Stable glutamine derivatives for oral and intravenous rehydration and nutrition therapy

A method for the treatment of dehydration or nitrogen deficiency-based malnutrition is provided which involves administering to a patient in need thereof an effective amount of a compound selected from oligopeptides formed from the coupling of one or more amino acids with glutamine, the product of coupling glucose with glutamine, the product of coupling glucose and one or more amino acids with glutamine, or the product from acylating glutamine with a carboxylic acid having from 2 to 6 carbon atoms.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to stable glutamine derivatives and their use 
in rehydration and nutrition therapy. 
2. Discussion of the Background 
Glutamine, the chief metabolic fuel of the small intestine, is an amino 
acid which cotransports Na.sup.+ across the enterocyte brush border 
membrane. It is known to be the major bowel nutrient and energy source and 
has been used in intravenous solutions to improve nitrogen balance, 
inhibit protein breakdown, stimulate the growth of epithelial cells, and 
reduce intestinal villous atrophy. 
Additionally, various researchers have shown that glutamine stimulates the 
absorption of sodium and chloride and has been tried in oral rehydration 
solutions to reduce cholera diarrhea. 
Viral enteritis is a leading cause of diarrhea in infants and toddlers less 
than 2 years old. Each year in the United States, about 22,000 infants are 
hospitalized for treatment of rotavirus-induced dehydration. Central to 
the pathophysiology of diarrhea in transmissible gastroenteritis (TGE), 
which is one experimental model of viral enteritis, are a number of 
intestinal abnormalities, including impaired glucose and amino 
acid-coupled Na.sup.+ transport, defective NaCl absorption, diminished 
disaccharide hydrolysis, and reduced mucosal absorptive surface. 
In a majority of cases, diarrheal disease morbidity and mortality is due to 
dehydration. The primary effect seen is the loss of fluid and electrolytes 
in diarrheal stools. An immediate effect in treatment of dehydration can 
be achieved by early oral administration of sugar (glucose) and 
electrolyte solution and continued feeding. However, conventional therapy 
by administration of oral rehydration formulations does not reduce stool 
volume or the duration of diarrhea. Thus, modifications of the oral 
rehydration therapy are needed to actually reduce stool volume or speed 
the recovery of normal mucosal function, which in turn would substantially 
enhance the acceptability and effectiveness of such therapy. 
The physiological principle of oral rehydration therapy was first observed 
by Schultz and Curran (Physiology Review, 50:637-718 (1970)). They 
demonstrated the intestinal cotransport of sodium with glucose. Others 
have demonstrated the active transport of organic compounds by the small 
intestine which, when coupled with sodium absorption, enhances the 
absorption of water and other salts. (See Lima et al, Bailliere's Clinical 
Tropical Medicine and Communicable Diseases, 3:627-636 (1988) and Soares 
et al, Brazilian Journal of Medical and Biological Research, 24:111-113 
(1991)). 
The effects of organic compounds on salt and water absorption were first 
applied successfully to the treatment of patients with cholera and 
thereafter it was shown experimentally that the salt-substrate cotransport 
was substantially intact in cholera patients and that oral therapy with 
sodium, chloride, potassium, bicarbonate and glucose in the same solution 
could restore and maintain normal blood volume and electrolyte 
concentrations. 
Organic molecules such as D-hexoses, neutral amino acids, dipeptides and 
tripeptides of neutral amino acids, and water-soluble vitamins can also 
enhance sodium absorption, followed by water absorption from the small 
intestine. The present inventors have previously shown the efficacy of 
glutamine in intestinal sodium absorption. (Lima et al, Brazilian J. Med. 
Biol. Res., 25:637-640 (1992)). However, the greatest limitation to the 
oral use of glutamine is its instability and tendency to degrade in water 
and acid, conditions which are found in the stomach. 
Bone marrow transplantation is being increasingly used in the treatment of 
hematologic malignancies. Patients undergoing bone marrow transplantation 
lose body protein because of the catabolic effects of chemotherapy, total 
body irradiation, and graft-versus-host disease. In addition, 
gastrointestinal toxicity often limits the consumption and absorption of 
enteral nutrients. Infectious complications also remain a major cause of 
morbidity in these patients. Infection accelerates protein loss, and 
protein-calorie malnutrition may decrease host resistance to microbial 
invasion. 
Parenteral nutrition is known to attenuate such protein losses and may 
prevent complications associated with malnutrition. Despite routine use in 
many centers, parenteral nutrition is also, unfortunately, associated with 
an increased incidence of infection in patients receiving chemotherapy 
with or without irradiation, and also in those receiving allogeneic bone 
marrow transplantation. Further, despite conventional nutritional support, 
these patients still suffer from markedly negative nitrogen balance. 
Modification of amino acid formulations may improve the clinical and 
metabolic efficacy of parenteral nutrition. Notably absent in all 
commercially available parenteral nutrient solutions is glutamine, because 
it has a shorter shelf-life than the commonly used amino acids and has 
been considered a nonessential amino acid. However, during catabolic 
states, glutamine concentrations in intracellular pools (primarily 
skeletal muscle) fall rapidly. This reduction in glutamine occurs due to 
use of glutamine for renal ammoniagenesis and as an oxidizable fuel for 
stimulated lymphocytes and macrophages and intestinal mucosal cells. 
Glutamine-enriched parenteral or enteral nutrition has been shown to 
enhance nitrogen balance, attenuate intestinal mucosal damage, decrease 
bacteremia, and improve survival after irradiation or chemotherapy when 
compared with glutamine-free nutrition. Limited clinical studies in 
postoperative patients have shown improved nitrogen retention with 
glutamine-enriched parenteral feeding. The clinical safety of L-glutamine 
added as a component of balanced parenteral nutrient solutions has 
recently been documented. (See Ziegler et al, Annals of Internal Medicine, 
116:821-828 (1992) and references cited therein). 
Masaki et al, U.S. Pat. No. 4,987,123, disclose the use of L-alanine, 
L-glutamine, L-alanyl-L-glutamine and salts thereof in the treatment of 
hepatic disorders. However, there is no indication as to the efficacy of 
using glutamine derivatives in treatment of disorders associated with 
dehydration or with nitrogen imbalance. 
While the above-noted studies have shown the efficacy of Glutamine in 
rehydration and nutrition therapies, the instability of glutamine in the 
digestive tract has diminished its usefulness. Accordingly, there is 
needed a method for administration of glutamine to patients which will 
provide effective treatment in oral rehydration therapy and nutrition 
therapy, while overcoming the difficulties of instability in an acidic 
environment. 
SUMMARY OF THE INVENTION 
Accordingly, one object of this invention is to provide new stable 
glutamine derivatives capable of delivering glutamine to the body in oral 
or intravenous rehydration or nutrition therapy. 
A further object of the present invention is to provide an improved method 
for treating conditions associated with dehydration or nitrogen deficiency 
based malnutrition using the stable glutamine derivatives of the present 
invention. 
These and other objects of the present invention have been satisfied by the 
discovery that coupling glutamine with one or more additional amino acids, 
coupling glutamine with glucose, coupling glutamine with glucose and one 
or more additional amino acids, or acylating glutamine with a C.sub.2 
-C.sub.6 carboxylic acid provides a compound capable of surviving the 
digestive system of patients while at the same time delivering sufficient 
amounts of glutamine to the patient to obtain effective amounts in the 
patient's system to treat conditions associated with dehydration or 
malnutrition due to nitrogen loss.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to new stable glutamine derivatives and their 
use in rehydration therapy a nutrition therapy. 
The stable glutamine derivatives of the present invention can be prepared 
by coupling glutamine with one or more additional amino acids to provide 
oligopeptides, or with glucose, or both, or acylating glutamine with a 
carboxylic acid having 2 to 6 carbon atoms, to provide a compound which is 
stable to degradation under acidic environments. While any naturally 
occurring amino acid may be used as the additional amino acid coupled to 
the glutamine, it is preferred to use alanine or glutamine, alone or in 
combination as the additional amino acids. A preferred number of total 
amino acid groups present in the compounds used in the present method 
ranges from 2 to 5 (formed from coupling from 1 to 4 amino acids with 
glutamine), with dipeptides and tripeptides most preferred. Most preferred 
compounds include alanyl-glutamine, alanyl-glutaminyl glutamine and 
gamma-glutamyl glutamine. The compounds used in the present invention are 
known and can be prepared using conventional peptide coupling reactions, 
such as on a solid phase peptide synthesizer or using 
1,3-diisopropyl-carbodiimide (DIPCDI) activation in solution coupling, as 
described in Hudson, J. Org. Chem., 53(3):617-624 (1988) and Bodansky et 
al, Synthesis, pp. 453-463 (1991). 
The compounds of the present invention have been shown to be much stabler 
in acidic water solutions (such as they would be expected to face in a 
patient's stomach or intestine) and to drive salt absorption comparable to 
if not better than glucose (see FIG. 1). FIG. 2 shows the pH dependent 
degradation of Glutamine under acidic conditions. By contrast, the acyl or 
alanyl derivatives of glutamine of the present invention were degraded 
&lt;10% even at pH=1 over 360 hours at room temperature. 
Since the ability to drive the intestinal sodium cotransport mechanism is 
known to directly correlate to efficacy in treatment of dehydration, 
especially when associated with diarrhea, these compounds provide an 
exciting approach to oral rehydration and nutrition therapy. 
These compounds are useful not only in malnourished children with diarrhea, 
but also in patients kept too long on parenteral (IV) fluids or tube 
feedings or in those with damaged intestinal mucosa from infection or 
chemotherapy. 
The present glutamine derivatives effectively block the degradation of 
glutamine in the highly acidic conditions which are encountered in the 
human stomach. In order to perform effectively in oral therapy, the 
compounds must be able to survive the conditions in the digestive tract 
while maintaining the ability to stimulate their absorption and maintain 
the integrity of the intestinal mucosa. 
The glutamine derivatives of the present invention have been found to 
provide the requisite acid stability. Additionally, these glutamine 
derivatives provide intestinal sodium cotransport which is comparable to 
or higher than the use of glutamine itself. 
The glutamine derivatives of the present invention can be administered 
either orally or intravenously. 
When administered orally, the compounds can be administered as a liquid 
solution, powder, tablet, capsule or lozenge. The compounds can be used in 
combination with one or more conventional pharmaceutical additive or 
excipients used in the preparation of tablets, capsules, lozenges and 
other orally administrable forms. 
When administered as an intravenous solution, the derivatives of the 
present invention can be admixed with conventional IV solutions containing 
various amino acids and nutrients, such as conventional parenteral therapy 
solutions. Such IV solutions are known in the art and used in rehydration 
and nutrition therapy. 
The compounds of the present invention are administered at a dose range 
effective to bring about improved intestinal sodium cotransport. 
A preferred dosage range of glutamine equivalent (Gln has a molecular 
weight of 146) is 0.05 to 0.8 g/kg/day of patient body weight, with 
approximately 0.5 to 0.6 g/kg/day or solutions of approximately 13 g/L 
glutamine equivalent (the solutions have sufficient glutamine derivative 
to provide an effective glutamine level equivalent to a solution of 13 g/L 
glutamine) or 1-10 mM glutamine derivatives being most preferred. 
Conventional therapy regimens are followed in oral or intravenous 
rehydration or nutrition therapy of patients in need thereof. Such 
patients include children who are malnourished and suffer from diarrhea, 
patients kept too long on IV fluids or tube feeding, or those having 
damaged intestinal mucosa from infection or chemotherapy. The need for 
starting treatment in rehydration or nutrition therapy is judged by 
conventional standards. The therapy is continued until the patient has 
improved beyond the minimum hydration requirements or as long as the 
increased nutritional demands require. 
Having generally described this invention, a further understanding can be 
obtained by reference to certain specific examples which are provided 
herein for purposes of illustration only and are not intended to be 
limiting unless otherwise specified. 
EXAMPLES 
To study intestinal sodium cotransport using the compounds of the present 
invention, Ala-Glu and Ala-Gln were tested according to the method of Lima 
et al, Brazilian J. Med Biol. Res., 25:637-640 (1992) for measuring 
intestinal sodium cotransport in rabbit ileal mucosa mounted in Ussing 
chambers. 
FIG. 1 shows the response in intestinal sodium cotransport using Ala-Gln 
and Ala-Glu in rabbit ileal mucosa. As shown the dose response increases 
dramatically in a non-linear fashion. Ala-Gln showed a maximum increase 
(E.sub.max) in Isc of 150 .mu.A/cm.sup.2, with a pD.sub.2 of 3.8. The 
pD.sub.2 value is related to the ED.sub.50 in accordance with the 
following formula: 
EQU -log ED.sub.50 =pD.sub.2. 
Meanwhile, Ala-Glu showed an E.sub.max =120 .mu.A/cm.sup.2, with a pD.sub.2 
of 4.1. Thus, the Ala-Gln compound of the present invention was found to 
provide comparable or improved intestinal sodium cotransport compared to 
Ala-Glu. 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein.