Note: Descriptions are shown in the official language in which they were submitted.
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_ IMPROVED INFANT FORMULA AND METHODS OF M'ROV1NG
INFANT STOOL PATTERNS
FIELD OF THE INVENTION
The invention relates to an improved enteral nutritional formula and more
particularly to infant formulas which contain ribo-nucleotide equivalents at a
level
of at least 10 mg/100 Kcal of formula and wherein the ribo-nucleotide
components
are at specific ratios.
This application is a division of Canadian Patent Application Serial No.
2,180,464, filed January 5, 1995.
BACKGROUND OF THE INVENTION
The composition of human milk serves as a valuable reference for
improving infant formula. However, human milk contains living cells, hormones,
active enzymes, immunoglobulins and components with unique molecular
structures that cannot be replicated in infant formula. Unlike human milk,
infant
formula must remain stable on the shelf for up to thirty-six (36) months.
These
fizndamental differences between human milk and infant formula often mandate
differences in the composition to achieve similar clinical outcome.
Human milk has served as a valuable reference for improving
infant formula. The investigation of human milk components has
stimulated many investigations into what constituents may be added
to infant formula. Greater knowledge of the composition of human
milk affords the opportunity to design infant formulas that are closer
to that of human milk. However, it becomes increasingly apparent
that infant formula can never duplicate human milk. Many
constituents in human milk are bioactive and because of synergies
among these components there is little reason to believe that the same
compound would have the same bioactivity in infant formula. The
likelihood of this possibility is further diminished when the impact of
heat treatment for sterilization and long-term storage of the formula is
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consi dered . The present i nventi on i s based , i n part , on the concept of
providing a formula which matches the performance of breast milk in most
parameters without attempting to actually duplicate the delicate balance of
human milk components.
The composition of human milk differs appreciably from that of other
speci es and much attenti on has been pai d to the vari ous components .
Several
investigators have reported on the nucleotide content of milk from humans
[Janas. L.M. et al: The Nucleotide Profile of Human Milk. Pediatr. Res.
16:659-662(1982) and Gil. A et al: Acid-soluble Nucleotides of Human Milk
at Different Stages of Lactation. Journal of Dairy Research. 49: 301-
307(1982).~ The numerous publications cited in the Janas and Gil references
al so rel ate to the nucl eoti de composi ti on of human mi 1 k and , i n
combi nati on .
leave one skilled in the art with a confused and conflicting understanding
of the nucleotide composition of human milk. None of the prior art
discloses the~minimum level of nucleotide equivalents taught by the present
invention nor the ratio of the four elements (adenosine, cytidine.
guanosine and uridine) to each other. Most importantly the prior art does
not suggest or disclose a formula that is superior to human milk in
enhancing the immune response of a human.
Nucleosides are nucleotides minus the one to three phosphate groups.
Nucl eosi des are a cl ass of chemi cal compounds that are of importance i n
physiological and medical research. They may be obtained from the partial
decomposition (hydrolysis) of nucleic acids. Nucleosides contain a purine
or pyrimidine base linked to either d-ribose, forming ribosides, or d-
deoxyribose, forming deoxyribosides. Nucleosides are nucleotides minus the
phosphorus group. Representative of the nucleosides are adenosine.
cytidine. guanosine, inosine and uridine.
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Nucleotides (nucleosides plus at least one phosphate group) are the
fundamental units of nucleic acids. The nucleotides found in nucleic acids
are phosphate esters of the nucleosides. The term nucleotides is also
someti mes appl i ed to compounds not found i n nucl ei c aci ds and whi ch
conta i n
substances other than the usual purines and pyrimidines. The nucleotides
inosine-5'-monophosphate and guanosine-5'-monophosphate are used as flavor
potentiators.~
Nucleotides are ubiquitous, low molecular weight compounds that
participate in energy metabolism and modulation of enzymatic reactions. In
addition, nucleotides are components of compounds that are crucial in the
synthesis and catabolism of carbohydrates, lipids, protein, and nucleic
acids. Clearly nucleotides and their metabolites are important determinants
of numerous cellular processes.
Adequate cellular supplies of nucleotides in humans and animals are
maintained by two pathways: the salvage pathway and de novo synthesis. The
salvage pathway involves recovery of nucleotides and nucleosides liberated
from metabolism (such as catabolized nucleic acids). De novo synthesis of
nucleotides requires the precursors aspartate, glutamine, glycine, and
carbamoyl phosphate. The salvage pathway generally supplies sufficient
quantities of nucleotides even in tissue with rapidly proliferating cells.
i ncl udi ng enterocytes . erythrocytes and i mmune cel 1 s . It i s al so
known that
addition of nucleotides to the diet inhibits the de novo pathway and
activates the salvage pathway in the liver and extrahepatic tissue,
especially in enterocytes.
Dietary sources rich in nucleotides include meats, fish, legumes. and
dairy products. Nucleotides are primarily present in polymeric forms (DNA,
RNA and nucleoproteins) in these foods and are degraded by ribonucleases.
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deoxyribonucleases and proteases, yielding nucleotides. Subsequent action
of phosphatases yi el ds nucl eosi des whi ch appear to be the preferred form
for
absorption. Some additional digestion to free purine and pyrimidine bases
may occur. Studies have been published that indicate that a specific
transport systems) exists for the absorption of nucleosides and bases.
Most dietary nucleotides are degraded, excreted, or utilized before
reaching the systemic circulation. Although dietary nucleotides appear to
have little access to the systemic circulation, they have been implicated
as having numerous systemic effects Reports indicate that dietary
nucleotides influence the response to sepsis, alter blood lipid profiles.
enhance brain function, and increase iron absorption, gut mucosal growth,
and gut bifidobacteria populations.
U.S. 3.231:385 discloses and claims an active phosphatase free cow's
milk which contains at least two of the respective disodium salts of (a)
cytidine 5'-monophosphate in the amount of 10 to 20 mg/L of cow's milk. (b)
guanosine 5'monophosphate in the amount of 0.2 to 0.4 mg/L of cow's milk.
( c ) uri di ne 5' -monophosphate i n the amount of 1. 2 to 1. 4 mg/L of cow'
s mi 1 k ,
(d) guanosine 5'-diphosphate in the amount of 0.4 to 0.6 mg/L of cow's milk,
( a ) uri di ne 5' -di phosphate gl ucose i n the amount of 0 . 5 to 1. 0 mg/L
of cow' s
mi 1 k . ( f ) uri di ne 5' -di phosphate gal actose i n the amount of 0 . 5
to 1. 0 mg/L
of cow's milk.and (g) uridine 5'-diphosphate glucuronic acid in the amount
of 1.0 to 3.0 mg/L of cow's milk.
U . S . 4 . 994 .442 di scl oses and cl ai ms the addi ti on of nucl eosi des
and/or
nucleotides to infant formula to provide a formula having enhanced
physiological properties and methods of stimulation or repair of intestinal
gut cells. This patent teaches and claims the use of at least one member
selected from the group consisting of uridine, uridine phosphate, and
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mixtures thereof: guanosine, guanosine phosphate and mixtures thereof;
adenosine. adenosine phosphate and mixtures thereof; cytidine, cytidine
' phosphate and mixtures thereof: and inosine, inosine phosphate and mixtures
thereof . Thi s patent al so cl ai ms a method for enhanci ng the i mmune
response
of T-cells and for providing specific fatty acid phospholipid profiles in
red blood cell membranes of infants. This patent fails to suggest the use
of the four specific ribo-nucleotides disclosed in the present invention.
Thi s reference al so fai 1 s to suggest the speci fi c rati os and 1 evel s
of ri bo-
nucleotides used in this invention and the surprising results relating to
the immune system and diarrhea that are achieved through the present
invention.
U.S. 5.066.500 discloses a non-milk based infant formula comprising
carbohydrates, a source of amino acids, vegetable oils. minerals, vitamins.
wherein the formula contains at least one of uridine, uridine phosphate or
mixtures thereof: guanosine, guanosine phosphate or mixtures thereof: or
adenosine, adenosine phosphate. or mixtures thereof: cytidine, cytidine
phosphate, or mixtures thereof, or inosine, inosine phosphate. or mixtures
thereof. This patent fails to disclose the four specific ribo-nucleotides
utilized in the instant invention, the levels and ratios of those
nucleotides in an enteral nutritional formula and the surprising results
that can be obtained through the use of the instant invention.
U.S. 4.544.559 discloses and claims a nucleotide enriched humanized
milk in powder form. The inventive aspect of this patent relates to the use
of five (5) nucleotides in the precise ratios as follows: adenosine mono-
phosphate (AMP) 1.32 mg/100g, cytidine-monophosphate (CMP) 1.12 mg/100g,
guanosine-monophosphate (GMP) 1.49 mg/100g, uridine-monophosphate (UMP) 3.42
mg/100g and inosine-monophosphate (IMP) 0.45 mg/100g of powdered formula.
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In contrast, the present invention only uses four (4) ribo-nucleotide
equivalents. (as will be defined infra) those being represented by:
' cytidine 5'- monophosphate (CMP). uridine 5' monophosphate (UMP) guanosine
5' monophosphate (GMP> and adenosine 5' monophosphate (AMP). Also critical
to the i nstant i nventi on , i s that these four ri bo-nucl eoti des ( or
nucl eoti de
equivalents) be present in the enteral formulation at a level of at least
milligrams of nucleotide equivalents per 100 Kcal of enteral formula.
An even more specific aspect of the present invention, which sets it apart
from the prior art is the requirement that the weight ratio of CMP to UMP
be at least 1.5:1: that the ratio of CMP to AMP be at least 2:1; and the
ratio of CMP to GMP be at least 1.75:1 (on a nucleotide equivalent basis.)
G.B. 2,216.416 discloses a method of stimulating the immune function
with the aid of a nucleobase source. the use of nucleobase sources for
immuno stimulation and compositions comprising such nucleobase sources.
Specifically, this patent relates to the administration of from 0.1 to 75
grams of RNA, DNA, nucleotides or nucleosides per day or an amount
equivalent thereto in nucleobase form. This reference fails to suggest or
disclose the specific benefits that can be realized through the use of four
ribo-nucleotides at specific levels and ratios.
The enteral formula of the instant invention provides a positive
advantage to the i nfant . The cl i ni cal studi es whi ch were conducted evi
dence
the unexpected advantages of the instant invention. An additional aspect of
the present invention is the overall balance of nutrient interactions and
bio-availability, which provide an improved nutritional product. Another
aspect of the present invention relates to an infant formula which meets the
requi rements of the Infant Formul a Act and to methods for its producti on
and
analytical techniques for the determination of nucleotide equivalents.
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Several investigators have reported that maternal milk contains
. ~ factors that protect against diarrhea. These investigators have also
reported that nucleotide-enriched formula have an effect on the incidence,
duration and etiology of acute diarrhea. These investigators have failed
to discover the specific nucleotides and ratios of the instant invention
that are effacious in the treatment/prevention of diarrhea.
There has been much interest in this area of enteral nutritional
formulations.. The prior art is replete with various formulations using
various ingredients. The general principle of adding RNA, DNA, nucleotides,
nucleosides and/or nucleobases to food products is disclosed within the
prior art. However, none of_the prior art either taken individually or in
any combination would suggest or predict, with any level of certainty, the
discoveries the Applicants have made herein.
DISCLOSURE OF THE INVENTION
The term "nucleotide equivalents" as used herein means the ribo-
nucleoside, ribo-nucleotides. RNA, phosphate esters and d-ribose adducts of
adenosine (A), cytidine (C), guanosine (G), and uridine (U). The various
forms of A.C.G, and U are determined, calculated and expressed as the
monophosphate esters: adenosine monophosphate (AMP), cytidine monophosphate
(CMP), guanosine monophosphate (GMP) and uridine monophosphate (UMP). These
are the free acid forms of the monophosphate esters as opposed to the salt
forms such as the mono or disodium salts. Some nucleotides are often sold
as the sodium salts. For example, the sum-of adenosine from RNA, the mono-,
di and triphosphate esters and the d-ribose adducts are stated as the
nucleotide equivalent of the mono phosphate ester of adenosine. This
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invention relates only to the use of ribo-nucleotides and does not
contemplate or claim the use of the deoxy form.
There is disclosed an enteral formula, said formula comprising:
1) protein, said protein being of a concentration of between 10 and 35
grams per liter of formula; (2) fat, said fat being of a concentration of
between 20 and 45 grams per liter of formula; (3) carbohydrates, said
carbohydrates being of a concentration of between 60 and 110 grams per
liter of formula; and (4) at least 10 mg of nucleotide equivalents per 100
Kcal of formula, said nucleotide equivalents consisting of RNA; mono-,
10 di-, and triphosphate esters of adenosine, cytidine, guanosine and uridine,
and the d-ribose adjuncts thereof; and wherein the weight ratio of
CMP:UMP is at least 1.5:1; of CMP:AMP is at least 2:1 and of CMP:GMP
is at least 1.75:1.
The minimum level of nucleotide equivalents in this invention is
mg per 100 Kcal of formula or 70 mg per liter of a formula having a
caloric density of about 687 Kcal per liter.
Thus, the protein, fat and carbohydrate concentrations in grams
per liter, indicated above correspond in g/100 Kcal of formula to about 1.5
to about 5 g of protein, about 3 to about 6.5 g of fat and about 8.7 to about
16 g of carbohydrate.
Levels of nucleotide equivalents at the claimed ratios above about
1.0 gms per liter of formula or about 100 mg/100 Kcal of formula is
outside the scope of this invention. From this maximum level of about 100
mg/100Kca1 of formula or 1.0 g/liter of formula and the ratios set out
above, the upper limits for each of the four-ribonucleotide components
may be calculated.
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There is also disclosed an enteral formula wherein the source of
protein is selected from the group comprising condensed skim milk, non-
fat milk, acid whey and cheese whey. In general, any appropriate source of
protein can be used in this invention, including hydrolyzed proteins. There
is further disclosed an enteral formula wherein the protein is 50-70% by
weight condensed skim milk or non-fat milk and the fat is selected from
the group consisting of soy oil, coconut oil, corn oil, high oleic safflower
oil, marine oils, egg yolk oils, high oleic sunflower oils, fungal oils and
mixtures thereof.
There is also disclosed an infant formula which comprises a
nutritionally adequate source of amino nitrogen, carbohydrates, edible fats,
minerals and vitamins; the improvement characterized in a composition
comprising at least one member selected from each of the groups (a), (b),
(c) and (d):
(a) uridine, uridine phosphates and mixtures thereof;
(b) guanosine, guanosine phosphates and mixtures thereof;
(c) adenosine, adenosine phosphate and mixtures thereof; and
(d) cytidine, cytidine phosphate and mixtures thereof; wherein
the total amount of the composition is at least 10 mg per 100 Kcal of
formula and wherein the weight ratio of CMP:UMP is at least 1.5:1 of
CMP:AMP is at least 2:1 and of CMP:GMP is at least 1.75:1.
Suitably, the weight ratio of CMP:UMP is from about 1.5:1 to
about 2.6:1, the weight ratio of CMP:AMP is from about 2:1 to about 3.9:1
and the weight ratio of CMP:GMP is from about 1.75:1 to about 2.8:1.
There is also disclosed an infant formula the improvement
comprising adding from 29 to 39 mg of CMP per liter of formula; 15 to 20
mg of UMP per liter of formula; 10 to 15 mg of AMP per liter of formula
and 14 to 20 mg of GMP per liter of formula.
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More specifically there is disclosed a formula wherein the protein is
50-70% condensed skim milk or non-fat milk and 30-50% cheese whey by
weight and the fat has as its source soy, coconut and high oleic safflower
oil.
The enteral formula according to the invention provides a source of
carbohydrates selected from sucrose, corn syrup, glucose polymers and other
carbohydrate sources. The formula may also contain dietary fiber.
In accordance with one aspect of the invention there is provided a
softer stool-forming enteral formula comprising protein, carbohydrates and
fat,
wherein said fat consists of a blend of high oleic safflower oil, soy oil and
coconut oil effective to establish a stool consistency in an infant, fed with
the
formula, comparable to that in a breast fed infant.
In accordance with another aspect of the invention there is provided
use of a fat consisting of a blend of high oleic safflower oil, soy oil and
coconut
oil in the manufacture of an enteral formula providing a stool consistency in
an
infant fed with the formula, comparable to that in a breast fed infant.
In accordance with still another aspect of the invention there is
provided a fat blend consisting of high oleic safflower oil, soy oil and
coconut
oil for use in producing a stool consistency, in an infant feed with an
enteral
formula containing the fat blend, comparable with that in a breast fed infant.
There is also disclosed a method of enhancing the immune system of a
human, said method consisting of feeding a human in need of treatment a
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formula, the improvement comprising a formula consisting essentially of:
1) protein, said protein being of a concentration of between 10 and 35 grams
per liter of formula: 2> fat, said fat being of a concentration of between
and 45 grams per liter of formula: 3) carbohydrates, said carbohydrates
being of a concentration of between 60 and 110 grams per liter of formula:
and 4> at least 10 mg of nucleotide equivalents per 100 Kcal of formula and
wherein the weight ratio of CMP:UMP is at least 1.5:1: of CMP:AMP is at
least 2:1: and of CMP:GMP is at least 1.75:1. There is also disclosed a
novel method of producing an enteral formula containing nucleotides and its
use to treat or prevent diarrheal disease and to novel analytical
techniques.
There is also disclosed a method for manufacturing infant formula,
said method comprising the steps of: 1) dispersing an appropriate quantity
of protein in water or oil sufficient to solubilize or suspend the protein.
thereby forming a protein solution: 2) dissolving carbohydrates in water,
thereby formi ng a carbohydrate sol uti on : 3 ) mi xi ng mi neral s i n water
or the
carbohydrate solution, thereby forming a mineral solution or a
mineral/carbohydrate solution: 4) combining appropriate quantities of said
protein solution, said carbohydrate solution, said mineral solution, and a
solution of oils containing oil soluble vitamins. 5) heat processing and
homogenizing the combined solution; 6) adding water soluble vitamins, iron.
choline and other nutrients to the combined solution: 7) adding water to
dilute the combined solution to the desired caloric density, approximately
400-725 kcal per liter of formula and 8) adding from 29 to 39 mg of CMP per
liter of formula: 15 to 21 mg of UMP per liter of formula: 10 to 16 mg of
AMP per liter of formula and 14 to 20 mg of GMP per liter of formula
directly to the batch or in the form of an aqueous solution.
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As used herein, the terms CMP, UMP. GMP and AMP mean not only the
monophosphates of adenosine, cytidine, guanosine and uridine but also their
' nucleotide equivalents which include polymeric RNA, ribo-nucleosides, ribo-
nucleoside containing adducts and di- and triphosphate ribo-nucleotides.
There i s al so di scl osed a novel analyti cal techni que that can quanti fy
the various forms of the four nucleotides in complex food matrices. The
analytical process comprises: 1) the enzymatic digestion of polymeric RNA
to nucleotide 2) the enzymatic co-digestion of nucleoside containing adducts
to nucl eosi des and the nucl eoti des to nucl eosi des 3) the coval ent
attachment
of the nucleosides to boronic acid that has been immobilized on a
polyacrylamide gel 4) the release of the nucleosides from the boronate
derivatized polyacrylamide gel via a pH shift 5) the separation of the
nucl eosi des vi a 1 ow pH reverse phase/i on pai ri ng HPLC usi ng octane sul
fonate
as the ion-pairing agent and 6) quantitation of the nucleoside via U.U.
absorbance using external standards or other means known in the art of
analytical chemistry.
There i s al so di scl osed a novel anti oxi dant system that i s used i n the
enteral formulas according to this invention. The antioxidant system
consists of ~-carotene. R,R.R, cx-tocopherol and selenium. The level of
R,R,R, cx-tocopherol can range from 10 to 30 IU per liter of formula. The
level of ~-carotene can range from 375 to 575 ug per liter of formula and
the 1 evel of sel eni um can range from 14 to 32 mcg per 1 i ter of formul a .
The
selenium used in this aspect of the invention may be delivered in the form
of selenate.
In actual use, the formula of this invention may be consumed by any
infant and should be in compliance with accepted levels of vitamins.
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minerals, micro-components and the like. The amount consumed does not
differ from that associated with the normal consumption of commercially
' available infant formula.
A representative formula for the enteral nutritional product of the
invention is set forth in Table I.
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TABLE I
FORMULA ACCORDING TO THE INDENTION
Nutrient . Concentration per liter of
formula
Protein . 13.0-20 g
Protein Source
CSM1 55-75%
7.15-15 g
WPCZ 25-45%
3.25-9.0 g
Lipid 13-21 g
H.O. Safflower Oil 35-55%
Soy Oil 20-40%
Coconut Oil 20-45%
Carbohydrate lactose 70-110 g
Nucleotides 70-100 mg
CMP 29-39 mg
UMP 15-21 mg
AMP 10-16 mg
GMP 14-20 mg
Iron 8-16 mg
R,R.R.cx
tocopherol 10-30 IU
Carotene 375-575 ug
Selenium 14-32 mcg
Calcium 475-850 mg
Phosphorus 240-700 mg
Ca:P Ratio ~ 1.4 to 2.4
'CSM is Condensed Skim Milk
2WPC is Whey Protein Concentrate
The pediatric nutritional formula of this invention is generally
prepared using the following method. An appropriate quantity of protein is
dispersed~in sufficient water or oil to solubilize or suspend it, thereby
forming a protein solution/suspension. Typically this protein source would
be intact milk proteins and/or hydrolyzed milk proteins. A carbohydrate
source such as one or more of corn syrup solids. lactose maltodextrins and
sucrose is dissolved in water, thereby forming a carbohydrate solution. A
source of dietary fiber, such as soy polysaccharide, may also be added.
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Appropriate minerals are dissolved in water, the carbohydrate solution or
oil, so as to form a mineral solution.
Once formed, the three solutions (protein, carbohydrate, and mineral)
are combined in appropriate quantities with oils and oil soluble vitamins.
This resulting solution is then heat processed and homogenized. Following
processing. water soluble vitamins, iron, choline and other nutrients are
added and then the nucl eoti des are added . The sol uti on i s then di 1 uted
wi th
water to the appropriate caloric density, approximately 670-725 kcal per
liter of formula. The formula is then dispensed into containers and
retorted to obtain commercial sterility or packaged aseptically using
commercially available techniques and equipment. As prepared, the formula
contains appropriate nutrients in compliance with the Infant Formula Act as
of the date of thi s appl i cati on . It shoul d al so be , recogni zed that
the
unique formula of this invention could be prepared for use in powdered form
or as a concentrated liquid.
The invention will be better understood in view of the following
examples, which are illustrative only and should not be construed as
limiting the invention.
DETAILED DESCRIPTION OF THE INDENTION
Analytical Techniques
One feature of the instant invention resides in the novel analytical
technique used to identify and quantify the nucleotide equivalents useful
in this invention. Analysis of certain starting materials. especially the
protein, will determine the actual amount of nucleotides to be added. This
analysis of the raw materials of the formula is critical to determine what
nucleotides, if any, are contained in the starting materials. The
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analytical method is also critical to determine the proper ratios of the
. nucleotides to each other. The analytical method according to this
. invention will determine nucleotide equivalent levels in complex food
matrices. The method in general exploits the enzymatic digestion of various
forms of ribonucleic acids to the simple monomeric ribonucleosides and the
ability of the cis-diol groups of ribonucleosides to form a pH dependent
covalent complex with boronic acid. Boronate derivatized polyacrylamide gel
is used to very selectively prefractionate ribonucleoside directly from
complex matrices. The isolated ribonucleosides are subsequently separated
via low pH reverse phase/ion-pairing HPLC using octanesulfonate as the ion-
pairing agent. Ribonucleosides are detected via UU absorbance, and the
corresponding levels are determined by comparison to external standards.
The method can be used to quantitate inherent levels of ribonucleoside in
foods. Because of the selective prefractionation, the method is essentially
matrix independent. It should be understood that the novel analytical
techni que of thi s i nventi on wi 11 not detect nucl eosi des from DNA or any
form
of nucleic acid that does not contain the cis-diol groups of ribose. It has
been used to determine ribonucleic acid types and levels in infant and
medical nutritional products, human milk, protein commodities, and clinical
and commercial animal chows.
The following is an example of the analytical technique of this
i nventi on that can be used to determi ne the presence and rati os of the
nucleotide equivalents.
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- EXAMPLE I
Analysis of Similac~ with Iron
To a 10 ml Reacti-Therm vial with stir bar was placed 2.0 ml of
Similac~ with Iron (a non-fat milk protein infant formula produced by the
Ross Products Division of Abbott Laboratories, ready to feed form, 676 Kcal
per liter) 3.0 ml of 50 mM sodium acetate at pH 5.1. 50u1 of 10 mM zinc
sulfate and 50 N1 of the enzyme preparation nuclease P1 (Sigma Chemical).
The enzyme preparation was 5 mg of dry enzyme powder, as received from
Sigma,and 4 ml of 50~M sodium acetate at pH 5.1. The mixture was heated to
37°C and stirred for 16 hours. This reaction converted the polymeric
RNA
to monomeric 5' mono-nucleotides.
To the same reaction vial was added 50 N1 of 30% ammonium hydroxide.
1 ml of 0.5 M ammonium acetate (pH 8.75), 50 ul of 1.0 M magnesium chloride.
50 ul of bacterial alkaline phosphatase (BAP) (Sigma Chemical as a
suspension) and 50 ul of a nucleotide pyrophosphatase enzyme preparation
(Sigma Chemical). The pyrophosphatase enzyme preparation was 5 mg of dry
powder in 4 ml of 0.5M ammonium acetate buffer. The mixture was incubated
at 37°C for three hours. This reaction converted the nucleoside
containing
adducts and the nucleotides to the ribonucleosides.
The reacti on mi xture was transferred to a 50 ml vol umetri c fl ask usi ng
25 ml of 0 . 5 M sodi um phosphate . pH 10 . 5. Water was added to a fi nal
vol ume
of 50 ml. The sample mixture was shaken and may be filtered to remove
insoluble protein.
grams of dri ed Affi -Gel -601, boronate deri vati zed ( from Bi o-Rad ) was
hydrated in 50 ml of 100mM phosphate buffer at pH 6.5. To a 10 ml open
column was added the hydrated Affi-Gel-601 to obtain a packed volume of
about 1 ml. The gel was converted to the basic form by washing with 5 ml
* T.M.
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al i quots of 0 . 25 M sodi um phosphate buffer , pH 10 . 5 . unti 1 the gel
no 1 onger
swelled. The gel was now about 2 ml in volume. The gel was resuspended in
the buffer to maintain adequate flow.
To the prepared gel was added 10 ml of the sampl a that was previ ously
treated with the enzymes and the eluant was discarded. At this point. the
nucleosides are covalently attached, through the cis-diol groups, to the
boronic acid gel. The gel was washed with 20 ml of 0.25 M sodium phosphate.
pH 10. 5 and the el uant was di scarded. The nucl eosi des were el uted and
collected in a 10 ml volumetric flask by adding 2 ml of 1.0 M phosphoric
aci d to the col umn fol 1 owed by 5 ml of 0.1 M phosphori c aci d . At thi s
poi nt
the nucleosides have been isolated from the sample and are now ready to be
characterized.
The vol umetri c fl ask was brought to a fi nal vol ume of 10 ml wi th
water. The sample was then placed on a HPLC for separation and
quantification of nucleosides using external standards. The nucleosides
were separated via low pH, reverse phase, ion pairing chromatography using
an acetonitrile gradient. The nucleosides were detected by U.V. absorbance
at 260 nm and~280 nm. Nucleosides were quantified by reference to external
standards and the results were converted to the corresponding monophosphate
nucl eoti de val ue by multi plyi ng the nucl eosi de val ue by the mol ecul
ar wei ght
rati o of the monophosphate nucl eoti de over the nucl eosi de . The resul is
were
expressed as mg/L, of mononucleotide.
NUCLEOTIDES IN SIMILAC~ WITH IRON
uridine - 3-5 guanosine - trace adenosine - trace
inosine -(trace. <0.5ppm) cytidine - 1-3
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It shoul d be noted that some sampl es have been found to be acti ve wi th
respect to nucleic acid degradation. Of particular concern is the enzymatic
conversion of AMP to IMP. Heat inactivation has proven to be effective in
rendering the sample inactive. The procedure for heat inactivation is to
heat the sampl a to over 100°C for at 1 east 15 mi nutes . After the
sampl a has
cool ed , buffer , enzyme , and zi nc are added and the fi rst hydrol ysi s i
s
carried out. '
This analytical technique was used on raw materials to determine base
line nucleotide content and on final clinical product to confirm the
presence and concentration of the four nucleotides used in the invention.
EXAMPLE II
Preparation of Enteral Formula
On a commerci al scal a , a control and an experi menta 1 formul a accordi ng
to the invention were prepared having the compositions set forth in Table
II. The two formula are as close as possible to being identical except for
the nucleotide components.
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TABLE II
COMPOSITION OF STUDY FEEDINGS
N
Nutrient (Control) (Formula of the Invention)
_____________________ per ~ er _________________
Protein, g 14.0 14.4
Fat,g 36.5 ~ 38.3
Carbohydrate, g 77.1 75.5
Calcium, mg 544.4 532.5
Phosphorus, mg 295.0 316.2
Magnesium, mg 73.5 77.7
Sodium, mg 170.1 179.2
Potassium. mg 931 948.6
Chloride, mg 487.7 493.2
Iron, mg 14.0 14.0
Zinc. mg 5.1 5.1
Copper, mcg 608 608
Iodine, mcg 61 61
Manganese, mcg 34 34
Vitamin A, IU~ 2930 2970
Vitamin D, IU 405 405
Vitamin E, IU 24.6 24.8
Vitamin K, mcg 54 54
Vitami n C , mg 170 172
~-Carotene mcg 450 450
Selenium, mcg 23 23
Thiamin, mcg 1350 1360
Riboflavin, mcg 1014 1014
Pyridoxine, mcg 480 480
Vitamin B12, mcg 1.7 1.7
Niacin, mcg 7095 7095
Folic acid.~mcg 101 101
Pantothenic acid, 3041 3041
mcg
Biotin, mcg 30 30
Taurine, mg 45 45
Choline, mg 108 108
Inositol, mg 32 32
Energy, Kcal 676 676
CMP, mg 2.72* 31.2
UMP, mg 4.19* 17.7
AMP. mg 0.57* 9.8
GMP, mg . . 0.45* 14.4
* - inherent levels from raw materials
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In this example, a 7711 Kg batch of the formula according to the
invention was prepared (NUC). The control formula (CON) was prepared in a
similar fashion except the addition of the nucleotides was omitted. The
list of ingredients and amounts are found in Table III.
TABLE III
In4redients and Amounts for NUC Formula
In4redient Amount
High Oleic Safflower Oil 120.2Kg
Coconut Oil 85.7 Kg
Soy Oil 80.3 Kg
Lecithin 2.92 Kg
Mono-and diglyceride 2.92 Kg
Oil Soluble Uit. Premix 0.365 Kg
Carotene 0.0137
Kg
Carrageenan 1.43 Kg
Whey Protein Concentrate 61.2 Kg
Lactose 476.3 Kg
Potassium Citrate 4~.6 Kg
Magnesium Chloride 0.735 Kg
Low Heat Condensed
Skim Milk 821 Kg
Calcium Carbonate 3.36 Kg
Ferrous sulfate 0.450 Kg
Water Soluble Vitamin
Premix Trace Minerals/
Taurine 1.11 Kg
Choline Chloride 0.600 Kg
Adenosine 5'monophosphate 0.113 Kg
Guanosine 5'monophosphate-Na20.197 Kg
Cytidine 5' monophosphate 0.259 Kg
Uridine 5'monophosphate-Na2 0.216 Kg
Ascorbic Acid 1.78 Kg
45% KOH 2.36 Kg
Total Yield 7711Kg
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The, first step is the preparation of the oil blend. To an
appropriately sized blend tank with agitation and heating soy oil, coconut
' oil and high oleic safflower oil were added. The mixture was heated~to
73.8-79.4°C. The lecithin and mono-and diglycerides (Myvero>* 18-06)
were
added to the blend tank with agitation. The oil soluble vitamin premix was
added with agitation. The premix container was rinsed with the oil blend
and transferred back ~to the blend tank to ensure complete delivery of the
vitamin premix. The beta-carotene was added to the oil blend and the
mixture agitated until the components were well dispersed. The beta-
carotene container was rinsed with the oil blend and the contents returned
to the bl end tank to ensure compl ete del i very of the beta-carotene sol uti
on .
Lastly, the carrageenan was added to the oft blend and the mixture was
agitated and held at 54.4-60°C until used.
The carbohydrate, mineral and CSM (condensed skim milk) protein slurry
was prepared next. To water heated to 68.3-73.8°C the lactose was added
and
the mixture agitated until the lactose was well dissolved. Potassium
citrate was then added followed by potassium chloride, sodium chloride and
magnesium chloride. The condensed skim milk (CSM) was then added. Tri-
calcium phosphate was added, the mixture agitated and held at 54.5-60°C
until used.
The protein-in-water (PIW) slurry was then prepared. The whey protein
concentrate was added to water at 54.5-60°C under mild agitation. The
PIW
slurry was held under mild agitation until needed. Also contemplated in
this invention is the use of protein-in-fat (PIF> slurries, wherein an
appropriate amount of protein is admixed with all or a portion of the oil
component.
(Myverol is a Trade-mark)
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The PIW slurry was then added to the prepared oil blend. The required
amount of the carbohydrate, mineral and CSM slurry was then added to the oil
- bl end . The pH of the mi xture was then determi ned and i f bel ow speci fi
cati on
it was adjusted using KOH to a pH of 6.75 to 6.85. The mixture was then
held at 54.4-60°C under agitation for at least 15 minutes.
The mixture was then heated to 68.3-73.8°C and deaerated under
vacuum.
The mixture was then emulsified through a single stage homogenizes at 6.21
to 7.58 MPa.
After emulsification, the mixture was heated to 120-122°C for 10
seconds and then 149-150°C for 5 seconds. The mixture was then passed
through a fl ash cool er to reduce the temperature to 120-122°C and
then
through a plate cooler to reduce the temperature to 71.1-79.4°C. The
mixture was then passed through a two stage homogenizes at 26.89 to 28.27
MPa and 2.76 to 4.14 MPa. The mixture was held at 73.9 to 83.2°C
for 16
seconds and then cooled to 1.1 to 6.7°C. At this point. samples are
taken
for microbiological and analytical testing. The mixture was held under
agitation.
A calcium carbonate solution may be prepared for use in adjusting the
calcium level of the mixture if outside of specification.
A vi tami n stock sol uti on was prepared . To water heated to 37 . 8 to
65.6°C was added potassium citrate and ferrous sulfate.. The vitamin
premix
was then added and the mixture agitated. The choline chloride was added and
then the required amount of this vitamin mixture was added to the batch.
The nucleotide solution was then prepared. The following nucleotides
were added to water with mild agitation in the following order: AMP, GMP.
CMP. UMP. Agitation was continued for about 10 minutes to dissolve the
nucleotides. The nucleotide solution was then added to the batch. This is
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one critical aspect of the invention. It is extremely important that the
nucleotides be added after the homogenizations and heat treatments.
Numerous experiments have been conducted that have shown the~addition of the
nucl eoti des at any other poi nt wi 11 resul t i n degradati on of the nucl
eoti des
and thereby change the specific levels and ratios as claimed. It is
believed that AMP is converted to IMP through the presence of adenosine
deaminase in the raw materials, especially the protein components.
Lastly , an ascorbi c aci d sol uti on was prepared and added sl owly to the
batch with agitation for at least 10 minutes. Final dilution with water to
meet specified levels of solids and caloric density was completed. The
batch was then packaged in 32 ounce metal cans and sterilized using
conventional technology.
EXAMPLE III
Clinical Studv of Enteral Formula
The purpose of the clinical investigation was to determine the effect
of a nucleotide-fortified formula according to the present invention on the
development of the neonatal immune system in infants as measured by the
antibody response to childhood vaccines.
This was a 12-month, randomized, controlled, blinded, mufti-site trial
of term infants. Infants enrolled into the study received human milk (HM)
or one of two clinically labelled formulas: 1) control formula (CON) or 2)
CON formula supplemented with nucleotides (NUC). The analyzed composition
~f each formula is set forth in Table II. A total of 311 infants completed
the study (107 CON. 101 NUC. 103 HM). Infants followed the immunization
schedule recommended by the American Academy of Pediatrics with single lots
of Hi b TITER~ Hemophi 1 us i nfl uenzae type b con j ugate vacci ne ( Di
phtheri a CRM
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197 and tetanus protein conjugate sold by Lederle, Inc.) and Diphtheria and
Tetanus Toxoids and Pertussis Vaccine Adsorbed. sold by Lederle. Inc.
Infants were full-term with a gestational age of 38-42 weeks, at or above
the 5th percentile for weight, length, and head circumference and were
enrolled between 2 and 10 days of age. All subjects were healthy with no
i ndi cati on of ~systemi c di sease and di d not recei ve any medi cati ons ,
mi nera l .
or vitamin supplements.
The primary outcome variable investigated was vaccine response at 6.
7, and 12 months of age. Also investigated were differential white blood
cell count. lymphocyte subset analysis. NK activity, and lymphoblast
transformation in response to specific and non-specific stimuli at 2. 6. 7,
and 12 months of age. Secondary outcome variables included intake,
anthropometry, and indices of tolerance (stool characteristics and incidence
of spit-up).
A1 so i nvesti gated was the anti oxi dant status of i nfants fed the formul a
according to the present invention which contained the novel antioxidant
system of: 10-30 IU of R. R, R, cx-tocopherol per liter of formula, 375-575
ug of ~-carotene per liter of formula and 14-32 mcg of selenium per liter
of formula.
During infancy, as in adulthood, the body has a number of antioxidant
systems to protect against injury from free radicals, the products of
oxidation. The antioxidant system of this invention was clinically proven
to promote the antioxidant status of the infant greater than currently
available infant formula. This improved antioxidant status was demonstrated
as a function of increased levels of plasma Vitamin E, reduced levels of
plasma lipid peroxides, and increased free radical trapping capacity.
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Experimental Desi4n
At 2.4, and 6 months of age DPT and Hib vaccines were administered.
Blood samples were obtained by venipuncture at 2, 6. 7, and 12 months~of
age. When vaccines were administered the blood sample was obtained before
the inoculation. Parents of the infants agreed to feed the infant only
study formula until 4 to 6 months of age when table foods were added to
supplement the study formula. The HM fed group were exclusively breast fed
up to 2 months of age and a mixture of HM and Similac~ with Iron (Ross
Products Division of Abbott Laboratories) after 2 months, if necessary.
Weight.length and head circumference were measured at 21 days of age
and at 2, 4. 6. 7. and 12 months of age. Three-day records of formula
intake: frequency of spit-up and vomiting and the frequency, color and
consistency of stools were used to assess tolerance. Blood samples (2 mL)
were drawn at 2 months of age and transferred directly into a heparin-
containing tube, and gently inverted. At 4. 6. 7, and 12 months of age 5
mL of blood was collected. Two and a half mL were transferred to
heparinized tubes and 2.5 mL to a plain tube without an anti-coagulant.
Tubes of blood were carefully packed in thermally insulated containers and
shipped to the laboratory for analysis.
Radial immunodiffusion assays were performed using standard kits
purchased from The Binding Site. Inc (5889 Oberlin Drive. Suite 101. San
Di ego , Cal i forni a 92121 ) for the measurement of serum or pl asma IgG and
IgA.
The detection of tetanus and diphtheria IgG was accomplished as
follows. Tetanus toxoid antigen (Connaught) was diluted in 0.05M carbonate
buffer (pH 9.6) to 2 ug/mL, added to the wells of microtiter plates at 200
uL per well, and incubated at room temperature for 1 hour. Diphtheria
toxoid antigen (Connaught) was diluted in the same manner to 15 ug/mL. The
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coated plates were washed three times in PBS containing 0.05% chicken egg
albumin and 0.1% Tween 20. Samples and positive control tetanus and
diphtheria toxoid immune globulin. were diluted in PBS/albumin/Tween, added
to triplicate wells at 200 uL/well, and incubated at room temperature for
1 hour. PBS alone was also added to triplicate wells to provide a blank.
Plates were again washed three times in PBS/albumin/Tween. Affinity
purified horseradish peroxidase-conjugated goat anti-human IgG (The Binding
Si to . Inc ) was di 1 uted i n PBS/al bumi n/Tween* added to the mi croti ter
pl ates .
and again incubated at room temperature for 1 hour. Tetramethylbenzidine
(TMB) substrate (Kirkegaard and Perry Laboratories) was added to all wells
at 100 NL/well, and incubated at room temperature for 10 minutes. The
substrate reaction was stopped by adding 100 uL of 1 M phosphoric acid per
well. Optical density of each well was measured using a wavelength of 450
nm. Sample units were calculated based on the tetanus and diphtheria toxoid
immune globulin standards. See Sedgurch and .Bolton; J CJin Microbiol.
1983:18:104-109.
Serum IgG directed against Haemophilus influenzae type b capsular
polysaccharide (Hib) antigen was detected using a modified version of the
procedure described by Anthony et al; J Clin MicrobioJ 1982;16:350-354. The
modifications are described in Granoff, et al: J Infect Dis 1986;154:257-
264.
Concentrati ons of total serum anti body to the Hi b anti gen were measured
by a radioactive antigen-binding assay (Hib Farr) using the procedure
described by Granoff et al; J Infect Dis 1986:154:257-264. The Hib antigen
was purified and labeled with iodine. A reference serum pool from the US
Bureau of Biologics (Rockville, Maryland) was used to standardize the assay.
*T.M.
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The smallest amount of immunoglobulin detectable was 0.025 Ng/mL serum, as
determined with this reference pool.
Natural killer cell (NK cell) activity was measured using Histopaque-
purified peripheral blood lymphocytes. The cytotoxicity of the NK cells was
measured using procedure described by Wierda et al., J Irrmunol. Methods
1989:122:15-24.
Statistical Methods
The immunological variables were analyzed in two different ways. For
the variables relating directly to vaccine response (Hib Farr, Hib IgG.
tetanus, diphtheria, total IgG and IgA) the variables were transformed by
taking logarithm base 10 and doing Analysis of Variences (ANOVAs). The
procedure is commonly used in the vaccine literature.
Anthropometric data were analyzed for each gender separately. Analysis
of Varience (ANOVA) was done at birth, initial. visit. 2, 4, 6. 7, and 12
months of age for wei ght . 1 ength and head ci rcumference . Wei ght gai n ,
1 ength
gain and head circumference gain were also analyzed by ANOVAs. Intake data
were ranked and analyzed by ANOVAs (number of feedings, volume intake,
percent of feedings with spit-up, vomits or both). Stool variables were
ranked and analyzed with ANOVAs (number of stools, mean rank consistency and
percent of stools with gas or unusual odor).
RESULTS
Substantial amounts of data were collected on each of the 311 infants
enrolled in this clinical investigation. Disclosure of all this information
is outside the scope of this document, however, the following is a sur~nary
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of the information that supports the novel and unobvious features of the
instant invention.
Vaccine antibody response data was statistically analyzed by two
methods. Table IV shows the medians of the variables in the original units.
The ANOVA was performed on medians of ranked data. Table V shows geometric
means. For this analysis, the variables were transformed by taking
logarithm base 10, and the ANOVA compared the mean of the logs. The mean
of the logs converted back to the original units is the geometric mean. Use
of~ geometric means is commonly used in the vaccine literature.
At 7 months of age, infants in the NUC group had a higher antibody (P
< 0 . 05) response than the CON or HM group to Hi b vacci ne (geometri c mean
of
7.24 vs 4.05 or 4.21 uG Ig/mL, respectively by the Hib Farr assay). The NUC
group had a higher response than the HM group to diphtheria toxoid vaccine
(geometric means of 1.77 vs 1.29 U diphtheria toxoid specific IgG/mL,
respectively). The enhanced antibody response to Hib vaccine persisted
through 12 months of age as seen in Table V.
There were no differences in NK activity at any time, and the
differential white count, lymphocyte subsets, and lymphoblast transformation
was very similar among all groups. The primary differences were at 12
months of age, when infants fed HM had more white blood cells, monocytes.
lymphocytes, CD3, and CD19 cells than CON (P < 0.05). The NUC group was
intermediate and not statistically different. Infants fed HM had greater
numbers of NK cells (CD3-. CD16+. CD56+) than formula-fed (CON or NUC)
infants (P < 0.05). The NUC group had a higher percent CD4 cells than HM-
fed infants (P < 0.05) throughout the study.
Growth of i nfants was si mi 1 ar i n al 1 three groups . Tol erance and i
ntake
was similar for the two formula groups.
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The similarity in growth and tolerance among all infants demonstrated
that both formulas are acceptable. Likewise. the similarity in measures of
' immune system components among infants fed formulas or HM demonstrates that
all feedings promote development of the immune system within normal ranges.
however, for the first time an immune enhancement as measured by vaccine
response to H. influenzae b and diphtheria toxoid is reported for infants
consuming infant formula (NUC).
The consistently enhanced vaccine response of infants fed NUC vs CON
suggests that nucleotides play an important function in irr~nunological
development of the infant.
DETAILED DISCUSSION OF RESULTS
Irr~nunol ogi ca 1 Parameters
Vaccine response data are provided in Table IV as reported from the
assays and Table V as geometric means. The antibody response to the Hib
vaccine was measured as Hib Farr (,ug Ig/mL). NUC-fed infants had
significantly higher levels of Hib Farr antibody than infants fed HM at 6
months (0.43 vs 0.30. P < 0.05) higher than infants fed CON or HM at 7
months (7.7 vs 3.62 and 5.40, respectively, P<0.05) and at 12 months (1.35
vs 0.68 and 0.82, respectively. P < 0.05). Hib response was also measured
as Hib specific IgG, and the results paralleled the Hib Farr values at 6 and
7 months. This parameter was not measured at 12 months.
Response to the diphtheria vaccine was measured as diphtheria toxoid
specific IgG. There were no differences between groups at 6 or 12 months.
but at 7 months infants fed NUC had a significantly (P <0.05) higher
response (1.77 U/mL) than infants fed HM (1.29 U/mL). See Table V). There
were no differences at any time point for tetanus specific IgG.
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TABLE IV
VACCINE SE
RESPON
Median(n)
6 months
NUC CON HM
Hib Farr (ug Ig/mL)1 0.43 (93)x 0.36(96)x' 0.30 (97)
Hlb I9G (mg/mL) 0.06 (94)x 0.06(101)x' 0.03 (99)
Diphtheria IgG (U/mL) 0.47 (78) 0.32(85) 0.36 (80)
Tetanus (IgG (U/mL) 0.71 (80) 0.72(82) 0.53 (80)
7 months
NUC CON HM
Hib Farr (ug Ig/mL) 7.70 (94)x 3.62(101) 5.40 (99)
Hib IgG (mg/mL)2 1.25 (93) 0.63(101) 0.60 (97)
Diphtheria IgG (U/mL) 1.70 (85)x 1.53(89)x' 1.42 (90)
Tetanus IgG (U/mL) 5.01 (86) 4.47(90) 4.75 (91)
12 months
NUC CON HM
Hib Farr (Ng Ig/mL) 1.35 (89)x 0.68(94) 0.82 (95)
Hib IgG (mg/mL) ND3 ND ND
Diphtheria IgG (U/mL) 0.30 (82) 0.24(87) 0.30 (84)
Tetanus IgG (U/mL) 0.92 (83) 0.84(87) 0.90 (85)
1 Values in the same horizontal row with different superscripts (a or b)
are significantly different, P < 0.05.
2 P < 0.05, no pairwise differences
3 ND = not determined
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TABLE V
VACCINE RESPONSE
Geometric Mean (n>'
6 months
NUC CON HM
Hib Farr (Ng Ig/mL) 1.30 (93)a 1.24 (96)a~1.23 (97)
Diphtheria IgG (U/mL) 0.36 (78) 0.28 (85) 0.33 (80)
7 months
NUC CAN HM
Hib Farr (ug Ig/mL) 7.24 (94)a 4.05 (101) 4.21 (99)
Diphtheria IgG (U/mL) 1.77 (85)8 1.38 (89)a~1.29 (90)
12 months
NUC ,~ HM
Hib Farr (ug Ig/mL) 1.41 (89)a 0.76 (94)° 0.85 (95)°
Diphtheria IgG (U/mL) 0.33 (82) 0.25 (87) 0.27 (84)
Values in the same horizontal row with different superscripts (a or b)
are significantly different: P < 0.05.
P < 0.05, no pairwise differences
It is generally accepted that a Hib FARR level of antibody greater
than 1 Ng of Ig/mL one month after immunization imparts protection to the
infant. The percent of infants who had this level of protection was
determined from the data set and is set forth in Table VI. The infants fed
the NUC formula consistently had a 10% greater protection rate than infants
in the other two groups.
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TABLE VI
HIB PROTECTION RATE
- (% of subjects with > 1 ug anti-Hib Ig (mL)
NUC CON HM
6 months 28% 18% 16%
7 months 90% 80% 80%
12 months 55% 44% 45%
Natural killer (NK) cell activity was similar in all three groups.
HM group had significantly higher numbers of NK cells (P < 0.05) than NUC
at 2, 6, and 12 months and CON at 2. 7, and 12 months. Formula-fed infants
had a higher percent CD4 cells at 2 months (NUC. CON > HM; P < 0.005). 7
months (CON. NUC > HM; P < 0.01), and 12 months (NUC > HM; P < 0.05). The
NK activity data are presented in Table VII.
TABLE VII
NK ACTIVITY1
NUC CON HM
2 months 11.2 8.0 9.0
6 months 9.0 12.6 9.0
7 months 13.9 14.3 13.0
12 months 19.4 21.3 21.4
' Values are % target cells killed at effector:target ratio of 50:1.
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Part of the impetus for this study and evidence that different ratios
and levels of nucleotides impact on different physiological parameters was
the report by Carver et al. (Pediatrics 1991:88:359) that infants fed
nucleotide-fortified SMAm (infant nutritional sold by Wyeth, Inc. believed
to contain 21 mg CMP: 6.0 mg AMP; 6.0 mg UMP: 6.0 mg AMP and 3.0 mg IMP per
liter of formula) had significantly higher NK activity than those fed
unfortified SMA. The present study, using the formula according to the
instant invention, shows no effect of nucleotides on NK activity at 2 months
and in fact no difference, among any of the groups at any time. Given the
small number of infants in the Carver study (42 degrees of freedom at 2
months) compared to this study (255 degrees of freedom at 2 months), it
woul d seem 1 i kely the Carver data are an aberrati on due to smal 1 sampl a
si ze
or , the addi ti on of nucl eoti des does not i ncrease number of NK cel 1 s
or , the
types and levels of nucleotides used by Carver produced only a cellular
response as opposed to the humoral response seen in this invention.
The anthropometric measurements indicate that growth was comparable
among all infants in the study. The fact that even before controlling for
birth values there were no differences among males for weight, length, or
head circumference gives assurance that growth was acceptable among all
groups.
The higher stool frequency and number of feedings per day of HM-fed
infants compared to formula-fed infants during the first 2 months is well
established. Softer stools of HM-fed infants are also common, although only
the NUC group was different at 2 months and by a small amount. Overall , the
measures of tolerance among all groups were very similar through 4 months
when half the infants were still being exclusively breastfed. These data
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demonstrate both formulas were extremely well tolerated and are set forth
in Table IX.
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TABLE IX
INTAKE AND TOLERANCE
Mean (SEM)1
2 months
NUC CON HM
100 ~ 107 103
Feedings (#/day) 6.2 (0.1) 6.4 (0.1) 7.7 (0.2)
Intake (mL/day) 831 (19) 823 (18) ND
Spit-up (% of feedings)8 (2) . 18 (2) 20 (2)
Stool Frequency /day)1.6 (0.1) 1.4 (0.1) 2.7 (0.2)
Stool Consistency' 2.0 (0.1) 1.9 (0.1) 1.7 (0.1)
4 months
CON HM
98 107 103
Feedings 5.9 (0.1) 6.0 (0.1) 6.6 (0.2)
(#/day)
Intake 987 (33) 926 (17) ND
(mL/day>
Spit-up (% of feedings)22 (2) 18 (2) 20 (2)
StoolFrequency ( /day)1.4 (0.1) 1.4 (0.1) 1.5 (0.1)
y
Stool 2.0 (0.1) 2.1 (0.1) 2.1 (0.1)
Consistenc
Values in the same row with different superscripts are significantly
different; P < 0.05.
Mean rank cons i stency , where 1 = watery , 2 = mushy , 3 = soft , 4 = formed
.
5 = hard.
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The differential white counts and lymphocyte subset numbers of all
infants receiving the formula according to this invention were well within
normal ranges throughout the first year of life.
The vacci ne response i n thi s study was i ntended to be an i mmunol ogi ca 1
probe or i ndi cator as to the responsi veness of the i mmune system i n
genera l .
On the humoral side, tetanus toxoid vaccine was selected because it is a
strong antigen, diphtheria toxoid was selected as a vaccine containing a
weaker antigen. and Hib vaccine was selected as a very weak antigen that
requires conjugation to a carrier protein to achieve a T-cell dependent
immune response to the Hib polysaccharide component of the vaccine to be
effective. It was thought that if nutritional intervention could evoke a
difference in response that could be measured. that difference would more
likely occur with the weaker antigens. While all infants would be expected
to respond well to a strong antigen, like tetanus toxoid, a less vigorous
response would be expected to a weak antigen. The Lederle Hib TITER~ was
selected specifically because the literature indicated that infants
responded rather weakly after the first and second immunizations.
Furthermore, the protein used as the conjugate in this vaccine, the CRM 197
protei n ( a non. toxi c mutant di phtheri a toxi n ) , i s anti geni ca 11 y
very s i mi 1 a r
to diphtheria toxoid. Diphtheria toxoid vaccination also represents a
response to a moderately weak antigen and correlates with immune response
to the H. influenzae conjugate vaccine with the CRM 197 protein carrier.
The vacci ne response at 6 months i s taken from bl ood drawn i rranedi ately
before the 6-month vaccination and represents the response 2 months after
the second imrnuni zati on gi ven at 4 months of age. A1 ready at that ti me
poi nt the Hi b response was si gni fi canal y hi gher i n NUC than HM for
both
anti -Hi b IgG and Hi b Farr anti body. At 7 months , one month after the thi
rd
CA 02299729 2000-03-08
~ WO 95/18618 PCTlUS95/00090
37
i mmuni zati on , NUC i s si gni fi cantly hi gher than CON and HM for Hi b
Farr. Hi b
IgG is higher at 7 months, and although there are not pairwise differences.
~ the NUC group is double CON and HM (1.25 vs 0.63 and 0.60, respectively).
The Hib Farr value was still significantly higher for NUCs at 12 months.
For this weak antigen, a difference was first seen at 6 months. The
difference became stronger at 7 months when the maximum response was
expected and was maintained through 12 months of age.
In response to the moderately weak antigen diphtheria vaccine, there
were no differences at 6 months, but at 7 months the NUC group was
significantly higher than HM. By 12 months this difference was no longer
present. For the moderately weak antigen, the direction of the present
difference was the same as with the weak antigen (Hib) but was different
only at the point of highest response.
For the strong antigen, tetanus, there were no differences among
feeding groups at any time point.
These data strongly support the instant invention of specific
nucleotide equivalents at specific levels and ratios to enhance the immune
system. In this example and in commercial production of enteral formulas
according to the invention, background levels of nucleotide equivalents are
determined and then the formula would be supplemented with appropriate
commodities, such as CMP, AMP. UMP and GMP, to the claimed levels and
ratios. It should be remembered that by nucleotide equivalents is meant
ribo-nucleotides, ribo-nucleosides. RNA, and ribo-nucleotide adjuncts, such
as activated sugars. The sum of all these elements determine the total
potentially available ribo-nucleotides equivalents.
Two additional pieces of data strongly support that the formula
according to this invention provides an unexpected result. The number of
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38
subjects who have achieved protective levels of anti-Hib immunoglobulin as
shown in Table VII is consistently 10% higher in the NUC group. The three-
way comparison does not show a statistical difference. However, a two-way
compari son between the NUC and CON formul a groups at 7 months i s si gni fi
cant
(P < 0.05).
An additional piece of data comes from two of the clinical sites which
chose to col 1 ect morbi di ty. data . As part of the study the i nci dence of
diarrhea was determined at the two clinical study sites. Of 26 infants fed
the NUC formula, only two reported diarrhea while 10 of 29 reported diarrhea
in the CON formula. The XZ analysis comparing the incidence of diarrhea in
infants fed the two formulas is significant (P < 0.05). In summary, the
improved response to vaccination, the higher percent of subjects who have
protective levels of antibodies, and the reduced incidence of diarrhea show
that infants consuming the nucleotide-fortified formula according to this
invention achieve enhanced immunological development as compared to those
consuming the control formula.
INDUSTRIAL APPLICABILITY
The results from these experiments demonstrate that the enteral
formula of this invention is effective in enhancing the irr~nune system and
treating diarrhea. The medical community is constantly searching for
nutritional formulas that will benefit the infant. The present invention
can clearly fill that need. The nucleotide equivalent level of the formula
in the study is about the minimum for efficacious effect. Additionally, the
formula is nutritionally complete as an infant formula. The manufacture of
the formula utilizes conventional equipment and may be readily accomplished.
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~ WO 95118618 PCT/US95/00090
39
While the infant formula and method of making said formula herein
described constitute a preferred embodiment of this invention, it is to be
understood that the invention is not limited to this precise formulation or
method and that changes may be made therein.
