Introduction

All over the world, oral drugs show a stable development trend. Oral drugs have the advantages of easy to carry, oral, easy to preserve and so on, and are widely accepted by people. Nowadays, there are more and more kinds of oral new drugs, but most of the prediction of the side effects of new drugs can only be completed by clinical trials. The process of drug absorption by the human body is a very complex process. The way of oral administration is safe and convenient, but after taking it, it may have a certain impact on the intestines and stomach, for the new drug that has just been put into production. The effect of medication on gastrointestinal function is often difficult to determine. Therefore, it is particularly important to find a suitable method to simply predict the side effects of oral drugs on gastrointestinal function.

Pepsin (PEP) is a digestive protease released by the main cells in the stomach and degraded into peptides by cleavage hydrophobicity and preferred peptide bonds between aromatic amino acids [1]. It has a single polypeptide chain with 324 amino acid residues with a molecular weight of 35000 Da [2]. PEP is stored as pepsinogen and released only when food protein enters the stomach [3]. PEP is a monomer L-protein composed of two homologous domains, and its most suitable pH is 1.5~2.0 [4]. Two aspartic acid residues Asp32 and Asp215 form the catalytic site of PEP, one of which must be protonated and the other deprotonated to make the protein active. The optimum temperature of PEP was between 37 ^ᵒC and 42 ^ᵒC [5]. The digestion of food in the stomach is mainly the preliminary decomposition of protein, dietary protein is difficult to absorb macromolecules. In order to let digestive tract cells, absorb nutrition in the protein, it is necessary to decompose the protein into smaller particles called peptides. PEP is a digestive enzyme that plays a major role in the digestion of proteins into polypeptides. At pH 1.5~5.0, it decomposes the ingested proteins into peptides, and the other part is broken down into amino acids necessary for human activity, such as tyrosine, phenylalanine and so on. Without PEP, the ability of the human body to metabolize protein will be severely inhibited.

Trypsin (TRP) is an important proteolytic enzyme secreted by the pancreas. It plays an irreplaceable role in protein digestion in the small intestine and contains 150-600 μg/ml in duodenal fluid. It can hydrolyze amino acid compounds linked by peptide chains, has esterase activity, and has little content in normal serum. TRP not only plays an important role as digestive enzyme in physiological process, but also plays an important role in hemostasis, apoptosis, signal transduction, reproduction and immune response [6,7]. The molecular weight of TRP is 23300, which is composed of 223 amino acid residues [8], of which 4 tryptophan, 10 tyrosine and 6 phenylalanine are its inherent fluorescent groups [9,10]. TRP is composed of two domains of almost the same size. the two domains are connected by disulfide bonds, and each domain is composed of six reverse parallel β-folds. Among them, His residue, Asp residue and Ser residue are the catalytic active centers of TRP, which constitute the structure of catalytic triplet [11]. TRP is excreted from the pancreas into the small intestine and is involved in the digestion of proteins in food and it is usually used as an important digestive protease model to study the interaction between drugs and proteins [12].

There are many reports on the interaction between small molecular drugs and digestive proteases by spectroscopy, but most of them focus on the binding mechanism between drugs and proteins. The binding constant and number of binding sites between small molecular drugs and digestive protease were used to quantitatively study the effect of the binding of small molecular drugs and digestive protease on the free concentration of digestive protease. Furthermore, it is inferred that the effect of combination on the efficacy and gastrointestinal digestive function of the human body has not been reported in the literature. In this paper, the binding constants K_a and binding sites n of some oral drugs with PEP and TRP were obtained by spectroscopy and the drug binding rate and protein binding rate were calculated by the established mathematical model. The effect of oral drugs combined with digestive protease on the efficacy of drugs and the effect of drugs on gastrointestinal digestive function of human body were quantitatively studied. The method is simple and practical, and the conclusions are basically consistent with the side effects shown in the drug instructions.

Mathematical Model

The binding constant and the number of binding sites between the protein and the drug were determined by spectroscopy. When the drug-protein binding reached a dynamic equilibrium, one part of the protein was a free protein and the other part was a binding protein. There is the following relationship between drug concentration Q, protein concentration B, binding constant K_a and number of binding sites n: [B] + n [Q] →[BQ_n], where n represents the number of equivalent and independent drug binding sites. If the interaction between B and Q accords with Langmuir monolayer adsorption model [13], the equilibrium constant K_a is:

When drug and protein are combined at 1:1, then:

Assuming that the total concentration of drug is Q, the total concentration of protein is B, and the concentration of drug-protein complex is x, then K_a is also expressed as:

By solving the univariate quadratic equation, the following results are obtained:

The drug combination rate can be expressed as:

The protein binding rate can be expressed as follows:

Results and Discussion

Under simulated human physiological conditions, the pH of PEP was about 2.0 and the pH of TRP was about 7.4. According to the commonly used measurement of the drug recorded in the drug instruction of different oral drugs (the drugs and digestive proteases involved in the literature report are from Sigma) and binding constant K_a calculated from fluorescence data at λ_ex=280 nm (n of the corresponding common oral drugs and digestive proteases reported in the literature was 1). The drug binding rate and protein binding rate were calculated by Equation (5) and Equation (6). The results were shown in Table 1 and Table 2. Based on the data, the effects of the combination of drugs and digestive proteases on the efficacy of these drugs and the digestive function of the stomach and small intestine were inferred.

The normal basal gastric juice volume was 10~100 ml, and the PEP concentration was calculated according to the basal gastric juice volume of 50 ml: 2.6×10^-8~4.3×10^-8 [26].

The most important reference in the study of drug-digestive protease binding rate is protein binding rate and drug binding rate. The higher the protein binding rate, the less the free digestive enzymes, the digestive function of the digestive system will also decrease; the greater the drug binding rate, the less the free drugs, the efficacy will also be reduced. After oral drugs enter the human body, their effective components will be absorbed in the human digestive system, most of the effective components of drugs through the gastrointestinal mucosa into the human blood circulation system to act on the affected area. As shown in Figure 1, taking the binding reaction of levofloxacin with PEP as an example, the binding model of levofloxacin and pepsin was obtained by nonlinear fitting: W(B) = -1.894×10⁴ Q² + 2.374×10²Q + 0.9029, r = 0.9985; W(Q) = 5.286×10² Q² - 0.6412 + 2.553×10^-4, r = 0.9978. In the range of the actual dosage of levofloxacin, the binding rate data of the drug and PEP, the free rate of the drug and the free rate of PEP can be obtained by combining the model. The effect of the interaction between levofloxacin and PEP on the efficacy and digestibility can be discussed by the data of binding rate and free rate. After oral administration of the tablet into the digestive system, the starch substance in the tablet is digested, and the effective component of the drug enters the gastric juice in contact with PEP and then binds within the actual dosage range of levofloxacin, The protein binding rate between the drug and PEP was 95.3%~97.6%, and the free PEP was only 4.7%~2.4%, indicating that the digestive capacity of the stomach was greatly reduced due to the binding of levofloxacin. The drug binding rate of levofloxacin was 0.012%~0.006%, and the free rate of levofloxacin was 99.988%~99.994%, which indicated that the efficacy of oral levofloxacin was almost not affected by PEP binding.

Table 1 lists data on the binding rate of some of the oral drugs often exposed to PEP in life. After the combination of oral drugs with gastric protein, the protein binding rate of most oral drugs, such as levofloxacin, gatifloxacin and so on, is relatively high. Based on the results of the data, it is speculated that taking these drugs has a greater impact on gastric digestive function. According to the adverse drug reactions recorded in the drug instructions, most of these drugs cause gastrointestinal dysfunction, the common manifestations are abdominal discomfort or pain, diarrhea, nausea or vomiting. However, there are also some oral drugs with low binding rate to PEP, such as baclofen, torvastatin and metformin. The free amount of PEP is less affected by the combination of drugs and PEP, so these drugs have little effect on gastric digestive function after oral administration. According to the adverse reactions recorded in the drug instructions, stomach digestive discomfort occasionally occurs after taking these drugs, but the symptoms are relatively mild.

Determination of TRP concentration in duodenal fluid by enzyme rate method: 6.3×10^-6~2.5×10^-5 mol/L in fasting state, the volume of intestinal fluid was 45~319 mL, the average value was 105 mL, and the concentration of trypsin in duodenal fluid was determined by enzyme rate method [34].

After oral drugs enter the human body, the main part of absorption is the small intestine. As shown in Figure 2, taking the binding reaction of enoxacin with TRP as an example, the binding model can be obtained by nonlinear fitting according to the obtained data: W(B) = -3.338×10⁴ Q² + 0.9450×10²Q + 0.9135, r = 0.9988; W(Q) = 1.827 ×10⁴ Q² – 0.5295×10²Q + 0.04982, r = 0.9982. In the range of the actual dosage of enoxacin, the binding rate data of the drug to TRP, the free rate of the drug and the free rate of TRP can be obtained by the binding model. When enoxacin interacts with TRP, the effects of efficacy and digestibility can be discussed by the binding rate and free rate data.

In the range of the actual dosage of the oral drug, after the drug molecule enters the small intestinal fluid with the digestive system, the TRP in the small intestinal fluid is in contact with the drug molecule. The free rate of TRP was reduced from 100% to 4.1%~2.0% due to the binding of enoxacin to TRP, which indicated that the number of free TRP was very small due to the binding of enoxacin to TRP. That is to say, enoxacin has a great effect on the digestive function of the small intestine. The content of free enoxacin was 97.58%~98.82%. The results showed that the efficacy of enoxacin was almost not affected by the binding effect.

From the data in Table 2, it can be seen that the protein binding rate of ciprofloxacin hydrochloride, ofloxacin, enoxacin, norfloxacin and vitamin B₉ is high, so that the amount of free TRP is very small, The results showed that the combination of drugs and TRP had a significant effect on the digestive function of the small intestine, so the common adverse symptoms after taking these drugs were abdominal discomfort or pain, diarrhea, nausea or vomiting, which were consistent with the drug instructions. Other oral drugs, such as vitamin B₁, α-tocopherol, etc., have relatively low protein binding rates to TRP, indicating that the binding of drugs to TRP has little effect on the digestive function of the small intestine. This is consistent with the fact that the occasional adverse symptoms after taking these drugs are anorexia and constipation, and the discomfort symptoms are relatively mild.

From the results of Table 1 and Table 2, it can be seen that the effects of common oral drugs on digestive function predicted by the protein binding rate of digestive proteases to drugs are consistent with the adverse drug reaction symptoms recorded in the drug instructions. Therefore, it is feasible to infer the effect of drugs on digestive function according to the protein binding rate between drugs and digestive proteases. In addition, the effect of digestive protease on the efficacy of common oral drugs can also be simply predicted. most of the effects of pepsin on drug efficacy can be ignored, and TRP has a greater impact on the efficacy of some drugs, such as astaxanthin, vitamin B₁, vitamin B₉ and so on.

In fact, it is a very complex process for drugs to be absorbed into the human body. Many drugs are actually absorbed in the digestive system in the stomach and small intestine. The spectral experiment is only a simple estimation of the binding rate of the system according to the drug dose, the binding constant and the number of binding sites of the drug and protein under simulated physiological conditions. This method is compared with equilibrium dialysis [35], high performance liquid chromatography [36], ultrafiltration [37], microdialysis [38] and liquid chromatography-tandem mass spectrometry [39], which are commonly used to study the binding rate of drugs and proteins. There will be some errors in the binding constant and the number of binding sites, resulting in a small number of adverse reactions predicted by this method are not completely consistent with the adverse reactions reported by the drugs. However, the experimental equipment of these commonly used binding rate research methods is expensive, the test period is long and the concentration range of the test drug is narrow. The method of using spectroscopy to study the protein binding rate is simple to operate and has a wide range of application, so this method is desirable. As far as the efficacy is concerned, there are many physiological factors that affect the efficacy, such as digestive system factors, circulatory system factors and disease factors. The effect of enzymes in gastric juice and intestinal juice on the efficacy belongs to the influence of digestive system factors on the efficacy, accounting for only a small part of the impact. Therefore, simply using the combination of digestive enzymes and drugs to study the drug efficacy and the effect of drugs on human digestive function, the conclusion will be different from the actual situation.

Conclusion

Under simulated physiological conditions, the binding rate of drugs to digestive proteins can be easily and quickly obtained by spectroscopy and the effects of some new oral drugs on the digestive function of human stomach and small intestine can be simply predicted by this method. Because the action process of drugs in human body is a very complex mechanism, there is a certain error between the predicted results of a small number of oral drugs and the actual reported results. The method provided in this paper provides a simple reference for the rapid judgment of the clinical safety of drugs, and has a certain reference value for the rapid judgment of the effect of new drugs on gastrointestinal function.

Acknowledgements

The authors gratefully acknowledge the financial support of National Science Foundation of China (Grant no. 21375032).

Figure 1: Binding model of levofloxacin and PEP.

Figure 2: Binding model of enrofloxacin and TRP.

1.       Gole A, Sastry M, Dash C, Rao M (2000) Encapsulation and biocatalytic activity of the enzyme pepsin in fatty lipid films by selective electrostatic interactions. Chemical Communications 16: 297-298.

2.       Spelzini D, Peleteiro J, Picó G, Farruggia B (2008) Polyethyleneglycol-pepsin interaction and its relationship with protein partitioning in aqueous two-phase systems. Colloids & Surfaces B Biointerfaces 67: 151-156.

3.       Shen LL, Xu H, Huang FW, Li Y, Xiao HF, et al. (2015) Investigation on interaction between Ligupurpuroside A and pepsin by spectroscopic and docking methods. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy 135: 256-263.

4.       Zhang HM, Cao J, Fei ZH, Wang YQ (2012) Investigation on the interaction behavior between bisphenol A and pepsin by spectral and docking studies. Journal of Molecular Structure 1021: 34-39.

5.       Wang RY, Xie YZ, Zhang YH, Kang XH, Wang XZ, et al. (2013) Comparative study of the binding of pepsin to four alkaloids by spectrofluorimetry. Spectrochim Acta A Mol Biomol Spectrosc 108: 62-74.

6.       Song H, Chen CY, Zhao SL, Ge F, Liu DQ, et al. (2015) Interaction of gallic acid with trypsin analyzed by spectroscopy. Journal of Food & Drug Analysis 23: 234-242.

7.       Zhang HM, Zhou QH, Cao J, Wang YQ (2013) Mechanism of cinnamic acid-induced trypsin inhibition: a multi-technique approach. Spectrochim Acta A Mol Biomol Spectrosc 116: 251-257.

8.       Wang RY, Kang XH, Wang RQ, Wang R, Dou HJ, et al. (2013) Comparative study of the binding of trypsin to caffeine and theophylline by spectrofluorimetry. Journal of Luminescence 138: 258-266.

9.       Li H, Pu JJ, Wang Y, Liu C, Yu J, et al. (2013) Comparative study of the binding of Trypsin with bifendate and analogs by spectrofluorimetry. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy 115: 1-11.

10.    Koutsopoulos S, Patzsch K, Bosker WTE, Norde W (2007) Adsorption of Trypsin on Hydrophilic and Hydrophobic Surfaces. Langmuir the Acs Journal of Surfaces & Colloids 23: 2000-2006.

11.    Zeng HJ, Wang YP, Yang R, You J, Qu LB (2016) Inhibitory effects of daidzein and genistein on trypsin: insights from spectroscopic and molecular docking studies. International Journal of Biological Macromolecules 89: 336-343.

12.    Liu YY, Zhang GW, Liao YJ, Wang YP (2015) Binding characteristics of psoralen with trypsin: Insights from spectroscopic and molecular modeling studies. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy 151: 498-505.

13.    Liu BS, Cao SN, Li ZY, Zhong BH (2013) A Forecast of The Binding Rate of Drug to Plasma Protein by Fluorescence Quenching Method. Chinese Journal of Luminescence 34: 488-493.

14.    Zeng HJ, Li MT, Li Q, Hu FF (2016) Mechanism of Interaction Between Levofloxacin and Pepsin by Spectroscopic and Molecular Docking Methods. Chinese Journal of Luminescence 37: 481-486.

15.    Lian SQ, Yang DZ, Zheng XX, Lian Q, Wang GR (2013) Study on Interaction of Gatifloxacin with Pepsin by Fluorospectrophotometry. Physical Testing & Chemical Analysis 49: 253-255.

16.    Li BH, Liu YF, Sun XM, Wang X (2011) Study of Interaction between Baclofen and Pepsin by Spectroscopy Methods. Journal of South-Central University for Nationalities (Natural Science Edition) 30: 21-24.

17.    Ji WR, Cheng K, Ge K, Shao CY (2014) Spectroscopic Study on the Interaction Between Oxacillin Sodium and Pepsin. Journal of Huaibei Normal University 3: 24-29.

18.    Liu C, He WJ, Chen SS, Chen J (2017) Interactions of digestive enzymes and milk proteins with tea catechins at gastric and intestinal pH. International Journal of Food Science & Technology 52: 247-257.

19.    Li X, Li P (2016) Study on the interaction of β‐carotene and astaxanthin with trypsin and pepsin by spectroscopic techniques. Luminescence the Journal of Biological & Chemical Luminescence 31: 782-792.

20.    Ma XL, He JW, Huang YM, Xiao Y, Wang Q, et al. (2016) Investigation and comparison of the binding between tolvaptan and pepsin and trypsin: Multi‐spectroscopic approaches and molecular docking. Journal of Molecular Recognition 30:1-9.

21.    Nan G, Wang P, Sun J, Lv J, Ding M, et al. (2016) Spectroscopy and molecular docking study on the interaction of daidzein and genistein with pepsin. Luminescence 31: 1524-1531.

22.    Pathak M, Sharma D, Sharma N, Sharma M (2018) Spectroscopic and thermodynamic studies of the binding mechanism of metformin to pepsin. Journal of Molecular Structure 1166: 183-189.

23.    Lian S, Wang G, Zhou L, Yang D (2013) Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin. Luminescence the Journal of Biological & Chemical Luminescence 28: 967-972.

24.    Jia Y, Yang HQ, Guo LQ, Wang Q, Huang YM, Li H (2016) Investigation of interaction between cefixime and pepsin by spectroscopic methods. Chemical Research & Application 28: 673-680.

25.    Liu BS, Wang JJ, Chen L, Li ZY, Ma LH, et al. (2018) Study of Interaction Between Cefetamet Pivoxil and Pepsin by Spectroscopic and Molecular Docking. Chinese Journal of Luminescence 39: 236-243.

26.    Feng JP, Chen X, Wang RL, Yin DT (2012) Assay of the pepsin content in the various gastric juice. China Medical Engineering 2012: 3-5.

27.    Wang GK, Yan CL, Lu XM, Cheng DJ, Lu Y (2009) Study on the Interaction of Ciprofloxacin Hydrochloride with Trypsin by Spectroscopic and Molecular Modeling Methods. Acta Chimica Sinica 67: 1967-1972.

28.    He J, Wu SQ, Li FX, Chen XG, Wu ZD (2004) A study of the interaction between fluoroquinolone antibiotics and proteins by fluoresence quenching measurements. Journal of Lanzhou University (Natural Sciences) 40: 54-61.

29.    Lu Y, Wang GK, Lu XM, Lv J, Xu MH, Zhang WW (2010) Molecular mechanism of interaction between norfloxacin and trypsin studied by molecular spectroscopy and modeling. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy 75: 261-266.

30.    Gao Y, Shao C, Ji W (2013) Studies on the Binding Mechanism of VB₁ and VB₉ with Trypsin. American Journal of Analytical Chemistry 4: 771-775.

31.    Li X, Ni T (2016) Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods. Journal of Biological Physics 42: 415-434.

32.    Zhao W, Lu S, Diao JZ, Lei F, Xie G, Liu KW (2006) Spectra Study on the Interaction between Colchicine and Trypsin. Natural Product Research & Development 18: 369-372.

33.    Deng J, Jia X, Yang J, Liu W, Qiu XD, et al. (2006) Spectroscopic Study on the Interaction between Rutin and Trypsin. Natural Product Research & Development 18: 968-971.

34.    Schiller C, Fröhlich CP, Giessmann T, Siegmund W, Minnikes H, et al. (2005) Intestinal fluid volumes and transit of dosage forms as assessed by magnetic resonance imaging. Alimentary pharmacology & therapeutics 22: 971-979.

35.    Liu X, Chen XH, Liu WT, Zhang J, Bi KS (2007) Determination of plasma protein binding rate of arbidol. Chinese Journal of New Drugs & Clinical Remedies 26: 115-119.

36.    Si LQ, Li G, Chen Y, Li F, Fu SJ, Huang JG (2006) High-performance liquid chromatographic determination MELE concentration and the binding rate of MELE to human plasma proteins. Chinese Journal of Hospital Pharmacy 26: 253-255.

37.    Ye Y, Zhou LL, Yan YL, Huang QJ (2010) Determination of plasma protein binding rate of tetramethylpyrazine phosphate by ultrafiltration. Journal of Chinese Medicinal Materials 33: 1282-1285.

38.    Zhang YF, Zhou LL, Li R (2006) Determination in vitro of rat plasma protein binding rate of sinomenine by using microdialysis method. Acta Pharmaceutica Sinica 41: 909-912.

39.    Yong H, Hui C, Lin Z (2011) Determination of Plasma Protein Binding Rates of Protocatechuic Acid, Isoorientin and Scutellarin in Compound Hongcao Freeze-Dried Powder for Injection by Ultrafiltration Combined with UPLC-ESI-MS/MS. Chinese Pharmaceutical Journal 46: 1200-1204.