Team:NTU-LIHPAO-Taiwan/Modeling/Pathways

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  • Bacteria Distribution
  • CPP-PYY Penetration Efficiency
  • Adsorption Into Bloodstream
  • Thrombin Cleavage Efficiency
  • Healthin Drug Effect
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Pathways

Bacteria Distribution

After orally ingested, certain amounts of modified probiotic L. casei can survive under acidic condition when passing through the stomach. Owning to gastric acid, bile acid and digestive enzymes, the presence of these bacteria in the intestinal tract only last for a limited time, and then are washed out in faeces.[1] During the process of transition and temporary colonization, CPP-PYY complexes are produced and exert the following effect.

In the first part of Modeling, bacteria distribution in the small intestine was investigated. Data from Y. K. Lee et al. showed bacteria number against time adhered on the duodenum, jejunum, and ileum in mouse after orogastric intubation of 10⁹ L. casei, which were listed in Table 1.[2] The ratio (%) of bacteria number to total ingested number were also indicated.

	Day 1	Day 2	Day 4
Duodenum	6050	3950	3100
Jejunum	18500	6000	5100
Ileum	62500	15400	13200
Total	87050	25350	21400
Ratio (%)	0.008705	0.002535	0.002140

[Table 1.] Total L. casei Shirota cell number adhered on various sections of the intestinal tract against time after orogastric intubation of 10⁹ L. casei

By summing up all the data, we constructed the continuous L. casei cell number and ratio distribution against time with curve fitting in MATLAB, as shown in Fig.1-1-1 and Fig.1-1-2. However, experimental data from suicide mechanism should be incorporated to complete the simulation for the survival of L. casei in the small intestine, here we hypothesized 4 days for bacteria survival. Therefore, the time-changing tendency could be utilized for further calculation.

[Fig.1-1-1] Total L. casei cell number adhered on the intestinal tract against time after orogastric intubation

[Fig.1-1-2] Ratio of L. casei cell number to total ingested cell number against time

CPP-PYY Penetration Efficiency

Knowing the L. casei cell number variation against time, we proceeded to simulate the penetration process for the cell penetrating peptide, TAT with PYY. Although the mechanism for TAT translocation depends on the delivery cargo and its sequence, most papers concluded that it is mediated via an as yet uncharacterized pinocytosis/endocytosis related mechanism, which is also receptor-independent.[3]

From the experimental results of CPP-PYY complex production rate, here we took 10^-10 μg/min for example, we hypothesized that one-fourth of the products secreted from the cell will be distributed in the direction toward the small intestine epithelial cells, not being digested by the intestine fluid. Liang JF et al. had provided the effective permeability (P_eff) of insulin-TAT conjugates by in vitro intestinal absorption assay on cultured Caco-2 cell monolayer, which was calculated based on the following equation.[4]

where J_ss is the flux, dC_R/dt is the change in conjugate concentration in the receiver chamber at steady-state, V_R is the volume of receiver buffer, A is the cross sectional area of the exposed tissue, and C_D is the conjugate concentration in the donor chamber. The result from Liang JF et al. was applied for modeling, where the value of P_eff was presented as 1.62×10^-5 (cm/s). Moreover, the results further implied that the product seemed to be through a transcytosis-like mechanism, which confirmed that our product can penetrate through the epithelial cells.[4]

The parameters for calculation were obtained from literatures, where L. casei cell size range = 0.7-1.1 x 2.0-4.0 micrometer[5], and the mean total mucosal surface of the small intestine interior averages 32 m² in recent research.[6] One-tenth of the bacteria height multiplied the mucosa surface area were taken as the donor chamber volume, where one-fourth of the CPP-PYY complexes without being digested were oriented toward the epithelial cells. Finally, the flux of the complexes through villi was achieved, giving us the result of mass rate per unit volume. Fig.1-2-1 demonstrates the amount of penetrating CPP-PYY complexes though villi against time.

[Fig.1-2-1] Amount of penetrating product(μg) through villi against time(min)

[Fig.1-2-2] CPP-PYY penetration through villi

Adsorption Into Bloodstream

The inside wall of the small intestine is lined with villi that contain blood capillaries to carry away the absorbed molecules for circulation. The large surface area contributed by these villi allows absorption to happen quickly and efficiently.

To evaluate the efficiency of the adsorption, we incorporated “Fick’s Law of Binary Diffusion”, where the diffusion coefficient was given by S R Chary and R K Jain.[7] Taken in calculation for our simulation, the measured interstitial fluid diffusion coefficient (D_AB) of bovine serum albumin in tissues was 5.8 x 10^-7 cm²/s as reference.

where J_A is the mass flux of CPP-PYY complex, ρ is the density of villus cavity, and ω_A stands for the mass fraction of the complex. The direction of diffusion was considered from the epithelial cells toward capillaries, signified as y direction, and the distance in between was viewed as 4 μm referred to literature.[8] Furthermore, the mass fraction was set “zero” in the capillaries owning to the rapid absorption, giving the value of gradient as “one” for the following estimation. The result of this effect of binary diffusion is illustrated in Fig.1-2-1.

[Fig.1-3] Mass diffusion flux(μg/m²•min) of CPP-PYY complex against time(min)

Thrombin Cleavage Efficiency

After CPP-PYY complexes are transported across intestinal cells, they enter capillaries with the help of CPP transmembrane protein and diffusion. However, to suppress appetite, CPP-PYY complexes should be cleaved. Therefore, in our design, we utilized a linker which has a thrombin-favor cleavage site and thrombin is ubiquitous in blood vessel.

From the literature by J. Y. Chang, the percentage of polypeptide cleaved by thrombin is applied to CPP-PYY complexes.[9] The amino acid sequence of CPP-PYY complexes is LEAGCKNFFPRSFTSCGSLE, and we sought to cut through Arg-Ser(R-S) site. Therefore, data of Arg-Thr(R-T) site in salmon calcitonin was taken for reference, because Ser and Thr are both polar and uncharged residue. The figure of time to percentage of Arg-Thr cleavage is shown below. However, due to the fluid motion in circulation, we supposed that nearly 100% of the complexes would be cleaved during 30 minutes.

[Fig.1-4] Percentage of Arg-Thr cleavage(%) against time(min)

Healthin Drug Effect

Steinert RE et al. had administrated the intragastric load of a mixed liquid meal on subjects for observing the plasma concentration of active PYY after meal, as shown in Fig.1-5-1.[10] The physiological PYY concentration in plasma is approximately 45 pg/ml, and it reaches maximum at 113 pg/ml after 30 minutes of food intake. To prevent the potential side effect of PYY resistance, as well as exert the optimal appetite suppresion effect at the same time, we sought to control the PYY concentration at the level of 100 pg/ml (0.1 μg/L) after taking Healthin.

[Fig.1-5-1] Plasma concentration of active PYY in response to an intragastric load of a mixed liquid meal

As the blood volume is about 78 ml per kilogram, we considered total average blood volume of 5 liter in human body for calculation.[11] The final concentration of PYY against time is presented in Fig.1-5-2, which exerts the optimal effect after one day of ingestion followed by gradual decrease during 4 days.

[Fig.1-5-2] Final concentration of PYY (μg/L) in the blood vessel

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Reference

Reference

[1] K.M. Tuohy and M. Pinart-Gilberga et al. Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora. J Appl Microbiol, ISSN 1364-5072. UK. (2006)

[2] Y. K. Lee and P. S. Ho et al. Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut. Applied and Environmental Microbiology, Vol. 70, No. 2. Finland. (2003)

[3] Lindsay MA. Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol, Vol. 2, p. 587-594. UK. (2002)

[4] Liang JF and Yang VC. Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency. Biochem Biophys Res Commun, Vol. 335, p. 734-738. USA. (2005)

[5] Axelsson, L. Lactic acid bacteria: classification and physiology, pp. 1-72. In, S. Salminen and A. Von Wright (eds). Lactic Acid Bacteria: Microbiology and Functional Aspects, 2nd ed. Marcel Dekker, Inc, New York. (1998)

[6] Herbert F Helander and Lars Fändriks. Surface area of the digestive tract – revisited. Scandinavian Journal of Gastroenterology, Vol. 49, p.681-689. Sweden. (2015)

[7] S R Chary and R K Jain. Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc. Nati. Acad. Sci, Vol. 86, p. 5385-5389. USA. (1989)

[8] Mamed Kadyrov et al. Epithelial-capillary distance of chorionic villi of normal-developed placenta. Placenta, Vol. 14. Russia. (1993)

[9] J. Y. Chang. Thrombin specificity - Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem, Vol. 151, p.217-224. (1985)

[10] Steinert RE et al. The role of the stomach in the control of appetite and the secretion of satiation peptides. Am J Physiol Endocrinol Metab, Vol. 302, p.666-673. Switzerland. (2012)

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