JP2008543790A - Treatment of short bowel syndrome - Google Patents

Abstract

  A pharmaceutical composition for treating or preventing short bowel syndrome is provided. The composition comprises an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor. The composition has been shown to increase body weight and increase intestinal absorption surface area in short bowel syndrome. Dosage units, methods of treatment, uses and kits are also provided.

Description

  The present invention generally relates to compositions and methods for treating or preventing conditions involving short bowel syndrome (SBS) or colon resection, small intestine resection, and large intestinal resection.

  Short bowel syndrome (SBS) is defined as various malabsorption that occurs after surgical removal of the main part of the small intestine. This syndrome is characterized by poor malabsorption of nutrients. SBS affects people of all ages, from premature infants to adults. Surgical removal of the small intestine of significant length is required and there are many conditions that cause SBS, including but not limited to necrotizing enterocolitis (NEC), abdominal wall defects, jejunal ileal closure, midgut torsion and inflammatory bowel Including disease.

  In infants, SBS can be caused by a number of events, for example after surgical intervention of necrotizing enterocolitis (NEC) or intestinal torsion. SBS causes stunting, growth disorders and severe developmental disorders, and reduced absorption of nutrients that can be fatal (Buchman, AL, et al., 1994; Strong, SA, et al., 1999). In addition, children can progress to the development of end-stage liver disease secondary to cholestasis induced by long-term complete parenteral nutrition.

  In adults, SBS can be caused by many factors, such as surgical intervention for Crohn's disease and other illnesses, resulting in reduced nutrient absorption and sometimes certain essential nutrient deficiencies. This occurs when the distal ileum is removed. The distal ileum develops mechanisms for the absorption of certain essential nutrients (eg, vitamin B12 and other fat-soluble vitamins) and the absorption of bile salts. In adults, SBS is less life-threatening than in infants, but to serious lifestyle changes such as chronic diarrhea, chronic dehydration, muscle spasms, total parenteral nutrition (TPN) Dependence, infections and dependence on exogenous nutritional supplements such as vitamin B12.

  Numerous researchers have demonstrated the role of EGF in intestinal adaptation after massive resection of the small intestine (Chaet, MS et al., 1994; Dunn, JC et al., 1997; O'Loughlin , EV et al., 1994; Sham, J. et al., 2002; Shin, CE et al., 1998; Thompson, JS, 1999). EGF treatment increases mucosal hyperplasia in the residual intestine in response to massive resection of the small intestine (Chaet, MS et al., 1994; Goodlad, RA et al., 1988; Shin, CE 1998), glucose absorption (Hardin, JA et al., 1997; O'Loughlin, EV et al., 1994) and digestive enzyme expression (Dunn, JC. Et al., 1997; O 'Loughlin, EV et al., 1994). The main source of endogenous production of EGF is from the salivary glands. Adaptability after resection of the small intestine is weakened by salivary gland excision, which can be recovered by using exogenous EGF (Helmrat, MA et al., 1998). In the remaining intestine, EGF receptor expression is increased (Avissar, NE et al., 2000; Warner, BW et al., 1997). Adaptability is weakened in animals with deficient EGF receptors (Helmrat, MA et al., 1997), and overexpression of EGF in the intestine enhances adaptability after small bowel resection (Erwin, CR et al. 1999). Furthermore, EGF treatment has been shown to suppress the rate of apoptosis of enterocytes after massive resection of the small intestine and contribute to the ability of EGF to maintain the integrity of the barrier (Stern, LE et al., 2000). Finally, in a recent human clinical trial, five SBS pediatric patients with normal <50% intestinal length were treated with oral recombinant human EGF. EGF treatment increased 3-O-methylglucose absorption and diet tolerance, and all patients gained weight. There were no adverse events associated with this therapy (Sigalet, D. et al., 2005).

  The major sources of endogenous arginine production, L-arginine and L-citrulline in plasma are reduced in pediatric patients who cannot be adapted after massive resection of the small intestine (Wasa, M. et al., 1999) without SBS It is also reduced in adult and pediatric patients who are dependent on TPN with SBS compared to patients who are dependent on total parenteral nutrition (TPN) (Wasa, M. et al., 1999). L-citrulline is produced mainly by degradation of glutamine in the intestine and decreased L-citrulline production (Chen, K. et al. 1996; Dejong, CH et al. 1998), decreased glutamine uptake in the intestine (Chen, K. et al., 1996) and reduced conversion of citrulline to arginine in the kidney (Dejung, CH. Et al., 1998). Resected rats fed an arginine deficient diet show weight loss, negative nitrogen balance, and markedly reduced muscle arginine concentrations, suggesting that arginine becomes an essential amino acid after massive resection of the small intestine (Wakabayashi, Y Et al., 1994).

  Several studies examining the role of arginine in post-resectal intestinal adaptation have produced conflicting results. In rats that underwent massive resection of the small intestine, supplementation with a basic diet with L-arginine increased structural fitness (Hebigichi, T. et al., 1997). Camli et al. (2005) reported increased body weight and increased intestinal adaptability after intraperitoneal arginine administration in a model of SBS. They also point to increases in serum, saliva and urine EGF after arginine administration, and conclude that the beneficial effects of arginine may be due to upregulation of EGF levels. Intraperitoneal L-arginine administration has been reported to increase body weight gain, cell proliferation and structural fitness in rats undergoing 90% small bowel resection (Ozturk, H. et al., 2002). Oral delivery of glutamine and arginine precursor, ornithine alpha-ketoglutarate, to the rat after massive resection of the small intestine is related to intestinal structural fitness, ornithine decarboxylase activity and ornithine content, and glutamine in plasma and muscle It has been reported to increase levels (Dumas, F. et al., 1998). Conversely, in the rat model of SBS, animals given L-arginine in drinking water have reduced jejunum and ileal weights, increased enterocyte apoptosis rate, and residual intestinal It has been shown that structural adaptability is reduced (Sukhotnik et al., 2003). Subcutaneous arginine administration in a rat model of SBS reduced protein synthesis and glutamine uptake in the intestine, indicating that although the intestinal permeability was improved in rats given arginine, the adaptation process was suppressed ( Welters, CF et al., 1999). In a model of SBS, parenteral administration of arginine weakened the intestinal adaptability compared to untreated excised animals (Sukhotnik et al., 2005). Administration of glutamine-reinforced TPN to rats undergoing massive resection of the small intestine significantly increased intestinal glutamine uptake, mucosal glutaminase activity, and mucosal DNA and protein content, but compared to controls, L-arginine levels were reduced (Chen, K. et al., 1996). Finally, stable SBS patients who were fed a arginine-free diet for 5 days and had an average elapsed time of 46 months after resection of the intestine had a significant decrease in plasma arginine, ornithine and hydroxyproline levels, and orotine It showed changes in urinary excretion of acids and nitrogen compounds, but with no adverse clinical events (Pita et al., 2004).

  However, current treatment options for SBS remain limited. In addition to drugs, a special diet is prescribed to suppress peristalsis and reduce gastric acid. For SBS that does not heal over time, lifelong treatment may be required. In severe cases, a liquid food or small intestine transplantation administered intravenously is considered.

  One aspect of the present invention comprises an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor, wherein the agonist and L-arginine, a biological equivalent of L-arginine or The ratio with the NO donor is 1: 4540000-1: 1 (mol: mol), or 1: 45400000 to about 1: 4500 (mol: mol), or 1: 4540000-1: 45000 (mol: mol). It is to provide a composition for treating or preventing short bowel syndrome. The composition can be in solid form, lyophilized form or solution form. The solution may be suitable for oral delivery to humans, for example enteral delivery to humans. The solution may also be suitable for intravenous administration. The EGF receptor agonist is EGF and can be synthesized, optionally produced by chemical synthesis or recombinant techniques, or derived from natural sources. The composition may comprise L-arginine. The composition can be used for weight gain purposes in persons with short bowel syndrome or at risk for short bowel syndrome. The composition can also be used for the purpose of increasing the absorbed surface area of the intestine in persons with short bowel syndrome or at risk for short bowel syndrome. The ratio of EGF receptor agonist to L-arginine, a biological equivalent of L-arginine or a NO donor can be 1: 100,000 (mol: mol).

  Another aspect of the invention is L-arginine, dissolved in a pharmaceutically acceptable liquid, suitable for oral administration to a subject at risk of short bowel syndrome or diagnosed with short bowel syndrome, It is to provide a dosage unit comprising a biological equivalent of L-arginine or a NO donor and an EGF receptor agonist. The liquid may be selected from the group consisting of water, saline, infant formula, buffer, milked milk, other suitable carriers, and combinations thereof. The ratio of agonist to L-arginine, the biological equivalent of L-arginine or the NO donor is 1: 4540000-1: 1 (mol: mol), or 1: 45400000 to about 1: 4500 (mol: mol). ), Or 1: 4540000 to 1: 45000 (mol: mol). The EGF receptor agonist is EGF and the dosage unit may comprise L-arginine.

  Another aspect of the invention is an L-arginine dissolved in a pharmaceutically acceptable solution and suitable for intravenous administration to a subject at risk of short bowel syndrome or diagnosed with short bowel syndrome. Providing a dosage unit comprising a biological equivalent of L-arginine or a NO donor and an EGF receptor agonist. The ratio of agonist to L-arginine, the biological equivalent of L-arginine or the NO donor is 1: 4540000-1: 1 (mol: mol), or 1: 45400000 to about 1: 4500 (mol: mol). ), Or 1: 4540000 to 1: 45000 (mol: mol). The EGF receptor agonist is EGF and the dosage unit may comprise L-arginine.

  Another aspect of the invention is L-arginine, dissolved in a pharmaceutically acceptable solution, suitable for parenteral administration to a subject at risk of short bowel syndrome or diagnosed with short bowel syndrome. Providing a dosage unit comprising a biological equivalent of L-arginine or a NO donor and an EGF receptor agonist. The liquid may be selected from the group consisting of water, saline, infant formula, buffer, milked milk, other suitable carriers, and combinations thereof. The ratio of agonist to L-arginine, the biological equivalent of L-arginine or the NO donor is 1: 4540000-1: 1 (mol: mol), or 1: 45400000 to about 1: 4500 (mol: mol). ), Or 1: 4540000 to 1: 45000 (mol: mol). The EGF receptor agonist is EGF and the dosage unit may comprise L-arginine.

  The dosage unit of the present invention can be used for weight gain purposes in persons with short bowel syndrome or at risk for short bowel syndrome. The dosage unit can also be used for the purpose of increasing the intestinal absorption surface area in persons with short bowel syndrome or at risk for short bowel syndrome.

  Another aspect of the present invention is a method for treating a patient diagnosed with short bowel syndrome or at risk for short bowel syndrome, comprising an EGF receptor agonist and L-arginine, the biological equivalent of L-arginine. Providing a product or NO donor to a patient in need thereof. The patient may be a patient who has already undergone a small bowel resection or will undergo a future small bowel resection.

  Another aspect of the present invention is a method for treating or preventing short bowel syndrome, preferably at least once a day, an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or It is to provide a method comprising administering NO donors enterally to a patient in need thereof. The EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor can be administered together as a mixture, for example, at least once a day. The EGF receptor agonist can be EGF. EGF can be synthesized by recombinant techniques or synthetic chemical processes. The method may include L-arginine.

  The methods of the invention can be used for weight gain purposes in persons with short bowel syndrome or at risk for short bowel syndrome. The method can also be used to increase the absorbed surface area of the intestine in people with short bowel syndrome or at risk for short bowel syndrome.

  Another aspect of the invention includes therapeutic amounts of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor and instructions for use in the treatment or prevention of short bowel syndrome. Is to provide a kit. An EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor can be formulated and supplied in solid form. The kit may also include instructions for dissolving the solid form in a solution suitable for oral administration, eg, intravenous administration. The agonist and L-arginine, the biological equivalent of L-arginine or the NO donor may be supplied separately. They can be in solid or solution form. The instructions may include mixing the agonist with L-arginine, a biological equivalent of L-arginine or a NO donor prior to administration.

  The kit can be used for weight gain purposes in persons with short bowel syndrome or at risk for short bowel syndrome. The kit can also be used to increase the absorbed surface area of the intestine in people with short bowel syndrome or at risk for short bowel syndrome.

  Another aspect of the present invention is a method of treating or preventing SBS in a patient comprising (i) an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or NO over the first treatment period. Administering a composition comprising a donor subcutaneously or intravenously to a patient; and (ii) an EGF receptor agonist and L-arginine, L-arginine, over a second treatment period after the first treatment period. It is to provide a method comprising enterally administering to a patient a composition comprising a biological equivalent or NO donor. The second treatment period may begin after ileus has been cured. Enteral administration can include enema administration or oral administration. The second treatment period includes two phases, where administration may be performed orally in the first phase and administration by enema in the second phase. For example, administration during the first treatment period can be performed intravenously, and administration during the second treatment period can be performed 4-6 days after resection by the oral route.

  Another aspect of the invention is a method of treating an SBS patient or a patient at risk for SBS, comprising delivering an EGF receptor agonist to the patient's intestine and producing NO in the patient's intestine. It is to provide a method comprising increasing in-vivo production. Increasing in vivo production of NO can include administering a nitric oxide synthase substrate to the patient, eg, administering a NO donor. The patient may already have the main part of the small intestine surgically removed.

  The methods of the invention can be used for the purpose of gaining weight in persons with short bowel syndrome or at risk for short bowel syndrome. The method can also be used to increase the absorbed surface area of the intestine in people with short bowel syndrome or at risk for short bowel syndrome.

  Another aspect of the invention is to provide the use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor for the treatment or prevention of short bowel syndrome. .

  Another aspect of the present invention is an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a biological equivalent for increasing weight in a person with short bowel syndrome or at risk for short bowel syndrome. Providing use with NO donors.

  Another aspect of the present invention is the biological of EGF receptor agonists and L-arginine, L-arginine to increase intestinal absorption surface area in persons with short bowel syndrome or at risk for short bowel syndrome. To provide use with an equivalent or NO donor.

  Another aspect of the present invention is the use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor for the preparation of a medicament for treating or preventing short bowel syndrome Is to provide.

  Another aspect of the invention is the use of an EGF receptor agonist and L-arginine, L-arginine for the preparation of a medicament for increasing body weight in a person with short bowel syndrome or at risk for short bowel syndrome. To provide for the use of a biological equivalent or NO donor.

  Another aspect of the invention is an EGF receptor agonist and L-arginine, L, for preparing a medicament for increasing the intestinal absorption surface area in a person with short bowel syndrome or at risk for short bowel syndrome. -To provide use of a biological equivalent of arginine or a NO donor.

  These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. In addition, various references are provided below that describe the specific procedures, equipment or compositions in more detail. All references described herein are hereby incorporated by reference as if the entire contents of each document were reprinted herein. Applicants reserve the right to directly incorporate any or all such documents at their discretion while the application is pending.

  Before describing the present invention, it will be helpful in understanding the present invention to explain the definitions of certain terms used below.

  As used herein, the term “comprising” means containing a particular component, including a particular component, or consisting of a particular component, but only to those components. It is not limited. This term is non-limiting. For example, the term “pharmaceutical composition comprising EGF and L-arginine” means any pharmaceutical composition containing EGF and L-arginine, the composition comprising other components such as another active ingredient, Perfumes, adjuvants and the like may also be included.

  As used herein, the term “short bowel syndrome” or SBS, or “short gut syndrome” refers to various symptoms resulting from malabsorption of nutrients, such as significant intestinal It refers to gastrointestinal syndrome, which is characterized by abdominal pain, diarrhea, fluid retention, unintentional weight loss and extreme fatigue that occurs after surgical removal of a long length. Thus, as used herein, the term “short bowel syndrome” also encompasses short gastrointestinal syndrome and large bowel resection. Intestinal hormonal reflexes and feedback loops may be disrupted, resulting in increased amounts of proximal stomach and small intestinal secretions, and altered motility patterns. The loss of moisture, sodium and magnesium can lead to electrolyte imbalances. Certain specific absorption functions unique to specific parts of the intestine, such as absorption of vitamin B12, bile salts and other fat-soluble vitamins by the ileum, may be impaired.

  As used herein, an “EGF receptor agonist” is any of the following effects when bound to any of the erbB (1-4) receptors, particularly the erbB1 receptor: Any molecule that produces a biochemical effect: increases intestinal glucose transport, alters the apical surface of enterocytes (cells covering the lumen of the small intestine), colonization of pathogens across the mucosal surface Or metastasis is suppressed and intestinal maturation is induced. The molecule is preferably an epidermal growth factor. Otherwise, the molecule can be an antibody, small molecule, protein, peptide, peptidomimetic or peptidomimetic.

As used herein, “epidermal growth factor” or EGF is a 53 amino acid protein that is synthesized in normal human duodenum and salivary glands and is known to be expressed in human breast milk. The amino acid sequence of human EGF is:
Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg;
(Boonstra et al., 1995).

The protein used in the experiments described herein had the sequence described above. The EGF used in the present invention is recombinant human EGF (purity> 97%) obtained from Protein Express (Higeta-Shoyu Co. Ltd., Japan). Non-human EGF sequences that act as EGF in humans are also envisioned. Thus, EGF variants, such as those reported for mice, rats and pigs (Jorgensen, PE et al., 1998; Nexo, E. et al., 1985; Pascall, J.C. et al., 1991). And Simpson, RJ et al., 1985), or bovine EGF cited in US Patent Application No. 20030059802, or so-called super-agonist chimeras of various different EGF receptor ligands (Lenferink). AE et al., 2000). This definition also applies to polypeptides having substantially the same sequence and activity as purified natural epidermal cell growth factor. This includes peptides or proteins chemically synthesized by recombinant techniques. This term also applies to proteins that differ from the native sequence by substitution with other amino acids or deletion of one or more amino acids, as long as the biological activity of EGF is substantially retained. The Further, this definition provides that fragments of EGF, peptide analogs, peptidomimetics, and acylated forms in US Pat. No. 5,070,188, so long as the biological activity of EGF is substantially retained. Is also included. The biological activity of EGF can be screened by receptor binding assays and confirmed using any of the methods indicated above for receptor agonists. Thus, for example, human EGF protein in which methionine (Met) at position 21 is substituted with isoleucine (Ile) falls within the range of “EGF”. Such proteins are generally expressed as _{hEGF-I 21,} when prepared by recombinant techniques is generally expressed as _{rhEGF-I 21} (chemically synthesized hEGF is "hEGF ”). Similarly, position 11 Asp is the hEGF substituted with Glu, generally expressed as _{hEGF-E 11.} Some EGF proteins cleaved near the carboxy terminus retain their biological activity and are generally represented by a subscript indicating the last peptide residue retained. Thus, EGF lacking the last two of the normal 53 peptides is generally designated EGF ₅₁ . Proteins with amino acid deletions, eg, proteins lacking Trp ₄₉ , generally have the term “del” (or .DELTA.) And a subscript indicating its position without changing the number of the remaining amino acids. It is represented by Thus, when Trp ₄₉ is deleted, the resulting protein is EGF-. DELTA. ₄₉ . Insertion that increases chain length is generally indicated as a substitution that replaces one amino acid with two or more amino acids, eg, rhEGF-L / G ₁₅ is a natural Leu ₁₅ followed by a Gly insertion. Indicates that Finally, regardless of whether with or without other modifications, if His ₁₆ is replaced by another amino acid, EGF of the present invention are generally represented as EGF-X ₁₆ collectively. For example, as described in US Pat. No. 6,191,106 (Mullenbach et al.) Issued February 20, 2001, EGF muteins also have essential EGF activity. Is within the scope of this definition.

  As used herein, “L-arginine” means a semi-essential amino acid (2-amino-5-guanidinovaleric acid) and salts thereof, eg, acid addition salts suitable for administration to mammals. A biological equivalent of L-arginine, like L-arginine, is a compound that is a substrate for nitric oxide synthase that produces NO in vivo, or L via an arginine-citrulline cycle or an enzyme of the urea cycle. A compound that can be converted to a substrate for nitric oxide synthase such as citrulline. The rate-limiting enzyme in endogenous L-arginine production is arginine succinate synthase. The major site of endogenous L-arginine production is the kidney that converts L-citrulline to L-arginine (Boger, RH et al., 2001). Glutamine is converted to L-citrulline in the small intestine (Pita, AM et al., 2003), and ornithine α-ketoglutarate is a precursor of glutamine (Dumas, F. et al., 1998). “NO donors”, typically small organic molecule compounds that can donate NO molecules when administered in vivo, can also be administered. Such compounds include S-nitroso-N-acetyl-penicillamine (SNAP), 3-morpholinoside nonimine (SIN-1), sodium nitroprusside (SNP), 4-phenyl-3-furoxanecarbonitrile (PFC). , Glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN), including but not limited to (Feelisch, M., 1998; Pacher, P. et al., 2003; Zell, R. et al., 2003). NO production is performed in vitro by the Griess assay (Marion, R., M. et al., 2003) or by chemiluminescence detection (Kikuchi, K. et al., 1993; Kojima, H. et al., 1998). Or in vivo by using manometry and an electronic nitric oxide sensor (Snygg, J. et al., 2003; Levine, DZ et al., 2001). Commercially available assays for in vitro detection of NO are available from Cayman Chemicals (Ann Arbor, Michigan).

Treatment or prevention of short bowel syndrome Materials and methods Animal model of disease Sprague-Dawley rats (250-300 g) were initially housed and acclimatized for 7 days, once daily for 4 days before surgery. The simulated food was supplemented with tubes.

Experimental Design Animals were randomly assigned to the following 5 groups: 1) excised and supplemented with vehicle (0.9% sterile saline) daily (SBS), 2) excised, arginine 1.5 mmol / kg / day tube replenished daily (Arg), 3) excised, EGF 100 μg / kg / day tube replenished daily (EGF), 4) excised, EGF 100 μg / kg / day and arginine 1.5 mmol Tube replenish daily / kg / day (EGF-Arg), and 5) Control reconstituted daily with vehicle (Con). EGF-Arg treatment is about 1: 100,000 mole: mole. The L-arginine used in this study was not pure L-arginine with a molecular weight of 174.2 but an amino acid hydrochloride with a molecular weight of 210.7. After overnight fast, as described above, surgery was performed under halothane anesthesia to secure a 25 cm residual small intestine (Sham, J. et al., 2002). Briefly, after a laparotomy, a 25 cm jejunum extending distally from the Treitz ligament is measured, the intestine is divided at that point, and the entire distal intestine is located in the middle of the ascending colon just below the right colonic artery The part was removed and an anastomosis was performed. Animals were tube supplemented with test compounds in 1 cc of vehicle daily for 9 days from 9 am to 10 am. The EGF used in the present invention is recombinant human EGF obtained from Protein Express ™ (Higeta-Shoyu Co. Ltd., Japan).

Measurements After excision, animals are allowed to ingest water for the remainder of the day after surgery, and then start pair feeding on the second day after surgery and freely dry food up to 20 g / day. Ingestion was allowed. The animals and food in each animal cage were weighed daily to quantify the amount of food consumed. The animals were fed a normal diet of 20 g per day. Each set of controls (resected and non-resected) + 3 treatment groups were grouped into 5 animals. If an animal in a group ate less than 20g, the entire set of 5 animals gained the weight the animal ate plus 10% each day thereafter. The number of animals in each group was as follows: arginine group, EGF group and EGF + arginine group n = 10, SBS (untreated) group n = 9, control n = 8.

  The animals were fasted overnight before the 10th day of lethality. There was a high mortality among these animals with SBS (finally n = 3 for untreated SBS animal group, n = 6 for Arg group and EGF + Arg group, n = 7 for EGF treated group, n = for control) 8). After the usual test compound dosing in the morning, all animals were injected intraperitoneally with bromodeoxyuridine (BRDU) (50 mg / kg) with oral tube supplementation of 2 ml Ensure ™. . One hour after BRDU injection, the animals were anesthetized with halothane and the intestines were removed and washed. Intestinal length was determined using the standardized weight and the measured residual jejunum weight and diameter. A 2 cm tissue fragment was taken 2 cm below the Treitz ligament and fixed for histological and BRDU evaluation.

Statistical analysis The results are reported as mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) using the Tukey post test. P <0.05 was considered significant.

Results Surgery resulted in severe SBS with significant weight loss and mortality in the resected group. For this reason, animal weights were thoroughly analyzed up to 5 days after surgery prior to significant mortality in the experimental group. As shown in FIG. 1, weight loss was expressed as a percentage of the original weight, and those data points were plotted to determine the slope of each growth curve and compared. Weight loss was significantly greater in all treated and untreated SBS groups compared to the control group (p <0.001). Furthermore, weight loss was significantly less in animals that received the EGF-arginine combination compared to the untreated SBS group (p <0.05). Weight loss was not different between treated groups or between untreated SBS animals and animals treated with EGF or arginine alone. Treatment with EGF or arginine alone did not have a statistically significant effect, whereas treatment with a combination of both EGF and arginine resulted in a statistically significantly smaller weight loss. Therefore, animals treated with a combination of EGF and arginine gained more weight than animals treated with EGF or arginine alone.

  The gut width is shown in FIG. 2 (as a measure of gut diameter). Intestinal width was significantly increased in animals treated with the EGF-arginine combination (p <0.05) compared to control animals. Intestinal width did not differ between any other groups. Thus, treatment with EGF or arginine alone did not have a statistically significant effect on intestinal width, while treatment with EGF and arginine resulted in a statistically larger intestinal width. An increase in intestinal width for the same intestine length results in an increase in the entire intestinal absorption surface area.

  FIG. 3 shows the effect of various treatments on overall intestinal parameters in post-mortem animals. Intestinal wet weight per length was significantly increased in all treated and non-treated SBS groups compared to the control group due to a significant increase in intestinal weight in all treated groups (p <0.01). . Intestinal length did not differ between any groups. There was a trend toward increased gut wet weight in the untreated SBS group and in the EGF-arginine combination group compared to the EGF group and the arginine group, but this trend did not reach statistical significance . Although not statistically significant, treatment with EGF and arginine tended to outweigh both EGF and arginine treatments.

  FIG. 4 shows villi length and crypt depth in jejunal tissue obtained from control, SBS, arginine, EGF and EGF-arginine treated animals. Villi length was significantly increased in EGF treated animals compared to the control group (p <0.05). The villi length was not different between any other groups. Crypt depth was significantly increased in EGF (p <0.05), EGF-arginine and untreated SBS (p <0.01) compared to controls. Crypt depth was not different between arginine treated animals and controls, or between any excised groups.

CONCLUSION EGF-arginine combined administration was significantly more effective in treating SBS than EGF or arginine administered alone. The EGF-arginine combination treatment resulted in increased body weight and increased intestinal absorption surface area in resected test animals compared to EGF or arginine given alone. Weight gain is the primary endpoint for this condition and indicates the clinical benefit of treatment. An increase in the intestinal absorption surface area is the second evaluation item, and is necessary to achieve the first evaluation item. This study demonstrates a significant improvement in both the first and second endpoints of such conditions.

  Single EGF and arginine were shown to provide some protection against weight loss, but only combination treatment was statistically significant compared to untreated SBS control animals. Moreover, only the combination treatment improved the intestinal absorption surface area. Thus, the present invention demonstrates the feasibility of combining EGF and arginine to treat short bowel syndrome. It is clearly shown that the combination actually works by increasing the weight of patients suffering from SBS and increasing the intestinal absorption surface area. Prior to the present invention, studies using arginine alone to treat SBS have shown the advantages of some studies, but are contradictory in that other studies have shown disadvantages. In addition, other studies have shown that glucagon-like peptide II, bombesin, insulin-like growth factor 1 octreotide ™, natural conjugated bile acids, low-fat diet and glutamine have beneficial and / or adverse effects. Show you get. However, the present invention clearly demonstrates that the combination of EGF and arginine is more effective in treating SBS than when EGF or arginine is administered alone.

Pharmaceutical composition for treating or preventing short bowel syndrome The treatment or prevention of short bowel syndrome comprises an EGF receptor agonist and L-arginine or a biological equivalent of L-arginine. SBS develops as the subject experiences malabsorption after surgery. Therefore, promoting absorption after surgery is expected to help prevent.

  The relative amounts of EGF receptor agonist and L-arginine are conveniently determined on a molar basis. A biological equivalent of L-arginine is, on a molar basis, a molar equivalent basis for the amount of NO that the biological equivalent can produce if it can produce more NO than L-arginine as a substrate for nitric oxide synthase. Determined by Similarly, the amount of NO released by the NO donor is determined on a molar equivalent basis. Accordingly, a method for treating or preventing SBS is provided by delivering an EGF receptor agonist to a patient's intestine and enhancing in vivo production of NO in the patient's intestine. This may include administration of a substrate for nitric oxide synthase or administration of a NO donor.

  The treatment of the present invention requires administration by an appropriate route. For administration of bioavailable substances to the free cavity side of the intestinal epithelial cells, administration by the oral route will be most preferred. As such, dosage units of these two substances are provided in a container suitable for easy opening and delivery and mixing into a pharmaceutically acceptable solution such as a dietary article. Thus, an appropriate amount of the ingredients of the present invention can be provided in powder or granular form that is added directly to milk or other food. A subject in need of total parenteral nutrition (TPN) can benefit from receiving these components as separate formulations or as components of a TPN composition. Alternatively, these components can be provided in a solution form suitable for immediate digestion by the patient or diluted with a suitable solution. The preceding solution can be administered to the patient via an oral gastric tube.

  Examples of suitable dietary products include water, saline, buffers, oral rehydration solutions, specialty formulas and milked breast milk, other suitable carriers, or combinations thereof. Any solution suitable for oral administration may be used. Additives may be added that protect EGF from enzymatic degradation by pancreatic proteases (Playford et al., 1993) and act as bystander proteins (ie, an inactive protein “filler”). For example, casein (milk protein) has been used experimentally for this purpose (Playford et al., 1993). Other approaches may include administering with a protease inhibitor to preserve EGF structure and activity.

  Alternatively, the treatment of the present invention may be administered orally, enterally, parenterally, by intravenous injection, subcutaneously, nasally, or by enema. For example, when initially treating SBS, during the initial period of treatment, some patients cannot receive oral treatment, so it is possible to select an intravenous route during this period. The composition can be formulated as a spray, solution, suspension, colloid, concentrate, powder, granule, tablet, compressed tablet, capsule (including coated or uncoated tablet or capsule), suppository, etc. . Sustained release or controlled release formulations are also included.

  The preparation may contain additives such as a viscosity adjusting agent, an osmotic pressure adjusting agent, a buffering agent, a pH adjusting agent, a fragrance, a stabilizer, a coloring agent, a preservative, etc.

  The dosage unit is a dose suitable for administration in a single administration, i.e. in a single ingestion of the patient. Thus, for example, the dosage unit of L-arginine administered as the hydrochloride salt of L-arginine is about 20 mg / kg / day (0.09 mmol / kg / day) to about 2000 mg / kg / day (9 mmol / kg / day). Or more preferably about 100 mg / kg / day (0.45 mmol / kg / day) to about 1000 mg / kg / day (4.5 mmol / kg / day), or more preferably about 200 mg / kg / day ( 0.9 mmol / kg / day) to about 500 mg / kg / day (2.4 mmol / kg / day) of L-arginine, or more preferably about 250 mg / kg / day (1.2 mmol / kg / day) to About 400 mg / kg / day (1.9 mmol / kg / day), more preferably about 300 mg / kg / day (1.4 mmol / kg / day) to about 350 mg / kg / day (1.6 mm l / kg / day), and about 0.2 μg / kg / day (0.032 nmol / kg / day) to about 2 mg / kg / day (0.32 μmol / kg / day), or more preferably about 1 μg / day kg / day (0.16 nmol / kg / day) to about 1 mg / kg / day (0.16 mmol / kg / day), or more preferably about 2 μg / kg / day (0.32 nmol / kg / day) to About 0.2 mg / kg / day (32 nmol / kg / day) of EGF receptor agonist. As used herein, “μg” means microgram, “μmol” means micromole, and so on. Typically, the ratio of L-arginine-EGF receptor agonist is about 1: 454000000 EGF receptor agonist (mol) / L-arginine (mol) to about 1: 1 EGF receptor agonist (mol) / L-arginine (mol), more preferably 1: 45400000 EGF receptor agonist (mol) / L-arginine (mol) to about 1: 4500 EGF receptor agonist (mol) / L-arginine (mol), more Preferably from 1: 4540000 EGF receptor agonist (mol) / L-arginine (mol) to about 1: 45000 EGF receptor agonist (mol) / L-arginine (mol). The treatment will probably be administered at least once a day, 3 or 4 times a day, or even continuously. Intermittent administration can be administered by any suitable route, such as bolus injection, oral formulations described elsewhere herein, subcutaneously, or by continuous intravenous infusion. More sustained administration will more typically be by intravenous infusion or controlled release implants.

  The treatment of the present invention can also be used to preventively treat a person at risk of developing SBS. Those at risk may include patients who have just undergone surgical resection of the main small intestine. Such treatment may begin as soon as possible after surgery, possibly after the surgery. After resection, the intestine usually begins to function after 3-4 days. Thus, oral treatment can be started 4-6 days after surgery. If prompt treatment after surgery is required, the therapy is administered intravenously, possibly followed by oral treatment when bowel function resumes. Thus, the treatment will include a first treatment period followed by a second treatment period.

  Generally speaking, EGF is prepared by a synthetic process and manufactured by conventional biotechnology or chemical techniques. Of course, EGF may be obtained from natural sources.

  Preferably, the combination of elements of the invention will be supplied as a single mixture and administered together, but they are provided as separate compartments and mixed for administration Or can be administered separately. Both may be bioavailable on the luminal side of the intestinal epithelial cells.

  When prepared for admixture with a liquid for delivery with water, the dosage unit may incorporate a solubility enhancer therein.

  The use of a coated composition that does not dissolve until it reaches the intestine often increases the effectiveness of the medication. See "Remington's Pharmaceutical Sciences" edited by Gennaro (Mack Publishing Company, 19th edition, 1995). Pharmaceutically acceptable salts of the active agent (eg, acid addition salts of L-arginine) are reported standard procedures known to those skilled in the art of synthetic organic chemistry (March, J., 1992). Can be prepared. It may also be desirable to include suitable pharmaceutically acceptable carriers, such as those commonly used with peptide-based agents such as diluents, excipients and the like.

  Of course, the product will be provided in a sealed, sterile package. Typically, EGF or equivalent polypeptide is provided as lyophilized material.

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FIG. 1 is a graph showing the weight loss expressed as a percentage of the animal's original body weight for the five test groups of rats. FIG. 2 shows untreated (SBS), treated with arginine (arg), treated with EGF (EGF), treated with EGF and arginine (EGF-arginine), and no SBS (control) SBS animals It is a bar graph which shows the width | variety of the intestine at the time of autopsy. Figures 3a, 3b and 3c are bar graphs showing the overall gut morphology at necropsy, representing gut length, gut wet weight and gut wet weight / length, respectively. FIGS. 4a and 4b are bar graphs representing jejunal villus length and crypt depth in control, SBS, arg treated, EGF treated and EGF-arg treated animals.

Claims (75)

  An EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor, wherein the ratio of the agonist to L-arginine, a biological equivalent of L-arginine or a NO donor is 1: 4540000 to about 1: 1 (mol: mol), or 1: 4540000 to about 1: 4500 (mol: mol), or 1: 4540000 to about 1: 45000 (mol: mol). A composition for treating or preventing.   The composition of claim 1 in solid form.   3. A composition according to claim 1 or 2 in lyophilized form.   The composition of claim 1 in solution form.   5. The composition of claim 4, wherein the solution is suitable for oral delivery to a human.   5. The composition of claim 4, wherein the solution is suitable for enteral delivery to a human.   5. A composition according to claim 4, wherein the solution is suitable for intravenous administration.   The composition according to any one of claims 1 to 7, wherein the EGF receptor agonist is EGF.   9. The composition of claim 8, wherein the EGF receptor agonist is a synthetic, optionally produced by chemical synthesis or recombinant techniques, or derived from a natural source.   The composition according to any one of claims 1 to 9, comprising L-arginine.   L-arginine, a biological equivalent of L-arginine or NO donor and EGF receptor agonist suitable for oral administration to a subject at risk for short bowel syndrome, dissolved in a pharmaceutically acceptable liquid A dosage unit comprising   L-arginine, a biological equivalent of L-arginine or an NO donor and an EGF receptor agonist suitable for oral administration to a subject diagnosed with short bowel syndrome, dissolved in a pharmaceutically acceptable liquid A dosage unit comprising   The pharmaceutically acceptable liquid is selected from the group consisting of water, saline, special infant formula, buffer, milked milk, other suitable carriers, and combinations thereof. The dosage unit described in 1.   The ratio of agonist to L-arginine, a biological equivalent of L-arginine or a NO donor is from 1: 4540000 to about 1: 1 (mol: mol), or 1: 4540000 to about 1: 4500 (mol: 14. A dosage unit according to claim 11, 12 or 13, which is from 1 mol.   The dosage unit according to any one of claims 11 to 14, wherein the EGF receptor agonist is EGF.   16. A dosage unit according to any one of claims 11 to 15, comprising L-arginine.   L-arginine, a biological equivalent of L-arginine or an NO donor and an EGF receptor suitable for intravenous administration to a subject at risk for short bowel syndrome, dissolved in a pharmaceutically acceptable solution A dosage unit comprising an agonist.   L-arginine, a biological equivalent of L-arginine or an NO donor and an EGF receptor suitable for intravenous administration to a subject diagnosed with short bowel syndrome dissolved in a pharmaceutically acceptable solution A dosage unit comprising an agonist.   The ratio of agonist to L-arginine, a biological equivalent of L-arginine or a NO donor is from 1: 4540000 to about 1: 1 (mol: mol), or 1: 4540000 to about 1: 4500 (mol: 19. A dosage unit according to claim 17 or 18, wherein the dosage unit is from 1: 4540000 to about 1: 45000 (mol: mol).   The dosage unit according to any one of claims 17 to 19, wherein the EGF receptor agonist is EGF.   21. A dosage unit according to any one of claims 17 to 20, comprising L-arginine.   L-arginine, a biological equivalent of L-arginine or an NO donor and an EGF receptor suitable for parenteral administration to a subject at risk for short bowel syndrome, dissolved in a pharmaceutically acceptable solution A dosage unit comprising an agonist.   L-arginine, a biological equivalent of L-arginine or an NO donor and an EGF receptor suitable for parenteral administration to a subject diagnosed with short bowel syndrome dissolved in a pharmaceutically acceptable liquid A dosage unit comprising an agonist.   24. A dosage unit according to claim 22 or 23, wherein the pharmaceutically acceptable liquid is selected from the group consisting of water, saline, buffer and complete parenteral nutritional formulation.   The ratio of agonist to L-arginine, a biological equivalent of L-arginine or a NO donor is from 1: 4540000 to about 1: 1 (mol: mol), or 1: 4540000 to about 1: 4500 (mol: 25) dosage unit according to any one of claims 22 to 24, wherein the dosage unit is from 1: 4540000 to about 1: 45000 (mol: mol).   26. A dosage unit according to any one of claims 22 to 25, wherein the EGF receptor agonist is EGF.   27. A dosage unit according to any one of claims 22 to 26, comprising L-arginine.   A method of treating a patient diagnosed with short bowel syndrome, comprising administering an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor to a patient in need thereof A treatment method comprising:   A method of treating a patient at risk for short bowel syndrome comprising administering an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor to a patient in need thereof. A treatment method comprising:   30. A method according to claim 28 or 29, wherein the patient has already undergone resection of the small intestine or undergoes resection of the small intestine in the future.   Preferably enterally administering the EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor at least once a day to a patient in need thereof. A method of treating or preventing short bowel syndrome in a patient.   32. The method of claim 31, wherein the EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor are administered together as a mixture.   35. The method of claim 32, wherein the mixture is administered at least once a day.   34. The method according to any one of claims 31 to 33, wherein the EGF receptor agonist is EGF.   35. The method of claim 34, wherein EGF is synthesized by recombinant techniques or synthetic chemical processes.   36. The method according to any one of claims 31 to 35, comprising L-arginine.   A kit comprising a therapeutic amount of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor and instructions for use in the treatment or prevention of short bowel syndrome.   38. The kit according to claim 37, wherein the EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor are supplied in a solid form.   40. The kit of claim 38, wherein the instructions comprise dissolving the solid form in a solution suitable for oral administration.   40. The kit of claim 38, wherein the instructions comprise dissolving the solid form in a solution suitable for intravenous administration.   40. The kit of claim 38, wherein the agonist and L-arginine, a biological equivalent of L-arginine or a NO donor are supplied separately.   42. The kit of claim 41, wherein at least one of the agonist and L-arginine, a biological equivalent of L-arginine or a NO donor is in solid form.   42. The kit of claim 41, wherein at least one of the agonist and L-arginine, a biological equivalent of L-arginine or a NO donor is a solution.   44. A kit according to any one of claims 41 to 43, wherein the instructions comprise mixing the agonist with L-arginine, a biological equivalent of L-arginine or a NO donor prior to administration. A method of treating or preventing SBS in a patient comprising:
(I) administering a composition comprising an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor over the first treatment period, subcutaneously or intravenously to the patient; and (Ii) Entering into the patient enteral compositions comprising an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor over a second treatment period after the first treatment period. A method of treating or preventing SBS, comprising administering.   46. The method of claim 45, wherein the second treatment period begins after ileus has healed.   47. The method of claim 45 or 46, wherein enteral administration comprises administration by enema.   47. The method of claim 45 or 46, wherein enteral administration comprises oral administration.   46. The method of claim 45, wherein the second treatment period comprises two phases, wherein the first phase administration is oral administration and the second phase administration is performed by enema.   50. The method of any one of claims 45 to 49, wherein administration during the first treatment period is performed by intravenous administration.   51. The method of any one of claims 45-50, wherein administration during the second treatment period is by the oral route and occurs 4-6 days after resection.   A method of treating SBS in a patient comprising delivering an EGF receptor agonist to the patient's intestine and increasing in vivo production of NO in the patient's intestine.   52. The method of claim 51, wherein increasing in vivo production of NO comprises administering a substrate for nitric oxide synthase to the patient.   52. The method of claim 51, wherein increasing in vivo production of NO comprises administering a NO donor.   A method of treating a person at risk for SBS, comprising delivering an EGF receptor agonist to the patient's intestine and increasing in vivo production of NO in the patient's intestine.   56. The method of claim 55, wherein increasing in vivo production of NO comprises administering a substrate for nitric oxide synthase to the patient.   56. The method of claim 55, wherein increasing in vivo production of NO comprises administering a NO donor.   58. The method of claim 55, 56 or 57, wherein the patient has already undergone surgical resection of the main small intestine.   The composition according to any one of claims 1 to 10, for use for the purpose of gaining weight in a person with short bowel syndrome or at risk for short bowel syndrome.   The composition according to any one of claims 1 to 10, for use for the purpose of increasing the intestinal absorption surface area in a person with short bowel syndrome or at risk for short bowel syndrome.   27. A dosage unit according to any one of claims 11 to 26 for use for the purpose of gaining weight in a person with short bowel syndrome or at risk for short bowel syndrome.   27. A dosage unit according to any one of claims 11 to 26 for use for the purpose of increasing the intestinal absorption surface area in persons with short bowel syndrome or at risk for short bowel syndrome.   37. A method according to any one of claims 27 to 36 for use for gaining weight in a person with short bowel syndrome or at risk for short bowel syndrome.   37. A method according to any one of claims 27 to 36 for use for the purpose of increasing the intestinal absorption surface area in a person with short bowel syndrome or at risk for short bowel syndrome.   45. Kit according to any one of claims 37 to 44, for use for the purpose of gaining weight in a person with short bowel syndrome or at risk for short bowel syndrome.   45. Kit according to any one of claims 37 to 44, for use for the purpose of increasing the intestinal absorption surface area in a person with or at risk for short bowel syndrome.   59. A method according to any one of claims 45 to 58 for increasing body weight in a person with or at risk for short bowel syndrome.   59. A method according to any one of claims 45 to 58 for use in increasing intestinal absorption surface area in a person with short bowel syndrome or at risk for short bowel syndrome.   Use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor for the treatment or prevention of short bowel syndrome.   Use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor for weight gain in persons with short bowel syndrome or at risk for short bowel syndrome.   Use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor to increase intestinal absorption surface area in persons with short bowel syndrome or at risk for short bowel syndrome .   Use of an EGF receptor agonist and L-arginine, a biological equivalent of L-arginine or a NO donor for the preparation of a medicament for treating or preventing short bowel syndrome.   EGF receptor agonist and L-arginine, biological equivalent of L-arginine or NO donor for the preparation of a medicament for weight gain in persons with short bowel syndrome or at risk for short bowel syndrome Use with.   An EGF receptor agonist and L-arginine, a biological equivalent of L-arginine, for preparing a medicament for increasing intestinal absorption surface area in a person with short bowel syndrome or at risk for short bowel syndrome or Use with NO donor.   11. The ratio of an EGF receptor agonist to L-arginine, a biological equivalent of L-arginine or a NO donor is 1: 100,000 (mol: mol). Composition.

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