DOI: 10.31038/JPPR.2022544

Abstract

Glimepiride, a second generation of sulfonylureas, is used in treating diabetes mellitus. The monograph does not include digestive enzymes, such as pancreatin, in the description of its dissolution tests. Because its aqueous solubility values in literature are inconsistent, this study firstly determined it. In order to gain insight how a glimepiride tablet acted in the gastrointestinal tract, a three stage dissolution study tested two commercial tablets. Pancreatin powder was added versus not added in the beginning of Buffer Stage 1. HPLC quantified samples at 228 nm. The US Similarity Factor ratified that (a) similar between commercial Products A and B release profiles (f₂ ≥ 50), and (b) different in drug releases between dissolution media containing versus not containing pancreatin. This study also found that the drug amount in the dissolution medium containing pancreatin decreased from 22 h to 24 h, while the same decline was not observed in the control group. Drug decomposition occurred in the pancreatin group was further expedited. After UV spectrophotometric scan and HPLC characterization of the 24 h dissolution samples, a small un-identified chromatographic peak was recorded at 235 nm and 240 nm respectively. This study highlights how endogenous mediated enzymes affects the free concentrations of lipophilic drugs.

Keywords

Glimepiride, Pancreatin, Three-stage dissolution, FDA similarity factor f₂, Release kinetics, Solubility descriptor

Introduction

For oral drug delivery to be successful, the aqueous solubility of the drug compound in the GI system should be either known from literature or assessed in the lab to determine if modifications are needed. Lipase is produced primarily in the pancreas. There is no lingual lipase in human [1]. The activity of the gastric lipase is not low. It is also stable in acid pH. Actually, its maximum activity is at pH 5, which is the immediate fed stage pH for many cases. The pH of a fed stomach lowers thirty min or so after food intake due to the secretion of gastric acid by parietal cells, which aids in food digestion, absorption of minerals, and control of harmful bacteria. Gastric lipase is stable in acid pH, but is not as active after the gastric pH is lowered. After being mixed with gastric lipase, powerful gastric juice and 2-4 h of peristaltic contractions (mechanical digestion churn), approximately 30% of fats/triglycerides in the food bolus is broken down into diglycerides and fatty acids in partially digested food mass (known as chyme). When the chyme passes to the small intestine, pancreatic lipase is secreted by pancreas into the duodenum through the duct system of the pancreas to continue the fat digestions [1]. The third lipase is hepatic lipase, which is produced by the liver [1]. Hepatic lipase is a lipolytic enzyme that contributes to the regulation of plasma triglyceride levels, but not in the intestinal lumen [2]. Over the past 50 years, dissolution testing has also been employed for different purposes, such as a quality assurance/quality control procedure, in research and development to detect the influence of critical manufacturing variables and in comparative studies for in vitro–in vivo correlation [2]. However, most compendium monographs describing dissolution tests as a part of performance tests do not include digestive enzymes in the media. Many are defined as one-stage (single medium pH) tests to serve quality control purposes. However, the pH of the gastrointestinal medium is a digestion parameter. Thereby, multistage in vitro dissolution tests should also be conducted during the preformulation and formulation stages. This project aimed at the examination of how pancreatin may impact the release of a lipophilic drug from its oral dosage form with food intake. Glimepiride (a long-action second generation of sulfonylurea) was chosen as the model drug, because its side chains are known to be less polar. Glimepiride is usually administered by mouth once a day by taken with the first meal of the day. The current market products for glimepiride are in tablet dosage form with strength availability as 1-, 2-, 3-, 4-, 6- and 8-mg. Unfortunately, the aqueous solubility values of glimepiride in the literature are not consistent, ranging from partly miscible to < 0.004 mg/mL [3-6]. That was why we set to test its aqueous solubility followed by the exploration of the solubility in different physiological pH media. Since the oral cavity, stomach, and small intestine function as three separate digestive compartments with differing chemical environments, a three-stage in vitro dissolution study is believed to be superior to one stage to answer the scientific questions of this study “How pancreatin may impact the release of a lipophilic drug?”, although one pH stage dissolution contributes in quality control. The statement regarding the quantity of pancreatin and the test solution for the pancreatin to add may be found in USP43-NF38 Intestinal Fluid, Simulated, TS in Reagents and Reference Tables > Solution [7]. This test solution is prepared first by dissolving 6.8 g of monobasic potassium phosphate in 250 mL of water, mix, and add 77 mL of 0.2 N sodium hydroxide and 500 mL of water. Second, add 10.0 g of pancreatin, mix, and adjust the solution with either 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to a pH of 6.8 ± 0.1. Last, dilute with water to 1000 mL [7]. Next, we searched for a protocol how to prepare each stage medium for the three-stage dissolution study and found the required description in Mesalamine Delayed-Release Tablets Monographs [7]. By doing so, the second project aim, which was to determine whether pancreatin impacts a highly lipophilic oral drug using in vitro dissolution study, became feasible. The dosage forms were two commercial glimepiride tablets in two different strengths, 2- and 4-mg. The percent of their in vitro drug release data was next entered an Excel worksheet according to the U.S. FDA Similarity Factor equation to compute into f₂ values for comparison [8-10].

Materials

(a) Model Drug and its Commercial Products

Glimepiride (TCI, JPOSL-FH) powder was purchased from VWR (Radnor, PA). Amaryl 2-mg and 4-mg Tablets (Lot FT00, products of Sanofi-Aventis), and Glimepiride 2-mg and 4-mg Tablets USP (Lots P2100682 and P2101988, Products of Accord Healthcare) were purchased from Cardinal Health (Dublin, OH). Amaryl Tablets were coded as Product A, while Glimepiride Tablets USP was coded as Product B.

(b) Reagents and Supplies

Monobasic sodium phosphate (Ward’s Science, AD-20224), phosphoric acid (VWR, 18H104005), acetonitrile (OmniSolv, 59135), tribasic sodium phosphate (Alfa Aesar, 10220493), hydrochloric acid (VWR, 2017062956), sodium hydroxide (EMD, 49124919), pancreatin (Ward’s Science) were purchased from VWR.

Methods

Glimepiride Solubility Study

The Glimepiride aqueous solubility values found from four different literature are not consistent ranging from partly miscible to < 0.004 mg/ml. Besides, “partly miscible” and “very poor solubility” are not the terms used in Solubility Table (Table 1). Therefore, its aqueous solubility was determined first. Ten mg of glimepiride powder was added into a water tank containing 10 liters of purified water with occasional shaking for up to 48 h. Three 10-mL samples were taken from different content locations of the 10-L tank. Sample of 5 mL was transfer into a 10 mL syringe and filtered through 0.2-micron nylon membrane syringe filter prior to being subject to UV-Visible spectrophotometer (Cary 50, Agilent Technologies) and HPLC for analyses. The other 5 mL were kept in culture tube without filtration. All experiments were performed in triplicate.

Glimepiride High-Performance Liquid Chromatography

The HPLC assay was adopted from Glimepiride Tablets Monograph in USP43-NF38, 2022 [7]. Diluent was acetonitrile and water in 9:1 v/v. The stock solution was prepared as 1 mg/mL Glimepiride. Standard solutions for building between day and within-day standard curves were constructed by taking aliquots of stock solution and diluting them with Diluent to prepare into the six standard concentrations between 0.004, 0.02, 0.1, 0.2, 0.5 and 1 mg/mL, and acquired the AUC of each standard concentration. The averaged AUCs of the same standard concentrations (n=3) were construct into a grand calibration plot. However, a shorter standard curve was plotted from 0.004 to 0.1 mg/mL for use in this project due to the low drug strengths (2 mg and 4 mg). The LC systems was Agilent Technologies Series 1260 Infinity consisted of an auto-sampler, a thermostatic column compartment, a degasser, a variable wavelength detector, and a quaternary pump with Chemstation^® software). After testing the peak shapes and retention times of three HPLC columns, chromatographic conditions adopting from USP Glimepiride Monograph [7] were eventually set as LiChrosorb RP18 column, column temperature 35°C, injection volume 20 μL, flow rate 1.2 mL/min, detection wavelength 228 nm, run cycle 12 min, dissolving 0.5 g of monobasic sodium phosphate in 500 mL of water, adjusting with 10% phosphoric acid to a pH of 2.1-2.7 and added 500 mL of acetonitrile to mix and serve as mobile phase.

Glimepiride UV-Visible Spectrophotometer

The wavelength ( λ_max ) where glimepiride had the highest absorption was determined by scanning the samples taken from the 10 mg glimepiride dissolved in 10 liters of water for 48 h using UV-Visible spectrophotometer (Cary 50, Agilent Technologies). If glimepiride is not soluble in water, these samples would further mixed with acetonitrile in two different ratios (water and acetonitrile in 1:4 and 1:9 v/v) and scanned from 200 to 800 nm to see whether organic solvent aided in glimepiride solubility.

Three-Stage In Vitro Dissolution Study Stimulating Human Gastrointestinal Fluids and Transition Time

Three different performance tests are described in Glimepiride Tablet Monograph USP43-NF38. These tests are similar in: (1) use of Apparatus 2 (Paddle), (2) pH 7.8 phosphate buffer (singe medium stage), and (3) 75 rpm stirring rate at 37.0 ± 0.5°C, but are different in dissolution times (15 min, 45 min, and 20 min respectively) [7,8]. A single medium pH performance test is good for use in QA/QC, but is not possible to assess whether pancreatin influences glimepiride release from a tablet and dissolved in the various simulated gastrointestinal media. Three dissolution stages containing one Acid Stage and two Buffer Stages modified from Mesalamine Delayed-Release Tablets [7]. The rational is that pancreatin should be added in the beginning of Buffer Stage 1 to mimic the enzyme being secreted from the pancreas and discharged from pancreatic duct into duodenum. Glimepiride Tablets Monograph only stated to use the phosphate medium at 7.8 pH, which is not the pH value of Buffer Stage 1 [7]. The 3-stage dissolution test with pancreatin added at the beginning of Buffer Stage 1 used in this study as the experimental group is described briefly as following. The medium preparation for each of the three stages in application to the control group were the same as that of the experimental group except without the addition of pancreatin.

Medium Preparations and Progression of Dissolution Study

The Gastric Fluid – Simulated in Reagents and Reference Tables > Solutions > Test Solutions and Indicator Solutions > Test Solutions only describe how to prepare as pH 1.2 without further information regarding fasting versus fed state. So are the performance tests of several tablet and capsule monographs. Since the smallest volume to set for Distek Dissolution Apparatus was 500 mL, we assumed it was for fasting state. We, thereby, selected 750 mL 0.1 N HCl into a 1-liter dissolution vessel and warmed to 37.0 ± 0.5°C as a medium size postprandial condition. A glimepiride tablet either 2- or 4-mg was placed into the vessel medium. The paddle stirred at 75 rpm for 4 h. At the end of this 4-h, Buffer Stage 1 was initiated by adding 200 mL of 0.20 M Tribasic Sodium Phosphate to adjust the pH to 6.4, while the paddle continued to stir at 75 rpm. For each pancreatin containing group (also called as experimental group, or experimental medium), 10.88 g of pancreatin was added (after 200 mL of 0.20 M tribasic sodium phosphate was added and mixed the 750 mL of 0.1 N HCl well in a dissolution vessel) to simulate the small intestinal fluid when food chyme arrives the duodenum and pancreatin secretion is activated. The color turned from clear to yellowish cloud. Figure 1 used 1-L beaker instead of 1 L of dissolution vessel to display phosphate buffer pH 6.4 after 10.88 g of pancreatin was added as the experimental group. At the end of the 4-h Buffer Stage 1, sodium hydroxide 2 N (that was, 1.04 g NaOH with a sufficient amount of water to make into 50 mL solution) was further added to bring the total medium volume at 1000 mL and pH was 7.4 (Stage 2 Buffer). The dissolution apparatus continued to stir during the Buffer Stage 2 for 16 h to simulate large intestinal pH and transition time.

Figure 1: Pancreatin powder in the ordered container with label as well as in a weight boat (right), and the buffer stage 1 medium of an experimental group in the beaker (left). See text.

The sampling schedule was 2 h, 4 h (Acid Stage), 6 h, 8 h (as 2 h and 4 h during Buffer Stage 1, pH 6.4), 9 h, 22 h, and 24 h (at 1 h, 14 h and 16 h during Buffer Stage 2, pH 7.4). Five mL of dissolution medium were collected from each vessel at the designated sampling time and replenished with equal volume of blank (same medium of each stage, but contained no drug). The first 2 mL out of these 5 mL were filtered through a 0.22-micron syringe filter were discarded. The remaining 3-mL was continued to filter through the syringe filter, collected into a culture tube, and capped. One mL of such sample was later placed into a HPLC vial for analysis when a 24-h dissolution cycle ended. The recorded AUCs were converted into concentrations using an prebuilt standard curve averaged out of 3 runs (Section 3.2) and further computed into the dissolved amount (in mg) as well as the percent of drug release. The drug dissolution profiles were constructed with the averaged percent of release (n=3) in the Y coordinate (ordinate) plotted against the dissolution time (in h) in the X coordinate (abscissa).

Tablet Dissolution Study

The feature was a 2 x 2 x 2 factorial design with three main effects. The first number in 2 x 2 x 2 stands for “two payloads 2-mg vs. 4-mg”. The second number stands for “with vs. without pancreatin”. The third number of 2 x 2 x 2 stands for “Tablet Product A and Product B”. A pretest showed all four glimepiride tablets (Products A and B in 2-mg and 4-mg) sank to the bottom of a dissolution vessel after a tablet was dropped into a dissolution vessel. They neither rise nor were hit by stirring paddles during tests. Therefore, no tablet sinker or use of dissolution apparatus 1 (basket method) was required.

Glimepiride Powder Study

Glimepiride powder of 2 mg and 4 mg as controls were subject to in vitro dissolution study using USP Apparatus 2 (Distek Premiere, model, 5100) at 75 rpm, 37.0 ± 0.5°C. The medium preparations between the experimental group and control group, dissolution conditions and sampling schedule were at same as Section 3.4.1.

The U.S. FDA Similarity Factor f₂ value

The equation of U. S. Similarity Factor, f₂ value used to compare two dissolution profiles is available in Guidance for Industry Dissolution Testing of Immediate Release Solid Oral Dosage Forms as well as M9 Biopharmaceutics Classification System-Based Biowaivers [9-12]. The Similarity Factor f₂ value, compared two dissolution profiles at a time using the following formula:

(Equation 1)

f₂ is the similarity factor;

n is the number of time points;

R_t is the mean percent reference drug dissolved at time t after initiation of the study;

T_t is the mean percent of test drug dissolved at time t after initiation of the study.

Two dissolution profiles are considered similar when the f₂ value is ≥ 50.

The evaluation of the similarity factor is based on the following conditions [9]:

1. A minimum of three-time points (zero excluded)
2. The time points should be the same for the two products
3. Mean of twelve individual values for every time point for each product.
4. No more than one mean value of ≥ 85% dissolved for any of the products.
5. To allow the use of mean data, the coefficient of variation should not be more than 20% at early time points (up to 10 minutes) and should not be more than 10% at other time points.
6. Two dissolution profiles are considered similar when the f₂ value is ≥ 50. When both test and reference products demonstrate that ≥ 85% of the label amount of the drug is dissolved in 15 minutes, comparison with an f₂ test is unnecessary, and the dissolution profiles are considered similar [9-12].

Results

Glimepiride Solubility and Descriptor Determination

After the three samples being collected from the 10-L tank containing 10 mg of glimepiride and 10 L of water which was allowed to dissolve up to 48 h in each experiment (Section 3.1) and subject to HPLC analysis, only a very small drug peak at limit of quantification (LOQ) magnitude out of the three sample injections was recorded (Figure 1a). The retention time of this small peak (Figure 2a) corresponds to that of the glimepiride peaks in a 0.04 mg/mL standard sample (8.6 min, Figure 2c). But the other two samples taken from the 10-L tank containing 10-mg glimepiride in 10-L water that did not show chromatographic peaks, we thereby took 1 mL out of the 5 mL unfiltered sample to dilute with 9 mL acetonitrile and then filtered to examine whether glimepiride was present but not soluble enough in water. A small but clear peak from each of these two injections was recorded (AUC < 3, Figure 2b). The retention times of these peaks also corresponded to the retention time of the glimepiride standard sample chromatogram at 0.04 mg/mL (8.6 min, Figure 2c). All solubility experiments were performed in triplicates.

Figure 2: The chromatograms of glimepiride aqueous solubility study illustrated that (a) a very small peak at the limit of quantification (LOQ) was recorded from one out of three sample chromatograms after 10 mg of glimepiride was placed into 10 L of water (no acetonitrile) for 48 h. (b) The drug peak appeared after a sample which did not showed drug peak in Figure (a) was further diluted with 9 parts of acetonitrile. (c) A 0.04 mg/mL standard sample prepared according to compendium LC method which Diluent was acetonitrile and water in 9:1 ratio.

Next task was to determine the term of glimepiride in the Description and Solubility Tablet (Table 1). The glimepiride aqueous solubility may now elucidate as “10 mg (0.01 g) of glimepiride requires more than 10 L (10,000 g) of water to dissolve.” The calculation showed

(Equation 2)

Based on one part of solute requires over 1 million part of solvent to dissolve, the descriptive terms in Description and Solubility (Table 1), glimepiride belongs to the category of “practically insoluble in water”.

Table 1: Description and solubility [7]

Descriptive Term	Parts of Solvent Required for 1 Part of Solute
Very Soluble	<1
Freely soluble	1-10
Soluble	10-30
Sparingly soluble	30-100
Slightly soluble	100-1000
Very slightly soluble	1000-10,000
Practical insoluble	>10,000

High Performance Liquid Chromatography

A grand standard curve was built based on averaging the AUCs of three individual standard curves from 0.004, 0.02, 0.1, 0.2, 0.5 to 1 mg/mL. Due to the low strengths of the tablets (2 mg and 4 mg) investigated in up to 1 L of dissolution medium in this project, a working range standard curve (containing 0.004, 0.02 and 0.1 mg/mL) was further plotted. The trendline was

AUC=55550 x Concentration (mg/mL) + 2.5394 R²=1 (Equation 3)

In Vitro Dissolution Results of Two Commercial Tablets in Two Strengths

The AUC collected from 7 sampling chromatograms in the 3-stage in vitro dissolution studies of two tablet groups (Products A and B) and 2 strengths (2 mg and 4 mg) were computed into percent of release as Mean ± SD using the aforementioned work range standard curve in Session 4.1 (0.04, 0.02 and 0.1 mg/mL) plus correction factor adjusted for volume withdrawal at each sampling point (Table 2). These percent of release were also plotted against time (in h) into X-Y plot with SD bars (Figure 3). Figures 3a to 3d showed that the percent of glimepiride releases in the experimental groups (pancreatin-containing) were lower than the control groups during the Buffer Stage 2 (pH 7.4 from 9 h to 22 h) in both tablets, Products A and B of the same strength. The differences between experimental and control groups are greater in 2 mg tablets (Figure 3a and 3c) than in 4 mg tablets (Figures 3b and 3d). Furthermore, the release decreased from 22 h to 24 h in the experimental group, but the release increased from 22 h to 24 h in the control group (Figure 3). Bar charts of the glimepiride dissolution in 0-8 h, 8-9 h, 9-22 h, and 22-24 h sampling intervals of Products A and B (not cumulative from 0 h to 24 h) were next constructed into bar charts (Figure 4). The percent increased or decreased in Figure 4 are among the four intervals of 0-8 h, 8-9 h, 9-22 h, and 22-24 h, not like the cumulative percent of release being reported in Figures 3. The FDA Similarity Factor, f₂ values, were further computed.

Table 2: Comparison of 24-h in vitro glimepiride release in percent (Mena ± SD, n=3) from 2-mg and 4 mg commercial Tablets A and B (a) 2 mg, and (b) 4 mg without versus with pancreatin added in the beginning of Buffer Stage 1.

A		Tab A 2 mg				Tab B 2 mg
Dissolution Stage	Time (h)		Without Pancreatin	With Pancreatin	Without Pancreatin		With Pancreatin
Acid	0		0	0	0		0
(0.1 N HCl)	2		0	0	0		0
	4		0	0	0		0
Buffer 1	6		16.6 ± 10.4	27.3 ± 16.2	31.3 ± 5.0		29.1 ± 2.8
(pH 6.4)	8		37.4 ± 8.9	46.5 ± 5.5	36.9 ± 2.6		31.2 ± 3.7
Buffer 2	9		112.6 ± 11.9	97.9 ± 41.4	112.2 ± 13.2		69.1 ± 5.7
(pH 7.4)	22		119.3 ± 11.8	120.5 ± 8.7	119.8 ± 13.2		106.7 ± 6.2
	24		111.1 ± 7.8	109.9 ± 1.66	123.9 ± 12.8		76.7 ± 23.5

B		Tab A 4 mg		Tab B 4 mg
Dissolution Stage	Time (h)	Without Pancreatin	With Pancreatin	Without Pancreatin	With Pancreatin
Acid	0	0	0	0	0
(0.1 N HCl)	2	0	0	0	0
	4	0	0	0	0
Buffer 1	6	16.2 ± 1.3	17.3 ± 1.8	10.9 ± 1.6	10.9 ± 4.0
(pH 6.4)	8	17.6 ± 1.0	19.4 ± 1.0	17.4 ± 1.8	23.0 ± 6.7
Buffer 2	9	89.8 ± 2.3	77.3 ± 19.3	90.5 ± 3.6	66.9 ± 20.8
(pH 7.4)	22	96.1 ± 4.0	101.7 ± 11.6	101.1 ± 4.9	97.3 ± 7.2
	24	102.9 ± 2.6	85.9 ± 17.4	104.8 ± 7.9	90.5 ± 11.6

Figure 3: The dissolution kinetic profiles in three medium pH stages of pancreatin and no pancreatin groups diverged at 2-mg strength starting 8 h (a and b) , however, this difference narrowed at 4-mg strength in Product A as well as in Product B (c and d). The most significant point was at 9 h samples, which was 1 h after the medium pH adjusted from 6.4 to 7.4.

Figure 4: Bar charts of the glimepiride dissolution percent in 0-8 h, 8-9 h, 9-22 h, and 22-24 h, four sampling durations of Products A and B (differed from the cumulative percent in Figure 4): (a) 2-mg tablets (Products A and B) in control group (pancreatin was not added in Buffer Stage 1), (b) 2-mg tablets in experimental group, (c) 4-mg tablets in control group, (d) 4-mg tablets in experimental group.

The USP Similarity Factor, f₂ Values

The f₂ values which compared two groups of the same strength at a time were computed according to Equation 1 and their results are listed in Table 3. Both Products A and B are FDA-approved products. The experimental group data were plotted as the test group in Equation 1, and the control group data as the reference group. Time point, n, were from 1 to 7. The second column from the left side of Table 3 compared the differences between 2 mg Product A and 2 mg Product B in the control medium (without pancreatin) while the third column compared the 2 mg Products A and B in experimental medium (with pancreatin). The fourth and fifth columns compared the differences of 4 mg Products A and B in control medium (without pancreatin) and in experimental medium (with pancreatin). The results of Table 3 indicated Products A and B are similar in dissolution profiles (all f₂ values in seven sampling points were ≥ 50).

Table 3: FDA similarity factor: comparison made between Products A and B while keeping strength and medium preparation the same.

	2 mg Product A vs. Product B		4 mg Product A vs. Product B
Time (h)	WO Pancreatin	With Pancreatin	WO Pancreatin	With Pancreatin
2	100.0	100.0	100.0	100.0
4	100.0	100.0	100.0	100.0
6	73.5	74.3	83.3	83.3
8	68.6	69.9	77.3	75.6
9	60.8	64.3	65.2	66.9
22	57.3	59.9	60.6	61.7
24	55.3	58.5	58.1	59.4

Product A as test tablet, while Product B as reference tablet

In Vitro Dissolution Results of Drug Powder in Pancreatin Presence vs. Not Presence

We desired to find out whether the drug released from commercial tablets and present in the dissolution medium containing pancreatin was impacted by the excipients incorporated into making tablets. In this section, we took 4 mg of glimepiride powder into a dissolution vessel to conduct 3-stage dissolution study as the procedures described in Section 4.3 for commercial tablets. The resultant XY plots of glimepiride drug powder dissolution is listed as Figure 5. For the experimental group, HPLC did not record AUC during Acid Stage and Buffer Stage 1 until the glimepiride (drug powder alone) dissolved in Buffer Stage 2 medium for 22 h, which was much slower (Figure 5) than the commercial tablet or commercial tablets about 20-25% were released and dissolved by the end of Buffer Stage 1 (8 h, Figure 3). In the experimental group, glimepiride powder dissolved 29.8 ± 4.9% at 22 h, but decreased to 25.90 ± 6.5% at 24 h when pancreatin was added in the beginning of Buffer Stage 1. In the control group (without pancreatin), glimepiride powder dissolved was 30.5 ± 6.7% at 22 h and further increased to 30.8 ± 7.1% at 24 h (Figure 3). Never the less, t-test indicates this difference in the 24 h samples between the experimental and control groups was not significant (p=0.46) reflecting the solubility of glimepiride drug powder was very low in 0.1 N HCl as well as in phosphate buffer pH 6.4.

Figure 5: The profiles of 4 mg glimepiride powder in three stage dissolution study displayed that the glimepiride release amount decreased from 22 h to 24 h in the group with pancreatin being added in Buffer Stage 1, but not in the control group (no addition of pancreatin) (n=3).

Checking for wavelength other than 228 nm in Buffer Stage 2 (pH 7.4 Phosphate Buffer) Containing verses Not Containing Pancreatin

The experimental medium (containing pancreatin) of Products A and B showed the trend of the percent of glimepiride decreased from 22 h to 24 h dissolution samples, we further subjected the 24 h glimepiride powder dissolution sample containing pancreatin to UV Spectrophotometric scans. The results confirmed that there were absorbance in the experimental group (containing pancreatin) at 235 nm and 240 nm in addition to the drug peak at 228 nm. We next inject the 24-h drug powder experimental samples to HPLC after syringe filtration. An unidentified peak was recorded at 235 nm and 240 nm, respectively (Figures 6a and 6b).

Figure 6: When a 24-h sample (pH 7.4) was subjected to HPLC using (a) 235 nm, and (b) 240 nm as suggested by UV spectrophotometric scans, a small unidentified peak was recorded at the retention time of 8.8 min (a) and 8.55 min (b) respectively.

Discussion

Standard Preparations of Glimepiride Monograph and Glimepiride Tablets Monograph

The standard preparations in Glimepiride Monograph and Glimepiride Tablets Monograph in USP-NF 2022 are different. The Diluent to prepare Glimepiride standard solution is described as acetonitrile and water (4:1), while the Diluent prepared sample solution in Glimepiride Tablets Monograph Assay is acetonitrile and water (9:1). Furthermore, in the same monograph, the diluting solution for sample solution in the Dissolution Test 1 is methanol and water (1:1), while the preparation of standard solution is described as acetonitrile, methanol and water. The problem lies on that sample solution in the compendium contains only one organic solvent (acetonitrile), while the standard solution contains two organic solvents (methanol and acetonitrile). Because of so, we kept the diluent between sample solution and standard solution the same, acetonitrile and water in 9:1. The dissolution medium of this project contained no emulsifying agent or organic solvent. Surfactants belong to a family of emulsifying agents [13]. Pancreatin tested in this project is a commercial grade digestive enzyme containing lipase, not an emulsifying agent. Bile juice which was not studied in the project is known to contain surfactant. The mobile phase followed the description in Glimepiride Tablets [7] also contained no emulsifying agent.

Excipients in Two Commercial Tablets

The inactive ingredients (also called as excipients) and pharmaceutical functions of Product A and Product B are listed in Table 4. Both Excipients NF and Handbook of Pharmaceutical Excipients [14] regard sodium starch glycolate as a disintegrant. They do not regard sodium starch glycolate as an emulsifying agent(s) for glimepiride. Since glimepiride powder has been proven as a practically insoluble drug in the early phase of this project. Further, the three-stage 24 h dissolution studies displayed that after glimepiride powder underwent 2 h of 0.1 N HCl (acid stage), 4 h of Buffer Stage 1 and 16 h of Buffer stage 2, it only yielded 30.8 ± 7.1% of drug release in the control group (no pancreatin added in Buffer Stage 1), and 25.9 ± 6.5% for the (pancreatin containing) experimental medium group for the same time point (Figure 5). We, thus, further asked what inactive ingredient(s) made the glimepiride of commercial tablets dissolve completely at the ends of 24-h in vitro tests (Figure 3). Emulsifying agents may be divided into carbohydrates, proteins, high molecular weight alcohols, surfactants, and solids [13]. Since none of the excipients in the two studied tablets of this project is regards as emulsifying agent (Table 3) it is fair to speculate that the increase in glimepiride solubility was due to the presence of disintegrants added during tablet manufacturing. Product A contains two disintegrants (sodium starch glycolate and microcrystalline cellulose), while Product B contains one disintegrant (sodium starch glycolate – grade A). There was no detectable glimepiride dissolved during the 4-h Acid Stage window (Figures 4a to 4d). Figure 5 further displayed that the solubility of 2 mg Product A without pancreatin incremented from 0% to 17.6 ± 1.0%, and 4 mg Product A without pancreatin incremented from 0% to 37.4 ± 8.9% during the 4-h Buffer Stage 1 window (from 4 h to 8 h in pH 6.4). The highest dissolution rate occurred between 8 h to 9 h which was one hour after pH was adjusted to Buffer Stage 2 (from pH 6.4 to pH 7.4). For the 4-mg control groups, Product A was from 17.6 ± 1.0% to 89.8 ± 2.3%, and Product B was from 17.4 ± 1.8% to 90.5 ± 3.6%. However, from 8 h to 9 h in pancreatin-containing group was lower than the control groups of the same product and same strength (Figure 4). For the 4-mg with pancreatin group, Product A was from 19.4 ± 1.0% to 77.3 ± 19.3%, and Product B was from 23.0 ± 6.7% to 66.9 ± 20.8%. The glimepiride dissolution was further increased from 9 h to 24 h in the control group without pancreatin (Figure 4 blue curves). In the pancreatin-containing group glimepiride also dissolved from 9 h to 22 h, but declined between 22 h to 24 h (Figure 4 orange curves). This in vitro prediction possibly differs from in vivo outcomes in three aspects. First, in vitro dissolution stirring rate was set at 75 rpm for the entire 24 h to simulate the fed state, while in vivo bowel peristalsis is slower than 75 rpm, but has a longer duration than 24 h (up to 48 h to 72 h from the stomach to the colon depending on the diet and fat composition in food) prior to the elimination of the indigestible food residues. Second, in the in vivo condition, in addition to the endogenic bile salt released from the gall bladder, emulsifying agents may come from vegetable hydrocolloids and animal derivatives, such as lecithin and cholesterol. This in vitro project did not add any emulsifying agent into any of the three dissolution stage. Neither did Glimepiride Dissolution in USP-NF and FDA Database suggest so. The main aim was to explore how pancreatin in dissolution medium affects a highly lipophilic drug. Third, population variation (gender, sex, age) plus social, psychologic, and economic behaviors are not easy to simulate correctly in an in vitro study.

Table 4: Excipients of the Products A and B

	Function	Product A		Product B
Glimepiride	API	2 mg	4 mg	2 mg	4 mg
Lactose (Hydrous)	Binder, Diluent	√	√
Lactose Monohydrate	Binder, Diluent			√	√
Sodium Starch Glycolate	Disintegrant	√	√
Sodium Starch Glycolate (Type A Potato)	Disintegrant			√	√
Povidone	Binder	√	√	√	√
Microcrystalline Cellulose	Diluent, Disintegrant	√	√
Magnesium Stearate	Lubricant	√	√	√	√
Ferric Oxide Yellow	Coloring Agent	√		√
FD&C Blue #2 Aluminum Lake	Coloring Agent	√	√	√	√

Specific Binding and Non-specific Binding

Glimepiride is a lipophilic, small molecule chemical compound, not a lipid. The lipase enzyme as a part of pancreatin mixture. Since glimepiride is not a lipid, we do not expect the lipase in pancreatin may digest it, but the data showed that glimepiride in the dissolution medium containing pancreatin decreased from 22 h to 24 h. Judging from the data, pancreatin is speculated to function as a carrier molecule for glimepiride (substrate) to adsorb on its surface starting from the time that pancreatin (a digestive enzyme, a protein) was added in Buffer Stage 1. Such an enzymatic binding is non-specific and substrate (glimepiride) concentration dependent (Figure 3). When the binding is not specific, glimepiride may leave the pancreatin binding site and return to bind in the intestinal lumen during the process of transition down from the stomach into the intestine just like albumin functions as a nonspecific binding carrier for lipophilic drugs in the circulation. As to how many binding sites a pancreatin molecule has, and which is a strong binding site or weak binding site will need to be further investigated by isotope-labeled binding assays or other technology, such as simulation. Data indicated that the nonspecific binding occurs between pancreatin and glimepiride supported by the lower dissolution profile in the pancreatin containing medium group, especially at 9 h sample. If possible, we recommend (1) add another dissolution sampling point between 9 h to 22 h; (2) evaluate whether the nonspecific binding is substrate concentration dependent; and (3) determine whether the decrease of glimepiride percent from 22 h to 24 h is caused by digestion or by hydrolysis degradation at pH 7.4.

Checking the Wavelengths of Potential Glimepiride Degradants

After UV spectrophotometric wavelength scans of 24-h glimepiride tablet dissolution medium containing pancreatin showed absorbance at 235 nm and 240 nm in addition to the drug (glimepiride) absorbance at 228 nm, endeavors to further explore these two absorbance were made. USP Reference Standards Catalog, as well as Glimepiride monography, was consulted first. There are glimepiride-related compounds A, B, C, and D. Compound A is a glimepiride cis-isomer. Compound B is glimepiride sulfonamide. Compound C is glimepiride urethane. Compound D is a glimepiride 3-isomer. To find out whether these compounds are related to the peaks seen in 235 nm and 240 nm, more studies may be conducted to investigate whether any of these four compounds are related to the compounds whose peaks were seen at 235 nm and 240 nm.

Project End Points of FDA Similarity Factor f₂ Values

Dissolution profiles may be considered similar by virtue of (1) overall profile similarity, and (2) similarity at every dissolution sample time point. The dissolution profile comparison may be carried out using model independent or model dependent methods. [11] For model independent method, a difference factor (f₁) and the more commonly used, a similarity factor (f₂) may be used. Please refer to Guidance for Industry Dissolution Testing of Immediate Release Solid Oral Dosage Forms for (1) Model Independent Multivariate Confidence Region Procedure and (2) Model Dependent Approaches. Tablet 3 reported the Similarity Factor, f₂ values of this project followed the conditions of the evaluation of the US FDA Similarity Factor in model independent approach. These conditions may be found in a Methods section (Section 4.4). For example, (1) zero time point was excluded, (2) time points were the same for the two products, (3) the used of mean data which the coefficient of variation were not more than 20% at early time points (up to 10 minutes) and were not more than 10% at other time points, (4) the mean percent dissolved in ≤ 15 minutes were also not ≥ 85. If the computation of f₂ to follow the condition of “No more than one mean value of ≥ 85% dissolved for any of the products” instead of the computation made to the entire dissolution duration, (that is 24 h for this project), it should end at 9 h to determine the profile similarity and difference between a test product and a reference product. In this study, the first f₂ comparison was Product A as the test product and Product B as the reference product (Table 3). The rationale is that by then there was one mean value of ≥ 85% dissolved for any of the products had reached. Based on 2004 USP dissolution workshop and 2019 Dissolution Similarity Workshop, dissolution is a critical tool for the evaluation of generic drug products. The Similarity Factor, f₂ value is useful to reject or support waiver request. However, challenges are present [12].

Conclusion

The solubility of glimepiride is clearly pH dependent. Among the four studied medium, water, 0.1 N HCl, pH 6.4 and pH 7.4 phosphate buffers, glimepiride is practically insoluble in the first two (water and 0.1 N HCl), while is soluble in phosphate buffers. Glimepiride, the model drug, was not found decomposed when the three-stage dissolution medium contained no pancreatin, but decomposed slightly in both pure powder form as well as the studied commercial tablets from 22 h to 24 h. According to the results, the kinetics of lipophilic drugs are impacted by the release of digestive enzymes with meal consumption. This is most likely caused by the drug’s non-specific binding to substrates (pancreatin in this case). This conclusion was supported by the substantial decrease of the soluble fraction of glimepiride simultaneously during Buffer Stage 2 (pH 7.4, 8 h to 24 h) after pancreatin was added in the beginning of Buffer Stage 1 (4 h after acidic stage). We recommend that the role of pancreatin, a digestive enzyme, in the in vitro dissolution be investigated through the uses of other drug substance and formulation products.

References

1. N’Goma JB, Amara S, Dridi K, Jannin V, Carrière F, et al. (2012) Understanding the lipid-digestion processes in the GI tract before designing lipid-based drug-delivery systems. Therapeutic Delivery 3: 105-124. [crossref]
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4. Du B, Shen G, Wang D, Pang L, Chen Z, et al. (2013) Development and characterization of glimepiride nanocrystal formulation and evaluation of its pharmacokinetic in rats. Drug Delivery 20: 25-33. [crossref]
5. Lestari M, Indrayanto G (2011) Glimpiride in Profiles of Drug Substances, Excipients and Related Methodology, Surabaya, Indonesia. Academic Press 169-204.
6. Li H, Pan T, Cui Y, Li X, Gao J, et al. (2016) Improved oral bioavailability of poorly water-soluble glimepiride by utilizing microemulsion technique. Int J Nanomedicine 11: 3777-3788. [crossref]
7. S. Pharmacopeial Convention 2022. USP Monographs: Mesalamine Delayed-Release Tablets; Reagents and Reference Tables > Solutions > Gastric Fluid, Simulated, TS; Intestinal Fluid, Simulated, TS; Reagents and Reference Tables > Reference Tables > Description and Solubility; and General Chapters: <711> Dissolution. In: USP43-NF38. Rockville MD: U.S. Pharmacopeia, pg: 2805, 6234, 6230, 6945.
8. Brodkorb A, Egger L, Alminger M, Alvito P, Assunção R, Balance S, et al. (2019) INFOGEST static in vitro simulation of gastrointestinal food digestion. Nature Protocols 14: 991-1014. [crossref]
9. Guidance for Industry Dissolution Testing of Immediate Release Solid Oral Dosage Forms.
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14. Sheskey P, Hancock B, Moss G, Goldfarb D (2020) Handbook of Pharmaceutical Excipients. London: Pharmaceutical Press, 9^th ed.

DOI: 10.31038/JPPR.2022523

Abstract

Acute kidney injury is defined as any of the following (not graded): increase in serum creatinine by ≥0.3 mg/dl (≥26.5 μmol/l) within 48 hours; or increase in serum creatinine to ≥1.5 times baseline, which is known or presumed to have occurred within the prior 7 days; or urine volume <0.5 ml/kg/ hour for 6 hours. The goal of a staging system is to classify the course of a disease in a reproducible manner that supports accurate identification and prognostication and informs diagnostic or therapeutic interventions. Risk factors for acute kidney injury include environmental, socioeconomic and/  or cultural factors, as well as factors related to the process of care, acute exposures and patients themselves. Loss of kidney excretory function implies disturbances in the main function of the kidneys (maintaining homeostasis), for example, through excretion of metabolic waste products. Patients were categorized according to the treatment for acute kidney injury as follows: conservative treatment; surgical treatment; and renal replacement therapy. The use of isotonic saline as the standard of care for intravascular volume expansion to prevent or treat AKI is thus based upon the lack of clear evidence that colloids are superior for this purpose, along with some evidence that specific colloids may cause AKI, in addition to their higher costs.

Keywords

Acute kidney injury, Definition, Pathophysiology, Risk factors, Stages, Treatment

Introduction

Acute kidney injury (AKI), previously called acute renal failure, is characterized by an abrupt decline in renal function, resulting in an inability to secrete wastes and maintain electrolyte and water balance. AKI has clinical manifestations ranging from a small elevation in serum creatinine (SCr) levels to anuric renal failure. The severity of AKI is defined by Risk Injury Failure Loss End Stage (RIFLE) and Acute Kidney Injury Network (AKIN) criteria, which are based on the presence of increased SCr levels and/or a decreased urine output. The AKIN definition also emphasizes the change in SCr levels over a short period of time (within 48 h). This serious disorder may aggravate pre- existing kidney disease, thus leading to a rapid loss of renal function [1]. AKI is a broad clinical syndrome encompassing various etiologies, including pre-renal azotemia, acute tubular necrosis, acute interstitial nephritis, acute glomerular and vasculitic renal diseases, and acute post renal obstructive nephropathy. More than one of these conditions may coexist in the same patient and epidemiological evidence supports the notion that even mild, reversible AKI has important clinical consequences, including increased risk of death. AKI can thus be considered more like acute lung injury or acute coronary syndrome. Furthermore, because the manifestations and clinical consequences of AKI can be quite similar (even indistinguishable) regardless of whether the etiology is predominantly within the kidney or predominantly from outside stresses on the kidney, the syndrome of AKI encompasses both direct injury to the kidney as well as acute impairment of function [2-4]. AKI is defined as any of the following (not graded): increase in SCr by ≥0.3 mg/dl (≥26.5 μmol/l) within 48 hours; or increase in SCr to ≥1.5 times baseline, which is known or presumed to have occurred within the prior 7 days; or urine volume <0.5 ml/kg/hour (oliguria) for 6 hours. The cause of AKI should be determined whenever possible (not graded) [2]. AKI was defined as an absolute increase in SCr of at least 26.5 mmol/l (0.3 mg/dl) or a 50% or above increase in SCr from baseline according to the AKIN criteria [3]. The severity of AKI was defined by the AKIN staging criteria as follows: Stage I, SCr increase to 1.5-2 fold of baseline; Stage II, SCr increase to 2-3 fold of baseline; and Stage III, SCr increase to 3 fold of baseline or an absolute increase of 356.6 mmol/l (4.0 mg/dl) with an acute increase of at least 44.2 mmol/l (0.5 mg/dl). All patients who needed dialysis were categorized into Stage III [3]. The global burden of AKI-related mortality exceeds by far that of breast cancer, heart failure or diabetes, with mortality remaining high during the past 50 years. In general, the incidence of AKI is reported as either community-acquired or hospital-acquired AKI. In high-income countries (HIC), AKI is predominantly hospital- acquired, whereas community-acquired AKI is more common in lower-income settings. These patterns apply to both adults and children globally. In HIC overall, patients with AKI tend to be older, have multiple comorbidities, and have access to dialysis and intensive care if needed. Post-surgical or diagnostic interventions or iatrogenic factors are the main causes of AKI in HIC. However, in low-income settings, numerous community-acquired causes exist, such as sepsis, volume depletion, toxins (bites, remedies) and pregnancy [5].

Staging/Classification

The goal of a staging system is to classify the course of a disease in a reproducible manner that supports accurate identification and prognostication and informs diagnostic or therapeutic interventions. The group recognized that a number of systems for staging and classifying AKI are currently in use or have been proposed. The RIFLE (Risk, Injury, Failure, Loss, and End-stage kidney disease) criteria proposed by the ADQI group were developed by an interdisciplinary, international consensus process and are now being validated by different groups worldwide [6,7] (Table 1).

Table 1: Classification/staging system for acute kidney injury [4]

 

(a) Modified from RIFLE (Risk, Injury, Failure, Loss, and End- stage kidney disease) criteria [6]. The staging system proposed is a highly sensitive interim staging system and is based on recent data indicating that a small change in serum creatinine influences outcome. Only one criterion (creatinine or urine output) has to be fulfilled to qualify for a stage. (b) 200% to 300% increase=2- to 3-fold increase.

(c) Given wide variation in indications and timing of initiation of renal replacement therapy (RRT), individuals who receive RRT are considered to have met the criteria for stage 3 irrespective of the stage they are in at the time of RRT.

Risk Factors

Risk factors for AKI include environmental, socioeconomic and/ or cultural factors, as well as factors related to the process of care, acute exposures and patients themselves. Environmental factors include inadequate drinking and waste water systems, insufficient control of infectious diseases and insufficient health care systems. Patient-related factors can be modifiable, for example, volume depletion, hypotension, anaemia, hypoxia and use of nephrotoxic drugs, or non-modifiable, for example, chronic kidney, heart, and liver or gastrointestinal disease, diabetes and severe infections and sepsis. Rarer causes include genetic predispositions to myoglobinuria, haemoglobinuria and urolithiasis Further important risk factors for AKI are severe diseases, acute infections, sepsis, malaria, severe trauma, hypovolaemia, old age, pre- existing CKD, acute organ failures, major surgeries (including cardiac surgery), being in the ICU with exposure to nephrotoxic drugs and opportunistic infections, chemotherapy for leukaemia or cancer, delayed graft function upon kidney transplantation, autoimmune disorders with rapid progressive kidney injury, cholesterol crystal embolism and urinary tract obstruction. In  HIC,  despite  severe  AKI occurring more frequently in the context of hospital-related  risk factors, such as major surgery, bleeding, septic shock or drug toxicity in older patients with multiple diseases, milder forms of AKI can also be community-acquired. The three main causes of AKI are summarized below as: Prerenal AKI is secondary to under perfusion of otherwise normal, functioning kidneys. The hallmark of prerenal AKI is rapid reversibility. Prerenal kidney injury can result from volume depletion that is the result of hypovolaemia (haemorrhage, volume depletion, renal or extrarenal losses, fluid sequestration, renal fluid loss (over-diuresis), third space (burns, peritonitis, muscle trauma)); Impaired cardiac function (congestive heart failure, inadequate perfusion pressures secondary to heart failure, acute myocardial infarction, massive pulmonary embolism; Systemic vasodilatation (anti-hypertensive medications, gram negative bacteraemia, cirrhosis, anaphylaxis, sepsis); Increased vascular resistance (anaesthesia, surgery, hepatorenal syndrome, NSAID medications, drugs that cause renal vasoconstriction (i.e. cyclosporine)). For patients with prerenal AKI, urinalysis is typically bland or with hyaline casts, urine sodium is low (ie, 1%), and urine osmolality is high. Intrinsic AKI can be challenging to evaluate because of the wide variety of injuries that can occur to the kidney. Generally, four structures of the kidney are involved including tubules, glomeruli, the interstitium, and intra- renal blood vessels. Acute tubular necrosis (ATN) is the term used  to designate AKI resulting from damage to the tubules. It is the most common type of intrinsic kidney injury. AKI from glomerular damage occurs in severe cases of acute glomerulonephritis (GN). AKI from vascular damage occurs because injury to intra-renal vessels decreases renal perfusion and diminishes GFR and finally acute interstitial nephritis occurs due to an allergic reaction to a variety medications or an  infection.  Tubular  (renal  ischaemia  (shock,  complications  of surgery, haemorrhage, trauma, bacteraemia, pancreatitis, pregnancy), nephrotoxic drugs (antibiotics, antineoplastic drugs, contrast media, organic solvents, anaesthetic drugs, heavy metals), endogenous toxins (myoglobin, haemoglobin, uric acid); Glomerular (acute post-infectious glomerulonephritis, lupus nephritis, IgA glomerulonephritis, infective endocarditis, good pasture syndrome, wegener disease); Interstitium infections ((bacterial, viral), medications (antibiotics, diuretics, NSAIDs, and many more drugs)), Vascular (large vessels (bilateral renal artery stenosis, bilateral renal vein thrombosis), small vessels (vasculitis, malignant hypertension, atherosclerotic or thrombotic emboli, haemolytic uraemic syndrome, thrombotic thrombocytopenic purpura)). Postrenal causes of  AKI are characterized by acute obstruction to urinary flow. Urinary tract obstruction increases intratubular pressure and thus decreases GFR. In addition, acute urinary tract obstruction can lead to  impaired renal blood flow and inflammatory processes that also contribute to diminished GFR. Obstruction of the urinary tract at any level may produce AKI. In general, obstruction must involve both kidneys and a solitary kidney to produce significant renal failure. However, a patient with pre-existing renal insufficiency may develop AKI with obstruction of only one kidney. Urinary obstruction may present as anuria or intermittent urine flow (such as polyuria alternating with oliguria) but may also present as nocturia or nonoliguric AKI. Causes of postrenal AKI include benign prostatic hyperplasia and prostate cancer  in men, gynecologic cancers especially cervical cancer in women, retroperitoneal fibrosis, ureteral stones, papillary necrosis, neurogenic bladder, and intratubular obstruction due to precipitation of various substances such as acyclovir or indinavir. Extrarenal obstruction (prostate hypertrophy, improperly placed catheter, bladder, prostate or cervical cancer, retroperitoneal fibrosis), Intrarenal obstruction (nephrolithiasis, blood clots, papillary necrosis) [5,8-13] (Figure 1).

Figure 1: Causes of AKI

Pathophysiology of Kidney Failure

Loss of kidney excretory function implies disturbances in the main function of the kidneys (maintaining homeostasis), for example, through excretion of metabolic waste products. Serum creatinine and urea nitrogen levels are often used as biomarkers of reduced kidney function, but their use skews awareness towards the kidneys’ excretory function. Fluid homeostasis is affected, as declining glomerular filtration rate (GFR) and activation of the renin angiotensin system promote fluid retention, which presents as peripheral oedema, third- space effusions, and pulmonary congestion, especially in those with heart failure. In addition, as urinary output determines potassium excretion, hyperkalaemia is a common complication of severe AKI. When hyperkalaemia leads to electrocardiogram changes, AKI constitutes a medical emergency and warrants immediate intervention. Both hyponatraemia and hypernatraemia may occur when the kidney loses the capacity for urine concentration or dilution as needed. Impaired phosphate clearance leads to hyperphosphataemia. AKI also affects acid-base homeostasis. A declining capacity for the excretion of fixed acids in patients with AKI causes tubular metabolic acidosis and respiratory compensation via an increased ventilatory drive. Although a hyperchloraemic metabolic acidosis develops initially, widening of the anion gap is often seen as the result of accumulation of phosphate, sulfate and small organic anions in the bloodstream. A decline in   the capacity to excrete metabolic waste products is indicated by azotaemia but implies disturbance of homeostasis of hundreds, if not thousands, of other metabolites that are not waste products, which all together account for symptoms of uraemia, such as fatigue, tremor or confusion. Importantly, kidney failure affects most organ systems of the body. Many of the AKI-related uraemic toxins originate from the intestinal microbiota, such as indoxyl sulfate or p-cresyl sulfate. The microbiota itself undergoes shifts in its composition, owing to AKI and the accompanying acidosis, azotaemia, intestinal ischaemia, and other alterations of the intestinal microenvironments, which affects the microbiota’s secretome and metabolites needed for normal human physiology. The lungs are affected by hyperpnoea to compensate for metabolic acidosis, hypervolaemia, cytokines, oxidative stress and cytotoxic elements of necrotic cell debris (released by parenchymal necrosis in the kidneys, causing microvascular injury, and eventually acute respiratory distress syndrome). AKI affects cardiac function via acidosis, hyperkalaemia, uraemic toxins, hypervolaemia, hypertension, and systemic inflammation46. Uraemic encephalopathy also involves systemic oxidative stress responses [5,14-20] (Figure 2).

Figure 2: Mechanisms of acute kidney injury: a molecular viewpoint. Cascade of events involved in the pathophysiology of acute kidney injury.

Treatment of AKI

As previously noted, treatment plans for patients with AKI are varied and depend on etiologic factors. Prerenal azotemia from volume depletion is usually responsive to isotonic saline repletion. Treatment of ATN requires the discontinuation of nephrotoxic agents, maintenance of optimum hemodynamics, and  close  surveillance  for complications of renal dysfunction (eg, acidosis, electrolyte abnormalities). Postrenal etiologies dictate obstruction removal. Patients were categorized according to the treatment for AKI as follows: conservative treatment; surgical treatment; and renal replacement therapy (RRT). RRT included intermittent hemodialysis (IHD), continuous renal replacement therapy (CRRT) and peritoneal dialysis (PD). Surgical treatment included surgical removal of obstructive tumors, stones, or prostate hypertrophy. Patients without RRT and surgical treatment were included in the conservative treatment  group. Treatment included correction of the primary cause of AKI, fluid replacement therapy, and nutritional support. If AKI was due  to serious trauma, heart failure and acute hemorrhage, patients were administered normal saline to correct intravascular volume depletion. In AKI patients with severe hyperkalemia, 5-10 units of insulin and 50% dextrose were used to promote uptake of potassium into cells. Supportive therapies such as antibiotics and adequate nutrition were given according to standard management practice. All medications that could potentially affect renal function were discontinued [5,22- 25]. Delayed antibiotic administration in septic shock was associated with early AKI development. However, certain nephrotoxic agents involving in the treatment such as aminoglycosides, vancomycin particularly in combination with piperacillin-tazobactam, and amphotericin B, as well as diagnostic agents such as intravenous radiocontrast media should be used with caution to prevent kidney injury according to the KDIGO AKI guidelines. Strict therapeutic drug monitoring should be considered when applicable [26,27].

Fluids Resuscitation

Fluid resuscitation followed by vasopressor medications is cornerstones in the treatment of shock. Hydroxyethylstarch (HES) is a widely used, relatively inexpensive alternative to human albumin for correcting hypovolemia. The use of isotonic saline as the standard of care for intravascular volume expansion to prevent or treat AKI is thus based upon the lack of clear evidence that colloids are superior for this purpose, along with some evidence that specific colloids may cause AKI, in addition to their higher costs. It is acknowledged that colloids may be chosen in some patients to aid in reaching resuscitation goals, or to avoid excessive fluid administration in patients requiring large volume resuscitation, or in specific patient subsets (for example, a cirrhotic patient with spontaneous peritonitis, or in burns). Similarly, although hypotonic or hypertonic crystalloids may be used in specific clinical scenarios, the choice of crystalloid with altered tonicity is generally dictated by goals other than intravascular volume expansion (for example, hypernatremia or hyponatremia). In addition, isotonic saline solution contains 154 mmol/l chloride and when administration in large volumes will result in relative or absolute hyperchloremia. All intravenous fluids can contribute to adverse renal and patient outcomes by fluid overload and renal edema. Fluids containing non- physiologic ratios of sodium and chloride may worsen AKI. Balanced electrolyte solutions, such as lactated Ringer’s solution, are preferable in most patients [2]. Albumin solutions have generally been found to be safe in sepsis resuscitation; however, evidence that hyperoncotic albumin might deteriorate AKI and ICU outcomes is growing [28,29].

Vasopressors

Norepinephrine is recommended as an agent of choice for septic shock treatment. Dopamine is not recommended for renal protection and is associated with more adverse events than norepinephrine is. Vasopressin does not appear to increase AKI risk [30-32].

Human recombinant alkaline phosphatase (AP) is an  endogenous enzyme that confers renal protection during sepsis via the dephosphorylation of various compounds, including bacterial endotoxins and proinflammatory mediators such as extracellular adenosine triphosphate, which is released by mitochondria in response to inflammation and hypoxia. Intravenous infusion of AP (bolus injection followed by continuous infusion for 48 hours or placebo) starting within 48 hours of AKI onset and showed improvement of endogenous creatinine clearance, requirement for and duration of dialysis, decreased urinary biomarkers of renal injury (KIM-1 and interleukin-18), and inflammatory biomarkers from baseline to day 28 in patients receiving AP. Human recombinant AP is a highly stable, biologically active enzyme [32,33].

Angiotensin II (ATII) is potent vasoconstrictor acting via angiotensin II type 1 receptors and appears to cause vasoconstriction of efferent more than afferent arterioles, resulting in increasing glomerular perfusion pressure and filtration rate. Sepsis leads to relative scarcity of ATII. In addition, ATII is a potent vasopressor without inotropic or chronotropic properties. Unlike norepinephrine, ATII may preserve medullary perfusion and oxygenation [34].

Diuretics

Loop diuretics (especially furosemide) have long been prescribed in the acute-care setting, and a number of RCTs have tested whether furosemide is beneficial for prevention or treatment of AKI. Specifically, prophylactic furosemide was found to be ineffective   or harmful when used to prevent AKI after cardiac surgery, and to increase the risk of AKI when given to prevent contrast-induced AKI. Furosemide may be useful in achieving fluid balance to facilitate mechanical ventilation according to the lung-protective ventilation strategy in hemodynamically stable patients with acute lung injury. Mannitol is often added to the priming fluid of the cardiopulmonary bypass system to reduce the incidence of renal dysfunction, but the results of these studies are not very convincing. Mannitol is beneficial in rhabdomyolysis by stimulating osmotic diuresis and by lowering the intracompartmental pressure in the affected crushed limbs [35,36].

Deferoxamine

A key early feature of AKI is the generation of reactive oxygen species. The iron chelator deferoxamine is a widely known free radical scavenger. In several models of AKI, deferoxamine was proved effective. The protective effect of deferoxamine in various models suggests the central role of free radicals in AKI. Studies in AKI are planned to test the efficacy of iron chelation [21].

Vasodilator Therapy: Dopamine, Fenoldopam, and Natriuretic Peptides

There is also limited evidence that the use of dopamine to prevent or treat AKI causes harm. Dopamine can trigger tachyarrhythmias and myocardial ischemia, decrease intestinal blood flow, cause hypopituitarism, and suppress T-cell function.  Fenoldopam mesylate is a pure dopamine type-1 receptor agonist that  has  similar hemodynamic renal effects as low-dose dopamine, without systemic αadrenergic or β-adrenergic stimulation. The guideline recommendation against using fenoldopam places a high value on avoiding potential hypotension and harm associated with the use of this vasodilator in high-risk perioperative and ICU patients, and a low value on potential benefit, which is currently only suggested by relatively low-quality single-center trials. Nesiritide (b-type natriuretic peptide) is the latest natriuretic peptide introduced for clinical use, and is approved by the US Food and Drug Administration only for the therapy of acute, decompensated congestive heart failure [37-39].

Caspase Inhibitors

Caspases are a family of proteases that are involved in the initiation and execution phase of apoptosis. Nonselective and selective caspase inhibitors are effective in attenuating renal injury in ischemia- or endotoxemia-induced AKI when administered before or at the time of injury [40].

Minocycline

Minocycline are second-generation  tetracycline  antibiotics  with proven human safety data. Minocycline is known to have antiapoptotic and anti-inflammatory effects. When administered 36 h before renal ischemia, minocycline reduced tubular cell apoptosis and mitochondrial release of cytochrome c, p53, and bax [41].

Ethyl Pyruvate

Pyruvate has been known as a potent endogenous antioxidant and free radical scavenger.In addition to an effect on mortality, ethyl pyruvate reduced kidney injury using the technique cecal ligation puncture as a model of sepsis. Ethyl pyruvate is a widely used food additive and has been shown to be safe in phase I clinical trials. It now is being tested in a phase II trial in patients who undergo cardiopulmonary bypass surgery [42].

Activated Protein C

Activated protein C (APC) is a physiologic anticoagulant that is generated by thrombin-thrombomodulin complex in endothelial cells. In addition to its effect on coagulation, APC has been shown to have anti-inflammatory, antiapoptotic effects. APC also attenuated renal IRI by inhibiting leukocyte activation. APC is approved by the Food and Drug Administration for treating patients who have severe sepsis and an Acute Physiology, Age, Chronic Health Evaluation (APACHE) score of 25 or higher [43].

Insulin

Insulin resistance and hyperglycemia are common in critically  ill patients, and intensive insulin therapy that targeted blood glucose level between 80 and 110 mg/dl reduced the incidence of AKI that required dialysis or hemofiltration [21,44].

Conclusion

Acute kidney injury (AKI), previously called acute renal failure, is characterized by an abrupt decline in renal function, resulting      in an inability to secrete wastes and maintain electrolyte and water balance. The severity of AKI was defined by the AKIN staging criteria as follows: Stage I, SCr increase to 1.5-2 fold of baseline; Stage II, SCr increase to 2-3 fold of baseline; and Stage III, SCr increase to 3 fold of baseline or an absolute increase of 356.6 mmol/l (4.0 mg/dl) with an acute increase of at least 44.2 mmol/l (0.5 mg/dl). Patient-related factors can be modifiable, for example, volume depletion, hypotension, anaemia, hypoxia and use of nephrotoxic drugs, or non-modifiable, for example, chronic kidney, heart, and liver or gastrointestinal disease, diabetes and severe infections and sepsis. Fluid homeostasis is affected, as declining glomerular filtration rate (GFR) and activation of the renin angiotensin system promote fluid retention, which presents as peripheral oedema, third-space effusions, and pulmonary congestion, especially in those with heart failure. In addition, as urinary output  determines  potassium  excretion,  hyperkalaemia  is a common complication of severe AKI. Hypotonic or hypertonic crystalloids may be used in specific clinical scenarios; the choice of crystalloid with altered tonicity is generally dictated by goals other than intravascular volume expansion (for example, hypernatremia   or hyponatremia). In addition, isotonic saline solution contains 154 mmol/l chloride and when administration in large volumes will result in relative or absolute hyperchloremia.

Abbreviations

AKI: Acute Kidney Injury; AKIN: Acute Kidney Injury Network; GFR: Glomerular Filtration Rate; HIC: High-Income Countries; ICU: Intensive Care Unit; RRT: Renal Replacement Therapy; RIFLE: Risk Injury Failure Loss End Stage; Scr: Serum Creatinine

Acknowledgments

The author would be grateful to anonymous reviewers by the comments that increase the quality of this manuscript.

Data Sources

Sources searched include Google Scholar, Research Gate, PubMed, NCBI, NDSS, PMID, PMCID, Scopus database, Science direct, Scielo and Cochrane database. Search terms included: definition, stages, risk factors, pathophysiology and treatment of acute kidney injury.

Funding

None

Availability of Data and Materials

The datasets generated during the current study are available with correspondent author.

Competing Interests

The author has no financial or proprietary interest in any of material discussed in this article.

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DOI: 10.31038/JPPR.2022521

Abstract

The aim of this investigation is was to review briefly the literature of the anti-viral activity for hydroxychloroquine, how has been used in the treatment of the novel coronavirus disease COVID-19, pathogenesis of COVID-19, and the mechanism of action for hydroxychloroquine in treatment of COVID-19. Hydroxychloroquine likely to attenuate the sever progression of COVID-19, Inhibiting the cytokine storm by suppressing T cell activation, also act by increase the pH of intracellular organelles. The study concludes and gave a well-supported document for quick review of the anti-viral activity of hydroxychloroquine used in the treatment of novel coronavirus COVID-19.

Keywords

Hydroxychloroquine (HCQ), Chloroquine (CQ), SARS-COV-2, Antiviral, Pathogenesis

Introduction

The novel corona virus COVID-19 was first reported on the month of December 2019 in Wuhan city-China and it has rapidly spread to all over the world [1]. On February 2020 the world health organization (WHO) named it as (COVID-19), simultaneously the international virus classification commission announced that the new corona virus was named as sever acute respiratory syndrome cornoavirus-2 (SARS-coV2) [2,3]. COVID-19 belongs to the family of genera betacoronaviridae, it is enveloped, single stranded, positive sense RNA virus, the name corona virus derived from the spike proteins that surrounded the outer member of virus [4,5]. COVID-19 is not the first virus from the family of coronaviruses, in the year 2003 a severe respiratory diseases known as sever acute respiratory syndrome (SARS) was reported as an epidemic and 8000 of people being infected with total 10% of cases death. In 2012 another corona virus has outbreak in middle east called as middle east respiratory syndrome (MERS) and infected about 2500 clinical cases with total 35% death. COVID-19 incubation period is about 4-14 days, the shedding period of virus 8-37 days but in severe cases takes about 19 days and in the critical condition takes 24 days [5]. Hydroxycholorquine used as anti-malarial drug and autoimmune diseases such as lupus and rheumatoid arthritis, recently hydroxychloroquine reported in the treatment of novel corona virus (SARS-coV19) [6]. Hydroxychloroquine is a derivative of chloroquine that chemically belongs to 4-aminoqinolines, chloroquine discovered in the year 1934 as anti-malarial drug. Chloroquine also used in the treatment of various infectious diseases [7]. Both Chloroquine as well as hydroxychloroquine acts by the same mechanism of action on coronavirus (COVID-19), that both of chloroquine and hydroxychloroquine are weak bases and they know to increase the intracellular organelles acidic pH, also prevent the binding of virus spike glycoprotein with ACE2 receptors [6].

Hydroxychloroquine (HCQ)

Hydroxychloroquine has similar structure to that of chloroquine (Figure 1). Chloroquine shows inhibitory action on the novel corona virus, but due its several adverse drug reactions chloroquine not suite for the treatment of SARS-coV-2. Hydroxychloroquine has proposed due to its antiviral activity, it is also shows better effect comparing to chloroquine [8]. chloroquine-(N4-(7-Chloro-4-quinolinyl)-N1,N1-diethyl-1,4-pentanediamine) is an amnio acidotropic form of quinine and first synthesized in Germany in the year of 1934 and used in the treatment of malarial and many other autoimmune diseases. Chloroquine has antiviral activity against several diseases like HIV, hepatitis A and C viruses, Influenza A and B viruses, Influenza A H5N1 virus, Dengue virus, chikungunya virus, poliovirus, lassa virus and Zika virus. Hydroxychloroquine sulphate is a derivative of chloroquine and was first synthesized in the year 1946 by addition of hydroxyl group (OH) into chloroquine structure, so produced hydroxychloroquine with less toxicity in animal. Hydroxychloroquine share the same therapeutic uses as well as mechanism of action to that of chloroquine [6,9,10]. Both Hydroxychloroquine and chloroquine have been shown anti-viral activity in SARS-coV-2 when administrated as prophylactic – pre-treated as well as post-viral entry, indicating multiple mechanisms of action. HCQ and CQ are weak bases so they know to increase the pH inside the intracellular organelles including (lysosomes, endosomes and golgi vesicles ), specific enzymes present in the lysosomes active only at low pH around 4.5, as the novel coronavirus SARS-coV-2 needs that enzymes for the process of replication and survive, Chloroquine and hydroxychloroquine increases the pH of lysosomes and interfere the activity of this enzymes and inhibits the SARS-coV-2, help in the treatment of the virus. In addition to that hydroxychloroquine can inhibit SARS-coV-2 entry through the changing the glycosylation of angiotensin converting enzyme-2 (ACE-2) receptor as well as spike protein, experimentally confirmed that hydroxychloroquine inhibits the entry and post-entry steps of the virus. hydroxychloroquine and chloroquine also block the transport of SARS-coV-2 from early endosomes (EEs) to endolysosomes (ELs) which is important required in the release of genome in SARS-coV-2. Due to the altered pH of the endosomes and interfere the binding between tall-like receptors ( TLR7, TLR9) and their RND/DNA ligands, TLR signals suppressed due hydroxychloroquine. In the cytoplasm hydroxychloroquine interfere with the interaction of cytosolic DNA and the nucleic acid sensor cyclic (GMP-AMP) synthase. Hydroxychloroquine acts as immune modulator, inhibit receptor binding and membrane fusion, also it has been shown to down regulate pro-inflammatory cytokines including IL6 levels, and minimize cytokines release syndrome /ARDS. Dose of hydroxychloroquine in COVID-19 was recommended as 400 mg twice in a day for two days, then 200 mg twice per a day for another 3 more days. Hydroxychloroquine has better therapeutic approach compared to chloroquine in the treatment of SARS-COV-2 infection [5-8,11-13] (Figure 1).

Figure 1: Structures of chloroquine and hydroxychloroquine

Pathogenesis

The pathogenesis of SARS-COV-2 not clearly understood, but the mechanism of SARS-COV, and MERS give us a huge information to understand the pathogenesis of novel coronavirus SARS-COV-2 [2]. The genome structure of coronaviruses are well known compared with other RNA viruses.coronaviruses RNA contain encodes viral polymerase, RNA synthesis material and non-structural polyproteins which not involved in host response modulation. Additionally the genome encodes also contain 4 structural proteins these proteins are, spike (S), envelope (E), membrane (M) and nucleocapsid (N) [14] (2019 NOVEL COVID-19). The novel coronavirus SARS-COV-2 mainly act by targeting the respiratory system and cause severe pneumonia and acute cardiac injury with high blood levels of cytokines and chemokines [15]. Virus Enter into host cell, the envelope spike glycoprotein bind to angiotensin converting enzyme-2 (ACE-2) for SARS-COV-2, CD29L (a C-type lectin also known as l-SIGN). After the entry of SARS-COV-2 into the cell the viral RNA genome is release into cytoplasm and converted to 2 polyproteins and structural proteins, then genome begins replication process. The genome that newly formed inserted into the membrane of the endoplamic reticulum, the vesicles carrying the virus particles fuse with the plasma membrane to release the virus. Due to antigen presentation the body stimulate humoral and cellular immunity which are mediated by virus specific B and T-cells that similar to common acute viral infections the antibody profile against SARS-COV virus has typical of IgM and IgG production. The infected patient with SARS-COV-2 shows the number of CD4 and CD8 T-cells in the peripheral blood significantly reduced. The main death cause of novel coronavirus SARS-COV-2 due to the high levels of cytokines and chemokines that released by body immunity. The different types of cytokines that released (IFN-a, IFN-g, IL-1b, IL-6, IL-12, IL-18, IL-33, TNF-a, TGFb) and chemokines (CCL2, CCL3, CCL5 CXCL8, CXCL9, CXCL10). High levels of these inflammation mediators lead to multiple organ failure and finally may lead to death in severe cases in SARS-COV-2 [2].

Conclusion

The authors succeeded in compiling the data and made the brief literature survey. In summary, Hydroxychloroquine serve as a better therapeutic approach than chloroquine for the treatment of COVID-19, due to inhibition of cytokine storm by reducing CD154 expression in T-cell. Hydroxychloroquine showed fewer side effects, safe during the pregnancy.

Conflicts of Interests

Authors do not have any conflicts of interest with the publication of the manuscript.

Acknowledgment

None

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