Abstract
Our project has two complementary sides - we want to produce decursin easily and in a cost-effective way, and we want to use it to treat chemotherapy induced alopecia (CIA). To prove that both our Design and our Proposed Implementation are in fact feasible we went in two routes. First, we wanted to make sure our construct is being expressed in our bacteria of choice - E. coli BL21. This was achieved by running qPCR for XimD & XimE enzymes. The results showed expression in our bacterial populations as expected. We were confident in the functioning of our plasmid and so we turned to proving the feasibility of our proposed implementation. We intend to treat CIA using decursin in products such as lotion or shampoo. To do so, we must prove that decursin is suitable to be a component of such products. We used the Parallel Artificial Membrane Permeability Assay (PAMPA) assay to ascertain if decursin can penetrate the skin and to assess its level of permeability. The results showed us that:
1) Our recombinant enzymes are being transcribed, hence our design appear to be manifesting.
2) Decursin has low permeability on which we can expand and improve in order to assist in its efficacy.
For more details about our POC's results, please refer to our Results page.
PAMPA
Introduction
Our interest in manufacturing decursin is due to its potential value to help with CIA.
For this purpose, we started to look to the future, past the current state of the project as suggested by the Pharmaseed company which can be seen on our Human Practices page.
We plan to incorporate our produced decursin into shampoo, to make the treatment for CIA more accessible. To investigate if this was feasible, we realized that it was necessary to check if the decursin molecules would successfully penetrate the scalp.
CIA significantly impacts hair matrix keratinocytes, causing the hair follicle to enter the resting telogen phase as opposed to cycling between the growth, regression, and rest phases[1].
Due to this, decursin needs to reach the dermis layer which contains the hair matrix and hair follicle bypassing the main barrier to permeation through the scalp, the stratum corneum (SC)[2][3].
Even more important was the need to check that decursin’s permeation was not too high. Decursin was shown to impact cell proliferation through various mechanisms[4][5][6].
However, if it penetrates too deep into the skin and accumulate in unwanted areas[7], such as spreading systemically throughout the body, it is possible that decursin could end up having an adverse effect instead of the curative one we sought[8].
This topic was further discussed in our Safety page. For this purpose, we began searching for effective methods for the measurement of the permeability of small molecules through the skin layer.
We conducted a search through the literature for similar situations and their solutions. We discovered that typically in-vivo or in-cellulo experiments are used in cases of permeability measurements, with the latter utilizing primary cell lines[9].
As a group, we were concerned about both the safety and ethical implications of using these solutions and therefore continued to search, hoping for a more suitable answer without compromising on the scientific side. This led us to the Parallel Artificial Membrane Permeability Assay (PAMPA).
PAMPA consists of two wells separated by a membrane that mimics the skin barrier. In the one well, the compound of interest is added, and following a specified incubation time the amount of said compound is quantified, showing how much passed through the barrier.
This assay is a cell-free, safe, inexpensive, and high throughput method that mimics passive diffusion through the main barrier to permeation through the scalp, the stratum corneum (SC)[3].
Results
Figure 1: Decursin’s low permeabilityDecursin's permeability was tested using a PAMPA kit, by introducing the metabolite to a system comprised of Skin Mimic solution and two plates with a membrane between them. Decursin’s absorbance was measured following 18 h incubation at a 330 nm wavelength. The absorbance was converted to permeability by using a known equation provided by the kit. The controls used are the standards that are supplied with the PAMPA kit. Methyl Paraben’s, Propyl Paraben’s and Theophylline’s absorbance was measured following 18 h incubation at 260, 260 and 270 nm wavelength respectively[10].
The PAMPA kit that we received arrived with three additional molecules that would serve as the permeability controls.
Methyl Paraben, Propyl Paraben and Theophylline act as the high permeability control, medium permeability control and low medium permeability controls, respectively.
In our hands, decursin demonstrated low permeability (fig.1).
This suggests decursin could be safe to use in a formulation that meets the skin, as it does not have excessive permeability as displayed by the assay.
However, we need to be sure that the permeability would be high enough to penetrate to the required depth of 4.16 millimeters at a concentration of roughly 10 nanomolar [11][12].
Due to the fact that the permeability is relatively low, we can now build on top of this and develop an optimized formulation to ensure the efficient and safe delivery of our product. In addition, we can look at the low permeability control, theophylline. Theophylline is a phosphodiesterase inhibitor used as an anti-ageing, anti-asthma, anti-cytotoxicity, and fat loss drug. For many of theophylline’s indications, it is delivered as a topical lotion, ointment, or cream as can be seen in product like ASPIRE’s Fat Dissolving Cream, Lipoxyderm™, and Lipo Sculpt Cream among others. This goes to show that theophylline and molecules with a similar permeability are capable of being delivered through the skin. We can learn from the development of Theophylline as a product when developing our product. Therefore, PAMPA not only proved that the concept was feasible but also provided an idea for the concentrations needed in the final formulation.
Enzymes Expression
Introduction
Our design is made up of three enzymes that together catalyze the formation of decursinol from umbelliferone. We divided our system to two designs on which we worked on in parallel.
This was done to better implement the two steps of the pathway: umbelliferone (UMB) to 7-demethylsuberosin (DMS) and 7-demethylsuberosin (DMS) to decursinol (DEC).
• The first step requires the cloning of a prenyltransferase (PT).
Unfortunately, because of time constraints we couldn't get to the point of measuring expression of PT in our bacteria.
• The second step is catalyzed by two enzymes - XimE & XimD.
Both were cloned into the same plasmid, and subsequently tested for their expression using quantitative PCR, a high-throughput, sensitive and reproducible technique to measure gene transcription[13]
Results
The results taught us two important things. The first thing we learned is that our enzymes are indeed being transcribed. The second thing we learned is that we succeeded in regulating the transcription of XimD. As part of our design, we cloned XimD under a T7 promoter regulated by a lac operator and XimE under a consitutive T7 promoter. We calculated the transcription fold-change following induction and learned that XimD transcription was 14-fold higher after induction with IPTG, while XimE transcription is similar between the same induced and non-induced samples, as intended (fig. 2). These results indicate that the enzymes are being transcribed and that we can control the temporal transcription of XimD. The significance of controlling XimD expression is elaborated on in our design and Model page.
Figure 2 : (A-C) Amplification curves of XimD, XimE and housekeeping gene transcripts. Five ml. of E. coli BL21 cells were allowed to grown over night in the presence (induced) or absence (non-induced) of 1mM IPTG, before RNA extraction. RNA was then subjected to one h of DNase (Invitrogen, #AM2238) and converted to cDNA (Invitrogen, #4311235). Finally, samples underwent quantitative PCR with gene-specific primers. IPTG presence resulted in the induction of XimD (a) by 14-fold (d), while XimE transcription remains relatively unchanged (b,d). rssA was used as a housekeeping gene. Threshold for analysis was set at 0.3. Dashed line: Ct value for each sample. NTC: non-template control. The XimD-XimE plasmid was used as a positive control. (D) Fold-change of XimD and XimE transcription. The Ct values acquired correspond to the initial total RNA value levels. Hence, the values were normalized to the Ct values of endogenous housekeeping gene, rssA, a known housekeeping gene[14]. Note that XimE is upregulated as well despite being constitutively transcribed, possibly due to the proximity of RNA polymerases in the proximal XimD locus. This figure was created with the assistance of user-created functions[15][16].