U.S. patent application number 13/213325 was filed with the patent office on 2011-12-15 for methods and formulations for increasing intestinal function.
This patent application is currently assigned to TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD.. Invention is credited to Raanan Shamir, Naim Shehadeh, Igor Sukhotnik.
Application Number | 20110306544 13/213325 |
Document ID | / |
Family ID | 34971125 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306544 |
Kind Code |
A1 |
Sukhotnik; Igor ; et
al. |
December 15, 2011 |
METHODS AND FORMULATIONS FOR INCREASING INTESTINAL FUNCTION
Abstract
A method for increasing intestinal function is provided. The
method comprising orally and/or enterally administering to a
subject in need thereof a therapeutically effective amount of
insulin, thereby increasing intestinal function.
Inventors: |
Sukhotnik; Igor; (Haifa,
IL) ; Shehadeh; Naim; (Kfar-Yassif, IL) ;
Shamir; Raanan; (Herzlia, IL) |
Assignee: |
TECHNION RESEARCH & DEVELOPMENT
FOUNDATION LTD.
Haifa
IL
|
Family ID: |
34971125 |
Appl. No.: |
13/213325 |
Filed: |
August 19, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11628316 |
Dec 4, 2006 |
8026211 |
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PCT/IL2005/000587 |
Jun 2, 2005 |
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13213325 |
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60575803 |
Jun 2, 2004 |
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Current U.S.
Class: |
514/5.9 ;
530/303 |
Current CPC
Class: |
A61P 3/00 20180101; Y02A
50/30 20180101; A61P 35/00 20180101; A61K 9/20 20130101; A61P 37/06
20180101; A61P 29/00 20180101; A61P 1/00 20180101; A61P 5/00
20180101; A61P 31/04 20180101; A61K 38/28 20130101; Y02A 50/471
20180101; A61P 1/12 20180101 |
Class at
Publication: |
514/5.9 ;
530/303 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61P 1/00 20060101 A61P001/00; A61P 1/12 20060101
A61P001/12; A61P 3/00 20060101 A61P003/00; A61P 5/00 20060101
A61P005/00; A61P 29/00 20060101 A61P029/00; A61P 31/04 20060101
A61P031/04; A61P 37/06 20060101 A61P037/06; C07K 14/62 20060101
C07K014/62; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating a disease or condition associated with
intestinal malfunction in a subject in need thereof the method
comprising, orally and/or enterally administering to the subject a
therapeutically effective amount of insulin thereby treating the
disease or condition associated with intestinal function, wherein
the disease or condition is selected from the group consisting of:
(i) intestinal developmental or structural abnormalities including
short bowel syndrome, intestinal failure, midgut volvulus,
intestinal atresia, age associated weight loss, postgastrectomy
syndrome, dumping syndrome, postvagotomy diarrhea, bariatric
surgery-associated diarrhea, chronic diarrhea associated with
maldigestion and malabsorption, chronic diarrhea in idiopathis
primary gastrointestinal motility disorders, cystic fibrosis,
cancer, gastrointestinal peptide tumors, endocrine tumors and
chronic diarrhea in gastrinoma; (ii) intestinal inflammation
including inflammation due to infection, inflammation of the bowel,
necrotizing enterocolitis, severe acute gastroenteritis, chronic
gastroenteritis, cholera, chronic infections of the bowel, AIDS,
infectious diarrhea, diarrhea due to bacterial overgrowth,
irritable bowel syndrome, chronic diarrhea associated with
collagenous colitis, ischemia reperfusion and sepsis; (iii)
intestinal developmental abnormalities or inflammation including
secretory diarrhea, acute diarrhea and chronic diarrhea; (iv)
autoimmune disease, including Crohn's Disease, chronic ulcerative
colitis, immunologic disorders affecting the small intestine; and
(v) radiotherapy-associated intestinal malfunction, tube-feeding
related diarrhea, parenteral feeding dependency, diarrhea
associated with thyroid disorders, antibiotic-associated diarrhea,
nutritional deficiency, anemia, and an eating disorder.
2. The method of claim 1, wherein the disease or disorder is
selected from the group consisting of secretory diarrhea, acute
diarrhea and chronic diarrhea.
3. The method of claim 1, wherein the subject is a human
subject.
4. The method of claim 1, wherein the subject is a non-human
mammal.
5. The method of claim 3, wherein the subject is a full-term
infant.
6. The method of claim 1, wherein the orally administering is
effected by an oral dosage unit.
7. The method of claim 6 wherein the oral dosage unit comprises
from about 1 mu to about 10,000 units of said insulin.
8. The method of claim 6, wherein the oral dosage unit is
solid.
9. The method of claim 6, wherein the oral dosage unit is selected
from the group consisting of a pill, a dragee, a tablet and a
capsule.
10. The method of claim 1, wherein the insulin is administered in
an amount ranging from about 1 mu/Kg body weight/day to about 100
u/Kg/day.
11. An oral dosage unit form comprising from about 1 mu to about
10,000 units of insulin.
12. The oral dosage unit form of claim 11, said dosage unit is
solid.
13. The oral dosage unit of claim 12, selected from the group
consisting of a pill, a dragee, a tablet and a capsule.
14. The oral dosage unit of claim 12, further comprising a
pharmacological agent.
15. A method of increasing intestinal absorption of a
pharmacological agent, the method comprising orally and/or
rectal-enterally administering to a subject in need thereof a
therapeutically effective amount of insulin prior to, concomitant
with or following administration of the pharmacological agent,
thereby increasing intestinal absorption of the pharmacological
agent.
16. The method of claim 15, wherein each of the insulin and the
pharmacological agent is formulated in a dosage unit.
17. The method of claim 15, wherein the insulin and the
pharmacological agent are formulated in a dosage unit.
18. The method of claim 16 or 17, wherein the dosage unit is for
oral administration.
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention relates to methods and formulations
for increasing intestinal function, which may be used for treating
short bowel syndrome.
[0002] Short bowel syndrome (SBS) is defined as an intestinal
failure following the loss of intestinal length or competence below
the minimal amount necessary for the absorption of nutrients and a
normal nutritional status [Sigalet D L. Short bowel syndrome in
infants and children: an overview. Semin Pediatr Surg 2001;
10:49-55; Vanderhoof J A. Short bowel syndrome. Neonat
Gastroenterol 1996; 23:377-86; Booth I W, Lander A D. Short bowel
syndrome. Bailliere's Clin Gastroenterol 1998; 12:739-72].
[0003] SBS typically follows resection of 50% or more of the small
intestine and is associated with diarrhea, steatorrhea,
dehydration, electrolyte disturbances, malabsorption and
progressive malnutrition [Vanderhoof J A. Short bowel syndrome.
Neonat Gastroenterol 1996; 23:377-86; Booth I W, Lander A D. Short
bowel syndrome. Bailliere's Clin Gastroenterol 1998; 12:739-72].
SBS is a common problem in pediatric surgery and occurs in newborns
and infants suffering from necrotizing enterocolitis (NEC),
intestinal atresia and volvulus requiring massive intestinal
resection. In adults, Crohn's disease, radiation enteritis and
massive resections due to catastrophic mesenteric vascular events,
intestinal obstruction, and trauma represent the more common causes
of SBS [DiBaise J K, Young R J, Vanderhoof J A. Intestinal
rehabilitation and the short bowel syndrome. Am J Gastroenterol
2004; 99:1386-95]. SBS remains a significant cause of infant
morbidity and mortality despite the availability of total
parenteral nutrition (TPN), advances in resuscitation, availability
of potent antibiotics, and modern techniques of organ support
[Coran A G, Spivak D, Teitelbaum D H. An analysis of the morbidity
and mortality of short bowel syndrome in the pediatric age group.
Eur J Pediatr Surg 1999; 9:228-30].
[0004] The key to survival after massive small bowel resection is
the ability of the residual intestine to adapt. In this setting,
adaptation means progressive recovery from intestinal failure
throughout which the small bowel increases its absorptive surface
area and its functional capacity in an attempt to meet the body's
metabolic and growth needs [O'Brien D P, Nelson L A, Huang F S,
Warner B W. Intestinal adaptation: structure, function, and
regulation. Semin Pediatr Surg 2001; 10:56-64]. Intestinal
adaptation constitutes the best option for patients with SBS. In
humans, intestinal adaptation begins within 24-48 hours of
resection and includes morphologic (structural adaptation) and
functional changes (functional adaptation) of the residual bowel.
Structural adaptation includes increasing bowel diameter and
length, villi elongation, deepening of the crypts, and increasing
the rate of enterocyte proliferation, finally resulting in
increased absorptive surface area and in increased numbers of
enterocytes. Functional adaptation entails modifications of the
brush border membrane permeability and up-regulation of
carrier-mediated transport, ultimately resulting in increased
nutrient absorption by isolated enterocytes. Although intestinal
transplantation (IT) has emerged as a feasible alternative in the
treatment of children with SBS during the last two decades,
intestinal adaptation remains the only chance for survival in a
subset of these patients. Considerable research over many years has
focused on the identification of those trophic factors that may
promote bowel absorption after massive intestinal resection and
provide a successful outcome in patients with SBS. These factors
include nutrients and other luminal constituents, gastrointestinal
secretions, hormones and peptide growth factors [O'Brien D P,
Nelson L A, Huang F S, Warner B W. Intestinal adaptation:
structure, function, and regulation. Semin Pediatr Surg 2001;
10:56-64].
[0005] Another area in which there has been a considerable research
effort over the last four decades is intestinal
ischemia-reperfusion. Restoration of blood flow following
intestinal ischemia is necessary to maintain cell function and
viability; however, the reintroduction of oxygen can initiate a
cascade of events that exacerbates intestinal tissue injury. The
mechanisms of intestinal injury following ischemia-reperfusion
event include nonspecific damage induced by ischemia per se and
damage caused by reperfusion. Intestinal ischemia induces
intestinal mucosal cell death, which is attributed mainly to a
reduction of oxygen supply relative to metabolic demands, depletion
of cellular energy stores and accumulation of toxic metabolites.
The reperfusion phase may significantly exacerbate ischemia-induced
mucosal injury via the formation of reactive oxygen species and
reactive nitrogen species [Carden, D. L., Granger, D. N. J.
Pathol., 2000, 190: 255; Granger, D. N., et al., Acta. Physiol.
Scand. Suppl. 548: 47, 1986] and changes in lipid mediator
synthesis [Tadros et al., Ann. Surg. 231: 566, 2000; Mangino et
al., Cryobiology. 33: 404, 1996]. Additionally, an infiltration of
intestinal wall by polymorphonuclear leucocytes and mast cells,
which release the cytokines, growth factors, or other molecules
leads to increased bowel permeability, gut barrier dysfunction,
translocation of bacteria and bacterial products into the systemic
circulation, causing multiple organ failure and death [Schoenberg
et al., 1991, Gut, 32: 905; Yamamoto et al., 2001, J. Surj
Research, 99:134].
[0006] Although necrosis is responsible for the intestinal cell
death during the ischemic phase, apoptosis has recently been
recognized to be a key phenomenon in enterocyte turnover and gut
barrier function following IR insult [Noda et al., Am J Physiol,
1998, 274: G270]. Thus, reduction of apoptosis and stimulation of
cell proliferation and differentiation following IR injury is a
potential target for therapeutic intervention.
[0007] A number of nutrient substances have been evaluated in an
attempt to maximize the adaptive response following IR injury and
following resection of the small intestine. Diets high in
glutamine, and high carbohydrate-low fat diets have been studied
[Byrne, T. P., et al., 1995, Annals of Surgery 222(3):254-5;
Scolapio, J. S. et al., 1997 Gastroenterology 113(4):1402-5; Sax,
H., 1998, Journal of Parenteral and Enteral Nutrition
26(2):123-8].
[0008] Formulas containing amino acids have been studied in an
attempt to avoid intact protein irritability and digestion [Bines,
J. F. et al., 1998, Journal of Pediatric Gastroenterology &
Nutrition 26(2):123-8]. Dietary restrictions of insoluble fiber,
oxalates, and lactose have also been proposed [Lykins, T. S. et
al., 1998, Journal of the American Dietetic Association
98(3):309-15] despite evidence that small amounts of lactose are
tolerated [Marteau, P. M. et al., 1997, Nutrition 13(1):13-16].
Compositions comprising arachidonic acid and docosahexanoic acid
have been proposed for improving the proliferative response during
adaptation of the gastrointestinal tract for use in short bowel
syndrome [U.S. patent application Ser. No. 0010047036].
[0009] Hormones, such as growth hormone [Weiming et al., 2004, JPEN
J Parenter Rectal-enteral Nutr. November-December; 28(6):377-81]
and hormone related peptides (e.g., Glucagon-like peptide 2 and
analogs thereof, U.S. patent application Ser. No. 0030162703) were
shown to have a trophic effect on the intestine.
[0010] There is also a growing body of evidence suggesting that
peptide growth factors may stimulate post-resection adaptive
hyperplasia or improve intestinal recovery following intestinal
ischemia. Peptide growth factors are divided into several families,
including epidermal growth factor family, the transforming growth
factor .beta. family, the insulin-like growth factor (IGF) family,
and the fibroblast growth factor family. In addition, a smaller
number of peptide growth factors without structural similarities of
the described families have also been identified and include
hepatocyte growth factor and platelet-derived growth factor.
[0011] The insulin-like growth factor family includes three
peptides: insulin, insulin-like growth factor I (IGF-I), and
insulin-like growth factor II (IGF-II). Several experimental
studies have suggested that both IGF-I and IGF-II are involved in
modulation of growth and differentiation of normal small bowel
[Laburthe M. et al., 1988, Am J Physiol; 254: G457-G462] and
following massive small bowel resection [Ziegler T R, Mantell M P,
Chow J C et al. (1996) Gut adaptation and the insulin-like growth
factor system: regulation by glutamine and IGF-1 administration. Am
J Physiol 271: G866-875].
[0012] Lemmey and co-workers have demonstrated a positive effect of
IGF-1 on body weight gain and intestinal absorptive function
[Lemmey A B., et al., 1991, Am J Physiol. February, 260(2 Pt
1):E213-9; Lemmey A B., et al., 1994, Growth Factors, 10(4):243-52]
following bowel resection in a rat model. IGF-1 was shown to
stimulate cell proliferation, increase villus height and promote
nutrient absorptive capacity in an animal model of SBS [Olanrewaju
H et cd., 1992, Am J Physiol, 263: E282-286]. Ileal IGF-1 mRNA
expression in rats rose nearly twofold during intestinal adaptation
after bowel resection, which was augmented with IGF-I
administration [Ziegler et al., 1996, Am J Physiol, 271:
G866-G875]. EGF and IGF-1 were shown to increase substrate
absorption after small bowel resection in rats, and this increase
in absorption persists after cessation of administration of these
growth factors [Lukish et cd., 1996, Gastroenterology, 110(Suppl):
A818].
[0013] However, animal experiments and clinical trials using the
above agents are at present inconclusive and there remains a widely
recognized need for an intestinal tissue growth promoting agent
which may be administered orally for the therapeutic treatment of
various intestinal disorders, intestinal ischemic damage, impaired
growth or loss of intestinal length.
[0014] The current advocacy of insulin therapy regimens involves
subcutaneous injections and intravenous administration, since like
other polypeptides, insulin is destroyed in the acidic environment
of the stomach and by digestive enzymes of the pancreas and small
intestine. Furthermore, insulin treatment is typically aimed at
increasing the level of insulin in the blood (such as for insulin
dependent diabetes), where the epithelial surface of the intestine
itself presents an effective barrier to the absorption of insulin
[Sukhotnik et al., 2002, J Surg Res. December; 108(2):235-42].
[0015] Accumulative evidence suggests a role of insulin in the
growth and development of the small intestine. For example, insulin
receptors are present on the luminal and basolateral membranes of
enterocytes [Buts J P, De Keyser N, Marandi S, Maernoudt A S, Sokal
E M, Rahier J, Hermans D. Expression of insulin receptors and of
60-kDa receptor substrate in rat mature and immature enterocytes.
Am J Physiol Gastrointest Liver Physiol 273:G217-226, 1997)].
Additionally, insulin is present in human and pig colostrum and
mature milk, substantiating its aforementioned potential role in
small intestine growth and development.
[0016] Oral insulin was shown to possess a trophic effect on
intestinal mucosa by stimulating ileal mass, mRNA and
disaccharidase activity in the newborn miniature pig [Shulman et
al., Pediatr Res. 1990 August; 28(2):171-512; Shulman R J, Tivey D
R, Sunitha I, Dudley M A, Henning S J 1992. Effect of oral insulin
on lactase activity, mRNA, and posttranscriptional processing in
the newborn pig. J Pediatr Gastroenterol Nutr 14:166-172)]. In a
recent clinical trial, the author has shown that enteral
administration of insulin to preterm infants (26-29 weeks of
gestational age) leads to a higher lactase activity and less
feeding intolerance [Shulman R J, et al., Arch. Dis. Child. Fetal
Neonatal Ed (2002); 88:F131-3].
[0017] Insulin was also shown to stimulate epithelial cell
proliferation and differentiation of intestinal epithelial cells in
vitro [Raj N. K. Sharma C. P., 2003, J Biomater Appl January
17(3):183-96]. Insulin accelerates enterocyte proliferation in the
intestinal mucosa of suckling mice [Malone et al., Diabetes Res
Clin Pract 2003 December; 62(3):187-95] and increases enzymatic
activity of the dissacharidases [Buts J P, Duranton B, De Keyser N,
Sokal E M, Maernhout A S, Raul F, Marandi S. Premature stimulation
of rat sucrase-isomaltase (SI) by exogenous insulin and the analog
B-Asp10 is regulated by a receptor-mediated signal triggering SI
gene transcription. Pediatr Res 1998; 43:585-91]. Furthermore,
insulin-receptor densities are selectively associated with
intestinal mucosa growth in neonatal calves [Kojima H. 1998, Assoc
Am Physicians, May-June; 110(3):197-206].
[0018] Moreover, Buts et al. had demonstrated preferential
localization of insulin binding sites to the intestinal crypt
cells, regardless of the age of the animal [Buts J P, De Keyser N,
Marandi S, Maernoudt A S, Sokal E M, Rahier J, et al. Expression of
insulin receptors and of 60-kDa receptor substrate in rat mature
and immature enterocytes. Am J Physiol Gastrointest Liver Physiol
(1997); 273:G217-26.]
[0019] Thus, prior art studies suggest that insulin is highly
active in promoting lactase activity, mRNA levels and ileal mass
when administered to healthy preterm infants or animal models, but
does not suggest oral or enteral administration of insulin for
increasing intestinal function in non-healthy infants.
[0020] The present inventors have previously shown that
physiological concentrations (i.e., about 100 .mu.u in maternal
milk and 700 .mu.u in colostrum) of insulin formulated in infant
formula can be used for the manufacture of formulas which are
similar to human milk. Such formulas are expected to protect new
born babies from the development of Type-1 diabetes and to improve
development and maturation of infants intestine (U.S. Pat. No.
6,399,090).
[0021] To date oral administration of insulin (not included in
infant formulas) has not been suggested for improving intestinal
function humans weaned of infant formula or non-human animal
subjects.
SUMMARY OF THE INVENTION
[0022] According to one aspect of the present invention there is
provided a method of increasing intestinal function comprising,
orally and/or enterally administering to a subject in need thereof
a therapeutically effective amount of insulin thereby increasing
intestinal function.
[0023] According to still further features in the described
preferred embodiment the subject suffers from intestinal
malfunction or malnutrition.
[0024] According to still further features in the described
preferred embodiment the subject suffers from a disease or a
condition selected from the group consisting of short bowel
syndrome, inflammation of the bowel, intestinal failure, chronic
ulcerative colitis, Crohn's Disease, necrotizing enterocolitis,
intestinal atresia, midgut volvulus, severe acute gastroenteritis,
chronic gastroenteritis, cholera, chronic infections of the bowel,
immunologic disorders affecting the small intestine,
chemotherapy-associated intestinal malfunction,
radiotherapy-associated intestinal malfunction, age associated
weight loss, postgastrectomy syndrome, dumping syndrome, AIDS,
diabetes, postvagotomy diarrhea, bariatric surgery-associated
diarrhea, tube-feeding related diarrhea, parenteral feeding
dependency, chronic secretory diarrhea, cancer, gastrointestinal
peptide tumors, endocrine tumors, diarrhea associated with thyroid
disorders, diarrhea due to bacterial overgrowth, chronic diarrhea
in gastrinoma, acute diarrhea, chronic diarrhea, infectious
diarrhea, antibiotic-associated diarrhea, irritable bowel syndrome,
chronic diarrhea associated with maldigestion and malabsorption,
chronic diarrhea in idiopathis primary gastrointestinal motility
disorders, chronic diarrhea associated with collagenous colitis,
nutritional deficiency, anemia, cystic fibrosis, and an eating
disorder, ischemia reperfusion and sepsis.
[0025] According to still further features in the described
preferred embodiment the subject is a human subject.
[0026] According to still further features in the described
preferred embodiment the subject is a non-human mammal.
[0027] According to still further features in the described
preferred embodiment the subject is an infant.
[0028] According to still further features in the described
preferred embodiment the orally administering is effected by an
oral dosage unit.
[0029] According to still further features in the described
preferred embodiment the oral dosage unit comprises from about 1 mu
to about 10,000 units of the insulin.
[0030] According to still further features in the described
preferred embodiment the insulin is administered in a amount
ranging from about 1 mu/Kg body weight/day to about 100
u/Kg/day.
[0031] According to still further features in the described
preferred embodiment the insulin has an amino acid sequence of
human insulin or functional equivalents thereof.
[0032] According to still further features in the described
preferred embodiment the insulin is recombinant insulin.
[0033] According to still further features in the described
preferred embodiment the insulin is synthetic insulin.
[0034] According to still further features in the described
preferred embodiment the insulin is a purified natural insulin.
[0035] According to another aspect of the present invention there
is provided an oral dosage unit form comprising from about 1 mu to
about 10,000 units of insulin.
[0036] According to still further features in the described
preferred embodiment the oral dosage unit form is solid.
[0037] According to still further features in the described
preferred embodiment the oral dosage unit is selected from the
group consisting of a pill, a dragee, a tablet and a capsule.
[0038] According to still further features in the described
preferred embodiment the oral dosage unit further comprising a
pharmacological agent.
[0039] According to yet another aspect of the present invention
there is provided a method of increasing intestinal function of a
pharmacological agent, the method to comprising orally and/or
rectal-enterally administering to a subject in need thereof a
therapeutically effective amount of insulin prior to, concomitant
with or following administration of the pharmacological agent,
thereby increasing intestinal absorption of the pharmacological
agent.
[0040] According to still further features in the described
preferred embodiment each of the insulin and the pharmacological
agent are formulated in a dosage unit.
[0041] According to still further features in the described
preferred embodiment the insulin and the pharmacological agent are
formulated in a dosage unit.
[0042] According to still further features in the described
preferred embodiment the dosage unit is for oral
administration.
[0043] According to still another aspect of the present invention
there is provided use of insulin for the manufacture of a
medicament for increasing intestinal function.
[0044] According to still further features in the described
preferred embodiment the insulin is formulated for oral or enteral
administration.
[0045] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
methods and formulations for increasing intestinal function.
[0046] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the patent specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0048] In the drawings:
[0049] FIG. 1 is a graph depicting body weight changes expressed as
% of preoperative weight (mean.+-.SEM) in control (Sham) and
resected rats untreated (SBS) or treated with oral insulin.
SBS-short bowel syndrome rats; INS-insulin, *p<0.05 SBS vs Sham
rats, .dagger.p<0.05 SBS-INS vs SBS rats.
[0050] FIGS. 2a-b are graphs depicting the effect of bowel
resection and oral insulin on crypt cell proliferation (FIG. 2a)
and enterocyte apoptosis (FIG. 2b) in jejunum and ileum
(mean.+-.SEM). 5-BrdU incorporation into proliferating jejunal and
ileal crypt cells was detected with a goat anti-BrdU antibody and
TUNEL assay was used to determine enterocytes apoptosis. SBS-short
bowel syndrome rats; INS-insulin, *p<0.05 SBS vs Sham rats,
.dagger.p<0.05 SBS-INS vs SBS rats.
[0051] FIGS. 3a-c are photographs depicting the effect of bowel
resection and oral insulin on enterocyte proliferation. These
representative sections demonstrate that cell proliferation is
increased following bowel resection (SBB) compared to sham animals
(Sham). Following administration of oral insulin, SBS-rats
(SBS-INS) demonstrated a marked increase in a number of
proliferating cells compared to SBS-nontreated animals.
[0052] FIG. 4 is a graph depicting the effect of
ischemia-reperfusion and oral insulin on the macroscopic intestinal
appearance. Values are mean.+-.SEM. IR-ischemia-reperfusion;
INS-insulin, *P<0.05 IR vs Sham rats, .dagger.P<0.05 IR-INS
vs IR rats.
[0053] FIG. 5 is a graph depicting the effect of oral insulin on
mucosal DNA and protein content following ischemia-reperfusion
injury. Values are mean.+-.SEM. IR-ischemia-reperfusion;
INS-insulin. *P<0.05 IR vs Sham rats, .dagger.P<0.05 IR-INS
vs IR rats.
[0054] FIG. 6 is a graph depicting the effect of
ischemia-reperfusion and oral insulin on the microscopic intestinal
appearance. Values are mean.+-.SEM. IR-ischemia-reperfusion;
INS-insulin. *P<0.05 IR vs Sham rats, .dagger.P<0.05 IR-INS
vs IR rats.
[0055] FIG. 7 is a graph depicting the grade of intestinal mucosal
injury after intestinal ischemia-reperfusion and administration of
oral insulin. Values are mean.+-.SEM. IR-ischemia-reperfusion;
INS-insulin. *P<0.05 IR vs Sham rats, .dagger.P<0.05 IR-INS
vs IR rats.
[0056] FIG. 8 is a graph depicting the effect of
ischemia-reperfusion and oral insulin on crypt cell proliferation
and enterocyte apoptosis in jejunum and ileum. 5-BrdU incorporation
into proliferating jejunal and ileal crypt cells was detected with
a goat anti-BrdU antibody, and TUNEL assay was used to determine
enterocyte apoptosis. Values are mean.+-.SEM.
IR-ischemia-reperfusion; INS-insulin. *P<0.05 IR vs Sham rats,
.dagger.P<0.05 IR-INS vs IR rats.
[0057] FIGS. 9a-c are photographs depicting the effect of IR and
oral insulin on enterocyte proliferation. These representative
sections demonstrate that cell proliferation is decreased following
IR compared to sham animals. Following administration of oral
insulin, IR-rats demonstrated a marked increase in a number of
proliferating cells compared to IR-nontreated animals.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0058] The present invention is of formulations and methods for
increasing intestinal function which can be used for diseases or
conditions associated with intestinal mal-function.
[0059] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0060] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0061] The etiology of inadequate intestinal function includes
intestinal loss due to development abnormalities such as intestinal
atresia and in utero midgut volvus; postnatal loss from surgical
resection after infarction (e.g., midgut volvulus or vascular
occlusion), trauma or tumor; inflammation, such as is due to
infection (e.g., necrotizing enterocolitis and acute
gastroenteritis) and autoimmune etiologies such as in Crohn's
Disease and ulcerative colitis.
[0062] To date, there is no effective treatment and current
management includes total parenteral nutrition, which itself is a
source of significant morbidity and mortality.
[0063] Accumulative evidence suggests a role for insulin in the
proliferation and differentiation of intestinal epithelial cells
vitro [Raj N. K. Sharma C. P., 2003, J Biomater Appl January
17(3):183-96].
[0064] The present inventors have previously shown that insulin
formulated in infant formula can be used for the manufacture of
formulas which are similar to human milk. Such formulas are
expected to protect new born babies from the development of Type-1
diabetes and to improve development and maturation of infants
intestine (U.S. Pat. No. 6,399,090).
[0065] To date oral or enteral administration of insulin (not
included in infant formulas) has not been suggested for improving
intestinal function and/or treating or preventing conditions
associated with intestinal malfunction (e.g., intestinal failure)
in humans (e.g., weaned of infant formula) or non-human animal
subjects.
[0066] While reducing the present invention to practice, the
present inventors uncovered that oral administration of insulin can
be used to improve intestinal function.
[0067] As is illustrated hereinbelow and in the Examples section
which follows, oral insulin administration to a rat model of short
bowel syndrome (SBS) caused dramatic adaptive gut growth (see
Example 1). These findings were substantiated in human infants
suffering from SBS, where oral administration of insulin reduced
stool output, decreased jaundice, increased the amount of oral
feeds and reduced the need for parenteral feeding (Example 2).
Finally, oral insulin accelerated intestinal recovery, enhanced
enterocyte proliferation and decreased cell death via apoptosis
following ischemia-reperfusion event.
[0068] Thus, according to one aspect of the present invention there
is provided a method of increasing intestinal function.
[0069] As used herein the phrase "increasing intestinal function"
refers to increasing at least one intestinal activity associated
with the heterogenic cellular environment of the intestine (e.g.,
small intestine). Examples of intestinal activities include, but
are not limited to, absorption (the transport of a substance from
the intestinal lumen through the barrier of the mucosal epithelial
cells into the blood and/or lymphatic systems e.g., nutrient
absorption), digestion, motility, adaptation (described in length
in the Background section), immunological function (e.g., antigen
recognition) and barrier function.
[0070] The method according to this aspect of the present invention
is effected by orally and/or enterally administering to a subject
in need thereof a therapeutically effective amount of insulin,
thereby increasing intestinal function of the subject.
[0071] As used herein the term "insulin" refers to the hormone
produced by the pancreas which is typically necessary for glucose
to be able to enter the cells of the body and be used for energy
(see U.S. Pat. No. 6,399,090). Insulin of the present invention
refers also to functional equivalents of insulin, such as fragments
thereof displaying insulin activity and functional peptide-mimetics
thereof.
[0072] As used herein the term "functional" refers to the ability
to increase intestinal function.
[0073] As used herein the term "mimetics" when made in reference to
peptides refers to molecular structures, which serve as substitutes
for the insulin of the present invention in increasing intestinal
function (Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252
for a review of peptide mimetics). Peptide mimetics, as used
herein, include synthetic structures (known and yet unknown), which
may or may not contain amino acids and/or peptide bonds, but retain
the structural and functional features of insulin. The term,
"peptide mimetics" also includes peptoids and oligopeptoids, which
are peptides or oligomers of N-substituted amino acids [Simon et
al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371]. Further
included as peptide mimetics are peptide libraries, which are
collections of peptides designed to be of a given amino acid length
and representing all conceivable sequences of amino acids
corresponding thereto. Methods for the production of peptide
mimetics are described hereinbelow.
[0074] According to a preferred embodiment of the present
invention, insulin is selected from the following insulin types:
recombinant insulin, synthetic insulin or active fragments or
functional mimetics thereof, purified natural insulin, and insulin
having an amino acid sequence of human insulin (e.g., human
insulin). Some of these types are overlapping and therefore the
insulin of choice may be categorized to more than a single type of
the types listed. Human recombinant insulin is available in a pure
form from Eli Lilly & Co, USA. Human natural purified insulin
is available in a pure form from Novo Nordisk, Denmark. Crude
extracts may also be useful, depending on the method of their
manufacturing. Synthetic insulin may be manufactured using
commercially available building units for solid-phase peptide
synthesis, as well known in the art.
[0075] As used herein the phrase "enteral administration" refers to
administration of a pharmacological agent through any part of the
gastro-intestinal tract, such as rectal administration, colonic
administration, intestinal administration (proximal or distal) and
gastric administration.
[0076] As used herein the phrase "subject in need thereof" refers
to a mammal of any age (e.g., infant such as term or preterm
infant, adult or old) or sex, preferably a human, which can benefit
from increased intestinal function. Examples of non-human mammals
include domestic animals such as cats, dogs, cattle, sheep, pigs,
goats, poultry and equines.
[0077] According to one embodiment of this aspect of the present
invention the subject is a non-healthy subject, which suffers from
intestinal malfunction (i.e., reduced function as compared to
intestinal function of a healthy subject of the same age, or even
complete intestinal failure) such as caused by a disease or
condition associated with inadequate intestinal function such as
due to reduced intestinal absorptive, anti-inflammatory, barrier,
digestive and/or motiliy functions and/or reduced tissue mass;
and/or malnutrition such as due to eating disorders, chemotherapy
or radio-therapy and the like.
[0078] For example, inadequate intestinal function can be due to
developmental abnormalities such as, intestinal atresia; in utero
midgut volvulus; postnatal loss from surgical resection such as due
to infarction (e.g., midgut volvulus or vascular occlusion) trauma;
inschemia (e.g., ischemia reperfusion) or tumor; or inflammation
such as caused by infection (i) necrotizing enterocolitis (ii)
acute gastroenteritis (e.g., cholera) or autoimmune causes (i)
Crohn's Disease (ii) ulcerative colitis.
[0079] Examples of diseases and conditions which are associated
with intestinal malfunction or malnutrition include, short bowel
syndrome, inflammation of the bowel, intestinal failure, chronic
ulcerative colitis, Crohn's Disease, necrotizing enterocolitis,
intestinal atresia, midgut volvulus, severe acute gastroenteritis,
chronic gastroenteritis, cholera, chronic infections of the bowel,
immunologic disorders affecting the small intestine,
chemotherapy-associated intestinal malfunction,
radiotherapy-associated intestinal malfunction, age associated
weight loss, postgastrectomy syndrome, dumping syndrome, AIDS,
diabetes, postvagotomy diarrhea, bariatric surgery-associated
diarrhea, tube-feeding related diarrhea, parenteral feeding
dependency, chronic secretory diarrhea, cancer, gastrointestinal
peptide tumors, endocrine tumors, diarrhea associated with thyroid
disorders, diarrhea due to bacterial overgrowth, chronic diarrhea
in gastrinoma, acute diarrhea, chronic diarrhea, infectious
diarrhea, antibiotic-associated diarrhea, irritable bowel syndrome,
chronic diarrhea associated with maldigestion and malabsorption,
chronic diarrhea in idiopathis primary gastrointestinal motility
disorders, chronic diarrhea associated with collagenous colitis,
nutritional deficiency, anemia, cystic fibrosis, liver disease, an
allergy, and an eating disorder (e.g., anorexia, bulimia), injury,
ischemia reperfusion and sepsis.
[0080] "Merck's Veterinary Manual" provides a detailed description
of animal's intestinal disorders, which can be treated according to
this aspect of the present invention.
[0081] As mentioned, the teachings of the present invention can be
used for treating the above described diseases or conditions.
[0082] As used herein the term "treating" refers to preventing,
curing, reversing, attenuating, alleviating, minimizing,
suppressing or halting the deleterious effects of a condition or
disorder associated with abnormal intestinal function symptoms
and/or disease state, as described hereinabove.
[0083] According to another embodiment of this aspect of the
present invention the subject can be a healthy subject, in which
case increasing intestinal function is effected to gain weight such
as for commercial reasons (such as in livestock e.g., farm animals
or poultry) or for prophylactic reasons, such as prior to
anti-cancer therapy, in which case the subject is expected to loose
weight. Alternatively, increasing intestinal function may be
beneficial for athletes, travelers or in combat to avoid weight
loss.
[0084] Insulin of the present invention is formulated in a
pharmaceutical composition or veterinary formulation for oral or
enteral (e.g., rectal) administration.
[0085] As used herein, a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients (e.g.,
insulin) described herein with other chemical components such as
physiologically suitable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration of a
compound to an organism.
[0086] As used herein, the term "active ingredient" refers to the
insulin accountable for the intended biological effect.
[0087] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier," which may be
used interchangeably, refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0088] Herein, the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils, and polyethylene glycols.
[0089] Techniques for formulation and administration of drugs may
be found in the latest edition of "Remington's Pharmaceutical
Sciences," Mack Publishing Co., Easton, Pa., which is herein fully
incorporated by reference.
[0090] It will be appreciated that insulin of the present invention
may be released in a any portion of the gastro-intestinal tract, as
peptide digestion fragments of insulin may exert the desired
activity in the intestine (i.e., increased intestinal
function).
[0091] According to a presently known configuration of the present
invention insulin is released in the intestine. Typically, to
achieve successful intestinal delivery, a drug needs to be
protected from absorption and/or the environment of the upper
gastrointestinal tract (GIT) and then be abruptly released into the
proximal intestine, which is considered the optimum site for
intestine-targeted delivery of drugs. Various strategies for
targeting orally administered drugs to the intestine include
covalent linkage of a drug with a carrier, coating with
pH-sensitive polymers, formulation of timed released systems,
exploitation of carriers that are degraded specifically by
intestineic bacteria, bioadhesive systems and osmotic controlled
drug delivery systems. Various prodrugs (sulfasalazine, ipsalazine,
balsalazine and olsalazine) have been developed that are aimed to
deliver 5-amino salicylic acid (5-ASA) for localized chemotherapy
of inflammatory bowl disease (IBD). Microbially degradable polymers
especially azo crosslinked polymers have been investigated for use
in targeting of drugs to intestine. Certain plant polysaccharides
such as amylose, inulin, pectin and guar gum remains unaffected in
the presence of gastrointestinal enzymes and pave the way for the
formulation of intestine targeted drug delivery systems. The
concept of using pH as a rigger to release a drug in the intestine
is based on the pH conditions that vary continuously down the
gastrointestinal tract. Times dependent drug delivery systems have
been developed that are based on the principle to prevent release
of drug until 3-4 h after leaving the stomach. Redox sensitive
polymers and bioadhesive systems have also been exploited to
deliver the drugs into the intestine.
[0092] Colonic release is expected to reduce inflammatory reactions
and increase motility, according to the present invention.
[0093] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing
processes.
[0094] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations that can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0095] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0096] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient. Oral
administration is preferred for administration to pre-term
infants.
[0097] Pharmacological preparations for oral use according to the
present invention to are preferably produced using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries as desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose;
and/or physiologically acceptable polymers such as
polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such
as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a
salt thereof, such as sodium alginate, may be added. Dragee cores
are provided with suitable coatings. For this purpose, concentrated
sugar solutions may be used which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene
glycol, titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for identification or to
characterize different combinations of active compound doses.
[0098] Pharmaceutical compositions that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules may contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0099] Insulin of the present invention can be formulated in a
solid dosage unit such as for oral administration or rectal
administration (as described below). Such a dosage unit form can
include, for example 1 mu (munit)-10,000 u (unit) of insulin (where
100 u=3.5 mg), preferably 1 mu (munit)-1000 u (unit) of insulin,
preferably 1 mu-500 u of insulin, 1 mu-200 u of insulin, preferably
1 mu-100 u of insulin, preferably 1 mu-10 u of insulin.
[0100] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, for example, conventional suppository
bases such as cocoa butter or other glycerides.
[0101] Pharmaceutical compositions suitable for use in the context
of the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a "therapeutically effective
amount" means an amount of active ingredients (e.g., insulin)
effective to prevent, alleviate, or ameliorate symptoms of a
disorder (e.g., ischemia) or prolong the survival of the subject
being treated.
[0102] As used herein in the specification and claims section that
follows, the phrase "therapeutically effective amount" refers to an
amount which is sufficient to improve intestinal function. The
"therapeutically effective amount" can be assessed by growth in
infants and children and by body weight in adults, by stool output,
assessment of nutrient of interest (e.g., Fe, Zinc, potassium,
etc.), patients general well being or alternatively in-vitro (e.g.,
biopsies exemplified in the Examples section which follows.
[0103] The "therapeutically effective amount" will, of course, be
dependent on, but not limited to the subject being treated, the
severity of the anticipated affliction, the manner of
administration, as discussed herein and the judgment of the
prescribing physician. [See e.g. Fingl, et al., (1975) "The
Pharmacological Basis of Therapeutics", Ch. 1 p. 1].
[0104] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art.
[0105] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro assays. For example, a dose can be
formulated in animal models (e.g. Example 1-Rat model for SBS) such
information can be used to more accurately determine useful doses
in humans.
[0106] Preferably, insulin is administered in an amount ranging
from 1 mu/Kg body weight/day to about 100 u/Kg/day, where 100 u=3.5
mg. The effective dosage will be determined according to the age of
the subject, the severity of the disease and the purpose of
treating (i.e., prophylilactic or therapeutic).
[0107] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human.
[0108] Depending on the severity and responsiveness of the
condition to be treated, dosing can be effected over a short period
of time (i.e. several days to several weeks) or until cure is
effected or diminution of the disease state is achieved.
[0109] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA-approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser device may also be accompanied by a notice in a form
prescribed by a governmental agency regulating the manufacture,
use, or sale of pharmaceuticals, which notice is reflective of
approval by the agency of the form of the compositions for human or
veterinary administration. Such notice, for example, may include
labeling approved by the U.S. Food and Drug Administration for
prescription drugs or of an approved product insert. Compositions
comprising a preparation of the invention formulated in a
pharmaceutically acceptable carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition, as further detailed above.
[0110] The small intestine is also an important site for the
absorption of pharmacological agents. The proximal part of the
small intestine has the greatest capacity for absorption of drugs.
Intestinal absorption of drugs is influenced to a great extent by
many of the same basic factors that affect the digestion and
absorption of nutrients, water and electrolytes. It is thus
suggested, that the teachings of the present invention can also be
used for increasing intestinal absorption of a pharmacological
agent, where insulin serves essentially as a carrier for increasing
bioavailability of the pharmacological agent.
[0111] Thus, according to another aspect of the present invention
there is provided a method of increasing intestinal absorption of a
pharmacological agent.
[0112] The method of this aspect of the present invention is
effected by orally and/or rectal-enterally administering to a
subject in need thereof a therapeutically effective amount of
insulin prior to, concomitant with or following administration of
the pharmacological agent, thereby increasing intestinal absorption
of the pharmacological agent.
[0113] As used herein the phrase "pharmacological agent" refers to
a medical drug.
[0114] It will be appreciated that each of the insulin and the
pharmacological agent can be formulated in a dosage unit.
[0115] Alternatively, the insulin and the pharmacological agent can
formulated together in a single dosage unit.
[0116] Examples of pharmacological agents which can be
co-administered with the insulin of the present invention include,
but are not limited to, antibiotics, chemotherapy, anti-diarrheal
and inti-inflammatory drugs. Growth factors such as Hepatocyte
Growth Factor (HGF), epidermal growth factor (EGF), Interleukin-11
(IL-11), glucagon-like peptide (GLP-2), and insulin-like growth
factors such as insulin-like growth factor-1 (IGF-1) may also be
included with the administration of insulin or provided
separately.
[0117] It will be appreciated that nutritional supplements, may be
administered as well with an effective dose of insulin. Nutritional
supplements or nutrients may include rectal-enteral formulas and
glutamine. The nutritional supplements may be administered along
with the insulin or alternatively the nutritional supplements may
be provided separately by the same or different administration
routes
[0118] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0119] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0120] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorpotaed by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Oral Insulin Supplementation in a Rat Model of SBS
[0121] Methods
[0122] Animal treatment: Animals were obtained from the Rappaport
Faculty of Medicine (Technion, Haifa, Israel). Male rats weighing
240-260 g were kept in individual stainless steel cages at constant
temperature and humidity, and a 12-hour light-dark cycle was
maintained. Rats were fasted 12 hours prior to the experiment with
free access to water. Animals underwent one of two surgical
procedures: bowel transection and re-anastomosis or 75% bowel
resection and anastomosis. General anesthesia was induced with
Pentobarbital (IP 40 mg/kg). Using sterile techniques, the abdomen
was opened using a midline incision. In sham rats, simple bowel
transection and end-to-end re-anastomosis was performed 15 cm
proximal to ileo-cecal junction. In SBS animals, small bowel was
resected from a point 5 cm distal to the ligament of Treitz to a
point 10 cm proximal to the ileo-cecal junction. Bowel continuity
was restored by end-to-end anastomosis using 5-0 Vicryl interrupted
sutures. For all operations, the abdominal cavity was closed in two
layers with a running suture of 3-0 Dexon. Prior to closure of the
abdomen, the rats were resuscitated with a 3 ml intraperitoneal
injection of warm normal saline. Rats were fasted for 24 hours but
were allowed free access to water.
[0123] Experimental design: Forty rats were randomly assigned to
one of three groups:
[0124] Group A--sham-rats underwent bowel transection (sham, n=14)
and than fed with regular chow diet.
[0125] Group B--SBS-animals underwent bowel resection (SBS, n=13)
and than fed regular chow diet.
[0126] Group C--SBS-Insulin (SBS-INS, n=13) rats underwent bowel
resection and were fed regular chow diet and water containing
insulin (ACTRAPID insulin, NOVO NORDISK, Denmark) at concentration
of 1 u/ml. Food and fluid intake of animals were monitored as well
as weight for a period of 14 days following treatment after which
the animals were sacrificed following intraperitoneal injection of
pentobarbital (75 mg/kg).
[0127] Intestinal adaptation parameters: The small bowel was
rapidly removed, rinsed with cold isotonic saline and divided into
two segments: jejunum proximal to anastomosis and terminal ileum.
Each segment was weighed, cut longitudinally, bowel circumference
was measured in three equidistant points as described by Dowling
[Dowling R H, Booth C C, 1967, Clin Sci; 32: 139-149] and the mean
circumference was calculated. Mucosa was scraped using a glass
slide, collected and weighed. Bowel and mucosal weight was
calculated per cm of bowel length per 100 g of body weight as
described previously [Sukhotnik I, et al., 2002, J Surg Res.,
December; 108(2):235-42]. Although bowel length may change due to
spasm or bowel distension, the calculation per unit of bowel length
is considered to be the gold standard in describing structural
changes in intestine. DNA and protein were extracted using TRIzol
reagent (Rhenium LTD, Jerusalem). The DNA concentrations were
recorded in a spectrophotometer and calculated per cm of bowel
length. Final protein concentration was measured
spectrophotometrically using a commercially available kit (Bio-Rad,
Protein Assay) and was calculated per cm of bowel length.
[0128] Histology: Histological sections were prepared from the
proximal jejunum and distal ileum and from comparable sites in
control animals. Segments of small bowel were fixed for 24 hours in
10% formalin and processed into standard paraffin blocks.
Five-micron tissue slices stained with hematoxylin-eosin. The
sections were studied microscopically using a micrometer eyepiece.
Histological images were loaded on a 760.times.570 pixels
resolution buffer using a computerized image analysis system
composed of a trichip RGB video-camera (Sony, Japan), installed on
a light microscope (Zeiss, Germany) and attached to an IBM
compatible personal computer (Pentium III, MMX, 450 mhz, 125 MB
RAM), equipped with a frame grabber. Images were captured,
digitized and displayed on a high resolution color 17 inch monitor.
The villus height and crypt depth were measured using the Image Pro
Plus 4 image analysis software (Media Cybernetics, Baltimore, Md.,
USA). Ten villi and crypts in each section were measured and the
mean reading was recorded in microns.
[0129] Crypt Cell Proliferation and Enterocyte Apoptosis: Rats were
injected with standard 5-bromodeoxyuridine (5-BrdU) labeling
reagent (Zymed Lab, Inc, CA) at dose 1 ml per 100 g body weight two
hours prior to sacrifice. Tissue slices (5 .mu.m) were
deparaffinized with xylene, rehydrated with graded alcohol, and
stained with a biotinylated monoclonal anti-BrdU antibody system
using BrdU Staining Kit (Zymed Lab, Inc, CA). An index of
proliferation was determined as the ratio of crypt cells staining
positively for BrdU per 10 crypts.
[0130] Apoptosis of enterocytes was assessed by terminal
deoxyuridine nick-end labeling (TUNEL) immunohistochemical assay
using the I.S. Cell Death Detection kit (Boehringer Mannheim GmbH,
Mannheim, Germany). 5 .mu.m thick paraffin-embedded sections were
deparaffinized, rehydrated in graded alcohol, and
microwave-pretreated in 10 mM citrate buffer (pH 6.0) for 10
minutes. After washing in phosphate-buffered saline (PBS), the
specimens were incubated in buffer containing a nucleotide mixture
with fluorescein-labeled deoxy-UTP and TdT (Boehringer Mannheim
GmbH, Mannheim, Germany) at 37.degree. C. for 1 h. Following
washing, the slides were incubated with blocking solution (3%
H.sub.2O.sub.2 in methanol) for 10 minutes and were stained with
anti-fluorescein antibody, Fab fragment from sheep, conjugated with
horse-radish peroxidase (converter-POD) at 37.degree. C. for 30
minutes. AES substrate (Zymed Laboratories) was applied for color
development. For each group, the number of stained cells was
counted in at ten villi in areas without necrosis. The apoptotic
index (AI) was defined as the number of apoptotic TUNEL-positive
cells per ten villi. All measurements were performed by a qualified
pathologist blinded as to the source of intestinal tissue.
[0131] Statistical analysis: The data are expressed as the
mean.+-.SEM. A paired Student's t-test and the non-parametric
Kruskal-Wallis ANOVA test were used as indicted. p<0.05 was
considered statistically significant.
[0132] Results
[0133] Body weight: The sham-operated control rats (Group A)
maintained constant body weight for the four first post-operative
days followed by a gradual increase in weight throughout the
two-week's observation period. Resected rats (Groups B and C)
demonstrated a significantly lower body weight from day 4 through
14 following operation compared to their sham-operated
counterparts. SBS-INS rats (Group C) gained weight at greater rate
from day 7 through 14 compared to SBS-nontreated animals (Group B)
(p<0.05).
[0134] Macroscopic bowel appearance: Two weeks following bowel
resection, there was an increase in intestinal thickness and
diameter. Compared to sham animals (Group A), SBS-rats (Group B)
showed a significantly greater bowel circumference in jejunum and
ileum. Exposure to oral insulin resulted in additional bowel
enlargement. SBS-insulin rats (Group C) demonstrated an additional
increase in jejunal and ileal bowel circumference compared to
SBS-untreated animals (Group B).
[0135] Overall mean bowel weight rose significantly in jejunum
(four fold increase, to p<0.05) and in ileum (two fold increase,
p<0.05) in SBS-rats (group B) compared to sham animals (group
A). Following oral insulin administration, (Group C) SBS rats
demonstrated an additional significant increase in jejunal (18%,
p<0.05) and ileal (40%, p<0.05) overall weight compared to
SBS-untreated animals.
[0136] Changes in mucosal weights were similar to those of bowel
weights. SBS-rats (Group B) demonstrated a three fold increase in
jejunal mucosal weight (p<0.05) and a two fold increase in ileal
mucosal weight (p<0.05) compared to sham animals (Group A). Oral
insulin supplemented group (Group C) demonstrated an additional 33%
increase in jejunal mucosal weight (p<0.05) and an almost two
fold increase in ileal mucosal weight (p<0.05) compared to
SBS-untreated counterparts.
[0137] Mucosal DNA and protein: Adaptation in residual bowel in the
resected group (Group B) was manifested by a 2.7-fold increase in
jejunal (p<0.05) and a 1.6-fold increase in ileal (p<0.05)
DNA content compared to sham animals. Oral insulin supplementation
resulted in an almost two-fold increase in ileal DNA content
compared to SBS untreated animals (p<0.05).
[0138] Mucosal protein content increased significantly following
bowel resection in both jejunum (three-fold increase, p<0.05)
and ileum (1.4-fold increase, p<0.05). Oral insulin
administration (Group C) induced an additional two-fold increase in
ileal (p<0.05) mucosal protein content compared to SBS-untreated
animals (Group B).
[0139] Microscopic bowel appearance: SBS-rats (Group B) showed a
marked increase in villus height in jejunum (785.+-.34 vs 529.+-.34
p<0.05) and ileum (672.+-.24 vs 462.+-.32 .mu.m, p<0.05) and
crypt depth in jejunum (209.+-.15 vs 163.+-.9 .mu.m, p<0.05) and
ileum (172.+-.11 vs 147.+-.7 .mu.m, p<0.05) compared to Sham
animals (Group A). SBS-insulin rats (Group C) demonstrated a 15%
increase in ileal villus height (p<0.05), a 15% increase in
jejunal (p<0.05) and 40% increase in ileal (p<0.05) crypt
depth compared to SBS-untreated animals (Group B).
[0140] Enterocytes proliferation and apoptosis: Bowel resection
(Group B) resulted in a significant increase in enterocyte
proliferation index in jejunum (258.+-.17 vs 154.+-.7 BrdU positive
cells/10 crypts, p<0.05) and ileum (263.+-.15 vs 182.+-.9 BrdU
positive cells/10 crypts, p<0.05) compared to sham animals. Oral
insulin administration (Group C) induced an additional 36% increase
in proliferation index in jejunum (p<0.05) and a 52% increase in
proliferation index in ileum (p<0.05) compared to SBS-untreated
animals (Group B).
[0141] Significantly greater numbers of apoptotic cells appeared in
the villi of jejunum (29.+-.7 vs 13.+-.4 TUNEL positive cells/10
villi, p<0.05) and ileum (33.+-.8 vs 14.+-.5 TUNEL positive
cells/10 villi, p<0.05) in SBS rats (Group B) compared to sham
animals. Exposure to oral insulin led to a significant decrease in
the apoptotic index in jejunum (11.+-.3 vs 29.+-.7 TUNEL positive
cells/10 villi, p<0.05) and did not affect apoptotic cells
number in ileum compared to SBS-untreated animals (Group B).
[0142] Conclusion
[0143] Oral insulin supplementation dramatically enhanced
structural intestinal adaptation. Overall bowel and mucosal weight
also increased with a synergistic increase in bowel circumference.
However, an approximately 10% increase in bowel diameter was
accompanied with a 20-50% increase in overall bowel weight and a
30-200% increase in mucosal weight in the remaining segments (see
FIG. 1). These data support the concept that mucosal hyperplasia
rather than bowel enlargement is responsible for increased bowel
and mucosal weights calculated per cm of bowel length.
[0144] Oral insulin significantly increased ileal mucosal DNA and
protein. Parallel increases in mucosal DNA and protein indicate
that the greater ileal mass of animals treated with oral insulin
can be attributed to cellular hyperplasia. Because the DNA and
protein content is directly proportional to mucosal cell number,
these measurements exclude such factors as edema or vascular
engorgement as responsible for differences in mucosal mass.
[0145] FIG. 2a and FIGS. 3a-c show an increase in the mucosal
proliferation of functioning intestine, as demonstrated by an
increased cell proliferation index following oral insulin
administration, suggests an activated enterocyte turnover and may
be considered as a main mechanism of mucosal hyperplasia in the
residual bowel. Increased villus height and crypt depth are the
result of increased proliferation and accelerated migration along
the villus, and are a marker for the increased absorptive surface
area. Most significant differences were observed in terminal ileum,
since hyperplasia in proximal jejunum was less prominent. Following
bowel resection, partial obstructive effects may explain the small
bowel enlargement in the jejunum. However, the significant increase
in mucosal parameters in the remnant of ileum must be considered an
indirect measure of true structural intestinal adaptation.
[0146] Although bowel and mucosal weight increased significantly in
the jejunum, to this change was not associated with an increase in
mucosal DNA, mucosal protein or villi height in this segment.
However, a marked increase in ileal bowel and mucosal weight was
accompanied by a two-fold increase in mucosal DNA and protein in
this area, a 15% increase in villi height and a 40% increase in
crypt depth compared to SBS-untreated animals, suggesting active
proliferating process.
[0147] Oral insulin resulted in decreased enterocyte apoptosis in
jejunum (see FIG. 2b). Both increased cell proliferation and
reduced cell apoptosis may be responsible for increased enterocytes
mass in jejunum during adaptation. In remaining ileum, oral insulin
administration led to much more significant increase in enterocyte
proliferation compared to jejunum without change in enterocyte
apoptosis. Increased enterocyte proliferation rather than apoptosis
is responsible for the bulk of enterocyte mass in remaining
ileum.
[0148] In summary, oral insulin supplementation caused dramatic
adaptive gut growth in a rat model of short bowel syndrome. Most
significant changes were observed in remaining ileum.
Example 2
Oral Insulin Supplementation in Infants with SBS
[0149] In view of the successful results obtained in the rat model
of SBS (Example 1, above), oral administration of insulin was
effected in human subjects suffering from SBS.
[0150] Experimental Procedures
[0151] Inclusion criteria:
[0152] 1. Infants with less than 30 cm of small intestine (with
ileo-cecal valve intact).
[0153] 2. Infants with less than 40 cm without ileo-cecal
valve.
[0154] 3. Infants and children younger than 10 years of age, with
SBS who are parrectal-enteral nutrition dependent and are not
expected to be weaned of parrectal-enteral nutrition
[0155] Exclusion criteria: Infants in an unstable condition such as
sepsis, acute gastroenteritis, pneumonia.
[0156] Dosage: 1 unit of insulin (Actrapid, Novonordisk,
Denmark)/kg body weight per dose.times.4/day (every 6 hours) was
administered for 28 days. Insulin was administered every six hours.
Insulin will be provided as 1 u/ml of 0.45 Nacl orally.
[0157] Glucose monitoring: Following the first insulin dose, blood
glucose levels were measured, immediately prior to the first and
second meal following the first dose of insulin. Measurements were
repeated for the first three days following supplementation and
then weekly (days 7, 14, 21 and 28)
[0158] Clinical evaluation:
[0159] The clinical evaluation of the patients' conditions included
the following parameters:
[0160] 1. Weight, length, head circumference, MAC (mid arm
circumference), TSF (triceps skin fold)--weekly
[0161] 2. Growth rate
[0162] 3. Rectal-enteral and parrectal-enteral intake of calories,
carbohydrates, lipids
[0163] 4. Blood concentration levels of glucose, albumin, ALT, AST,
GGT, ALP, total cholesterol, triglycerides (fasting),
HDL-cholesterol, insulin (fasting, before insulin
administration)
[0164] 5. Blood amino acid levels
[0165] 6. Insulin antibodies. 1 ml of serum was removed prior to
treatment and at day 28 following treatment. The samples were
frozen at -70.degree. C. and sent, on dry ice, to Division of
Pediatric Gastroenterology and Nutrition, Meyer Children's Hospital
of Haifa, Bat-Galim 31096, Haifa for analysis.
[0166] Results
[0167] Patient history: Male, Full Term, AGA, birth weight 3700
grams, suffered from malrotation and volvulus at 2 days of age. On
laparotomy, severe ischemia was observed in the gut. Following 24
hours, another laparotomy was performed with 10 cm of proximal
jejunum and 10 cm of terminal ileum recovered. An additional
re-laparotomy was performed following a further 48 hours where most
of the bowel was recovered, but 15 cm of terminal ileum was found
to be necrotic. Partial small bowel resection and ileostomy were
performed. The infant developed cholestatic jaundice post-surgery,
with non-functional ileostomy and intestinal obstruction. An
additional re-laparotomy was performed and 70% of the small bowel
was found to be sick, necessitating massive bowel resection with 35
cm of terminal ileum and 10 cm of proximal jejunum preserved. One
week later, yet another re-laparotomy was performed for recurrent
intestinal obstruction with resection of 10 cm of structured bowel.
When fully on parenteral nutrition and with severe cholestatic
jaundice, oral insulin was started at the age of 3 months. Stool
output, a decrease in jaundice, an increase in oral feeds and a
reduced need for parenteral nutrition were observed within a few
days after the initiation of treatment. Two months following the
addition of oral insulin, parenteral nutrition was stopped and
three months following the addition of insulin, the child was
discharged from hospital, thriving and on full oral feeds.
Example 3
Effects of Oral Insulin on Intestinal Recovery Following
Ischemia-Reperfusion Injury in Rat
[0168] The purpose of the present study was to evaluate the effect
of oral insulin supplementation on structural mucosal changes in
the small bowel induced by ischemia and reperfusion (IR) injury in
rats and to evaluate the mechanisms by which insulin might
influences intestinal recovery including its effect on enterocyte
proliferation and death via apoptosis.
[0169] Methods
[0170] Animals: Animals were obtained from the Rappaport Faculty of
Medicine (Technion, Haifa, Israel). Male rats weighing 240-260 g
were kept in individual stainless steel cages at constant
temperature and humidity, and a 12-hour light-dark cycle was
maintained. Rats were fasted 12 hours prior to the experiment with
free access to water.
[0171] Experimental Design: Thirty rats were randomly assigned to
one of three groups: Group A--sham-rats underwent laparotomy (sham,
n=10); Group B--IR-animals underwent occlusion of superior
mesenteric artery and portal vein for 30 minutes followed by 24
hours of reperfusion (IR, n=10); Group C--IR-INS animals were
pretreated with insulin given in drinking water (2%) for 3 days
prior to and 24 hours following IR event (IR-INS, n=10).
[0172] Surgical procedure: Following overnight fasting, the animals
were anesthetized with intraperitoneal injection of pentobarbital
(IP 40 mg/kg). Using sterile techniques, the abdomen was opened
using a midline incision. Sham underwent laparotomy and
mobilization of superior mesenteric artery (SMA) and portal vein
without their clamping. IR underwent laparotomy, mobilization and
occlusion of SMA and portal vein by vascular clamp for 30 minutes
followed by a reperfusion period of 24 hours. Before closure of the
abdomen, the rats were resuscitated with a 3-ml intraperitoneal
injection of warm 0.9% saline. For all operations, the abdominal
cavity was closed in two layers with a running suture of Dexon "S"
Polyglycolic Acid 3/0. Rats were allowed free access to water and
food. Twenty-four hours later, the rats were anesthetized with
intraperitoneal pentobarbital (75 mg/kg) and were sacrificed by
open pneumothorax.
[0173] Intestinal Mucosal Parameters: The small intestine from the
pylorus to the ileo-cecal valve was removed and divided into two
segments: proximal jejunum and distal ileum. The intestine was
split on the antimesenteric border, washed with cold saline, dried,
and each segment was weighed. The mucosa was scraped from the
underlying tissue with a glass slide and weighed. Bowel and mucosal
weights were calculated as mg/cm bowel length/100 g body weight.
Mucosal samples were homogenized with TRIzol reagent (Rhenium LTD,
Jerusalem). DNA and protein were extracted by the method of
Chomczynski [BioTechniques. 15:532, 1993] and were expressed as
micrograms per centimeter of bowel per 100 g of body weight. In
brief, 100 mg tissue was mixed with 1 ml of TRIzol reagent and
homogenized for 2 min. Following a three minute incubation period
and centrifugation the contents separated into three phases. DNA
was isolated from the interphase using ethanol, washed with sodium
citrate and was stabilised with 75% ethanol. Protein was isolated
from the lower phase using isopropyl alcohol, washed with guanidine
hydrochloride, and stabilised with 100% ethanol. Quantitation of
DNA was performed with a spectrophotomer. Concentration of the
final protein concentration was detected using Bio-Rad Protein
Assay technique.
[0174] Intestinal Histology: Intestinal samples from the proximal
jejunum and distal ileum were fixed in 10% formalin, dehydrated in
progressive concentrations of ethanol, cleared in xylene, and
embedded in paraffin wax. Deparaffinized 5 .mu.m sections were
stained with haematoxylin and eosin. Ten villi and crypts were
selected for the microscopic analysis, using a 10.times.4
magnifying lens. Histological images were loaded on a 760.times.570
pixels resolution buffer using a computerized image analysis system
composed of a trichip RGB video-camera (Sony, Japan), installed on
a light microscope (Zeiss, Germany) and attached to an IBM
compatible personal computer (Pentium III, MMX, 450 mhz, 125 MB
RAM), equipped with a frame grabber. Images were captured,
digitized and displayed on a high resolution color 17 inch monitor.
The villus height and crypt depth were measured using the Image Pro
Plus 4 image analysis software (Media Cybernetics, Baltimore, Md.,
USA).
[0175] The degree of intestinal tissue injury was evaluated on a
grading scale from 0 to 8 as described previously by Park et al.,
[Surgery. 107: 574, 1990]: 0--normal mucosa, 1--subepithelial space
at villus tip, 2--more extended subepithelial space, 3--epithelial
lifting along villus sides, 4--denuded villi, 5--loss of villus
tissue, 6--crypt layer infarction, 7--transmucosal infarction,
8--transmural infarction.
[0176] Enterocyte proliferation and apoptosis: Crypt cell
proliferation was assessed using 5-bromodeoxyuridine (5-BrdU).
Standard BrdU labeling reagent (Zymed Laboratories, Inc, San
Francisco, Calif.) was injected intraperitoneally at a
concentration of 1 ml/100 g body weight 2 hours prior to sacrifice.
After paraffin removal, rehydration, and peroxidase inhibition,
sections (5 .mu.m) were successively incubated with a biotinylated
monoclonal anti-BrdU antibody system provided in a kit form (Zymed
Laboratories, Inc, San Francisco, Calif.). An index of
proliferation was determined as the ratio of crypt cells staining
positively for BrdU per 10 crypts.
[0177] Apoptotic cells were identified using the terminal
deoxynucleotidyl transferase-mediated, dUTP nick end-labeling
(TUNEL) assay. Terminal deoxynucleotidyl transferase (TdT)
(Boehringer Mannheim GmbH, Mannheim, Germany) was used to label DNA
strand breaks. Incorporation of fluorescein was detected by
anti-fluorescein antibody Fab fragments from sheep, conjugated with
horse-radish peroxidase (POD) (Boehringer Mannheim GmbH, Mannheim,
Germany). Briefly, five-micrometer paraffin-embedded sections were
dewaxed and rehydrated with xylene and graded alcohol. Tissue
sections were microwave-pretreated in 10 mM citrate buffer (pH 6.0)
and incubated with TUNEL reaction mixture containing nucleotide
mixture with fluorescein-labeled deoxy-UTP and TdT at 37.degree. C.
for 60 min. Following incubation with blocking solution at room
temperature for 30 minutes, the sections were incubated with
Converted-POD at 37.degree. C. for 30 minutes. TUNEL-positive color
development was obtained by incubating the sections with AES
substrate (Zymed Laboratories). The apoptotic index (AI) was
defined as the number of apoptotic TUNEL-positive cells per 10
villi.
[0178] Statistical analysis: All data are given as mean.+-.SD.
Differences between experimental groups were tested for statistical
significance (p<0.05) using the nonparametric Kruskal-Wallis
ANOVA test, followed by the corrected Mann-Whitney test.
[0179] Results
[0180] Intestinal mucosal parameters: 80% of IR-animals and all
sham animals survived the experimental protocol. There was no
effect of oral insulin on post-operative mortality. IR rats (Group
B) showed a significant decrease in bowel weight in jejunum
(20.1.+-.0.7 vs 22.7.+-.1.0 mg/cm/100 g, p<0.05), mucosal weight
in jejunum (7.0.+-.0.6 vs 9.1.+-.0.5 mg/cm/100 g, p<0.05) and
ileum (5.9.+-.0.6 vs 7.9.+-.0.6 mg,/cm/100 g, p<0.05) (FIG. 4),
mucosal DNA in ileum (6.2.+-.1.2 vs 9.6.+-.0.5 mg/cm/100 g,
p<0.05) (24.8.+-.1.1 vs 30.7.+-.2.7 .mu.g/cm/100 g, p<0.05),
mucosal protein in jejunum (18.+-.3 vs 30.+-.6 .mu.g/cm/100 g,
p<0.05) and ileum (18.+-.3 vs 30.+-.4 .mu.g/cm/100 g, p<0.05)
(FIG. 5) compared to sham animals (Group A). Administration of oral
insulin (IR-INS, Group C) resulted in an increase in jejunal
(23.+-.0.8 vs 20.1.+-.0.7 mg/cm/100 g, p<0.05) intestinal
weight, jejunal (8.9.+-.0.6 vs 7.0.+-.0.6 mg/cm/100 g, p<0.05)
and ileal (7.3.+-.0.4 vs 5.9.+-.0.6 mg/cm/100 g, p<0.05) mucosal
weight (FIG. 4), mucosal DNA content in ileum (8.7.+-.0.5 vs
6.2.+-.1.1 mg/cm/100 g, p<0.05), mucosal protein in jejunum
(28.+-.3 vs 18.+-.3 .mu.g/cm/100 g, p<0.05) and ileum (25.+-.2
vs 18.+-.3 .mu.g/cm/100 g, p<0.05) (FIG. 5) compared to IR
animals (Group B) rats.
[0181] Microscopic bowel appearance IR rats exhibited a significant
decrease in villus height in jejunum (341.+-.29 vs 543.+-.52 .mu.m,
p<0.05) and ileum (225.+-.25 vs 363.+-.39 .mu.m, p<0.05),
crypt depth in jejunum (167.+-.8 vs 216.+-.27 .mu.m, p<0.05) and
ileum (131.+-.7 vs 184.+-.21 .mu.m, p<0.05) compared to sham
animals (FIG. 6). Insulin treated rats (Group C) demonstrated a
significant increase in jejunal (622.+-.91 vs 341.+-.29 .mu.m,
p<0.05) and ileal (421.+-.40 vs 225.+-.25 .mu.m, p<0.05)
villus height as well as in jejunal (264.+-.13 vs 167.+-.8 .mu.m,
p<0.05) and ileal (219.+-.11 vs 131.+-.7 .mu.m, p<0.05) crypt
depth compared to IR-animals (Group B).
[0182] IR injury (Group B) led to significant increase in the mean
intestinal injury grade (Park's criteria) in jejunum and ileum
compared to sham animals (FIG. 7). Oral insulin did not
significantly change the injury grade in both jejunum and ileum
compared to IR-animals.
[0183] Enterocyte proliferation and apoptosis: IR rats (Group B)
demonstrated a significant decrease in enterocyte proliferation
index in jejunum (212.+-.18 vs 252.+-.16 BrdU positive cells/10
crypts, p<0.05) and ileum (173.+-.8 vs 233.+-.10 BrdU positive
cells/10 crypts, p<0.05) compared to sham animals (FIG. 8). Oral
insulin administration (Group C) induced a significant increase in
enterocyte proliferation index in jejunum (298.+-.21 vs 212.+-.18
BrdU positive cells/10 crypts, p<0.05) and ileum (289.+-.26 vs
173.+-.8 BrdU positive cells/10 crypts, p<0.05) compared to IR
animals (Group B) (FIGS. 9a-c).
[0184] Significantly greater numbers of apoptotic cells appeared in
the villi of jejunum (2.2.+-.1 vs 0.4.+-.0.2 TUNEL positive
cells/10 villi, p<0.05) and ileum (2.8.+-.0.7 vs 0.4.+-.0.2
TUNEL positive cells/10 villi, p<0.05) in IR rats (Group B)
compared to sham animals (FIG. 8). Exposure to oral insulin led to
significant decrease in apoptotic index in ileum (1.2.+-.0.4 vs
2.8.+-.0.7 TUNEL positive cells/10 villi, p<0.05) compared to
IR-untreated animals (Group B).
CONCLUSION
[0185] Results show that IR caused a direct intestinal mucosal
injury (as evident from increased Park's intestinal injury score)
and lead to mucosal hypoplasia. The observed decreased bowel and
mucosal weight, decreased mucosal DNA and protein, and decreased
villus height and crypt depth in this model support this
conclusion. Therefore, it should be emphasized, that mucosal
hypoplasia rather than edema, vascular engorgement or intestinal
muscle hypotrophy is responsible for the decreased intestinal mass
after IR. Decrease enterocyte turnover, which is evident from
decreased enterocyte proliferation and increased cell death via
apoptosis, is responsible for this negative effect. Decreased
villus height is presumably due to decreased cell proliferation and
migration along villus axis or due to the specific arrangement of
the villus microvasculature, which results in an oxygen tension
gradient along the villus [Bohlen, H. G., 1980, Am J Physiol. 238,
H164, 1980].
[0186] Oral insulin produced various beneficial effects.
Pretreatment with oral insulin did not protect the intestinal
mucosa from damage caused by IR. IR-INS group manifested similar to
IR-untreated animals intestinal mucosal injury grade, suggesting
similar degrees of intestinal damage. However, exposure to oral
insulin accelerated intestinal mucosal repair and enhanced
enterocyte turnover. This is evident from the significant increase
in bowel and mucosal weight, increased DNA and protein content,
increased villus height and crypt depth in this model. Increased
mucosal DNA and protein suggests accelerated cell metabolism, which
is consistent with the enhanced epithelial cell proliferation.
Histologically, marked increased villus height and crypt depth in
both jejunum and ileum suggests expended absorptive surface area
and closely correlate with large cell mass. The increase in cell
proliferation of crypt cells increased significantly following oral
insulin administration and closely correlated with increased crypt
depth. Cell apoptosis rate decreased significantly in ileum of
insulin treated rats, which may represent an additional mechanism
that maintains mucosal integrity following IR.
[0187] In conclusion, administration of oral insulin did not
prevent ischemic damage but accelerated intestinal recovery,
enhanced enterocyte proliferation and decreased cell death via
apoptosis following ischemia-reperfusion event.
[0188] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0189] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
REFERENCES CITED
Additional References are Cited in the Application
[0190] 1. Coran A G, Spivak D, Teitelbaum D H (1999) An analysis of
the morbidity and mortality of short bowel syndrome in the
pediatric age group. Eur J Pediatr Surg 9: 228-230
[0191] 2. Robinson M K, Ziegler T R, Wilmore D W (1999) Overview of
intestinal adaptation and its stimulation. Eur J Pediatr Surg 9:
200-206
[0192] 3. Sigalet D L, Martin G R (1998). Mechanisms underlying
intestinal adaptation after massive intestinal resection in the
rats. J Ped Surg 33: 889-892
[0193] 4. Podolsky D K. Peptide growth factors in the
gastrointestinal tract. In: Johnson L R, eds. Physiology of the
gastrointestinal tract. Third edition. Raven Press, New York, 1994:
129-167
[0194] 5. Laburthe M, Rouyer-Fessard C, Gammeltoft S. Receptors for
insulin-like growth factors I and II in rat gastrointestinal
epithelium. Am J Physiol 1988; 254: G457-G462
[0195] 6. Lund P K. Molecular basis of intestinal adaptation: the
role of the insulin-like growth factor system.
[0196] 7. Lemmey A B, Martin A A, Read L C, Tomas F M, Owens P C,
Ballard F J. IGF-I and the truncated analogue des-(1-3)IGF-I
enhance growth in rats after gut resection. Am J Physiol. 1991
February; 260(2 Pt 1):E213-9
[0197] 8. Lemmey A B, Ballard F J, Martin A A, Tomas F M, Howarth G
S, Read L C. Treatment with IGF-I peptides improves function of the
remnant gut following small bowel resection in rats. Growth
Factors. 1994; 10(4):243-52
[0198] 9. Olanrewaju H, Patel L, Seidel E R. Trophic action of
local intraileal infusion of insulin-like growth factor I:
polyamine dependence. Am J Physiol 1992; 263: E282-286
[0199] 10. Ziegler T R, Mantell M P, Chow J C, Rombeau J L, Smith R
J. Gut adaptation and the insulin-like growth factor system:
regulation by glutamine and IGF-1 administration. Am J Physiol
1996; 271: G866-G875
[0200] 11. Lukish J, Yu D, Kato Y, Schwartz M Z. The effect of
certain growth factors on intestinal function and adaptation
following massive small bowel resection. Gastroenterology 1996;
110(Suppl): A818
[0201] 12. Shulman R I. Oral insulin increases small intestinal
mass and disaccharidase activity in the newborn miniature pig.
Pediatr Res. 1990 August; 28(2):171-5
[0202] 13. Shulman R I, Tivey D R, Sunitha I, Dudley M A, Henning S
J. Effect of oral insulin on lactase activity, mRNA, and
posttranscriptional processing in the newborn pig. J Pediatr
Gastroenterol Nutr. 1992 February; 14(2):166-72
[0203] 14. Shehadeh N, Wies R, Eishach O, Berant M, Etzioni A,
Shamir R. Influence of oral insulin supplementation on
carbohydrate, lipid and protein metabolism in weaned Balb/c mice. J
Pediatr Endocrinol Metab. 2003 16(3):431-7.
[0204] 15. Shulman R J. Effect of rectal-enteral administration of
insulin on intestinal development and feeding tolerance in preterm
infants: a pilot study. Arch Dis Child Fetal Neonatal Ed. 2002
March; 86(2):F131-3.
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