea, if feasible, would make all of the current blood stocks capable of being donated to any patient! That's pretty amazing!

HOW DOES IT WORK?

The basic science behind this is involves the application of a well established biochemical technique called enzymatic hydrolysis. Enzymes are protein molecules which have the ability to add or remove chemical components to specific molecules, I've mentioned them before in a previous blogpost so I'll skip over a detailed explanation  here. There are pre-existing enzymes in nature which will selectively remove the N-acetylgalatosamine and Frucose sugars from blood cells. Interestingly, the first attempts were with an enzyme found in coffee beans, but subsequent attempts using different versions of the same enzyme resulted in much better efficiency. The image below shows how these sugars actually look and demonstrates how the conversion of group A or B to group O involves the removal of just one molecule of sugar. The sugar removed in each case is highlighted by a coloured star.

Conversion of blood groups A and B to group O. Image adapted and modified from the original research paper on this topic, "Bacterial Glycosidases for the production of universal blood cells" 

There are two enzymes used for this, once called α-N-acetylgalactosaminidase for removing the sugars associated with blood group A, and a second called α-galactosidase for removal of sugars associated with blood group B. The actions of enzymes result in blood group O cells. The structure of α-N-acetylgalactosaminidase enzyme bound to a single sugar molecule is shown below. On the left is an image of the whole enzyme/sugar complex (shown in grey, red and blue), with the sugar shown in purple. The image on the right is the same enzyme/sugar complex zoomed in to show the the complex in more detail. Enzyme often have small clefts or holes in their structure into which their substrates fit. All the chemistry associated with the removal of this sugar molecule happens in and around this small cleft area, called the active site.

The α-N-acetylgalactosaminidase enzyme, bound to a sugar residue. Image made using YASARA, and PDB ID 2IXA.

The α-N-acetylgalactosaminidase enzyme, bound to a sugar residue. Image made using YASARA, and PDB ID 2IXA. 

WHY THIS IDEA IS COOL

Biological material like blood is sensitive to large changes in pH, or temperature. In addition, it's important to keep it free from any external contamination. Biochemists are familiar with this so we add reagents to control the pH, we keep solutions cold, because it helps preserve the integrity, and slow bacterial degradation of the components and we often work in sterile conditions to prevent any contamination. This is time consuming and expensive. But one of the cool things about the procedure used to make this blood is that it is relatively easy. There are no difficult or expensive conditions required. The enzymes required were simply added to a 200mL volume of blood which had been washed with buffer solution, the solution was then mixed gently for 1 hour at room temperature, after this the blood was washed with a salty solution to remove the enzymes. In addition, even though there are different enzymes responsible for converting blood groups A and B it was possible to simply add both enzymes to a single unit of AB blood and let both of them operate together to produce a unit of blood group O.

THE DISADVANTAGES

There are some disadvantages with the enzymatic hydrolysis of blood cells. The research group responsible for demonstrating this modification of blood cells has shown it works on a small scale, but for this to be really useful it would need to be able to convert thousands of units of blood every week. Such large scale conversion would require large quantities of the enzymes used. Specifically, for the conversion of 1 unit of blood group A1 to blood group O the researchers mention they used 60mg of enzyme. Recently the Irish Blood Transfusion Service (IBTS) advertised they needed 1,500 units of group O blood each week. To convert 1,500 units of blood would therefore require 90,000mg (90g) of enzyme every week.

It's a little difficult to put this in perspective, but I used to make enzymes as part of my Ph.D work. This is done via the genetic modification of microorganisms, bacteria or yeast cells for example, which are then grown in what is called a bioreactor, or fermentation vessel. Typical yields of protein are pretty low, in the order of 100mg/L of cell culture material. So the ability to make 90g of enzyme every week is demanding, and expensive, but by no means impossible. Biotechnology companies are used to this problem, and utilise large scale bioreactors, in the order of 20,000L, to mass produce therapeutic proteins for other medical reasons. This is currently being done for diseases like diabetes, where insulin is required in large quantities for worldwide supply, so there's no technological reason why this could not be done for these enzymes.

SUMMARY

Blood donations are always needed, and in particular blood group O is in high demand because it is accepted by any blood group. Supply is always going to be restricted due to legitimate medical reasons such as disease, so anything that can be done to ease the strain on supply is definitely worth considering. The research presented here is simple, but clever, and uses well established biochemistry techniques. It might not be feasible to produce quantities of enzyme that are required, but there's a few more tricks that biochemists can perform the make it better. We can modify the protein using genetic engineering, to make it behave more efficiently, removing the sugar molecules more quickly. We can also try different ways to manufacture the enzyme which might improve the quantity we obtain. In short, this is a fantastic idea, and worth pursuing further, and I'm still annoyed I didn't think of it first!