阅读说明：本技术 一种二乙二醇丁醚溶石剂 (Diethylene glycol monobutyl ether litholytic agent ) 是由 陈梓衡 张�杰 倪磊 宋红红 贺安琪 凌晓峰 徐怡庄 冯娟 于 2020-04-09 设计创作，主要内容包括：本发明提供了一种胆结石的溶石剂,通过将二乙二醇丁醚与水配合使用进行溶石,溶石效果好,减少醚类的使用,降低溶石剂的毒副作用。本发明中的溶石剂不但能够溶解胆固醇型胆结石,还可以很好的溶解胆色素型和混合型胆结石,溶石剂为水体系,便于清洗,不受胆汁残留的影响,有望在临床开展使用。(The invention provides a litholytic agent for gallstones, which is used for litholytic by matching diethylene glycol monobutyl ether with water, has good litholytic effect, reduces the use of ethers, and reduces the toxic and side effects of the litholytic agent. The litholytic agent can dissolve cholesterol gallstones and can well dissolve bile pigment type and mixed gallstones, is a water system, is convenient to clean, is not influenced by bile residues, and is expected to be clinically used.)

1. A gallstone litholytic agent, comprising diethylene glycol monobutyl ether.

2. The litholytic agent of claim 1, further comprising water.

3. The litholytic agent according to claim 1 or 2, wherein the volume ratio of diethylene glycol monobutyl ether to water is 1 (0.1-10) when dissolving cholesterol-type gallstones.

4. The litholytic agent of claim 3, further comprising methyl tertiary-butyl ether having a mass solubility in litholytic agent of 0.1% to 5% for dissolving cholesterol-type gallstones.

5. A litholytic agent according to any one of claims 1 to 4, wherein the volume ratio of diethylene glycol monobutyl ether to water is 1 (0.1 to 5) in the dissolution of bile pigment type and mixed type gallstones.

6. A litholytic agent according to any of claims 1-5, wherein said litholytic agent is administered by percutaneous transhepatic catheterization or endoscopic retrograde cholecystectomy to perfuse the litholytic agent into the gallbladder.

7. A litholytic agent according to any one of claims 1 to 6, further comprising a surfactant, a proteolytic agent, or a complexing agent.

8. Use of a litholytic agent according to any one of claims 1 to 7, wherein said litholytic agent is used for the preparation of a medicament for the dissolution of cholesterol-type gallstones, bile pigment-type gallstones or mixed gallstones.

9. The use according to claim 8, wherein the volume ratio of diethylene glycol monobutyl ether to water is 1 (0.1-10) in dissolving cholesterol-type gallstones.

10. The use according to claim 8, wherein the volume ratio of the diethylene glycol monobutyl ether to water is 1 (0.1-5) in dissolving bile pigment type and mixed type gallstones.

Technical Field

The invention relates to a novel litholytic agent for dissolving gallstones, in particular to a diethylene glycol monobutyl ether litholytic agent.

Background

Gallstones, also called cholelithiasis, are the leading place in biliary tract diseases in China and are the key point for treating biliary tract diseases in China, and treatment means of the gallstones include operations and various litholytic methods. Mainly comprises oral litholysis, extracorporeal shock wave lithotripsy and direct contact litholysis. Among them, the direct contact litholytic method has the advantages of small damage, little pain, definite curative effect, etc., and is particularly suitable for patients who cannot tolerate anesthesia, surgery and refuse surgery.

In 1981, Walker firstly directly perfuses a gall bladder with ether to dissolve gallstones, but because the boiling point of the ether is lower than body temperature, the ether is evaporated to generate high pressure after entering a human body, so that severe abdominal pain is generated for a patient and the patient is not widely accepted, and in the next decades, people successively use various solvents such as chloroform and the like to dissolve stones, but the solvents are not popularized due to various adverse reactions or poor effects.

Among them, monooctyl lipid was found to be a good cholesterol dissolving agent and was developed as a commercial formulation Capmul-8210. The mono-octyl grease is metabolized into glycerol and caprylic acid after entering the intestinal tract, the caprylic acid is decomposed and metabolized in the liver, and the main side effect after entering the human body is local stimulation to cause inflammation or ulceration of the gallbladder, bile duct base and gastric mucosa. However, this litholytic agent is difficult to dissolve in calcified bile pigment gallstones.

At present, Methyl Tertiary Butyl Ether (MTBE) is the gallstone dissolving agent which is most studied so far, and shows excellent stone dissolving effect and operation. Methyl tert-butyl ether has special odor and is volatile, the boiling point is 55 ℃, the solubility of cholesterol in MTBE is 20g/100mL, but the cholesterol-type gallstone dissolved by MTBE is 50 times of that of single octyl grease. The cholelithiasis inhibitor can completely dissolve human gallstones with cholesterol content of 40-94% in 60-100 minutes in vitro, can also be used for mixed gallstones with a small amount of bile pigment, but has poor dissolving effect on bile pigment type gallstones, and MTBE is insoluble in blood, so that when bile remains in a gall bladder, a litholytic agent cannot be in full contact with the gallstones, and litholytic effect is influenced. In addition, MTBE dissolves medical latex tubes in the treatment and use process, so that various medical rubber tubes cannot be used in the use process, and the toxic and side effects of MTBE on human bodies are large. The cholechrome-type gallstones in the cholelithiasis of China are more, the composition of the gallstones is more complex, and the litholytic agent with small toxic and side effects of the existing litholytic agent has relatively poor dissolving effect.

Based on the above problems, there is a need for a litholytic agent with less side effects, which is safe, and can shorten litholytic time and reduce side effects to relieve pain of patients during treatment.

Disclosure of Invention

The present inventors have conducted intensive studies in order to solve the above problems and found that diethylene glycol monobutyl ether as a litholytic agent provides a very good litholytic effect and can dissolve not only cholesterol-type gallstones but also bile pigment-type gallstones and mixed gallstones, thereby completing the present invention.

The invention aims to provide a gallstone litholytic agent, which comprises diethylene glycol monobutyl ether.

The litholytic agent can be used to dissolve cholesterol-type gallstones, bile pigment-type gallstones and mixed gallstones.

The invention also aims to provide the application of the litholytic agent in preparing a medicament for treating gallstones.

The administration mode of the litholytic agent comprises percutaneous transhepatic cholangiocatheterization or retrograde gallbladder intubation under an endoscope, and the litholytic agent is infused into the gallbladder to be directly contacted with gallstones.

The stone dissolving agent has the following beneficial effects:

(1) the invention takes the diethylene glycol monobutyl ether as the litholytic agent, and the diethylene glycol monobutyl ether litholytic agent with lower concentration is obtained by matching with water, thereby reducing the side effect of the human body and simultaneously improving the litholytic capacity of the litholytic agent.

(2) The litholytic agent can dissolve cholesterol gallstones, bile pigment gallstones and mixed gallstones, and can be used for non-operative treatment of clinical gallstone diseases.

(3) The stone dissolving agent has low toxicity, is easy to dissolve in water and convenient to clean, can dissolve stone well under the condition of residual bile and is not influenced by environments such as gall bladder and the like.

Drawings

FIG. 1 shows a schematic representation of the litholytic agent administration mode of the present invention;

FIG. 2 is a graph showing the effect of the litholytic agent 1-3 of the present invention on dissolving bile pigment type gallstones;

FIG. 3 is a graph showing the effect of the litholytic agent 1-5 of the present invention in dissolving mixed gallstones.

Detailed Description

The present invention will now be described in detail by way of specific embodiments, and features and advantages of the present invention will become more apparent and apparent from the following description.

The litholytic agent comprises diethylene glycol butyl ether and also comprises water. In experiments, the diethylene glycol butyl ether has a good stone dissolving effect, and can dissolve cholesterol-type and mixed-type gallstones and also can dissolve bile pigment-type gallstones. In addition, the diethylene glycol monobutyl ether is matched with water for use, so that the concentration of the litholytic agent can be reduced, the damage to a human body is reduced, and the litholytic agent can still be well contacted with gallstones to dissolve the gallstones under the condition that bile remains. In addition, after completion of the litholytic treatment, the residual litholytic agent can be removed by washing out the residual litholytic agent with an aqueous solvent such as physiological saline. Additionally, butyl diglycol ether was less toxic and LD50 rats were dosed orally with 4500mg/kg (data source: European chemical administration ECB), LD50 rabbit skin 4120mg/kg (data source: Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ.2004., p.1269).

In the invention, a large number of experiments show that the dissolution rate of gallstones is gradually increased initially to slowly reach a highest value along with the increase of the content of the diethylene glycol butyl ether in the litholytic agent, and then the change of the litholytic rate is not obvious when the concentration of the diethylene glycol butyl ether is increased.

When the mixed solution of diethylene glycol monobutyl ether and water is used as a litholytic agent, when the water addition amount is changed from 10mL to 0.1mL relative to 1mL of diethylene glycol monobutyl ether, the time for dissolving a certain amount of cholesterol-type gallstones is firstly shortened, when the volume ratio of the diethylene glycol monobutyl ether to the water is 3:4, the shortest dissolving time is reached, the concentration of the diethylene glycol monobutyl ether is continuously increased, and the litholytic time is firstly constant and then slightly increased. The diethylene glycol monobutyl ether can achieve the best stone dissolving effect at a lower concentration, so that the using amount of the diethylene glycol monobutyl ether is reduced, the side effect of the diethylene glycol monobutyl ether on a human body can be reduced, and the pain of a patient is relieved.

In addition, in the experiment, it is found that if the amount of the dissolved cholesterol gallstone is increased, the dissolving time is increased, and when the litholytic agent with low concentration of diethylene glycol butyl ether is used (for example, the volume ratio of the diethylene glycol butyl ether to water is 1:10), the dosage of the litholytic agent is also increased, which shows that the dissolving amount of the fixed concentration of the diethylene glycol butyl ether litholytic agent to the cholesterol gallstone has a certain limit. The action time and dosage of the litholytic agent on cholesterol gallstones need to be coordinated by adjusting the concentration of diethylene glycol butyl ether so as to achieve the use state with relatively good litholytic effect and relatively low side effect.

In the cholesterol gallstone dissolving agent, the volume ratio of the diethylene glycol butyl ether to water is 1 (0.1-10), preferably 1 (0.3-5), more preferably 1 (1-2), such as 1: 1.3.

In a preferred embodiment of the present invention, dissolution of cholesterol gallstones is accelerated by adding methyl tert-butyl ether to the litholytic agent, the mass solubility of the methyl tert-butyl ether in the litholytic agent being 0.1% to 5%, preferably 0.5% to 3%, more preferably 0.8% to 1.2%.

At present, the research on the litholytic agent mainly focuses on the litholytic agent for dissolving cholesterol-type gallstones, and the litholytic agent has no ideal effect on the dissolving of bile pigment-type gallstones and mixed gallstones.

It is worth noting that the diethylene glycol monobutyl ether litholytic agent also has good litholytic effect on bile pigment type gallstones. When a fixed mass of the cholestain type gallstone is dissolved, a law similar to that of dissolving the cholestain type gallstone is found through a large number of experiments, when the volume ratio of the diethylene glycol butyl ether to the water is 3:4, the used litholysis time is shortest, the litholysis time is longer than the litholysis time of the cholestain type gallstone, and the results show that the cholestain type gallstone is more difficult to dissolve, and the difficulty is higher in the actual litholysis treatment process.

In addition, the diethylene glycol monobutyl ether litholytic agent can also be used for dissolving mixed gallstones. When the mixed gallstones are dissolved by using the diethylene glycol butyl ether litholytic agent with different concentrations, the fact that a mutation process exists between the volume ratio of the diethylene glycol butyl ether to water of 3:4 and 1:1 in the dissolving time is found, the volume concentration of the diethylene glycol butyl ether is slightly increased, and the litholytic time can be shortened by nearly half. In the practical application process, the litholytic time can be greatly shortened by utilizing the mutation process of the concentration.

In the aspect of dissolving bile pigment type and mixed gallstones, the volume ratio of the diethylene glycol monobutyl ether to water in the diethylene glycol monobutyl ether litholytic agent is 1 (0.1-5), preferably 1 (0.5-3), more preferably 1 (1-1.5), such as 1: 1.3. When the content of diethylene glycol butyl ether in the litholytic agent is too low, the bile pigment type gallstones cannot be well dissolved even if the dissolution time is increased and the litholytic agent is used in an increased amount.

In a preferred mode of the invention, the litholytic agent further comprises a surfactant, a proteolytic agent or a complexing agent.

The surfactant is added into the litholytic agent, so that the litholytic agent not only can solubilize, but also has the effects of emulsification, wetting, decontamination, sterilization and the like. The surfactant is selected from benzalkonium bromide, lecithin, polyethylene glycol, polysorbate and poloxamer, preferably selected from lecithin, polyethylene glycol, polysorbate and poloxamer, and more preferably selected from lecithin or poloxamer.

Lecithin can regulate serum lipid level, reduce cholesterol level, protect liver, and enhance immunity and anti-fatty liver activity. The lecithin is added into the litholytic agent, so that a certain liver protection capability can be provided, and the damage of the litholytic agent to the liver can be reduced.

Poloxamers are polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymers in which the polyoxyethylene chains are relatively hydrophilic and the polyoxypropylene chains are relatively lipophilic. According to the invention, poloxamer is added, so that on one hand, the uniform and stable litholytic agent can be formed with water, and on the other hand, due to the addition of poloxamer, the use amount of the polyalkylol butyl ether can be reduced, and the side effect of the litholytic agent on a human body can be reduced.

At present, more than 70 percent of cholesterol in cholesterol gallstones also contains a small amount of bilirubin, cholic acid, phospholipid, protein, free fatty acid, calcium salt, magnesium salt and the like; in the mixed gallstone, 30-70% of cholesterol is cholesterol, and also contains cholesterin, calcium salt and the like; the main components of bile pigment type gallstone include free bile pigment, bile pigment polymer, various calcium salts and mucus glycoprotein, calcium salt, bacteria, worm egg, cell, etc. Therefore, in the present invention, it is preferable to add a protein dissolving agent in order to promote the litholytic effect of gallstones.

The protein dissolving agent is selected from guanidine hydrochloride, urea, thiourea, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonate inner salt (CHAPS) and Dithiothreitol (DTT), preferably guanidine hydrochloride, urea or CHAPS, more preferably one or two of guanidine hydrochloride and urea.

Guanidine hydrochloride and urea have solubilization on hydrophobic amino acid residues, and a high-concentration (4-8 mol/L) aqueous solution of guanidine hydrochloride and urea can break hydrogen bonds, so that the hydrophobic residues in protein molecules are stretched, and the solubility is further increased.

At room temperature, 3-4 mol/L guanidine hydrochloride can transform globular protein from natural state to intermediate denaturation state, usually increasing the concentration of denaturant can increase the denaturation degree, and about 6mol/L guanidine hydrochloride can transform protein into denaturation state completely. Since guanidinium hydrochloride has ionic properties, in 8mol/L guanidinium hydrochloride solution, they are generally present in a random coil conformation.

The urea has slower and weaker dissolving capacity than guanidine hydrochloride, the protein solubility is 70-90%, and the urea can be cracked to form cyanate when the acting time is longer or the temperature is higher, so that the amino group of the recombinant protein is subjected to covalent modification, but the urea has the advantages of no ionization, neutrality, low cost and the like.

The dissolving capacity and the denaturation capacity of guanidine hydrochloride are stronger than those of urea, covalent modification of recombinant protein cannot be caused, but the cost is high, and precipitation is easy to generate under an acidic condition; the urea has relatively weak protein dissolving capacity, but has the advantages of no ionization, neutrality, low cost, no protein precipitation after renaturation, etc. In practical application, the stone dissolving agent is selected for use according to the use environment of the stone dissolving agent.

The concentration of guanidine hydrochloride in the litholytic agent is 0.1-8 mol/L, preferably 1-7 mol/L, and more preferably 2-6 mol/L.

The concentration of urea in the litholytic agent is 0.1-8 mol/L, preferably 1-7 mol/L, and more preferably 2-6 mol/L.

The complexing agent is selected from ethylene glycol diethyl diamine tetraacetic acid (EGTA), Ethylene Diamine Tetraacetic Acid (EDTA), disodium ethylene diamine tetraacetic acid (EDTA-2Na) or nitrilotriacetic acid (NTA), preferably ethylene glycol diethyl diamine tetraacetic acid (EGTA), disodium ethylene diamine tetraacetic acid (EDTA-2Na) or Ethylene Diamine Tetraacetic Acid (EDTA), and more preferably disodium ethylene diamine tetraacetic acid (EDTA-2 Na).

EDTA, EDTA-2Na and EGTA are good solvents for bile pigment type gallstones, and because the contents of bile pigment and calcium in the pigment type gallstones are high, calcium in the bile pigment type gallstones can be effectively dissolved, and the bile pigment type gallstones can be complexed with calcium ions in the presence of magnesium ions. The calcium ion complexing agent is added into the stone dissolving agent, so that gallstones, particularly bile pigment type gallstones, can be cracked, calcium ions in the gallstones are dissolved out of the gallstones, the surfaces of the gallstones are damaged layer by layer, and gradual dissolution is realized.

In addition, under the action of the solubilizer, the litholytic agent with weak polarity and EDTA-2Na molecules with strong polarity have mutual solubilization function, and the synergistic effect of the components is exerted. The concentration of the complexing agent is 0.005-0.1 g/mL, preferably 0.008-0.08 g/mL, and more preferably 0.01-0.05 g/mL.

The administration mode of the litholytic agent comprises percutaneous transhepatic cholangiotomy or retrograde gallbladder intubation under an endoscope, etc., the litholytic agent is infused into the gallbladder to directly contact with gallstones for dissolving the gallstones, and the schematic diagram of the catheterization or intubation is shown in figure 1.

The test shows that the stone-dissolving agent has very low swelling ratio to the medical latex tube, and the stone-dissolving agent has no latex tube component, which indicates that the stone-dissolving agent in the invention can not dissolve out the components in the latex tube, thereby causing harm to human body.

The litholytic agent can dissolve cholesterol gallstones and can well dissolve bile pigment type and mixed gallstones, the litholytic agent is an aqueous system, is convenient to clean and not influenced by bile residues, the using concentration of the diethylene glycol butyl ether is low, and the litholytic effect is good.

Examples

Example 1

1mL, 5mL and 7.5mL of diethylene glycol butyl ether are respectively added into 10mL of deionized water, and magnetic stirring is carried out for about 2min at the stirring speed of 100r/min to form uniform and stable litholytic agent 1-1, litholytic agent 1-2 and litholytic agent 1-3.

Three cholesterol gallstones (gallstones of 44 years old from patients, women) weighed about 0.1g respectively are put into the mixed solution and are crushed and dissolved in an ultrasonic cleaner (ultrasonic power 240W).

When 30min, the gallstone in the stone dissolving agent 1-3 is completely dissolved, and the stone dissolving agent is changed into a light yellow transparent solution from the original colorless transparent solution; when 50min, the gallstone in the stone dissolving agent 1-2 is completely dissolved, and the stone dissolving agent is changed into light yellow suspension from the original colorless transparent solution; at 70min, gallstone in litholytic agent 1-1 is completely dissolved, and the litholytic agent is changed from colorless transparent solution to yellowish and whitish suspension. After standing, a syringe with a needle head of 0.7 mm in diameter is used for sucking the stone dissolving agent in the beaker, and the stone dissolving agent 1-3 corresponds to the beaker and has no impurity residue. The litholytic agent 1-1 and the litholytic agent 1-2 correspond to a beaker, and a small amount of powdery fine particles are remained.

In clinical use, because the residual powdery particles after stone dissolution are very fine, the residual powdery particles and the stone dissolving agent can be cleaned after stone dissolution.

Example 2

1mL of deionized water and 10mL of deionized water are respectively added into 10mL of diethylene glycol butyl ether for mixing, magnetic stirring is carried out for about 2min, the stirring speed is 100r/min, and uniform and stable litholytic agents 1-4 and 1-5 are formed.

Two cholesterol-type gallstones (gallstones of 44 years old from patients, women) weighing about 0.1g each were placed in the above solution and then disrupted and dissolved in an ultrasonic cleaner (ultrasonic power 240W).

When 30min, gallstone in the litholytic agent 1-4 is completely dissolved; at 40min, the gallstone in the litholytic agent 1-5 is completely dissolved, the color of the litholytic agent is changed from colorless clarification to yellow clarification, which shows that the cholesterol type gallstone is completely dissolved in the litholytic agent, and the whole gallstone is firstly broken into small particles under the action of dissolution and ultrasound, and then gradually dissolved and disappeared. After standing, the litholytic agent in the beaker was aspirated out using a syringe with a needle diameter of 0.7 mm, and no residue remained at the bottom of the beaker.

Example 3

Weighing about 0.1g black cholechrome type gallstone (gallstone is from patient, male, 67 years old, cholelithiasis) respectively in litholytic agent 1-3 and litholytic agent 1-5, and adding gallstone. Crushing and dissolving gallstone by using an ultrasonic cleaner (ultrasonic power 240W).

At 230min, bile pigment type gallstone in litholytic agent 1-3 is completely dissolved; at 310min, gallstone in 1-5 of the litholytic agent is completely dissolved. The litholytic agent is a reddish brown transparent solution of a colorless transparent solution in the litholytic process of the two litholytic agents, and the whole gallstone is firstly crushed into small particles under the action of dissolution and ultrasound and then gradually dissolved and disappeared. The effect of the litholytic agent 1-3 on the process of dissolving bile pigment type gallstones is shown in figure 2. After standing, the litholytic agent in the beaker is sucked out by using a syringe with a needle head diameter of 0.7 mm and a needle head of 10ml, and the bottom of the beaker is free from impurities.

Example 4

Two particles of about 0.15g of intact brown mixed gallstones (gallstones from patients, women, 33 years old) are weighed and placed in litholytic agent 1-3 and litholytic agent 1-5, respectively. Crushing and dissolving gallstone by using an ultrasonic cleaner (ultrasonic power 240W).

When the time is 60min, the gallstone in the stone dissolving agent 1-5 is completely dissolved; the gallstone in the litholytic agent 1-3 is completely dissolved in 100 min. After standing, the litholytic agent in the beaker was aspirated out without impurities remaining using a syringe with a needle diameter of 0.7 mm. In the dissolving process, the litholytic agent is changed into a reddish brown transparent solution from a colorless transparent solution, and the whole gallstone is firstly crushed into small particles under the action of dissolution and ultrasound and then gradually dissolved and disappeared. The process and effect of the litholytic agent 1-5 for dissolving brown mixed gallstones is shown in figure 3.

Example 5

The stone dissolving agent has swelling effect on the latex tube used in the gall-stone dissolving operation. Different solvents cause different swelling degrees of the latex tube, and serious swelling appears to bring great negative effects to the gall-stone dissolving operation.

The effect of the following four litholytic agents on the degree of swelling of the latex tube was studied:

1-3 of litholytic agent: water, 7.5mL, 10 mL;

1-5 parts of litholytic agent: 10mL of diethylene glycol butyl ether and 10mL of water

Two sections of latex tubes with equal length are taken and completely soaked in the two stone dissolving agents for 24 hours, and then the changes of the mass, the volume and the length of the latex tubes before and after soaking are compared. Three sections of latex tubes with equal length are taken, deionized water is used as a negative control group, and traditional litholytic agents isoamyl acetate and methyl tertiary butyl ether are used as positive control groups.

Before soaking, the mass of the latex tube is weighed, and the length of the latex tube is measured by a steel plate ruler. And adding a fixed amount of deionized water into the measuring cylinder, immersing the latex tube into the measuring cylinder, and reading the difference to obtain the volume of the latex tube.

The latex tube was allowed to dry naturally for 1 hour at room temperature, then placed in the above-mentioned litholytic agent, and the comparative latex tube was soaked in sufficient amounts of deionized water, isoamyl acetate, and methyl t-butyl ether for 24 hours. After completion of the soaking and after natural drying at room temperature for 1 hour, the mass, length and volume of the latex tube were measured in the same manner as described above, and the results are shown in Table 1.

TABLE 1 emulsion tube Change ratio before and after immersion

It can be seen from the data change before and after latex tube soaking that the existing stone-dissolving agent isoamyl acetate and methyl tert-butyl ether can obviously cause the swelling of the latex tube, and the swelling of the latex tube by the stone-dissolving agent provided by the invention is obviously reduced.

The invention has been described in detail with reference to specific embodiments and/or illustrative examples and the accompanying drawings, which, however, should not be construed as limiting the invention. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.