0:01
Everyone, thank you for the introduction and thank you to the organisers for inviting me.
0:06
So my talk is solubility limited absorption and the impact of particle drift and oral fraction absorbed.
0:13
So the outline, the things I will cover is solubility limits oral absorption, the concept of the particle drift effect.
0:20
We'll look at some in vitro tools that we have available and some in vitro evidence.
0:25
And then how do we translate this to in vivo performance?
0:29
And then if we have time, we can follow up with some Q&A.
0:34
So I'm sure many of you have probably seen graphs like this where, you know, a plot of the administered dose, you know, during an ascending dose study, for example, versus the absorbed dose or the area under the curve concentration profiles.
0:49
And then and in the best case example drug that's completely soluble, maybe BCS class one, you'll get complete absorption, and it will be dose proportional.
1:00
So if you increase the administer dose, everything will be absorbed, and you'll get something like the green line shown here.
1:06
If you have a dissolution limited absorption, you may not achieve the absorption that relates to the maximum dose.
1:16
But then many examples of BCS Class 2 molecules.
1:20
You may see something relating to the red line where you see solubility limited absorption.
1:26
Above a certain administered dose, the area under the oral absorption curve will start to level off.
1:33
Well, that's what you might think you should see in theory, but in practise when you actually do many of these studies, you might see something like this in the in vivo results.
1:44
And then it can be even more pronounced when you have micro and nano sized particles in your formulation.
1:51
And this is noticeable especially at high doses in toxicology studies and first in human studies.
1:56
So how can this be explained?
1:59
Well you may be able to think of a few examples.
2:01
Maybe you have a super saturating dosage form, and you have super saturation followed by precipitation.
2:08
The last speaker mentioned some examples.
2:12
Maybe you have efflux transporters and you're saturating the efflux transporters, so you get higher than expected absorption.
2:21
But another theory that could explain this concept is the theory of the particle drifting effect.
2:28
So when your drugs approach, when you approach the in gastrointestinal walls, the membrane barriers of gastrointestinal walls, there's something known as the unstirred or unstirred water layer or the aqueous boundary layer.
2:42
Now typically it's only dissolved drugs that will migrate through that unstirred water layer.
2:47
And so it's the dissolved molecularly dissolved drug that is able to diffuse through unstirred water layer and then permeate across the gastrointestinal membrane walls and achieve oral drug absorption.
2:58
But in the case of very high drug loadings or more particularly in the case of very tiny particles such as nano suspensions, these may be able to diffuse into the unstirred water layer barrier and provide a reservoir for additional absorption.
3:13
And this is so this could be another mechanism that explains, you know, what I just showed you in the previous slide.
3:21
So this effect was first published by Kiyohiko Sagano.
3:27
He was working at Pfizer at the time.
3:29
He's now at Ritsumeikan University in Japan and he published it back in the International Journal Pharmaceutics back in 2010.
3:40
So that gives you a little bit of background.
3:43
Now what Pion does is we actually manufacture and supply combined solution permeation tools and this can give us the ability to study effects such as this.
3:55
So some of the tools that we have available, if we're doing this kind of dynamic combined dissolution, solubilization absorption tools are shown on this slide.
4:05
And we have different scales setups depending on the stage of the drug development process you're in.
4:10
So in very early stages of development, we have the μFlux shown on the left hand side, which is small volumes, it's compound sparing.
4:20
It's typically used in preclinical or early development setting when you want to rank order your API and some preparations for combined dissolution absorption studies.
4:33
And each of these setups has a donor compartment separated from a receiver compartment with a membrane.
4:39
And the membrane we use has been developed to correlate to human intestinal permeability.
4:47
So in the μFlux on the left you see a donor cell on the left to the acceptor cell on the right and a membrane in the middle which is coated with a phospholipid mixture.
4:55
In all of the other setups are sort of scaled to later stages and development process.
4:59
We go to larger volumes.
5:01
We have the MiniFlux which works volumes from about 130 to 250 millilitre volumes.
5:07
These are representative of gastrointestinal tract volumes.
5:10
So when you're starting to scale up to make your first sort of tablets and capsule development and then we can go up to larger scale setups where we are working with the United States Pharmacopoeia dissolution type vessels.
5:22
But we have the absorption chamber inserted into the top.
5:25
You can stick your tablets in the main dissolution vessel and then there's the membrane barrier and then the absorption chamber.
5:33
And the method of quantitation we use is called the Rainbow Dynamic Dissolution Monitor.
5:38
It's the apparatus shown there on the right and it's a fibre optic spectrometer platform.
5:46
Each probe has its own individual spectrometer.
5:48
There's a deuterium lamp.
5:50
We shine UV lights along the fibres.
5:53
The probes can be inserted into those dissolution permeation setups.
5:57
There's an open window at the bottom of the probe and a course mirror so you can set them in the donor receiver sides.
6:03
The media can pass through the open window.
6:07
The UV light gets reflected off the course mirror and back to detectors back at the detection unit on the right.
6:14
And this gives us the ability to do real time data collection and it's really information rich as well.
6:20
We can capture data every two to three seconds in all of the vessels.
6:25
So we can build up a really detailed profile about the dissolution, donor dissolution and the permeation characteristics into the receiver compartment and a little bit about the kind of membrane mixture.
6:40
So it's supplied in an ampule, it's a mixture of phospholipids.
6:44
We just coat it onto the filter support of one of the absorption chambers.
6:49
It spreads out, impregnates the pores of the filter support, it complete, creates a complete intact membrane barrier, has high electrical resistance if you put do tear measurements.
6:59
So we can assume that isn't, you know, the phospholipid barrier is the major resistance to migration of molecules through to the receiver compartment and you can tell when it works because it's made of a completely translucent barrier.
7:12
And then the receiver compartment.
7:13
We also use something called acceptor sync buffer, which is a chemical scavenger and maintains sync conditions.
7:19
So any molecules that permeate through the barrier do not back permeate. And it's a mixture of phospholipids that's been developed to be representative of human jejunal permeability and the correlation to human jejunal permeability as shown on the right.
7:33
So we typically use these setups for kind of rank ordering of formulations in early development setting, maybe looking at impact of biorelevant media such as simulating intestinal fluids, screening for excipients.
7:44
And then as we go to the larger scale setups, we can start to evaluate whole sort of tablets and capsules and formulated products.
7:53
But what I wanted to do in this particular case, just to give an example or a case study relating to investigating the particle drift effect.
8:01
So I'll look at the case study.
8:03
And then how do we take that in vitro data and be able to scale it to in vivo performance, which we do through a piece of software that we call Predictor, which enables us to calculate maximum absorbable dose and oral fraction absorbed?
8:24
So here is our first example.
8:29
So this is celecoxib, a poly soluble drug, BCS Class 2.
8:34
Here are the physchem properties.
8:37
The solubility in an aqueous buffer is only about 3 micrograms per mil in the presence of simulated intestinal fluid and it's around 46 micrograms per millilitre.
8:48
It's a neutral compound that physiological pH is and we were, we say we sourced the mark one, one of the marketed formulations of this of this molecule.
9:00
And the marketed formulation is known to contain a large proportion of nanoparticles in the formulation.
9:10
And here's some of the in vivo data.
9:13
And what we should point out here is as you can see on the on the plasma concentration profiles, as you increase the administered dose that the absorption and the area under the curve is still increasing.
9:25
But nevertheless, if you look at you know, even at the lowest dose administered here, the 50 milligramme dose, the dose number is you know is greater than 9 for the lowest dose.
9:37
Now the dose, the dose number of one or lower would mean that the dose is completely soluble in the intestinal fluid volume.
9:45
The dose number of two means you need 2 intestinal fluid volumes to dissolve the entire dose.
9:51
So dose number of nine means you need 9 intestinal fluid volumes to dissolve that dose and that's for the lowest dose.
9:57
By the time you get up to the highest dose you you've got lots of it.
10:02
It's so poly soluble, you need massive amounts of intestinal fluid.
10:06
But you can see the plasma concentration time profiles, it's increasing with dose.
10:11
So, so what is going on?
10:13
And we think this is relating to the particle drifting effect of the nanoparticles in the formulation.
10:20
And we can actually evaluate this, as I say, using the dissolution permeation setup.
10:24
So here is the μFlux study.
10:27
What we've done is we've scaled the dose to be kind of dose appropriate.
10:32
So this is our small volume setup where we're doing the study in about 15 millilitre volumes.
10:38
We're assuming the volume of the intestinal, the gastrointestinal tract volume is around 150 millilitres and so we scaled it down tenfold.
10:47
So 50 milligramme dose, we take 5 milligrammes, you know 400 milligramme dose we take 40 milligrammes.
10:54
And these are the results in the graph on the right shown of how much drug makes it through to the receiver compartment.
11:00
So you would expect if it was just solubility limited that that's going to all pretty much give the same amount of drug appearing on the receiver side because the permeability or the flux through the membrane should be concentration driven.
11:14
So it would reach the solubility limit, and you should theoretically just see the same amount of drug appearing on the receiver side.
11:22
But in actuality, with that, you can see how much increase in appearance or total mass of drug being transported or permeating through the membrane as you increase the dose levels.
11:34
And we believe that this is due to that particle drifting effect of nanoparticles into that unstirred water layer or aqueous boundary layer.
11:42
So we can see from the in vitro results, we also have this effect.
11:46
We think it's been mirrored in vivo, but can we take our in vitro data and translate it into in vivo outcome?
11:55
So we developed a piece of software that goes along with the flux data, you know, flux apparatus for taking the data and using it to calculate human in vivo outcome.
12:08
So this is called the predictor software.
12:10
So we basically take our artificial man, our donor permeation in vitro cell, but we need to scale it up to the in vivo situation.
12:20
So in our in vitro setting, we actually have a relatively small membrane surface area.
12:24
You can see it's about this big.
12:29
But in the human and gastrointestinal tracts, we know that there's a massive surface area available for drug absorption to take place.
12:37
You know, in the upper intestine, it's estimated I think to be about 120 square metres of membrane surface area.
12:45
But also if you look at the structure of the gusto intestinal tract, it consists of circular folds and these finger like structures, the intestinal villi.
12:53
And depending on the permeability of the drug, some molecules will have access to high permeability molecules only may need to touch the fingers or the top of the villi and they will be absorbed straight away.
13:07
Poor permeability drugs may have time to descend into the valleys and the crypts and have access to a larger surface area, so we need to make some corrections for available surface area available for drug absorption, which can partly depend on the permeability of the molecules.
13:23
You may need to make some corrections for the difference between the assumed unstirred water layer thickness in the in vitro setting, which will depend on the hydrodynamics and stirring of the in vitro platform versus the unfair water layer thickness in the human gastrointestinal tract.
13:41
We also need to make some corrections for the clearance of the drug from the donor side.
13:48
So again, in the in vitro setting with a small surface membrane surface area, there's likely to be a lot less clearance of dose from the in vitro setting versus in the in vivo setting when you have a huge surface area available for absorption.
14:01
But we can make these kinds of corrections based on sort of the flux data.
14:05
And also we make adjustments for intestinal transit time such that, you know, the outcome is effectively that we can say that the mass absorbs shown on the equation on the right hand side on the bottom is a function of the in vivo scaled flux multiplied by the surface area of membranes available for drug absorption to take place, multiplied by the transit time over which drug absorption can take place.
14:28
And the transit time in in the human upper intestine is typically like 3 to 4 hours.
14:34
So we take our in vitro data, and we do all these kind of scaling to the in vivo situation.
14:40
And what we find is that, you know, the use for the in vivo situation.
14:45
Effective permeability is dominated by the massively increased surface area available for drug absorption in the human intestinal wall, which is a consequence of the circular folds in the intestinal villi.
14:56
The API permeability properties will have a significant influence on the surface area available.
15:02
As I mentioned in highly permeable when you need to touch the top of the villi, maybe less permeable can descend further into the into the valleys.
15:12
And then compounds that are on stead water layer, you know, barrier resistant, you know, limited compounds, they may only encounter the surface expansion with respect to, if you just assumed it was a straight cylinder we need to account for the surface expansion.
15:34
But compounds that may drift through as nanoparticles, they will be able to descend deeper and have access to a much larger surface area.
15:45
And what this translates to is that from the in vitro flux assays where we may see an example of just a twofold improvement in the in vitro scaled flux and we seek to see big differences in those curves.
15:59
What it actually translates to is when we do the surface expansion, it could be actually a tenfold improvement in overall drug absorption, and this is the outcome of the results from the celecoxib.
16:13
So the table shows the results with the doses we show.
16:19
If we just do a calculation on the flux, but we don't account for particle drift, we end up really underestimating the amount of drug that was absorbed.
16:29
The third column shows the corrections when we account for the effect of the particle drift, you know, of the nanoparticles and then we can compare to the, you know, the published in that, you know, literature where I got the data from.
16:41
In the literature, the in vivo fraction absorbs, and it's more recently seen on the bar graph on the right.
16:46
So in vivo results in light green without taking into account particle drift, it's in dark green.
16:54
But when we incorporate particle drift into the into the calculation and make the corrections, we get the orange bars which you can see are much more in line with the actual in vivo outcome.
17:07
So I think that was kind of what I wanted to present.
17:12
But just summarising, you know, from the flux data, it's possible to estimate the absolute in vivo absorbed fraction of a drug using in vitro flux measurements.
17:25
But in when we do experiments with ascending doses, we can isolate the proportion of an in vitro flux arising from particle drift performed above the solubility limit.
17:36
So we do some tests below solubility limit and then we do some ascending doses above the solubility limit of the compound, and we can isolate the impact of the particle drift.
17:45
And then we can compensate for this when we scale up the in vitro flux to the in vivo condition by applying all of those corrections using the Predictor software.
17:56
So it can help support the development of enabling formulations.
18:00
We can identify any present influence from the particle drifting by doing these in vitro flux assays at appropriate dose volume ratios.
18:09
But beyond that we can also assess the possibility of bioavailability improvement due to particle drifting from potential surface access improvement to unstirred water layer penetration for those compounds that are unstirred water layer limited absorption.
18:27
So if your compounds just membrane permeability limited, you know making nano size may not be you many advantages, but if it's an unstirred water layer limited compound, then going for nano particle formulation or suspension could give you some big advantages.
18:44
So there is lots more publications in this area.
18:48
So say the first one was by Sagano in about 2010, but there's starting to be a lot more recent publications which is some of them are listed here.
18:57
So there's a lot of interest in this area based from a mechanistic understanding of all absorption and also for improvement of all absorption through enabling formulation technology.
