The Half Decent Pharmaceutical Chemistry Blog

In a city where nearly everything revolves around the university, this is a period of the year when it could be really hard to find people to hang out in a pub with for a couple of refreshing pints. Friday night before Easter is, apparently, a lonely one when your so-called best friend is catholic (much more than what you ever thought).
To sum up, yesterday I faced the dilemma of whether to spend the night staring at a computer or to go out on my own. Actually, there was no dilemma at all, as I have always had wonderful times at pub counters. Especially when rough and full of depressed members of the lower-classes.

While sitting at the centre of one of these naturalistic paintings, unwinding in the dim light and finally reconsidering all the things that had happened to me in the last ten days, I began to reconsider an idea one of my colleagues at the chemist’s gave: the World’s Biggest Rectal Suppository ever made! This really could have been an ambitious but gratifying target for a pharmacy and, because we were in a small town, something the locals could have been proud of for the years to come, a tale grandparents would bring out to entertain children.
And, who knows, an attraction for tourists: Visit the land of the Massive Suppository!

I know suppositories are a laughing stuff, but to make them is far from being trivial: there is actually much more science involved than you might expect. It is so complicated that, despite the cheap ingredients, rectal suppositories are being quickly dismissed as means of administration. Compliance is another issue, but not so fast on that front. Just take oral administration, which has rapidly taken over old rectal formulations because of superior compliance: despite the unquestionable user-friendliness of introducing the drug through the right orifice, you can’t think about administering anything through the oral route to an unconscious person, while you could rely on rectal suppositories. What’s more, even in less dire conditions, nausea might make ingestion of oral tablets impossible: therefore, a suppository is just what you need.
When embarking on an ambitious project such as crafting an immense rectal suppository, compliance is obviously not an issue. What could be annoying, on the other hand, is the fragile equilibriums taking place in this type of formulation, which result in them being a lab-course exercise only these days.
When you design a suppository you cannot but start with the environment where it will be absorbed. The rectum is rich in mucous: therefore, hydrophilic excipients such as PEGs or glycogelatin are, theoretically, excellent solutions, dissolving easily and releasing the active ingredients. PEGs are, indeed, fantastic: they give you the opportunity to try incredibly concentrated formulations, highly recommended when defecation is to be promoted (increasing osmotic pressure leads to rectal irritation).
However, if absorption and systemic effects are required, instead of a banal local effect, only lipophilic active substances could be introduced into the aforementioned hydrophilic suppository, as a double partition equilibrium takes place: one between the drug and the mucous (hydrophilic) and the other between the mucous and the mucous membrane.
To sum up, the content and the excipients must have opposite partition coefficients to yield a systemic effect, following a decent absorption.

The same goes for hydrophilic active ingredients, generally expected to reach the blood stream and, thus, frequently combined with lipophilic substances such as triglycerides or, more often, cocoa butter. The latter is the traditional excipients for rectal suppositories, as it’s incredibly cheap and certainly not smelly. These suppositories must melt in the rectum, where the average temperature is 37°C, yielding a huge, oil bubble, through which a partition equilibrium is in place.
However, the main problem with cocoa butter is its polymorphism: there are up to 4 different types of crystals, covering a range of melting temperature from 17°C to 37°C. In particular, the desired 37°C crystal is also the most stable of the lot, but, upon overheating of the mixture to get the water in oil emulsion that we will solidify in our casting mould, a conversion to a less stable form occurs: this form is characterised by an awfully low melting point (26°C), which ruins the whole product, making it unsuitable for the job.
This problem, as every good technician knows, is worsened by freezing point depression of cocoa butter when it gets mixed with the active ingredients, which makes preventing overheating an even tougher task.
Have a nice Easter (whatever your religion).

Yesterday I wanted to write something about the ongoing rubbish crisis in Naples, focusing mainly on the danger linked to people burning uncollected garbage in the streets and the resulting overproduction of carcinogenic dioxins. This issue is having a huge impact on the Italian public opinion. Moreover, because Italy is surprisingly a member of the EU and Naples is a major tourist spot, the crisis is causing a lot of embarrassment to the government and institutions, with international press pointing out what inefficiencies and lack of organization has resulted in.

There are two reasons why such a juicy post wasn’t written, though. The former is that the person who should have lectured me on dioxins and their nasty, toxic effect, my professor of Toxicology, was an ape and when I look at my notes and the book she wrote for a decent bunch of information on these chemicals, I realised she actually told us nothing about it.

The second reason is that, when I got back home in the evening, I was fighting to keep my eyes open, so, writing was beyond consideration. Don’t think I’m making it up: I agree that no assay could make you feel so tired. What wore me out was cleaning the autoclave with my supervisor. So, instead of dioxins, of which I have no knowledge whatsoever, I am here to introduce you to the mysteries of autoclaves and steam sterilization.

This is it: a Fedegari (I told you this post was sponsored) autoclave. Last afternoon, despite the articles I must read, my task was to clean it; something no one had ever done, although almost a year ago, while sterilizing a bag of Petri’s plates before throwing them away, the content of one leaked out and because of the high temperature solidified at the bottom of the chamber. At least this is what everybody guessed, as they said the leak wasn’t too bad and the machine remained sterile, albeit a bit dirty.

The cleaning procedure (sorry for the picture quality: they were taken with a 2 Mega pixels camera from a mobile phone) took a couple of hours and, towards the end, when the machine was empty, I literally got the upper part of my body entirely inside it to remove the rubbish our cheap detergent had only softened. Meanwhile, my supervisor, not tall enough to do this, had to keep the autoclave down to the ground as the machine is remarkably light when empty and my weight was sufficient to make it eventually fall to the ground on one side and begin to roll.

Fortunately, as proved by the pictures, the autoclave wasn’t that dirty, after all. That’s not a surprise when you think that stuff remained immerged in water which is heated up to 121°C for at least 16 minutes. These parameters aren’t arbitrarily chosen, but result from a series of consideration whose aim is to guarantee the most efficient sterilization in the worst case (a common assumption when it comes to good manufacturing practices).

In fact, there are mainly three parameters a good…autoclavist must always bear in mind when optimizing the settings for a cycle. A degradation reaction follows a first order kinetic and, therefore, you can easily calculate how many of the initially present micro organisms have been destroyed. This, in turn, leads to the simple and very practical assumption that the relationship between the number of contaminants and time has the characteristics of an exponential decrease, which means the more organisms you had at the beginning, the longer it’ll take for having an acceptable sterilization. By the way, the first parameter which regulates a sterilization is the D-value, or decimal decay time, which measures the interval required to achieve a reduction of microbial content of a logarithmic unit, at a given temperature. Generally, at 121°C, this time is a minute.
This might look a pretty useless value, though, given that the temperature is everything but constant throughout the process. In fact, the second value, z, is often called temperature coefficient, as it tells us how many degrees are needed to have a 10-fold variation of D. It’s practical to assume z = 10, which means 1°C varies D of 24%. Mind you, it’s not only very useful, but also consistent with empirical observation.
Finally, both (arbitrary) assumptions (D = 1 minute and z = 10) come together in the last value: F0. This parameter is the equivalent exposure time at 121°C for an ideal organism, whose z is 10. The importance of F0 is massive: it allows you to determine the efficiency and effectiveness of a sterilization carried out in the real world, where temperature isn’t constant. For example, a sterilization cycle seems to last less than what it should because a simple computer can easily include in the theoretical 16 minutes at 121°C, both heating and cooling. Here is the formula with which you can enjoy yourself during cold, rainy evenings:

 

There is only one way of manufacturing drugs in the small lab of a chemist’s that yields exactly the same result you’d obtain in an industry: capsules. I might annoy you with a technical description of the machinery you find in a massive, pharmaceutical factory: this was, to be honest, my intention when I first considered talking about capsules, but then I realised 99% of you would have either thrown the monitor in a swimming pool or erased your entire hard disk to avoid the risk of finding this post again.

Fortunately, I thought that the only pros of my internship is showing something no one has ever dared to post on a blog ever: in a nutshell, to literally take you to the chemist’s and have fun with drugs (hmm, sounds a bit ambiguous, doesn’t it?).
So, what I’ve devised is a gorgeous, two-posts series about making capsules: from the raw materials to a finished product I crafts.

Gelatine is the essential ingredient to prepare ordinary telescopic capsules. This polymer is indeed a great material: it’s water soluble and its solubility increases with temperature (whereas viscosity decreases) and  moderate cooling is sufficient to solidify it. Still, without a plastifier it wouldn’t be absolutely flexible: sorbitol and glycerol are often added (5% each) to the big mix.
The result is rather hygroscopic and that’s why it’s important not to touch capsules too much.

This mixture is for a 10% made of water and is solid at room temperature. Not at 80°C, though, and that’s great because, with no need for a mind-blowing heating, we yield both halves of the capsule with a series of nails of two types, given that they must be slightly different or they wouldn’t fit.
Inside the nail there’s a tube so that compressed air can be employed to discharge the solid gelatine and complete the process.

And that’s it. This means capsules are easy to produce industrially and, as a result, cheap. Every chemist’s shop could afford to buy a complete set of coloured, telescopic capsules: from the huge 000 to the smallest 5.

There is another and much more important characteristic of capsules that makes them the best option for a pharmacy, when it comes to selling “home-made” powders: it doesn’t matter if the capsule was manufactured by a big pharmaceutical firm or, well, by me, because, in the end, both will look exactly the same.

Here, for example, is an old photo I took two years: even if they were placed in a paper sack, those capsules had been made during a lab course, so you can’t say they are a professional work, but they are pretty good-looking, thanks to the choice of two different colours, aren’t they?
To get nice, colourful products, all you need is to separate the two solutions that you use to yield each part of the capsule and add colourants. You can even choose natural substances if you’re a freak.

Coming up: time for the first crop ever of  half-decent capsules! And we will save the planet too…

On my first week at the chemist’s, I helped an employee to tidy up the drawers: we worked together on any given afternoon trying to reorganize them last week. In the end, the result is pretty satisfactory: although we didn’t manage to empty any drawer, we left a lot of space in each one, so that there’s room enough for any new product to be launched on the market.

Fortunately, however, I’m not going to describe how you tidy up drawers. What I’m going to do, instead, is to talk about either an Italian habit and a pharmaceutical practice: oddly, both have a lot to do with weddings.

The reason why I mentioned such a boring task is because the said colleague is to get married on Saturday. Traditionally, at Italian wedding feasts you eat a lot and rarely dance. Before you leave (or escape: that depends on your attitude), the bride thanks you for coming with a wedding keepsake full of colourful and polished sugared almonds.

Almond aren’t the only thing to undergo a sugar coating process: in the past, most of oral drugs were filmed using this technique. These days, on the contrary, this is widely considered the last resort, when it comes to coating: although you can carry it out in a simple coating pan, it is a discontinuous and time-consuming procedure to go through and the final product will be colourful, smooth, with excellent taste-masking properties and good-looking but without any logo or inscription and, predictably, unable to release the drug with controllable and customisable kinetics.

Not to mention the problems that can lead to an unacceptable results. So the vast majority of drugs currently sold as sugar coated tablets were patented a long time ago and the manufacturer doesn’t want to spend money on a new version to patent.

Still, this doesn’t mean that sugar coating isn’t interesting. What’s more, it reminds you of the good, old times of pioneering pharmaceutical engineering, when, with little money and lots of new drug recently discovered, people had to bring out all their ingenuity. Those were the times when making a tablet was a precise, hand-made activity, because you could just push buttons and let machinery do everything…

Basically, when a tablet undergoes sugar coating, a thick coat is laid using aqueous solutions with up to 50% sugar content. The coat will cause a 50-100% weight increase of the original tablet and, whatever shape the original product was, it will come out as an oval disk.

Sugar coating is now done in coating pans (solid wall ones, to be precise). The pan is usually at 40°C when he cores are inserted. Given that we will spray an aqueous solution, the very first step must provide a protection for the sensitive core against humidity: in the past, wax and tar were the most utilized insulators.
Then it is time for the long build-up phase, which consists of spraying, pan rolling and drying. A solution of sugar (50%), gum Arabic, coloured powders, talc or chalk powder or even cocoa is sprayed all over the bed of tablets. The pan rolls for 15-20 minutes, so that the solution is widely distributed and each item is dried.
This can be repeated 30-times, so you can see how long this can be.

Finally there is the smoothing step, when imperfections on the surface are corrected by just rolling the pan. This is followed by polishing: waxes or PEGs (or both) are added to the cores and the pan is turned on once again.

The whole procedure may look a little bit dull, but you shouldn’t think the only drawback is time. When the pan is opened, in fact, nasty surprises could be found: irregular final weight distribution, erosion, twinning or explosion of the products (caused by insufficient drying or bad insulation) are somehow the best ones, as they can be easily detected.
Much worse are those hard to recognise quickly: products whose surface is not enough smooth or too opaque or whose colour is not uniform require careful inspection of all the products.

These days there are many innovative drug delivery systems but none of them are as customisable as liposomes.

In the sixties, it was observed that when phospholipids are introduced in water, microparticles (smaller than 10 µm) tend to form. These particles are actually vesicles, a bilayer membrane containing a liquid solution: an ideal environment for many drugs.

Obviously the phospholipids can be chosen among an incredibly wide spectrum: the base bound to the hydrophilic head as well as the length and number of double bonds of the lipophilic part can all vary, in order to optimise the characteristics of the vehicle.
The great thing about liposomes is that they are 100% biocompatible and, thanks to their size, can be injected.

Very practical. A liposome can not only carry hydrophilic substances, but lipophilic molecules too: these drugs will interact with the lipophilic chains of the phospholipids. Amphiphilic substances, unsurprisingly, can be easily loaded in a liposome as well.

So many options lead to four different kinds of release: a drug can diffuse away from the vesicle, pass in the aqueous medium and finally enter the target cell following its gradient. The liposome can undergo endocitosis. Whenever the drug tightly binds to the phospholipids, this can be introduced in a cell through lipids exchange between liposome and cell. Finally, a liposome can release its content in the cell as a result of a membrane fusion.

Like the membrane of our cells, cholesterol can be introduced in order to achieve the optimal fluidity of the vesicle. But, in this case, what cholesterol does is actually to reduce the fluidity and increase the stability of the vehicle.
This requires a lot of care: you see, for each liposome made from a unique type of phospholipid there is a temperature (called main transition temperature) which has to be accurately known before we add cholesterol.
In fact, if we work below that temperature, the lipids will behave like a tough gel and the insertion will go terribly wrong: cholesterol will interact with the hydrophilic heads solely and disturb the order of the membrane, making the vesicle too fluid and permeable.
On the contrary, when the temperature is raised above the main transition temperature, we end up with liquid crystals. Cholesterol can therefore move freely and find its normal position in the bilayer membrane.
Generally up to 50% of cholesterol can be added.

In a nutshell, all you need to synthesize liposomes are water, the right quantity of cholesterol and the correct ionic strength. And make sure you’re working above the main transition temperature of your phospholipids(s)!

There are four types of liposomes, according to their size: SUV (small unilamellar vesicle), LUV (large unilamellar vesicle), MLV (multilamellar vesicle) and MVV (multivesicular vesicle). 

LUVs are my favourite ones (fortunately I had the opportunity to talk about them during my last exam): they are simply perfect delivery systems. Larger than SUVs, they can be loaded with higher doses, but, because they are still unilamellar systems, they are synthesized using a rather simple technique called solution casting (which yields MLVs), followed by high pressure extrusion (according to the diameter of the pores SUVs or LUVs can be produced). 

Solution casting begins with lipids destroyed by solvents such as dichloromethane or chloroform. This operation is carried out in a round-bottom flask: the solvent is then rapidly evaporated, leaving a film of phospholipids on the inner surface. An aqueous solution of the drug is added and, to increase the chances of yielding vesicles, glass microspheres are also employed: vesicles should form spontaneously, as the system tries to minimize its energy, but to increase the yield, you have to work this way. 

My favourite thing about LUVs, however, is one of their applications: the so-called stealth liposomes. Thing is, liposomes work wonderfully when it comes to endogenous physical targeting: pH-dependent, temperature-dependent and immunoliposomes are all valid vehicles, but stealth systems really rock.

PEGs cover the vesicles: as a result, the half-life is dramatically increased, because the immune system is unable to detect it. Stealth liposomes have been successfully used to parentally administer doxorubicin hydrochloride.Last but not least, liposomes are probably the best choice as carrier in non-viral gene therapy.