U.S. patent application number 14/656486 was filed with the patent office on 2015-10-08 for method to increase the growth velocity of human infants.
The applicant listed for this patent is Swedish Orphan Biovitrum AB (Publ). Invention is credited to Olle HERNELL, Maria OHMAN, Birgitta OLSSON, Patrik STROMBERG, Lennart SVENSSON, Kristina TIMDAHL, Marten VAGERO.
Application Number | 20150283215 14/656486 |
Document ID | / |
Family ID | 44201316 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150283215 |
Kind Code |
A1 |
HERNELL; Olle ; et
al. |
October 8, 2015 |
METHOD TO INCREASE THE GROWTH VELOCITY OF HUMAN INFANTS
Abstract
The present invention relates to a method to increase the growth
velocity of a human infant, said method comprising the enteral
administration to said infant of recombinant human
bile-salt-stimulated lipase (rhBSSL). Such method has particular
utility for underweight or preterm human infants, particular those
in medical need of increasing their growth velocity. The invention
also relates to compositions, including infant feeds, kits,
packaged-pharmaceutical-products and pharmaceutical compositions,
and also to methods to prepare infant feeds. In another aspect, the
present invention relates to methods to: (X) protect the small
bowel mucosa of a human infant from damage; to (Y) protect an
immature intestinal epithelium of a human infant from the
deleterious effects of incompletely digested and/or excess fat
and/or lipid; and/or to (Z) limit accumulation of incompletely
digested and/or excess fat and/or lipid in the ileum of a human
infant; said methods in each case comprising the step of enteral
administration of rhBSSL.
Inventors: |
HERNELL; Olle; (Umea,
SE) ; OLSSON; Birgitta; (Stenhamra, SE) ;
STROMBERG; Patrik; (Sollentuna, SE) ; SVENSSON;
Lennart; (Solna, SE) ; TIMDAHL; Kristina;
(Tullinge, SE) ; VAGERO; Marten; (Saltsjo-Boo,
SE) ; OHMAN; Maria; (Pitea, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Swedish Orphan Biovitrum AB (Publ) |
Solna |
|
SE |
|
|
Family ID: |
44201316 |
Appl. No.: |
14/656486 |
Filed: |
March 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13278662 |
Oct 21, 2011 |
8986759 |
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14656486 |
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61405277 |
Oct 21, 2010 |
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Current U.S.
Class: |
424/94.6 |
Current CPC
Class: |
A23L 33/17 20160801;
C12Y 301/01003 20130101; A61K 38/465 20130101; A23V 2002/00
20130101; A23L 5/00 20160801; A61P 1/00 20180101; A61P 43/00
20180101; A23L 33/40 20160801; C12Y 301/01013 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A23L 1/29 20060101 A23L001/29 |
Claims
1. A method to increase the growth velocity of a human infant, said
method comprising the step of enteral administration of recombinant
human bile-salt-stimulated lipase to said infant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/278,662, filed Oct. 21, 2011, now U.S. Pat. No. 8,986,759,
issuing Mar. 24, 2015 which claims priority to and the benefit of
U.S. Provisional Application Ser. No. 61/405,277, filed on Oct. 21,
2010, entitled "Method to Increase the Growth Velocity of Human
Infants," the entire contents of which are incorporated herein by
reference in their entirety.
SEQUENCE LISTING
[0002] Incorporated by reference herein in its entirety is the
Sequence Listing co-submitted with the instant application,
entitled "Sequence_Listing.sub.-- ST25.txt", created Oct. 17, 2011,
size of 10 kilobytes.
TECHNICAL FIELD
[0003] The present invention relates to a method to increase the
growth velocity of a human infant, said method comprising the
enteral administration to said infant of recombinant human
bile-salt-stimulated lipase (rhBSSL). Such method has particular
utility for underweight or preterm human infants, particular those
in medical need of increasing their growth velocity. In other
aspects, the invention relates to compositions, including infant
feeds, kits, packaged-pharmaceutical-products and pharmaceutical
compositions, and also to methods to prepare infant feeds, in each
case useful for increasing the growth velocity of a human infant.
In another aspect, the present invention relates to methods to: (X)
protect the small bowel mucosa of a human infant from damage; to
(Y) protect an immature intestinal epithelium of a human infant
from the deleterious effects of incompletely digested and/or excess
fat and/or lipid; and/or to (Z) limit accumulation of incompletely
digested and/or excess fat and/or lipid in the ileum of a human
infant; said methods in each case comprising the step of enteral
administration of recombinant human bile salt stimulated lipase to
said infant. The present invention also relates compositions,
including infant feeds, kits, packaged-pharmaceutical-products and
pharmaceutical compositions, and also to methods to prepare infant
feeds, in each case useful for these protective or
accumulation-limiting methods.
BACKGROUND
[0004] Babies born weighing less than 2,500 g are considered low
birth weight (LBW), and are at increased risk for serious health
problems as neonates, lasting disabilities and even death. Certain
LBW babies can be further classified into Very Low Birth Weight
(VLBW) babies, born at less than 1,500 g, and Extremely Low Birth
Weight (ELBW) babies, born at less than 1000 g. The rate of LBW
neonates shows differences around the world. For example, the World
Health Organization (WHO) estimated that 16.5% of births in less
developed regions in the year 2000 were LBW. In contrast, around 1
of every 12 (8.3%) babies born in 2005 in the United States was
born LBW (Martin et al, 2007; National Vital Statistics Reports, 56
(6)) and in England and Wales the overall rate of LBW babies has
been reported as 7.3% (Doyle, 2000; BMJ, 320: 941-942). The rate of
LBW babies is increasing, particularly in more developed regions
such as the United States, believed to result predominantly from an
increase in preterm delivery of artificially conceived multiple
pregnancies.
[0005] Many LBW babies require specialized care in Newborn
Intensive Care Units (NICUs) as they are especially susceptible to
health problems (for example, as reported in McIntire et al, 1999;
N Engl J Med, 340: 1234-1238) including respiratory distress
syndrome (RDS), cyanotic attacks, bleeding in the brain
(intraventricular hemorrhage, IVH), cerebral palsy, heart problems
such as patent ductus arteriosus (PDA), hypocalcemia,
hypoglycaemia, intestinal problems such as necrotizing
enterocolitis (NEC), jaundice and retinal-development problems such
as retinopathy of prematurity (ROP). A number of studies have found
that the cost of care for neonatal care rises steeply with
decreasing birth weight, with a US study estimating neonatal costs
to be $224,400 for a newborn with a birth-weight of 500-700 g,
compared to only $1000 for a baby born with a birth-weight over
3,000 g, and one estimate of over $50 billion in annual costs for
such care provided in the United States. Beyond such acute care,
being born underweight has been reported to be associated with a
number of mid-term health problems, including: (a) poor weight gain
and head growth in infancy (Gutbrod et al, 2000; Arch Dis Child
Fetal Neonatal Ed, 82: 208-214); (b) developmental delay and later
language problems in early childhood (Marlow et al, 2005; N Engl J
Med, 352: 9-19); (c) neurological abnormalities; and (d) increased
incidence of deafness. Some studies also suggest that individuals
born LBW may be at increased risk for certain chronic conditions in
adulthood, including high blood pressure, type-2 diabetes and heart
disease.
[0006] There are two main reasons why a baby may be born with low
birth weight: (1) premature birth--a normal pregnancy lasts for
about 40 weeks (38-42 weeks), and the WHO defines prematurity as a
baby born before 37 full-weeks from the first day of the last
menstrual period. The earlier a baby is born, the less it is likely
to weigh; and (2) fetal growth restriction--babies that may be
full-term but are underweight, also known as small-for-gestational
age (SGA) or small-for-date babies. Some of these babies are small
simply because their parents are small (and these babies are often
healthy), while others have low birth weight because something has
slowed or halted their growth in the uterus (or intra-uterine
growth retardation, IUGR). Some babies are both premature and have
suffered IUGR, and these babies are particularly at high risk for
health problems such as those described above.
[0007] Recently, the WHO has systematically reviewed the worldwide
incidence of preterm birth (Beck et al, 2010; Bull World Health
Organ, 88: 31-38), and they estimate that in 2005 12.9 million
births, or 9.6% of all births worldwide, were preterm.
Approximately 11 million (85%) of these preterm births were
concentrated to Africa and Asia, while about 0.5 million occurred
in each of Europe and North America (excluding Mexico) and 0.9
million in Latin America and the Caribbean. The highest rates of
preterm birth were in Africa and North America (11.9% and 10.6% of
all births, respectively), and the lowest were in Europe (6.2%).
The relatively high rate of preterm births estimated for North
America equates to an estimated absolute number of 480,000 preterm
births in 2005, and despite the relatively lower rate, still
equates to an estimated 466,000 preterm births in Europe during the
same year. Preterm birth rates available from some developed
countries, such as the United Kingdom, the United States and the
Scandinavian countries, show a dramatic rise over the past 20 years
(e.g., Callaghan et al, 2006; Pediatrics, 118: 1566-1573). Factors
possibly contributing to but not completely explaining this upward
trend include increasing rates of multiple births, greater use of
assisted re-production techniques, increases in the proportion of
births among women over 34 years of age and changes in clinical
practices, such as more frequent use of elective Caesarean
section.
[0008] Preterm babies are generally susceptible to the same health
problems as LBW babies, with the severity of the problems
increasing with degree of prematurity: a baby born at 36 weeks will
probably be a little slow to feed; a baby born before 33 weeks will
have more serious problems including, possibly, immature lungs;
while a birth before 28 weeks causes very significant problems but
the survival rate is quite remarkable. Data suggest 90% survival if
born over 800 g, 50% survival if over 500 g and 80% survival if
born before 28 weeks; although these figures may also hide
significant disability in survivors. For example, severe problems
such as cerebral palsy, blindness and deafness may affect as many
as 10 to 15% of significantly premature babies, about 1 in 4 babies
with birth weight below 1.5 kg has peripheral or central hearing
impairment or both (Jiang et al, 2001; Acta Paediatr, 90 1411-1415)
and 66% of babies under 1.25 kg develop ROP (Allin et al, 2006;
Pediatrics, 117: 309-316).
[0009] Pancreas and liver functions are not fully developed at
birth, and in premature infants this is particularly notable.
Lindquist and Hernell (1990; Curr Opin Clin Nutr Metab Care, 13:
314-320) have recently reviewed the subject of lipid digestion and
absorption in early life. Breast-fed infants digest and absorb fat
(and importantly long-chain polyunsaturated fatty acids, LCPUFAs)
more efficiently than formula-fed infants (Bernback et al, 1990; J
Clin Invest, 85:1221-1226; Carnielli et al, 1998; Am J Clin Nutr,
67: 97-103). In addition to infant formulas of similar fat
composition, mother's milk also contains a broad-specificity
lipase, bile-salt stimulate lipase (BSSL) (EC 3.1.1.13) that
promotes highly efficient fat absorption from human milk.
[0010] BSSL is a naturally occurring pancreatic enzyme which is
activated by bile salts in the duodenum and participates in the
hydrolysis of lipids together with other lipases. In early infancy,
and especially in the preterm infant, pancreatic exocrine functions
are not fully developed (Manson & Weaver, 1997; Arch Dis Child
Fetal Neonatal Ed, 76: 206-211). Hence, in the preterm pancreas,
expression of pancreatic lipases is low compared to adult pancreas
(Lombardo, 2001; Biochim Biophys Acta, 1533: 1-28; Li et al 1007;
Pediatr Res, 62: 537-541). Therefore, the BSSL present in breast
milk is an important lipase for these infants; the low level of
pancreatic lipase is compensated for by expression of BSSL in the
lactating mammary gland and secretion of the enzyme with the
mother's milk. The human lactating mammary gland synthesizes and
secretes BSSL that, after specific activation by primary bile salts
in the lower intestine of the baby, contributes to the breast-fed
infant's endogenous capacity for intestinal fat digestion.
[0011] BSSL is believed to have a broader substrate specificity
than most lipases. Not only is the enzyme capable of completely
hydrolyzing all three fatty acids of triglycerides
(triacylglycerols, TGs), but also fat soluble vitamin esters such
as vitamin A as well as cholesteryl esters. Thus, BSSL drives the
intraluminal lipolysis toward completion and results in the
formation of glycerol and free fatty acids (FFAs), including
long-chain polyunsaturated fatty acids (LCPUFAs), the latter being
indispensable building blocks for the developing central nervous
system (Hernell, 1975; Eur J Clin Invest, 5: 267-272; Bernback et
al, 1990; Hernell et al, 1993; J Pediat Gastro Nutr, 16: 426-431;
Chen et al, 1994; Biochem Biophys Acta, 1210: 239-243). BSSL shows
optimal activity at a pH of 8-8.5 and is more stable in acid
environments than pancreatic lipase. BSSL is resistant to
degradation by pepsin at physiological concentrations. BSSL
accounts for about 1% of the total protein in milk and is present
at concentrations from 0.1-0.2 g/L (Blackberg et al, 1987; FEBS
Lett, 217: 37-41; Wang & Johnson, 1983; Anal Biochem, 133:
457-461; Stromqvist et al, 1997; Arch Biochem Biophys, 347: 30-36).
The levels of BSSL in human milk are similar throughout the day
(Freed et al, 1986; J Pediatr Gastroenterol Nutr, 5: 938-942) and
BSSL production in human milk is maintained for at least 3 months
(Hernell et al, 1977; Am J Clin Nutr, 30: 508-511) although
concentrations of BSSL may decline with duration of lactation
(Torres et al, 2001; J Natl Med Assoc, 93: 201-207). Triglycerides
comprise about 98% or more of all lipids in human milk or formula
and thereby account for about 50% of the energy content.
[0012] The superiority of human milk as a nutritional source for
term as well as preterm infants has been manifested in many studies
and expert group recommendations. Accordingly, the recommended
feeding method world-wide is breastfeeding. Neither is however,
breastfeeding nor feeding the mother's own breast milk always
possible or recommended for medical reasons--and breastfeeding may
not be practiced for a number of other reasons--in each case as
discussed elsewhere herein.
[0013] Despite dropping from about 60% to about 50% in the 1980s
(Foss & Southwell, 2006; Int Breastfeeding J 1: 10), the
percentage of US women initiating breastfeeding increased from the
1990s and was reported to be about 74% in 2004 (Scanlon et al,
2007, in CDC Morbidity and Mortality Weekly Report, 2rd Aug. 2007).
However, the percentage of women continuing breast feeding appears
to drop substantially after initiation in the early postpartum
period, with this study reporting that in 2004 only 42% and 21% of
women were still breastfeeding after 6 and 12 months, respectively.
Data from Sheffield in the UK showed the same trends, with a drop
from about 70% to 50% during the 1980s but that only 35% to 30%
(during the same period) of such women were actually doing so at
one month after birth (Emery et al, 1990, Arch Dis Childhd, 65:
369-372). The percentage of women who exclusively breastfeed is
even lower, and overall for infants born in the US in 2004 only
about 31% and 11% of women were exclusively breastfeeding through
ages 3 and 6 months, respectively, and with significant disparities
between subgroups of these women; rates of exclusive breast feeding
through age 3 months were lowest among black infants (20%) and
among infants of mothers who were aged <20 years (17%), had a
high school education or less (23% and 24%, respectively), were
unmarried (19%) resided in rural areas (24%) and had an
income-to-poverty ratio of <100% (24%) (Scanlon et al, 2007).
Indeed, significant national and cultural differences in
breastfeeding exist. Emery & coworkers (1990) reported a
significantly lower percentage of Asian women than white women
intending to breastfeed in Sheffield UK. Furthermore, Singh (2010;
Eur J Sci Res, 40: 404-422) has reported that in Brazil, the mean
duration of exclusive breastfeeding is only 28.9 days, in Malaysia
only 25% or infants are exclusively breast fed at 2 months and in
Bogota and Nairobi this percentage is 12% and 21% or infants,
respectively.
[0014] In cases where the infant is not breast-fed, infant formula
or banked and non-banked pasteurized and/or frozen breast milk is
often used. All are, however, in some respects nutritionally
suboptimal for newborn infants.
[0015] Due to risks of viral infection (human immunodeficiency
virus [HIV], cytomegalovirus [CMV], hepatitis) and to a lesser
degree transmission of pathogenic bacteria, donor milk used in
so-called milk banks is generally pasteurized before it is used.
However, BSSL is inactivated during pasteurization of human milk
(Bjorksten et al, 1980; Br Med J, 201: 267-272); nor is it present
in any of the many different formulas that exist for the nutrition
of pre- or full-term neonates. It has been shown that fat
absorption, weight gain and linear growth is higher in infants fed
fresh compared to pasteurized breast milk (Andersson et al. 2007;
Acta Paediatr, 96: 1445-1449; Williams et al, 1978; Arch Dis Child
43: 555-563). This is one reason why it has been advocated that
newborn infants, particularly preterm infants, that cannot be fed
their own mothers milk should be fed non-pasteurized milk from
other mothers (Bjorksten et al, 1980).
[0016] Hamosh (1983; J Ped Gastro Nutr, 2: 248-251) reported that
BSSL enzyme activity is present in fresh breast milk of women who
delivered at 26 to 30 weeks. This report further described that
milk specimens stored at -20 or -10.degree. C. showed a slow loss
in BSSL activity, but a more dramatic loss of bile-salt dependency
on activity after only three weeks storage at -10.degree. C. which
may contribute to hydrolysis of milk lipids even during storage of
breast milk at -20.degree. C.
[0017] Milk bile-salt-stimulated lipase has been found only in the
milk of certain species, namely humans, gorillas, cats and dogs
(Freed, et al, 1986; Biochim Biophys Acta, 878: 209-215). Milk
bile-salt-stimulated lipase is not produced by cows, horses, rats,
rabbits, goats, pigs or Rhesus monkeys (Blackberg et al, 1980;
Freudenberg, 1966; Experientia, 22: 317).
[0018] Native human milk BSSL (hBSSL-MAM) has been purified to
homogeneity, as reported by Blackberg & Hernell (1981; Eur J
Biochem, 116: 221-225) and Wang & Johnson (1983), and the cDNA
sequence of human BSSL was identified by Nilsson (1990; Eur J
Biochem, 192: 543-550) and disclosed in WO 91/15234 and WO
91/18923. Characterization and sequence studies from several
laboratories concluded that the proteins hBSSL-MAM and the pancreas
carboxylic ester hydrolase (CEH) (also known as pancreatic BSSL)
are both products of the same gene (for example, Baba et al, 1991;
Biochem, 30: 500-510 Hui et al, 1990; FEBS Lett, 276: 131-134; Reue
et al, 1991; J Lipid Res, 32: 267-276).
[0019] Following the isolation of the cDNA sequence, recombinant
human BSSL (rhBSSL), as well as variants thereof, has been produced
including in transgenic sheep (rhBSSL-OVI); such as described in
U.S. Pat. No. 5,716,817, WO 94/20610 and WO 99/54443. Production of
proteins for therapeutic use using transgenic animals has been met
with significant safety, scientific, regulatory and ethical
resistance. Indeed, to date there is no approved therapeutic
product on the US or EU market that has been produced from
transgenic sheep, and only two medical products produced from other
transgenic animals have so far been approved: ATRYN (recombinant
antithrombin) produced from transgenic goats, and RUCONEST
(recombinant component 1 esterase inhibitor) produced from
transgenic rabbits. Proteins produced in such a manner (to be
expressed in mammary tissue and excreted in milk) can be
contaminated with components naturally found in the milk of these
animals, such as whey or non-human milk or whey proteins, which may
cause safety issues if such proteins are used for human use in
certain individuals, such as those intolerant or allergic to
milk-based components or products.
[0020] It has long been known (at least by the mid-1960s) that the
addition of lipase or esterase-containing tissue-extracts to
milk-based food is useful in the treatment of scours in animals (CA
662815 & U.S. Pat. No. 3,081,225). Also, U.S. Pat. No. 3,261,50
suggests the use of exogenous lipases for the treatment of celiac
disease or malabsorption syndrome in humans, including in young
children. The methods described therein involve the extraction and
use of a largely uncharacterized mixture of enzymes from tissues
such as the tongue and other oral tissues of calves, kid-goats and
lambs.
[0021] With reference to infant feeding practice, in particular the
feeding of LBW infants, it has long been promoted that fresh human
breast milk is the most suitable feed for LBW infants. This is
based on studies such as the early work by Williams et al (1978)
who showed that heat-treatment of human milk reduced fat absorption
by approximately one-third (compared to raw human milk) in an
experimental study of seven VLBW preterm infants (less than 1.3 Kg)
aged between 3 and 6 weeks, fed for three consecutive weeks with
raw, pasteurized and boiled human milk, each for one week. This
study made the suggestion that the improvement in fat absorption
may be related to the preservation of milk lipases in the raw,
compared to the heat-treated, human milk. Of note is that this
study described that all infants gained weight most rapidly during
the week in which they were fed raw milk; with the mean weight gain
(reported in g gained per week per 100 mL milk consumed) during
this period approximately one third greater than the similar
periods during which pasteurized or boiled milk was administered.
In a larger (but shorter) study reported by Alemi (1980;
Pediatrics. 68: 484-489), fat excretion was studied in 15 VLBW
infants, born with a birth-weight of between 660 and 1,695 g and a
gestational age of 26 to 33 weeks, and the study started at 7 to 44
days after birth. Fat excretion was lower in those infants fed a
mixture of human milk and formula for 72 hours compared to the
infants fed formula only. More recently, Andersson & coworkers
(2007) reported in a randomized study that pasteurization of
mother's own milk reduced fat absorption and growth in preterm
infants, and proposed that these effects were due to inactivation
of milk-based BSSL by pasteurization. Of note is that the reported
range of coefficient of fat absorption (CFA) from a number of
studies, including those above, are wide; both from human milk and
from formulas. This can partly be explained by the amount and
composition of fat given, and partly by large interindividual
differences in the capacity to utilize dietary fat in preterm
newborns, but it also reflects a considerable difficulty in
correctly assessing CFA (Hernell, 1999; J Pediatr, 136:
407-409).
[0022] One animal model study has attempted to investigate the
effects on infant growth by the addition on exogenous BSSL to
neonatal food (Wang et al, 1989; Am J Clin Nutr, 49: 457-463). This
study involved the addition of purified human BSSL (0.1 mg/mL) to
kitten-formula (mixed three to one with cow milk) to six
bottle-feed kittens for 5 days. This study reported that kittens
fed with kitten-formula supplemented with hBSSL had a growth rate
of twice that of those fed with formula alone. Of note is that the
formula was supplemented with cow milk, the kittens were not
preterm or of low birth weight, they were breast fed for the first
48 hours of their life and the study was conducted with purified
native hBSSL. The authors suggested that the kitten could be
utilized as an animal model in the investigation of the functional
role of BSSL, and on the basis of this study related patent
applications were filed (including, U.S. Pat. No. 4,944,944, EP
0317355 and EP 0605913) that disclose (amongst other aspects): a
method for fortifying a fat-containing infant formula which is poor
in bile-salt-activated lipase comprising adding to the formula an
effective amount of an isolated bile-salt-activated lipase selected
from the group consisting of milk bile-salt-activated lipase [BSSL]
and bile-salt-activated pancreatic carboxylesterase [now known also
to be BSSL] to increase fat absorption from the formula and growth
of the infant; and a method for feeding an infant a dietary base
from a first source comprising fats consisting of administering an
isolated bile-salt-activated lipase selected from the group
consisting of milk bile-salt-activated lipase [BSSL] and
bile-salt-activated pancreatic carboxylesterase [also BSSL] to the
infant in an amount sufficient to improve the infant's digestion
and absorption of the fats in the base and increase the growth of
the infant, wherein the lipase is derived from a second source. No
data supporting an improvement in fat absorption were disclosed,
nor any data obtained from any study that involved human infants.
Another study (Lindquist et al, 2007; J Pediatr Gastroenterol Nutr
44: E335) has been reported by Lindquist & Hernell (2010) as
artificially feeding purified human BSSL to BSSL-knock-out mice
pups nursed by BSSL-knock-out dams to restore normal fat absorption
and preventing the formation of intestinal lesions.
[0023] Following the cloning of the hBSSL cDNA and the disclosure
of various approaches to produce large quantities of recombinant
human BSSL (rhBSSL), numerous disclosures have been made, and
claims to, various infant formulas comprising rhBSSL (for example,
U.S. Pat. No. 5,200,183, WO91/15234, WO 91/18923, and U.S. Pat. No.
5,716,817) and various methods or uses of such formula or rhBSSL,
including as an infant supplement, for the improvement of
utilization of dietary lipids, treatment of fat malabsorption,
certain pancreatic abnormalities and cystic fibrosis (for example,
WO91/18923, WO 94/20610 and WO 99/54443). However, as with the
earlier suggestive studies, no supporting data obtained from
experiments supplementing human infants with recombinant
bile-salt-stimulated lipase are disclosed. Indeed, in 1996 after
all these suggestions, associative studies and disclosures, leading
workers in the area were still questioning: "Should bioactive
components of human milk [such as BSSL] be supplemented to
formula-fed infants?"; and further stating that: "There are no data
on attempts to supplement digestive enzymes [such as BSSL]"
(Hamosh, at Symposium: Bioactive Components in Milk and Development
of the Neonate: Does Their Absence Make a Difference? Reported in J
Nutr, 12: 971-974; 1997). More recently, Andersson & coworkers
(2007) have speculated that supplementing pasteurized milk with
recombinant human milk BSSL may restore endogenous lipolytic
activity of the milk.
[0024] The 722 amino-acid native BSSL is heavily glycosylated
(30-40% carbohydrate) (Abouakil et al, 1989; Biochem Biophys Acta,
1002: 225-230), with extensive O-glycosylation sites within the
C-terminal portion of the molecule that in its most abundant form
contains 16 proline-rich repeats of 11 residues with O-linked
carbohydrates (Hansson et al, 1993; J Biol Chem, 268: 26692-26698).
The role of the extensive O-glycosylation is unproven, but based on
its sequence composition the large C-terminal tail is predicted to
be very hydrophilic and accessible (Wang et al, 1995; Biochemistry,
34: 10639-10644).
[0025] Differences in glycosylation patterns can have dramatic
differences in the activity or other properties of many proteins,
especially proteins used in medicine. For example, ARANESP
(darbepoetin alpha) is a specifically engineered variant of
erythropoietin that differs from PROCRIT (epoetin alpha) by 2 amino
acids that provides the molecule with 5 N-linked oligosaccharide
chains rather than 3, and which significantly alter the
pharmacokinetic properties; with darbepoetin showing a threefold
increase in serum half-life and increased in vivo activity compared
to epoetin (Sinclair and Elliot, 2005; J Pharm Sci 94:
1626-1635).
[0026] Different recombinant production systems (such as mammalian
cell, yeast, transgenic animal), and even seemingly minor changes
in production process from the same expression system, can lead to
changes in the glycosylation of the same protein/polypeptide
sequence. For example, recombinant human alpha-galactosidase A is
used in enzyme replacement therapy for Fabry's disease, and the
commercial drug product is produced in two ways, having the same
amino acid sequence but each having a different glycosylation
pattern: REPLAGAL (agalsidase alfa) and FABRAZYME (agalsidase
beta). REPLAGAL is produced in a continuous line of human
fibroblasts while FABRAZYME produced in Chinese hamster ovary (CHO)
cells, and each product has different glycosylation. In common with
other proteins produced from CHO cells, FABRAZYME is a sialyated
glycoprotein, and has differences in the degree of sialyation and
phosphorylation compared to REPLAGAL (Lee et al, 2003;
Glycobiology, 13: 305-313). The qualitative and quantitative
differences in the sialylation of glycoproteins produced in CHO
cells in comparison with natural human glycoproteins have
consequences for both the level of biodistribution and immunogenic
potency. In fact, the presence of IgG has been reported in almost
all patients treated with agalsidase beta compared to only 55% of
patients treated with agalsidase alfa (Linthorst et al, 2004;
Kidney Int, 66: 1589-1595). Moreover, in some cases, an allergic
type reaction to treatment with agalsidase beta has been recorded,
with the presence of IgE in the circulation and/or a positive
intradermal reaction (Wilcox et al, 2004; Am J Hum Genet, 75:
65-74).
[0027] Indeed, while their peptide maps are very similar, the
glycosylation patterns of native BSSL does differ substantially
from that of rhBSSL produced in mouse C127 and hamster CHO cell
lines, and also in the ability to bind to certain lectins including
concanavalin, Ricinus communis agglutinin and Aleuria aurantia
agglutinin suggesting that native BSSL contains considerably more
fucose and terminal beta-galactose residues than the recombinant
forms (Stromqvist et al, 1995; J Chromatogr, 718: 53-58). Landberg
et al (1997; Arch Biochem Biophys 344: 94-102) further
characterized these two recombinant forms, and reported that both
recombinant forms had a lower molar percent of total monosaccharide
(20% and 15% for C127- and CHO-produced rhBSSL, respectively,
compared to 23% for native hBSSL), and that while native hBSSL
reacted to certain Lewis antigen-detecting antibodies, the
C127-rhBSSL did not.
[0028] Although the C127- and CHO-produced rhBSSL reported above
were generally similar to each other in terms of molecular mass,
glycosylation and lectin binding, in contrast, the rhBSSL isolated
from the milk of transgenic mice showed a lower apparent molecular
mass on size-exclusion chromatography (SEC) and no detectable
interactions with a panel of lectins, indicating a significantly
lower degree of O-glycosylation of rhBSSL in milk from transgenic
mice than found for the other recombinant forms (Stromqvist et al,
1996; Transgen Res 5: 475-485).
[0029] Clinical studies in specific indications conducted with one
particular form of rhBSSL have been reported; namely early-phase
exploratory studies of exocrine pancreatic insufficiency (PI) due
to chronic pancreatitis or cystic fibrosis (CF). In 2004, a phase
II trial was reported that showed that CF patients (aged 12 to 39
years) with PI had a more rapid and efficient lipid uptake when
supplemented with rhBSSL at a single dosing of 0.2 g or 1 g as a
complement to 25% of their regular Creon dosing, as compared to
Creon alone given at their regular dose, or at 25% dosage
(Strandvik et al, 2004; 18th North American Cystic Fibrosis
Conference, St Louis Mich.; abstract published in Pediatr Pulmonol,
S27: 333), and in 2005 the results of a second phase II trial were
reported as rhBBSL showing a greatly improved ability of a group of
Swedish patients with CF suffering from PI to digest fat (press
release from Biovitrum, reporting Strandvik et al, 2005; 28th
European Cystic Fibrosis Society (ECFS) Conference, Crete). In both
clinical trials, these clinical results were obtained using
rhBSSL-OVI. More recently, it has been announced that a further
phase II trial with an oral suspension of rhBSSL (described therein
as "bucelipase alpha"), dosed at 170 mg 3 times daily for 5-6 days,
to evaluate the effect on the fat absorption in adult patients with
CF and PI has been completed, but no efficacy results from this
have to date been published (clinicaltrials.gov identifier
NCT00743483).
[0030] It has been disclosed since at least 2008 that two phase II
trials using rhBSSL were planned and ongoing, each to investigate
the coefficient of fat absorption, and change in length and body
weight, in preterm infants born before 32 weeks gestational age
treated with 0.15 g/L rhBSSL or placebo for one week each, added to
infant formula (clinicaltrials.gov identifier NCT00658905) or to
pasteurized breast milk (clinicaltrials.gov identifier
NCT00659243).
[0031] In light of the prior art, and the long felt need for a
solution, it is therefore an object of the present invention to
provide a method of increasing the growth velocity of a human
infant, such as an underweight or preterm human infant. Said method
should overcome one or more of the disadvantages of the prior art,
that include: that an active ingredient that can be reliably and/or
reproducibly produced in large quantities; that the active
ingredient has been manufactured by a scientifically, regulatory
and/or ethically acceptable method; and/or that the method or the
active ingredient used in the method, has been demonstrated within
a randomized clinical trial involving human infants to be
efficacious and safe.
[0032] The solution to the above technical problem is provided by
the various aspects and embodiments of the present invention as
defined or otherwise disclosed herein and/or in the claims.
SUMMARY
[0033] In one aspect, the invention relates to a method to increase
the growth velocity of a human infant, said method comprising the
step of enteral administration of recombinant human
bile-salt-stimulated lipase to said infant.
[0034] In another aspect, the invention relates to a therapeutic
method to treat a human infant suffering from underweight or
premature birth, said method comprising the step of enteral
administration of recombinant human bile-salt-stimulated lipase to
an infant in medical need thereof.
[0035] Another aspect of the invention relate to a method of
preparing a modified infant formula or modified pasteurized breast
milk for increasing the growth velocity of a human infant, said
method comprising the steps:
[0036] i. providing a first quantity of recombinant human
bile-salt-stimulated lipase and a second quantity of an unmodified
infant formula or unmodified pasteurized breast milk; and
[0037] ii. adding an amount of said lipase to said unmodified
infant formula or unmodified pasteurized breast milk,
so as to form a modified infant formula or modified pasteurized
breast milk that includes an amount of lipase effective to increase
the growth velocity of a human infant when said modified infant
formula or modified pasteurized breast milk is fed to said infant
for at least one feed per day over at least around 4 days, for at
least one feed per day over at least around 5 days, or for at least
one feed per day over at least around 7 days.
[0038] In yet other aspects, the invention relates to: (a) a
modified infant formula that includes recombinant human
bile-salt-stimulated lipase in an amount effective to increase the
growth velocity of a human infant when said modified infant formula
is fed to said infant for at least one feed per day over at least
around 4 days, for at least one feed per day over at least around 5
days, or for at least one feed per day over at least around 7 days;
and/or relates to (b) a modified pasteurized breast milk that
includes recombinant human bile-salt-stimulated lipase in an amount
effective to increase the growth velocity of a human infant when
said modified pasteurized breast milk is fed to said infant for at
least one feed per day over at least around 4 days, for at least
one feed per day over at least around 5 days, or for at least one
feed per day over at least around 7 days.
[0039] One further aspect of the invention relates to a kit for the
preparation of a modified infant formula or modified breast milk
for increasing the growth velocity of a human infant, said kit
comprising the components:
[0040] (a) at least one first container that includes a first
amount of recombinant human bile-salt-stimulated lipase, such as in
a lyophilized formulation; and
[0041] (b) at least one second container, which is distinct from
the first container, that includes a second amount of unmodified
infant formula or unmodified pasteurized breast milk;
[0042] where said lipase and said unmodified infant formula, or
unmodified pasteurized breast milk, are each in an amount
sufficient to prepare a modified infant formula or modified
pasteurized breast milk, respectively, that includes an amount of
said lipase effective to increase the growth velocity of a human
infant when said modified infant formula or modified pasteurized
breast milk is fed to said infant, such as is fed to said infant
for at least one feed per day over at least around 4 days, for at
least one feed per day over at least around 5 days, or for at least
one feed per day over at least around 7 days.
[0043] In yet another aspect, the invention relates to a method to
increase the growth velocity of a human infant, said method
comprising the steps of:
[0044] i. preparing or otherwise providing a modified infant
formula or a modified pasteurized breast milk of the invention, or
preparing a modified infant formula of the invention or a modified
pasteurized breast milk according to the method of the invention or
by using the kit of the invention;
[0045] ii. feeding the modified infant formula or modified
pasteurized breast milk so prepared or otherwise provided to said
infant; and
[0046] iii. repeating the preceding steps for at least one feed per
day over at least around 4 days, for at least one feed per day over
at least around 5 days, or for at least one feed per day over at
least around 7 days.
[0047] In a yet further aspect, the invention relates to a
packaged-pharmaceutical-product comprising a pharmaceutical
composition that includes an amount of recombinant human
bile-salt-stimulated lipase, wherein said
packaged-pharmaceutical-product further comprises instructions that
describe the steps of:
[0048] i. preparing a modified infant formula or modified
pasteurized breast milk that contains said lipase in an amount
effective to increase the growth velocity of a human infant when
said modified infant formula or modified pasteurized breast milk is
fed to said infant for at least one feed per day over at least
around 4 days, for at least one feed per day over at least around 5
days, or for at least one feed per day over at least around 7 days;
and
[0049] ii. enteral administration of said amount of lipase by
feeding said modified infant formula or modified pasteurized breast
milk to a human infant, such as for at least one feed per day over
at least around 4 days, for at least one feed per day over at least
around 5 days, or for at least one feed per day over at least
around 7 days.
[0050] In a particular aspect, the invention also relates to a
pharmaceutical composition in a unit dose that includes between 0.1
and 100 mg of recombinant human bile-salt-stimulated lipase, such
as between 1.5 and 75 mg of said lipase, between 5 and 45 mg of
said lipase, or about 20 mg of said lipase.
[0051] In alternative aspects, the present invention also relates
to: methods for; methods of preparing infant feeds useful for;
infant feeds useful for; kits useful for;
packaged-pharmaceutical-packages for; pharmaceutical compositions
for; and recombinant human bile-salt-stimulated lipase for, (X)
protection of the small bowel mucosa of a human infant from damage;
(Y) protection of an immature intestinal epithelium of a human
infant from the deleterious effects of incompletely digested and/or
excess fat and/or lipid; and/or (Z) limitation of accumulation of
incompletely digested and/or excess fat and/or lipid in the ileum
of a human infant; in each case involving recombinant human bile
salt stimulated lipase to said infant.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1.1 shows a schematic presentation of the structure of
rhBSSL, also showing sites for potential glycosylation.
[0053] FIG. 2.1 shows a schematic plan of the clinical studies of
rhBSSL added to infant formula or to pasteurized breast milk.
[0054] FIG. 2.2 shows correlation between differences in growth
velocity (g/kg/day) and CFA (%), combined data, for the PP
population.
DETAILED DESCRIPTION
[0055] In one aspect, the invention relates to a method to increase
the growth velocity of a human infant, said method comprising the
step of enteral administration of recombinant human
bile-salt-stimulated lipase to said infant.
[0056] Recombinant human bile-salt-stimulated lipase (rhBSSL)
useful in the invention is described, defined or referred to
herein. For example, it includes polypeptides recognizable by a
person of ordinary skill in the art as being human
bile-salt-stimulated lipase, wherein said human lipase has been
produced by or isolated from a non-human source, such as a
non-human organism, adapted or modified (for example by recombinant
genetic technology) to produce such polypeptide.
[0057] Human bile-salt-stimulated lipase (BSSL) is an enzyme known
by various identifiers or aliases; for example, "carboxyl ester
lipase (CEL)", "bile-salt-activated lipase (BAL)",
"bile-salt-dependent lipase (BSDL)", "carboxylesterase",
"carboxylic ester hydrolase" (CEH), and a number of other alias and
descriptions as will be readily available to the person ordinarily
skilled in the art from information sources such as "GeneCards"
(World Wide Web at genecards.org). A number of natural amino acid
sequences and isoforms of human BSSL have been identified from
human milk (and pancreas), and a number of different amino acid
sequences (typically, predicted from cDNA or genomic sequence) have
been described; all of which herein are encompassed within the term
"human bile-salt-stimulated lipase." For example, human
bile-salt-stimulated lipase is naturally produced first as a
precursor sequence including a 20 to 26 amino acid signal sequence,
and the mature full-length form of the protein described as having
722 to 733 amino acids (for example see, Nilsson et al, 1990;
WO91/15234; WO91/18923; the polypeptide predicted from cDNA
sequence GenBank submission ID: X54457; GenBank ID: CAA38325.1;
GeneCards entry for "CEL/BSSL"; GenBank ID: AAH42510.1; RefSeq ID:
NP.sub.--001798.2; Swiss-Prot ID: PI9835). In further examples,
other shorter isoforms of human bile-salt-stimulated lipase are
described in Venter et al (2001; Science, 291: 1304-1351); GenBnk
ID: AAC71012.1; Pasqualini et al (1998; J Biol Chem, 273:
28208-28218); GenBank ID: EAW88031.1; WO 94/20610 and Blackberg et
al (1995; Eur J Biochem, 228: 817-821).
[0058] In particular embodiments, the human bile-salt-stimulated
lipase comprises a protein having an amino acid sequence
comprising, or as shown by, SEQ ID NO:1. In other particular
embodiments, the (recombinant) human bile-salt-stimulated lipase
has an amino acid sequence of either the mature or precursor forms
of BSSL selected from those disclosed in Nilsson et al, 1990;
WO91/15234, WO 91/18923; RefSeq ID: NP.sub.--001798.2; GenBank ID:
AAH42510.1; GenBank ID: CAA38325.1; GeneCards entry for "CEL/BSSL";
Swiss-Prot ID: P19835. In further such embodiments, the
(recombinant) human bile-salt-stimulated lipase comprises a protein
with an amino acid sequence that is at least 720 consecutive amino
acids of any of the sequences disclosed in the preceding references
or of SEQ ID NO:1. In other embodiments the (recombinant) human
bile-salt-stimulated lipase comprises a protein having at least the
amino sequence from position 1 to 101 of that disclosed in SEQ ID
NO:1 or WO 91/15234, or at least the amino acid sequence from
position 1 to 535 of that disclosed in SEQ ID NO:1, such as
"Variant A" disclosed in Hansson et al, 1993; J Biol Chem, 35:
26692-26698, wherein such protein has bile salt binding and/or
bile-salt-dependent lipase activity, as for example may be
determined by the methods disclosed in Blackberg et al (1995; Eur J
Biochem 228: 817-821).
[0059] It will now therefore be apparent to the person ordinarily
skilled in the art that in certain embodiments of the present
invention one or more of these described forms of (recombinant)
human bile-salt-stimulated lipase may be useful in the various
aspects of the invention. Further, it will be apparent to such
person that other (recombinant) proteins that have
bile-salt-dependent lipolytic activity (for example, as may be
determined by the methods disclosed in Blackberg et al, 1995) and
that are similar in amino acid sequence to those polypeptide
sequences described, defined or referred to herein may also have
utility in the present invention, and hence are also encompassed by
the term "human bile-salt-stimulated lipase". In certain such
embodiments, a protein that shows more than 90%, 95%, 98%, 99%,
99.5% sequence identity over at least about 30, 50, 100, 250, 500,
600, 700, 711, 720, 722, 733 or 750 amino acids to a sequence
described, defined or referred to herein. In other embodiments, one
or more amino acid substitutions may be made to one of the BSSL
polypeptide sequences disclosed, defined or referred to herein. For
example, one, two, three, four, five or up to 10 amino acid
substitutions, deletions or additions may be made to the sequence
disclosed in SEQ ID NO:1. Such amino acid changes may be neutral
changes (such as neutral amino acid substitutions), and/or they may
affect the glycosylation, binding, catalytic activity or other
properties of the protein in some (desired) manner. Proteins with
such substitutions, providing they have bile-salt-dependent
lipolytic activity, will also be recognized by the person
ordinarily skilled in the art as being "human bile-salt-stimulated
lipase" in the sense of the present invention.
[0060] In other embodiments the human bile-salt-stimulated lipase
is expressible from or otherwise encoded by a nucleic acid having a
suitable nucleic acid sequence. By way of non-limited example, said
lipase is expressible from or otherwise encoded by a nucleic acid
comprising the sequence between positions 151 and 2316 of SEQ ID
NO:2, or that disclosed in WO 94/20610 or Nilsson et al (1990). As
will also be appreciated by the person of ordinary skill, a
"suitable nucleic acid sequence" will also encompass variants of
the preceding nucleic acid sequences. For example, changes in one
or more nucleotide bases that do not change the amino acid encoded
by a triplet-codon (such as in the 3.sup.rd codon position) will
also be "suitable". Sub-fragments of such nucleic acid sequences
will also be "suitable" if they encode a (short) isoform of human
bile-salt-stimulated lipase as described herein. Furthermore,
nucleic acid sequences that encode a protein having a variant of
the amino acid sequence shown by SEQ ID NO:1, such as those
described above, will also be "suitable". Accordingly, the present
invention envisions embodiments whereby the (recombinant) human
bile-salt-stimulated lipase is a protein that is expressible or
otherwise encoded by a nucleic acid that hybridizes to a nucleic
acid comprising the sequence between positions 151 and 2316 of SEQ
ID NO:2 or to one comprising the sequence between positions 151 and
755, and wherein said protein has bile-salt-dependent lipolytic
activity. In certain such embodiments, the hybridization is
conducted at stringent conditions, such as will be known to the
person of ordinary skill, and is described in general text books
for example "Molecular Cloning: A Laboratory Manual", by Joe
Sambrook and David Russell (CSHL Press).
[0061] In a particular embodiment, the (recombinant) human
bile-salt-stimulated lipase is produced by expression from a
nucleic acid described, defined or referred to herein.
[0062] A human bile-salt-stimulated lipase described, defined or
referred to herein, in the context of the present invention is a
recombinant bile-salt-stimulated lipase (rhBSSL); i.e., where said
human lipase has been produced by or isolated from a non-human
source, such as a non-human organism, adapted or modified (for
example by recombinant genetic technology) to produce such lipase.
In particular embodiments, the rhBSSL is produced using cell-free
and/or in-vitro transcription-translation techniques from an
isolated nucleic acid molecule described, defined or referred to
herein. Alternatively, a recombinant non-human organism is used,
wherein said non-human organism includes at least one copy of such
a nucleic acid, and where said nucleic acid is expressible by said
non-human organism to produce the desired protein: rhBSSL. For
example, recombinant bacterial, algae, yeast or other eukaryotic
cells may be used, and the rhBSSL is, in certain embodiments,
produced from the culture of such recombinant cells. In other
embodiments, the rhBSSL may be produced by extra-corporal culture
of modified or specifically selected human cells, for example by
their in-vitro culture. In yet other embodiments, rhBSSL may be
produced by its isolation from the milk of transgenic animals; such
as transgenic cattle, sheep, goats or rabbits. The skilled person
will be aware of the numerous technologies available to produce
human bile-salt-stimulated lipase using recombinant technology.
[0063] Recombinant human bile-salt-stimulated lipase has been shown
to be producible from recombinant cell culture including the
culture of E. coli, mouse and hamster (Hansson et al, 1993), and P.
pastoris (Trimple et al, 2004; Glycobiol, 14: 265-274) cells.
Recombinant human bile-salt-stimulated lipase has also been shown
to be producible and isolatable from the milk of transgenic mice
(Stromqvist et al, 1996; Transgen Res, 5: 475-485) and from the
milk of transgenic sheep (WO99/54443). In certain embodiments of
the present invention, the recombinant human bile-salt-stimulated
lipase is isolated from the culture of such recombinant cells or
from the milk of such transgenic animals. In an alternative
embodiment, the recombinant human bile-salt-stimulated lipase is
not one isolated from the milk of a transgenic sheep or a
transgenic mouse.
[0064] In a particular embodiment of the present invention, the
recombinant human bile-salt-stimulated lipase is isolated from an
expression product of a recombinant Chinese hamster ovary (CHO)
cell line, is produced by a recombinant CHO cell line, or is
expressible by, or isolatable from, a recombinant CHO cell line.
Use of a recombinant CHO cell line expression system to produce
such lipase can produce rhBSSL that exhibits particular structural,
activity or other characteristic features, such as one or more of
those described herein. By way of non-limiting example, the rhBSSL
useful in the present invention may be isolated using a process
and/or exhibit characteristics analogous to, or substantially as
described in, the Exemplification herein.
[0065] In certain embodiments of the present invention, the
recombinant human bile-salt-stimulated lipase is identified by the
International Non-proprietary Name (INN) stem "bucelipase" (see WHO
Drug Information, 21: 62, 2007), for example because it has the
amino acid sequence shown therein. The recombinant human
bile-salt-stimulated lipase, when used in the present invention
may, with reference to SEQ ID NO:1, have one or more disulfide
bridges at the locations Cys64-Cys80 and Cys246-Cys257, and/or is
glycosylated at one or more of the possible glycosylation sites at
Asn-187, Thr-538, Thr-549, Thr-559, Thr-576, Thr-587, Thr-598,
Thr-609, Thr-620, Thr-631 and Thr-642 (in one such embodiment,
schematically represented in FIG. 1.1). In certain such
embodiments, the rhBSSL is in a glycoform, and may for example,
have the INN of "bucelipase alfa".
[0066] In other particular embodiments of the present invention,
the recombinant human bile-salt-stimulated lipase has structural,
composition and/or other properties that are different to those of
native human bile-salt-stimulated lipase (BSSL-MAM) and/or
different from that form of recombinant bile-salt-stimulated lipase
that has been produced by isolation from the milk of transgenic
sheep (rhBSSL-OVI), such as described in WO 99/54443.
[0067] Accordingly, in certain such embodiments, the recombinant
human bile-salt-stimulated lipase useful for the present invention
is (substantially) free of other milk proteins or milk components.
As will be apparent upon the disclosure of the present invention,
in certain embodiments the rhBSSL is added to a milk-based infant
feed before administration to the human infant. Accordingly, in
such embodiments, the "free of other milk proteins or milk
components" will apply to that form, composition or formulation of
the recombinant bile-salt-stimulated lipase that exists shortly
before (such as immediately before) addition of said lipase to said
milk-based infant food. For example, in such embodiments the
pharmaceutical compositions or kits components of the invention
containing rhBSSL, or that amount of rhBSSL that is provided ready
for addition to any infant formula and/or pasteurized breast milk,
are free of such milk-based contaminates. In certain such
embodiments, the rhBSSL is free of milk casein and whey proteins,
such as lactoferrin, or free of other contaminates native to milk,
in particular where such milk-derived proteins or other
contaminates are derived from the milk of humans, sheep or mice. In
these embodiments, the "free of" any particular such protein or
contaminant means that no material amounts of such protein or other
contaminate can be detected by routine detection methodologies.
Alternatively, any such particular impurity may be present at a
level of less than about 5%, such as less than about 2%, 1%, 0.5%
or 0.1%, or is essentially or effectively absent, or that the total
of all such milk-derived proteins or other contaminates are present
at a level of less than about 5%, such as less than about 2%, 1%,
0.5% or 0.1%, or are essentially or effectively absent. As will be
understood by the person ordinarily skilled in the art, recombinant
human bile-salt-stimulated lipase produced & isolated from cell
culture, such as from recombinant CHO cells will be considered
"free of" such milk-based contaminates.
[0068] In other certain such embodiments of the present invention,
the recombinant human bile-salt-stimulated lipase has a purity of
greater than about 70%, such as a purity of greater than about 80%,
90% or 95%. In particular such embodiments, such percentage purity
is a percentage purity of total protein. As described above, in the
applicable embodiments such purity measure is that of the
composition comprising said lipase before addition to any infant
feed or other administration medium. Such purity values may be
determined by RP-HPLC, SE-HPLC or SDS-PAGE (with SYPRO.RTM. Ruby or
silver staining) techniques.
[0069] In other embodiments of the invention, particularly if the
recombinant human bile-salt-stimulated lipase is produced using
(expressed from) recombinant CHO cells, the rhBSSL when used in the
present invention may be characterized by one or more structural,
activity or other properties such as those described in the
following.
[0070] In further certain such embodiments of the invention, the
recombinant human bile-salt-stimulated lipase has a level
(overall/total) of glycosylation that is less than that of native
human bile-salt-stimulated lipase (BSSL-MAM) and/or has a level
(overall/total) of glycosylation that is more than that of
recombinant human bile-salt-stimulated lipase isolated from the
milk of transgenic sheep (rhBSSL-OVI). The levels of glycosylation,
such as the level of monosaccharide and/or sialic acid content of
BSSL (or a sample thereof) may be measured using high pH anion
exchange chromatography with pulsed amperiometric detection
(HPAEC-PAD). In particular embodiments of the present invention,
the total monosaccharide content of the recombinant human
bile-salt-stimulated lipase (moles monosaccharide per mole rhBSSL)
is between about 20 and 100, between about 25 and 65 or between
about 25 and 55, such as between about 40 to 45 mole/(mole rhBSSL),
In certain embodiments of the invention the total sialic acid
content of the rhBSSL (moles sialic acid per mole rhBSSL) is
between about 20 and 35, such as between about 25 and 30 mole/(mole
rhBSSL).
[0071] In yet other certain such embodiments of the present
invention, the recombinant human bile-salt-stimulated lipase has a
glycosylation pattern, for example of O-glycans, that is different
to that of BSSL-MAM and/or different to that of rhBSSL-OVI. Such
differences may be detected using capillary electrophoresis with
laser induced fluorescence detection (CE-LIF) and/or HPAEC-PAD. In
particular embodiments of the invention, the rhBSSL may have
between about 20 and 50 mole of N-acetyl neuraminic acid
(NANA=Neu5Ac) per mole rhBSSL [mole/(mole rhBSSL)], such as between
about 25 and 40 mole/(mole rhBSSL). The rhBSSL used in the
invention may have less than about 5 mole N-glycosyl neuraminic
acid (NGNA=Neu5Gc) per mole rhBSSL, such as less than about 2
mole/(mole rhBSSL), or where NGNA is essentially undetectable. The
rhBSSL used in the invention may have less than about 20 mole
fucose per mole rhBSSL, such as less than about 10, less than about
5, less than or about 2 mole/(mole rhBSSL), and in certain
embodiments fucose is essentially undetectable. The rhBSSL used in
the invention may have between about 5 and 25 mole galactosamine
per mole rhBSSL, such as between about 10 and 20 or between about
15 and 18 mole/(mole rhBSSL). The rhBSSL used in the invention may
have less than about 10 mole glucosamine per mole rhBSSL, such as
less than about 5, less than about 3 or about 2 mole/(mole rhBSSL).
The rhBSSL used in the invention may have between about 5 and 25
mole galactose per mole rhBSSL, such as between about 10 and 20 or
between about 15 and 18 mole/(mole rhBSSL). The rhBSSL used in the
invention may have less than about 5 mole glucose per mole rhBSSL,
such as less than about 2 mole/(mole rhBSSL), or where glucose is
essentially undetectable. The rhBSSL used in the invention may have
between about 2 and 8 mole mannose per mole rhBSSL, such as between
about 4 and 6 mole/(mole rhBSSL). In particular embodiments of the
invention, the rhBSSL may have a profile of monosaccharide and/or
sialic acid content about that as, or substantially as, represented
in Table 1.1.
[0072] In other embodiments of the invention, the recombinant human
bile-salt-stimulated lipase useful for the present invention is
different from BSSL-MAM and from rhBSSL-OVI in the profile or
amount of lectin binding or Lewis-antigen binding tests, such as
those assays and profiles described in Blackberg et al (1995) and
Landberg et al (1997) respectively. Such lectin binding or
Lewis-antigen binding tests can indicate differences in
glycosylation pattern between these different forms of BSSL. Other
techniques may be used to identify and/or characterize recombinant
human bile-salt-stimulated lipase useful for the present invention.
For example, rhBSSL may be characterized (and/or differentiated
from BSSL-MAM or from rhBSSL-OVI) by endoprotease Lys-C digestion
followed by analysis of the resulting peptides with reverse-phase
HPLC with quantitative UV detection (at 214 nm), and
recording/inspection of the resulting chromatogram. Differences in
the resulting chromatogram may be due to--and hence further
reflect--unique features of glycosylation of specific peptides
comprising the rhBSSL that have specific differences in retention
time.
[0073] In yet further such embodiments of the present invention,
the recombinant human bile-salt-stimulated lipase has a molecular
mass of between 90 KDa and 75 KDa. In particular such embodiments
the molecular mass of said lipase is between about 84 and 86 KDa,
such as about 85 KDa. The molecular mass may be determined by
routine techniques including MALDI-MS. By way of comparison, using
the same detection techniques the molecular mass of BSSL-MAM is
measured as being substantially greater (for example, around 100
KDa) and that of rhBSSL-OVI is measured as being substantially
smaller (for example, around 78 KDa).
[0074] In other further such embodiments of the present invention,
the recombinant human bile-salt-stimulated lipase can comprise a
population of recombinant human bile-salt-stimulated lipase
molecules having sequences of different amino acid lengths. In
certain of such embodiments, the amount of lipase molecules that
are present in a form that is shorter at the C-terminal end by one,
two, three, four, five or up to ten amino acids, compared to the
longest or (predicted) full-length form (such as that shown by SEQ
ID NO:1) is greater than 50% of the amount of lipase molecules
present in such longest or (predicted) full-length form. In certain
such embodiments, between about 100% and 500% of the amount of the
longest (or predicted full-length) lipase molecule is the amount
present as a shorter lipase molecule, such as by one or two amino
acids from the C-terminal end. In particular such embodiments
between 200% and 400%, for example about 300%, of the amount of the
longest (or predicted full-length) molecule (for example, that
shown by SEQ ID NO:1), is the amount present as a shorter lipase
molecule such as by one or two amino acids from the C-terminal end.
In particular embodiments or the foregoing, less than 1% of the
amount of the longest (or predicted full length) said lipase
molecules is present as a lipase molecule shorter by two amino
acids. In other embodiments, between two- to five-fold, such that
about three-fold, the number of longest (or predicted) said lipase
molecules are present in a form that are shorter than such longest
(or predicted) molecule from the C-terminal end by one, two, three,
four, five or up to ten amino acids.
[0075] In yet other further such embodiments of the present
invention, the recombinant human bile-salt-stimulated lipase may
have a specific activity that is greater than BSSL isolated from
human milk and/or rhBSSL-OVI. For example, the specific activity of
the rhBSSL may be between about 15% and 35% higher, such as about
20% or 25% higher specific activity than that of BSSL-MAM and/or
rhBSSL-OVI (based on mass). Techniques to measure specific activity
of human BSSL will be known to the person of ordinary skill and
include using the 4-nitrophenyl ester butyric acid (PNPB) assay as
generally described in the Exemplification herein. Other in-vitro
assays for BSSL are known, for example by use of trioleoylglycerol
emulsified in gum Arabic as the substrate for BSSL and sodium
cholate (10 mM) as activating bile salt (for example, as described
by Blackberg and Hernell, 1981; Eur J Biochem, 116: 221-225). In
particular embodiments, prior to measuring specific activity, the
BSSL may be purified to high purity, such as by using the
techniques of heparin-affinity chromatography and size exclusion
chromatography.
[0076] As will be understood by the person of ordinary skill, the
recombinant human bile-salt-stimulated lipase used in the present
invention may be characterized by more than one of the
distinguishing features described or defined herein, such as those
above. For example, a combination of two or more (such as three,
four, five or more) of such features may together characterize a
particular embodiment of the recombinant human bile-salt-stimulated
lipase for use in the present invention.
[0077] In the present invention, the amount of recombinant human
bile-salt-stimulated lipase enterally administered to the human
infant may vary. In certain embodiments, the amount of said lipase
is an effective amount, such as an amount effective to increase the
growth velocity of the human infant when said lipase is
administered to the infant according to present invention. Suitable
amounts of recombinant human bile-salt-stimulated lipase that may
be administered to the infant in any given day may range from an
amount per day of between 1 and 100 mg of said lipase per Kg weight
of infant. In particular embodiments between 5 and 50 mg of said
lipase per Kg weight of infant or between 15 and 40 mg of said
lipase per Kg weight of infant may be administered over a day, such
as between about 22.5 and 27 mg of said lipase administered per Kg
weight of infant per day. By way of non-limiting example, a 1.5 Kg
infant dosed at 25 mg/Kg/day may be administered with a total of
about 37.5 mg of recombinant human bile-salt-stimulated lipase per
day. In certain embodiments of the present invention, the mass of
rhBSSL used or referred to herein, instead of being given as an
absolute mass, is given as the mass of active rhBSSL molecules.
Since different production or storage batches of rhBSSL may vary in
enzymatic activity, the absolute mass of rhBSSL administered may be
varied in order to compensate for such variations in activity and
hence to provide a more uniform amount of active rhBSSL. The
activity of rhBSSL may be easily determined using the PNPB assay as
described herein, with reference to an active standard BSSL
molecule. Suitable masses of active rhBSSL are within the ranges of
masses given above. As the molecular mass of a complex protein such
as rhBSSL may vary, for example due to differences in
glycosylation, the amount of said lipase may be defined in ways
other than in terms of mass, such as in terms of (active) molar
amounts. The skilled person will be readily able to make other
conversions from specific mg amounts to the corresponding micro
mole amount. Alternatively, the amount of recombinant human
bile-salt-stimulated lipase may be expressed in terms of the
activity of the lipase in enzyme units (U), such as defined as the
amount of said lipase that catalyzes the formation of 1 micro mole
of product per minute under the conditions of the assay, for
example as determined in an in vitro assay for BSSL activity such
as one described herein.
[0078] A will be appreciated by the person of ordinary skill, a
human infant is typically (unless for example on a glucose drip)
regularly fed with a nutritional base that contains a source of fat
such as triglycerides. The infant may be fed the nutritional base
orally or via tube-feeding. The nutritional base (feed or food) is
commonly an infant formula or human breast milk. Accordingly,
certain embodiments of the invention the recombinant human
bile-salt-stimulated lipase is administered to a human infant that
receives a nutritional base containing a source of fat such as
triglycerides. In particular such embodiments said nutritional base
is an infant formula and/or pasteurized breast milk; both known by
the person of ordinary skill to contain a substantial proportion of
fat in triglyceride form. In various such embodiments of the
invention, the enteral administration of the rhBSSL may be prior
to, after or concomitant to when said infant receives the
nutritional base. If administered prior to or after the receiving
the nutritional base, then the rhBSSL may be administered within
about 1 hour of said infant receiving the nutritional base, such as
within about 30 mins, 15 mins or 5 mins, or within a period of less
than about 2 min of the infant receiving the nutritional base.
Should the period between receiving the nutritional base be within
about 1 min of administration of the rhBSSL, then this may
effectively be considered administration of the rhBSSL concomitant
to said infant receiving the fat-containing nutritional base (such
as an infant formula and/or pasteurized breast milk). Such
concomitant (or co-) administration will occur if the rhBSSL is
first added to an infant formula or breast milk, which is then fed
to the human infant.
[0079] As is generally known, it is preferable to exclusively feed
fresh breast milk from the infant's own mother. However, for
various reasons the infant may be fed pasteurized breast milk from
other mothers, such as from a breast milk bank. Alternatively, the
infant may be fed, as is common, infant formula instead of or in
addition to (non-fresh) breast milk. The prevalence of not breast
feeding (or ceasing to breast feed) a human infant is described
elsewhere herein. That a human infant is not fed its mother's fresh
milk, but one of these alternatives, may be due to one or more
causes. For example: (i) the mother may not produce enough breast
milk because of health reasons such as previous breast surgery or a
prolactin deficiency; (ii) the mother may suffer from mastitis,
eczema, or a plugged milk duct making breast feeding painful; (iii)
the infant may suffer from a disorder in the mouth, such as a cleft
lip or palate; (iv) the mother may not have sufficient knowledge to
breastfeed, may choose not to feed fresh breast milk, such as for
reasons of culture or convenience; or (v) the mother may be advised
not to feed her own fresh breast milk in order to protect the
infant from potentially harmful components of her own breast milk,
including the transmission of infective agents such as HIV virus,
CMV virus, T-cell lymphotropic virus or tuberculosis mycobacteria,
dangerous medication or drugs (or their metabolites) such as from
illicit drug-use, retroviral or chemotherapy drug therapy, or if
the mother is undergoing radiation therapy. Finally, the infant may
be too weak to feed from the breast, which can be a particular
problem for preterm or underweight infants.
[0080] Accordingly, in certain embodiments of the invention the
human infant is not exclusively fed fresh mothers' milk, for
example the infant is not exclusively fed fresh milk from its own
mother such as by exclusive breastfeeding or feeding of fresh
expressed breast milk. An infant that is not fed exclusively
breastfed or not exclusively fed from expressed (fresh) breast milk
from its own mother will receive milk from other sources, such as
infant formula or pasteurized and/or (previously) frozen breast
milk from a breast milk bank. In particular embodiments of the
present invention, the infant is not fed fresh mother's milk, for
example the infant is exclusively fed with infant formula,
pasteurized and/or frozen breast milk such as from a breast milk
bank. This may occur immediately upon birth, i.e., the human infant
never receives its mother's fresh breast milk, or very soon
thereafter such as within the first, second, third, fourth, fifth
or sixth day of birth. In other embodiments, the human infant may
cease to be fed its mother's fresh milk within about one week, two
weeks or three weeks of birth, or within about one month, two
month, three month or up to 6 months of birth.
[0081] The recombinant human bile-salt-stimulated lipase may be
enterally administered according to the present invention by
various means, including oral administration. For example, the
administration may be performed using a paste, syrup, electuary,
bolus, powder, granules, elixir, suspension, solution or other
liquid form of the lipase. Oral administration may include buccal
and sublingual administration of the lipase. Other forms of enteral
administration may include methods that directly administer the
lipase to the gastrointestinal tract, such as administering
directly to the stomach by use of a gastric feeding or gastrostomy
tube or placed into the small intestine using a duodenal feeding
tube. For especially small, preterm or week infants such tube-based
forms of administration may be more practical, or may be necessary,
to administer the recombinant human bile-salt-stimulated lipase
according to the instant invention.
[0082] Depending on the particular method of enteral
administration, the formulation in which the recombinant human
bile-salt-stimulated lipase is administered may differ. Liquid
dosage forms for enteral administration of rhBSSL include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the rhBSSL, the
liquid dosage forms may contain inert diluents commonly used in the
art, such as, for example, water or other solvents, solubilizing
agents and emulsifiers, and mixtures thereof. Besides inert
diluents, the compositions for enteral administration can also
include additives such as wetting agents, emulsifying and
suspending agents, bulking agents and stabilizers. Suspensions, in
addition to the active inhibitor(s) of the present invention, may
contain suspending agents.
[0083] Whilst the most suitable means and formulation for enteral
administration to a human infant for any specific circumstance may
differ, a particularly suitable means of administration of the
recombinant human bile-salt-stimulated lipase is to administer said
lipase as part of the regular feed to said human infant, either
orally or by tube-feeding. Accordingly, in a particular embodiment
of the present invention the recombinant human bile-salt-stimulated
lipase is first added to infant formula or to non-fresh (such as
[previously] pasteurized) breast milk which is then fed to said
infant. Feeding of this modified infant formula or modified
non-fresh breast milk to the infant thereby provides enteral
administration of said lipase. This means of administration is of
particular relevance as it provides that the lipids comprised in
the milk-based feed are present at the same time and location in
the gastrointestinal tract as the (co)administered rhBSSL. In a
certain particular embodiment of the invention, the recombinant
human bile-salt-stimulated lipase is (co)administered with infant
formula, such as by being first added to the formula before feeding
said infant. The infant formula may have a composition analogous or
substantially similar to one disclosed elsewhere herein.
[0084] As will be understood by the person of ordinary skill, the
infant formula or (previously) pasteurized breast milk modified by
the addition of recombinant human bile-salt-stimulated lipase will
be commonly fed to said infant by use of a feeding bottle fitted
with an appropriate teat or nipple to simulate the natural nipple
and hence provide more effective feeding. Alternatively, the
modified infant formula or modified non-fresh breast milk may be
fed using other means; for example, by use of a dropper, syringe,
spoon or a soaked-cloth, such as may be required if the infant has
a deformity of the mouth. In certain embodiments, such as with
extremely underweight, preterm or weak infants, the feeding may be
made directly to the gastrointestinal tract via a gastric,
gastrostomy, or duodenal tube.
[0085] In certain embodiments of the invention, the non-fresh
breast milk to which the recombinant human bile-salt-stimulated
lipase is added to pasteurized breast milk. In other embodiments
the breast milk has been frozen, such as after pasteurization. In
particular embodiments, the breast milk used in the instant
invention has come from a breast milk bank. Breast milk banks may
include the National Milk Bank (NMB), a nationwide organization
that collects donated human milk, ensures milk safety and quality
and makes it available for infants in need, or the Human Milk
Banking Association of North America (HMBANA), a non-profit
association of donor human milk banks established in 1985 to set
standards for and to facilitate establishment and operation of milk
banks in North America.
[0086] As will be appreciated by the person of ordinary skill, it
is particularly suitable that the breast milk used in the present
invention is human breast milk. However, in alternative
embodiments, particularly with older infants, the breast milk is
obtained from a domesticated large animal such as a cow, sheep,
goat or horse. Such embodiments may be practiced in certain
cultures or countries that do not always feed human milk or infant
formula, but may feed a human infant (at least partially) with milk
obtained from such an animal. Such milks may not include sufficient
animal BSSL to aid lipase digestion in a human infant--and
certainly will not contain human BSSL--regardless of whether the
milk has been pasteurized. Accordingly, the breast milk, when used
in such an embodiment of the invention, may comprise fresh animal
breast milk, i.e., milk that has not been heat-treated and/or
frozen.
[0087] In yet another alternative embodiment of the present
invention, the recombinant human bile-salt-stimulated lipase is
added to an infant formula. The skilled person will be aware of the
many infant formulae that are commercially available, which
include: Enfamil.TM., Pregestimil.TM. Nutramigen.TM., and
Nutramigen AA.TM. (all marketed or made by Mead Johnson);
Similac.TM., Isomil.TM., Alimentum.TM., and EleCare.TM. (all
marketed or made by Abbott Laboratories, Ross division); Nestle:
12%, the largest producer of formula in the world, makes
GoodStart.TM. (marketed or made by Nestle/Gerber Products Company);
Farex1.TM. and Farex2.TM. (marketed or made by Wockhardt
Nutrition). For preterm infants, other infant formulae such as
Similac.TM., Neosure.TM., Entramil.TM. Premature.TM., Similac.TM.
Special Care, Cow & Gate Nutriprem.TM. 2 and Entramil.TM.
Enfacare.TM. are also available Common to all infant formula is
that they contain a source of lipids that are the substrates to
lipases such as rhBSSL. In a particular embodiment, the infant
formula has the composition (before addition of rhBSSL) generally
in conformance with, or substantially as the specifications shown
in Exhibit A, or as one recommended by the ESPGHAN Coordinated
International Expert Group (Koletzko et al, 2005; J Ped Gastro Nutr
41: 584-599). In certain embodiments, the infant formula contains
one or more of the ingredients, and at approximately the levels,
shown in Exhibit B. In particularly advantageous embodiments, the
infant formula contains at least 0.5% (of total fat) that is DHA
and/or AA, and in further such embodiments where the concentration
of AA should reach at least the concentration of DHA, and/or if
eicosapentaenoic acid (C20:5 n-3) is added its concentration does
not exceed the content of DHA.
[0088] For particular reasons, such as for convenience, safety and
efficient distribution, the recombinant human bile-salt-stimulated
lipase may be added to a bulk amount of (non-fresh) breast milk in
a central location (such as at a milk bank) and then stored and/or
distributed to infants. Analogously, the rhBSSL may be added to a
bulk amount of infant formula at a central location, such as by a
manufacturer of an infant formula, and then packaged and
distributed (for example by being sold) to parents or
care-providers of the human infants. This particular embodiment has
particular utility when the modified formula (including rhBSSL) can
be stored and shipped as a dry powder. Alternatively, and
particularly should an infant-specific dose be desired, the
recombinant human bile-salt-stimulated lipase may be added to the
infant formula or breast milk shortly before feeding and in amounts
sufficient for such feeding, or in a ratio and amounts specific to
that particular infant. For example, an appropriate amount of
rhBSSL may be added to a feed-sized quantity of non-fresh breast
milk or to infant formula.
[0089] A suitable ratio between the amounts of recombinant human
bile-salt-stimulated lipase and the other components in the infant
feed for the present invention lies wherein said lipase is added to
infant formula or (previously) pasteurized and/or frozen breast
milk to a final concentration of between about 0.03 and 0.5 g/L
formula or milk. For example, said lipase may be added to infant
formula or non-fresh breast milk to a final concentration of
between about 0.05 and 0.3 g/L formula or milk. In particular
embodiments the recombinant human bile-salt-stimulated lipase is
added to a final concentration of between about 0.1 and 0.2 g/L
formula or milk, such as around 0.15 g/L formula or milk. As will
appreciated from the description of certain earlier embodiments,
suitable (absolute) concentrations may be adapted to provide a
given concentration of active rhBSSL (suitable amounts being within
those ranges given above), and/or such concentrations may
alternatively be expressed in terms of the (active) molar (or micro
mole) amounts of rhBSSL per unit volume of milk, such as the
resulting molarity (M) of the rhBSSL in said milk, or in terms of
the enzyme activity (U) per unit volume of milk (e.g., U/mL). In
particular embodiments of the invention, the rhBSSL is administered
as between about 15 and 300 units, between about 50 and 150 units
rhBSSL per mL infant formula or milk (U/mL), between about 80 and
90 or about 87 U/mL infant formula or milk.
[0090] In particular embodiments of the present invention, the
human infant is an underweight human infant. The human infant may
be underweight upon birth, such as a Low Birth Weight (LBW) infant
born weighing less than 2,500 g, a Very Low Birth Weight (VLBW)
infant born weighing less than 1,500 g or an Extremely Low Birth
Weight (ELBW) babies, born at less than 1000 g. Alternatively, the
underweight infant may have a low birth mass (one that is below the
average birth weight for a given gestational age) or is small for
gestational age (SGA) (mass is below the 10th percentile of birth
weight for a given gestational age). Alternatively, the infant may
be underweight as it is not growing at a typical rate, such as an
infant that is failing to thrive (FTT).
[0091] Various possible causes for, and the prevalence of, an
infant to be underweight are described elsewhere herein. In
particular, an infant is often underweight because it is born
preterm. While not all preterm infants are underweight, preterm
infants do have not fully developed their pancreas and liver
functions, and can often not thrive as well as full-term babies.
Accordingly, in another particular embodiment of the present
invention, said human infant is a preterm human infant, i.e., one
that is born before the normal pregnancy duration of about 40
weeks, or in particular is one born before about week 37 of
gestation. In certain such embodiments, said preterm human infant
is one born between about week 37 and about week 32 of gestation.
In particular such embodiments, said preterm human infant is one
born between about week 32 and about week 25 of gestation, or one
born between about week 25 and about week 22 or gestation. In other
particular such embodiments, said preterm infant is one born before
about week 37 but after about week 21, week 22 or week 23, of
gestation.
[0092] As will be appreciated by the person ordinarily skilled in
the art, gestational age is commonly calculated by starting to
count from the first day of the mother's last menstrual period
(LMP), although in certain circumstances, such as in-vitro
fertilization, gestational age can be calculated from the date of
conception using a method known as fertilization age, embryonic
age, conceptional age or intrauterine developmental (IUD) age. This
method makes an infant appear about 2 weeks younger than if
gestation was calculated by the more common LMP method.
[0093] In particular embodiments of the present invention said
human infant is between 0 and 200 days of postpartum age. For
example, the first administration of the recombinant human
bile-salt-stimulated lipase may be made upon the day or birth,
within one, two, three, four, five or six days of birth, or up to
about the sixth month after birth. In certain such embodiments said
human infant is less than four weeks of age, such as less than
about three, two or one week of postpartum age upon first
administration of recombinant human bile-salt-stimulated lipase
according to the present invention. In other such embodiments, said
human infant is between about one and two months or age, or is
between about two and four months of age, such as about five months
of age, upon first administration of recombinant human
bile-salt-stimulated lipase according to the present invention.
[0094] Once first administered, in certain embodiments of the
instant invention the recombinant human bile-salt-stimulated lipase
is administered at least once per day (for example with at least
one feed) for more than one day. For example, rhBSSL may be
administered at least once per day according to the instant
invention for a duration lasting at least about 4 days. In certain
such embodiments, the recombinant human bile-salt-stimulated lipase
is administered at least once per day (such as with at least one
feed), for at least around 5 days, such as for a duration lasting
at least around 7 days. In particular such embodiments, the
recombinant human bile-salt-stimulated lipase is administered with
(or as part of) most feeds given to said infant in any given day,
for example between about 4 or 12 feeds per day, such as between
about 4 and 10 feeds per day such as about 6, 7 or 8 feeds per day.
In another non-limiting embodiment, the infant may be sometimes fed
(such as once, twice or three-times per day) without
(co)administration of the recombinant human bile-salt-stimulated
lipase. In alternative such embodiments, the infant is
(co)administered recombinant human bile-salt-stimulated lipase with
every feed given to said infant; i.e., the infant is administered
the rhBSSL for all feeds per day.
[0095] In certain embodiments the administration regimen for
recombinant human bile-salt-stimulated lipase lasts for a period of
time that is at least about one or two weeks. In particular such
embodiments this duration is at least around 3 weeks, such as at
least about 4 weeks. In alternative embodiments of the present
invention, the recombinant human bile-salt-stimulated lipase is
administered, such as part of a course of medical therapy, until
the human infant is transferred out of intensive care, until
discharged from hospital, until no longer under medical care or
supervision or until said infant has obtained a medically
acceptable weight.
[0096] As will be appreciated by the person of ordinary skill,
growth of a human infant may be monitored by any common or
acceptable method, in order to investigate, monitor, follow and/or
check for an increase, or otherwise an improvement or enhancement,
of growth velocity. For example, the growth velocity of a human
infant is, or may be monitored, for the purposes of the present
invention by regular measurement and recording (such as daily) of
head circumference, body mass (weight), body-length or leg length
(such as knee-to-heel length). Other methods of measuring size
and/or growth of a human infant are generally known. Such regular
measurements can readily be converted to growth velocity; i.e., an
amount of growth in a unit period (such as per day). In certain
embodiments of the present invention, said increase in growth
velocity of the human infant is, or is measured as (or otherwise
monitored as), an increase in the rate of weight gain of said
infant, such as a growth rate expressed as grams per day, a growth
rate expressed as grams per Kg body weight per day (g/Kg/day), a
growth rate expressed as grams per day per 100 Kcal energy consumed
(g/day/100 kcal), or a growth rate expressed as grams per day per
100 mL milk/formula consumed (g/day/100 mL). Measuring body mass
(weight) is a particular convenient method to monitor growth of an
infant, and such second method of expressing growth rate (g/Kg/day)
has particular utility as it seeks to normalize the absolute growth
rate for different sized infants, as larger infants typically
increase in weight by a larger absolute amount than smaller infants
over the same period. Accordingly, in certain such embodiments, the
rate of weight gain achieved by, observed in or desired from said
human infant when administered rhBSSL is between about 10 and 30 g
increase in weight per Kg body weight of said infant per day
(g/Kg/day). In particular such embodiments such rate of weight gain
is between about 15 and 25 g/Kg/day, such as about 20 g/Kg/day or
about 18 g/Kg/day.
[0097] In other embodiments of the present invention, the increase
in growth velocity in the human infant administered recombinant
human bile-salt-stimulated lipase is a weight gain that is between
1 g/Kg/day and 8 g/Kg/day, such as about 2, 3, 4 or 5 g/Kg/day
greater than a human infant not administered rhBSSL. In an
alternative embodiment of the invention, the increase in growth
velocity is a weight gain that is between about 5% and 40% greater
than the value of the growth velocity of a human infant not
administered rhBSSL, such as between about 10% and 30% greater or
15% and 25% greater, including about 20% greater.
[0098] As will be appreciated, the weight of a human infant may
fluctuate from day-to-day for various reasons, including those
unrelated to administration of rhBSSL. Accordingly, the growth
velocity stated herein as a per-day amount (or relative or
percentage) may not be achieved by, observed in or desired from
said human infant each and every day, and may only be so achieved
by, observed in or desired from if measured and estimated over a
number of days, such as over 3, 5 or 7 days, or for longer periods
such as two, three or four weeks, or for example, over the period
the infant during which the infant is being administered rhBSSL or
receiving medical care such as within an NICU.
[0099] In other embodiments of the present invention, an increase
in growth is measured (or otherwise monitored) as an increase in
leg length; for example an increase in knee-to-heel length, as may
be expressed as mm growth in a unit period, such as a week. In yet
another embodiment of the present invention, the growth velocity of
the human infant is monitored relative to its own size such as by
use of the infant's Weight-for-Height percentage (W/H %) or
Standard Deviation (SD) score (also known as Z-score) which enables
an infant's growth to be monitored with reference to the Global
Database on Child Growth and Malnutrition of the WHO.
[0100] As described elsewhere herein, the inventors observed that
the present invention--as exemplified by two controlled clinical
trials and an analysis of combined data from these two
trials--resulted in an increase in growth rate of human infants
administered recombinant bile-salt-stimulated lipase, while
observing only a limited increase in the overall absorption
coefficient of (i.e., all or the most abundant) fatty acids, as
measured by overall CFA (coefficient of fat absorption). As set out
in more detail within the Exemplification below, infants in the
per-protocol data-set (PP) showed a statistically significant
increase in growth velocity upon administration of rhBSSL compared
to placebo (LS mean difference of 2.08 g/Kg/day; p=0.019) but with
a less pronounced and non-significant increase in overall CFA (LS
mean difference of 3.56%; p=0.069). In terms of relative (%)
increases of the effects (in the PP data set) compared to the LS
mean effects for placebo, administration of rhBSSL increased growth
velocity by 13.8% (17.15 compared to 15.06 g/Kg/day), but only
increased overall CFA by 5.4% (69.06 compared to 65.50% CFA). Such
an observation was more pronounced in the subset of infants fed
with infant formula (PP); showing a high and statistically
significant increase in growth velocity upon administration of
rhBSSL compared to placebo (LS mean difference of 2.30 g/Kg/day;
p=0.038) but with little concomitant (and non-significant) increase
in overall CFA (LS mean difference of 2.08%; p=0.462); and the
relative (%) increase compared to the LS mean effects for placebo,
upon administration of rhBSSL for formula-fed infant increased
growth velocity by an increase of 14.9% (17.75 compared to 15.45
g/Kg/day), but with only an increase in overall CFA of 3.1% (69.46
compared to 67.38% CFA). Furthermore, and also set out in more
detail within the Exemplification herein, there was very little
(non-significant) correlation between intra-individual differences
in growth velocity (rhBSSL-placebo) of individual infants vs their
corresponding difference in overall CFA (R.sup.2 linear=0.041;
p=0.177), with little of the variance observed in intra-individual
differences in growth velocity accounted for by variance in the
corresponding individuals' increase in overall CFA values (ANOVA
following linear regression). Other analysis approaches or
methodologies may be used to further investigate and/or present
results from the two clinical trials disclosed herein, including
analysis approaches or methodologies that investigate and/or
present results related to the limited concomitance between an
increase in growth velocity and an increase in overall CFA for
infants administered recombinant bile-salt-stimulated lipase.
[0101] Methodologies to measure growth velocity are disclosed
elsewhere herein. Fat absorption may be investigated, monitored or
observed by various means known in the art. For example, by
inspection of the fat-balance between fat-input and fat-excretion
of total fatty acid quantified through the use of gravimetric
analysis of fatty acids, such as used by Andersson & coworkers
(2007). Alternatively, quantification of individual fatty acids may
be conducted using gas chromatographic methods such as described in
the Exemplification herein. Sidisky & coworkers (1996; The
Reporter [Supelco/Sigma-Aldrich], 15(1):1-4) describe the
properties of various capillary columns to aid the selection of
appropriate columns to separate and hence detect key fatty acid
methyl esters. The degree of fat absorption may be quantitatively
expressed as a coefficient of fat absorption (CFA) for any
individual, sub-group of similar or related fatty acids, or for
all/overall fatty acids by appropriate summing of values for
individual fatty acids such as is described in more detail in the
Exemplification below. As a further example of methodology, for an
individual human infant (or group thereof), an improvement in fatty
acid absorption, such as the absorption of DHA or AA, may be
investigated, monitored, followed and/or checked, for example by
analysis of the absolute or relative fatty-acid content, over time
or during treatment, of plasma or red blood cell membrane
phospholipids (Carlson et al, 1996; Pediatr Res, 39: 882-888; Boehm
et al, 1996; Eur J Pediatr 155: 410-416), including the use of
chromatographic (GC) separation of individual fatty acids followed
by identification/quantification for example by using mass
spectrometry.
[0102] Also of note from clinical trials disclosed herein is that
despite the average increase in growth velocity being comparable
with other infant growth studies (for example, see Andersson et al,
2007), the mean overall CFA values observed are lower (mean overall
CFA in the PP data set: 69.08% for rhBSSL and 65.66% for placebo)
than those that have generally been observed in other infant CFA
studies (for review, see Lindquist and Hernell, 2010). However, the
variation in overall CFA values for individual infants (Standard
Deviation in the PP of 14.68% for rhBSSL, 16.13% for placebo and
13.19% for the intra-individual difference) generally conformed to
those values generally observed in other infant CFA studies
(Williamson et al, 1978; Morgan et al, 1998; Acta Paediatr 87:
318-324; Andersson et al, 2007). BSSL is known as a broad spectrum
lipase that can hydrolyze many kinds of lipids and lipid-like
molecules (for review, see Lindquist and Hernell, 2010), and since
over half of the energy available to an infant comes from
hydrolyzed lipids contained in milk, it may have been expected by
the person of ordinary skill in the art that the most striking
result would have been an increase in overall CFA--and that any
increase in growth velocity would not be as striking as (since it
would have been expected to strongly depend upon) an increase in
overall CFA.
[0103] Accordingly, in certain embodiments of the present
invention, the increase in growth velocity that is achieved,
observed or desired in said human infant, is so achieved, observed
or desired without observing and/or achieving a concomitant
increase in the overall coefficient of fat absorption (i.e., for
all or the most abundant fatty acids) in said infant. In particular
such embodiments of the invention, said increase in growth velocity
is not concomitant with, indicated by and/or correlated to an
increase in the overall coefficient of fat absorption (i.e., for
all or the most abundant fatty acids). In other particular such
embodiments, the increase in growth velocity is not fully
explainable by (or caused by) an increase in overall CFA. For
example, the increase in overall CFA may be less than that which
can account for, such as energetically, calorifically, numerically
(such as by percentage increases) or statistically account for, the
increase in growth velocity.
[0104] In other embodiments of the present invention, any
difference in overall CFA for the human infant administered
recombinant human bile-salt-stimulated lipase (i.e., all or the
most abundant fatty acids) is less than about 5% percentage-points
greater than, such as less than about 4%, 3%, 2% or 1%
percentage-points greater than any increase in absolute overall CFA
for a human infant not administered rhBSSL. In an alternative
embodiment of the invention, any relative increase in overall CFA
for the human infant administered rhBSSL is a value that is less
than about 106% of the CFA value of a human infant not administered
rhBSSL, such as less than about 105%, 104%, 103%, 102% or 101% of
the absolute CFA value of a human infant not administered
rhBSSL.
[0105] In another particular embodiment of the invention, the
relative increase (such as a percentage increase) in growth
velocity of the human infant administered recombinant human
bile-salt-stimulated lipase compared to the growth velocity of a
human infant not administered rhBSSL is greater than the relative
increase (such as a percentage increase) of overall CFA (i.e., all
or the most abundant fatty acids) of the human infant administered
rhBSSL compared to the overall CFA of a human infant not
administered rhBSSL. In certain of such embodiments, the relative
increase in growth velocity of the human infant administered rhBSSL
(compared to that of an infant not administered rhBSSL) is about
10-fold, such as about 5-fold, 3-fold or 2-fold greater than that
of the relative increase in overall CFA (i.e., all or the most
abundant fatty acids) of the human infant administered rhBSSL
(compared to that of an infant not administered rhBSSL). For
example, in a specific non-limiting example, the relative increase
in growth velocity of the human infant administered rhBSSL may be
about 15% (compared to the absolute growth velocity of an infant
not administered rhBSSL), but the relative increase in overall CFA
in the infant administered rhBSSL may be only about 3% (compared to
the absolute CFA of an infant not administered rhBSSL).
[0106] As described elsewhere herein, a common pathological
condition suffered by preterm and/or LBW infants is transmucosal
necrosis (NEC), which is characterized by mucosal and transmucosal
necrosis and inflammation, usually involving the terminal ileum or
colon. The inflammation and loss of mucosal integrity is often
accompanied by rupture of the intestinal wall and sepsis. Despite
advances in the diagnosis and treatment of this disease, NEC
remains a major cause of morbidity and mortality in nurseries
caring for premature and LBW infants (Foglia et al, 1995; Curr
Probl Surg, 32: 757-823; Grosfeld et al, 1996; Surgery, 120:
650-656; Uceda et al, J Pediatr Surg, 30: 1314-1316).
[0107] There have been experimental suggestions that the
significance of milk-BSSL in infants may be not just to aid
absorption of fatty acids. For example, Miller & Lowe (2008; J
Nutr 138: 927-930) observed that in CEL-(BSSL) deficient mice, only
the absence of both mother's milk and pancreatic CEL (BSSL)
produces at malabsorption; the absence of only mother's milk CEL
(BSSL) did not affect the efficacy of dietary fat absorption, and
that even with increased fecal fats, the CEL-(BSSL) deficient mouse
pups had normal weight gain. Also, and in particular, Howles and
coworkers (1999; Am J Physiol, 277: G653-G661) have
speculated--following experiments using CEL-(BSSL) deficient
mice--that CEL (BSSL) may prevent fat-derived intestinal injury in
neonatal mice, in particular due to the accumulation of excess
lipid in the epithelium of the distal small intestine (see also,
Lindquist et al, 2007; J Pediatr Gastroenterol Nutr 44: E335, as
reported by Lindquist & Hernell, 2010).
[0108] Dietary fat has been shown to influence both the morphology
and the function of the intestinal epithelium. Several
investigators have found that the type and amount of lipid in
formulas and milk significantly affect the transport properties and
permeability of the neonatal intestinal epithelium and the rate at
which it proliferates and matures (Neu et al, 1987; Pediatr Res 22:
330-334; Udall et al, 1981; Pediatr Res. 15: 245-249; Weaver eta 1,
1987; Pediatr Res, 22: 675-678). In a neonatal pig model of
transmucosal necrosis, Crissinger & co-workers (Crissinger et
al, 1994; Gastroenterology, 106: 1215-1222) showed that the degree
of permeability and intestinal damage directly relates to the
presence and type of fat in various neonatal formulas. Luminal
lipids were also found to exacerbate intestinal injury in a rat
model of NEC (Bhatia et al, 1996; J Surg Res 63: 152-156).
[0109] Indeed, it was observed in the clinical trials disclosed
herein that infants (in the PP data set) fed with infant formula
were exposed to a larger amount of total fat (and excreted more fat
in their stools) between the food tracer markers of each treatment
period (mean total fat exposure: 29.12 g fat for rhBSSL and 28.50 g
fat for placebo) compared to the infants fed with breast milk
(19.00 g fat for rhBSSL and 20.51 g fat for placebo) [figures not
corrected for any differences in body weight].
[0110] In an alternative aspect therefore, the invention also
relates to a method to protect the small bowel mucosa of a human
infant from damage, said method comprising the step of enteral
administration of recombinant human bile-salt-stimulated lipase to
said infant. In a related aspect, the invention also relates to a
method to protect an immature intestinal epithelium of a human
infant from the deleterious effects of incompletely digested and/or
excess fat and/or lipid, said method comprising the step of enteral
administration of recombinant human bile-salt-stimulated lipase to
said infant. In another related aspect, the invention relates to a
method to limit accumulation of incompletely digested and/or excess
fat and/or lipid in the ileum of a human infant, said method
comprising the step of enteral administration of recombinant human
bile-salt-stimulated lipase to said infant. In certain embodiments
of these aspects, said human infant is a preterm infant, is fed by
enteral or gastric tube and/or is fed infant formula.
[0111] The term "protect", in the context of these aspects of the
present invention will be understood by the person of ordinary
skill, and includes embodiments of these aspects of the present
invention wherein the administration of said lipase prevents or
reduces the likelihood, severity and/or prevalence of the undesired
effects in said infant. The protective effects of administration of
recombinant human bile-salt-stimulated lipase, or its effect to
limit the accumulation of incompletely digested and/or excess fat
and/or lipid, may be further supported from results of additional
clinical studies in human infants. For example, the prevalence, or
mortality caused by, intestinal damage to infants--such as the
prevalence, or mortality caused by necrotizing enterocolitis or
ileal perforation--may be investigated by analysis of safety data
from clinical trials that administer rhBSSL to human infants. In
other clinical studies, such effects upon administration of rhBSSL
may be further investigated by endoscopic, biopsy or postmortem
analysis of human infants. In alternative, and less invasive,
methodologies, these advantageous effects of rhBSSL may be
investigated by following an indicative biomarker, for example one
that may be present in the blood or plasma of the human infant, and
which the presence/absence or level/concentration of said biomarker
correlates with the start, diagnosis, presence or severity of one
or more of the deleterious effects listed herein. The start,
diagnosis, presence and/or severity of such deleterious effects may
also be monitored using other medical techniques, such as
manipulation of the abdominal region of the infant, body
temperature and/or the behavior/sleep patterns of the infant.
Necrotizing enterocolitis, in particular, may be diagnosed and/or
evaluated--either as part of a clinical trial, or as part of a
medical examination--by abdominal radiograph or using Bell's
necrotizing enterocolitis staging system (Bell et al, 1978; Ann
Surg, 187:1-7). Alternatively, stool patterns, presence of occult
blood or presence of specific pathogens may be used as indicators
of NEC risk, or gastric residuals may be used as a predictor of
necrotizing enterocolitis.
[0112] The disclosure by the inventors herein--that in clinical
trials that administer recombinant human bile-salt-stimulated
lipase to human infants, the growth velocity of said infants was
more pronounced than the increase in the overall coefficient of fat
absorption (i.e., all or the most abundant fatty acids)--suggested
that mechanisms additional to that of lipid digestion by rhBSSL may
be factors to explain such results. The human infants fed infant
formula were exposed to an increased amount of total fat compared
to those fed with breast milk, and the disconcordance between an
increase in their growth velocity and any increase in their overall
CFA upon administration of rhBSSL was more pronounced. As may be
supported by the indirect evidence provided from these data of the
clinical trials (particularly in those infants ingesting more fat
from infant formula), administration of recombinant human
bile-salt-stimulated lipase to human infants may enable more lipids
to be digested--resulting in less incompletely digested and/or
excess fat/lipid, and hence lead to less damage to their lower
intestines, such as to their microvilli thereof, caused by
incompletely digested and/or excess fat/lipids. An intestine that
is less damaged will be more able to absorb nutrients from the
infant food other than fatty acids, such as carbohydrates, proteins
and their digestion products including monosaccharides and amino
acids. Carbohydrates and proteins, which as well as fats also
provide a substantial portion of the energy requirements to a human
infant, may hence be more readily absorbed after digestion and thus
such infants may show increased growth (for example, due to a
generally more healthy lower intestine) that is not explainable
only by an increase in the digestion of TG by rhBSSL and the
absorption of the resulting fatty acids.
[0113] In certain embodiments of these aspects of the present
invention, administration of recombinant human bile-salt-stimulated
lipase to said infant protects the small bowel mucosa from the
deleterious effects of incompletely digested and/or excess fat
and/or lipid, such that derived from an infant formula and/or from
pasteurized breast milk.
[0114] In other certain embodiments of the invention, said human
infant is fed infant formula and/or pasteurized breast milk, and in
certain such embodiments the infant is not fed fresh mother's
breast milk.
[0115] In particular embodiments of these aspects of the present
invention, administration of the recombinant human
bile-salt-stimulated lipase protects the mucosa of the jejunum
and/or the ileum of said infant, and in particular protects the
infant's villus epithelium from damage.
[0116] In more specific embodiments of the invention,
administration of the recombinant human bile-salt-stimulated lipase
protects the small bowel mucosa of said infant from mucosal and/or
transmucosal necrosis and/or inflammation.
[0117] In a more particular embodiment, the administration of said
lipase protects said infant from certain pathological conditions
such as necrotizing enterocolitis and/or ileal perforation. In
certain of such embodiments, the administration of rhBSSL prevents
or reduces the likelihood, severity and/or prevalence of
necrotizing enterocolitis and/or ileal perforation in said
infant.
[0118] Consistently predisposing factors for NEC are prematurity,
LBW and enteral feeding, suggesting that the immature intestinal
mucosae of these infants are unable to withstand the stress
associated with processing a complex diet (Kliegman et al, 1993;
Pediatr Res 34: 701-708). Accordingly, in certain embodiments of
these aspects of the invention (and also for certain embodiments of
the other aspects of the invention), the human infant is fed by
enteral (or gastric) tube. In particular, the infant fed by enteral
(or gastric) tube may be an infant born premature and/or LBW.
[0119] As will now be readily apparent to the person of ordinary
skill, one or more of any of the embodiments described earlier--for
example those describing the various recombinant human
bile-salt-stimulated lipases, dosage amounts, administration modes
and/or regimens, infant sub-populations, and also that
administration with rhBSSL can result in an increase of growth
velocity and a limited increase in overall CFA--may also further
characterize these protective (or accumulation-limiting) methods of
the present invention. For example, such protective (or
accumulation-limiting) methods may use a rhBSSL isolated from an
expression product of a recombinant hamster ovary cell, and/or may
be administered in an amount per day of between 1 and 100 mg of
said lipase per Kg weight of infant, such as administered in an
infant formula to a preterm infant born before about week 37 of
gestation.
[0120] In certain embodiments of the various aspects of the present
invention, the recombinant human bile-salt-stimulated lipase is
administered prior to, after or concomitantly with at least one
other food supplement and/or milk fortifier. Several such food
supplements or milk fortifiers are approved, sold or otherwise used
to help increase the growth of, or otherwise benefit, human infants
and will be well known to the skilled person. By way of non-liming
example, such food supplements and/or milk fortifiers include:
Nutriprem.TM., Milupa.TM., Eoprotin.TM., Enfamil.TM. Human Milk
Fortifier and Similac.TM. Human Milk Fortifier In certain other
embodiments of the present invention, the recombinant human
bile-salt-stimulated lipase is administered prior to, after or
concomitantly with at least one other lipase, such as another
recombinant human lipase.
[0121] In alternative embodiments, the recombinant human
bile-salt-stimulated lipase is administered without administration
of additional food supplements and/or milk fortifiers (such as
those described or defined herein), or without administration of
any other lipase.
[0122] As will be appreciated, the relative ease at which the
present invention may be practiced--in one embodiment
administration merely by addition of the recombinant human
bile-salt-stimulated lipase to an infant formula for oral feeding
to the human infant--lends the invention to be practiced at the
infant's home without medical intervention, supervision, support or
advice. For example, the recombinant human bile-salt-stimulated
lipase may be generally sold as a dietary supplement to aid the
growth or general health of babies, such as to increase the growth
rate of an infant where it is culturally desirable for the infant
to reach its own growth potential, for protection from damage to
the small bowel mucosa or for limiting the accumulation of
incomplete digested and/or excess fat/lipid. As a further
non-limiting example of an embodiment of the present invention, an
infant formula may be manufactured and distributed for domestic use
that already includes an appropriate amount of rhBSSL. Accordingly,
in a certain aspect the invention relates to a non-medical method
to increase the growth velocity of a human infant, or relates to
one of the protective (or accumulation-limiting) methods described
above.
[0123] Alternatively, the present invention may be practiced, or
instructed to be practiced, by qualified medical staff, or
otherwise under or with medical intervention, supervision or
advice, such as in a hospital or medical clinical, for example in
an intensive care unit caring for underweight, LBW and/or preterm
human infants. Accordingly, in such an alternative aspect, the
method relates to a medical method to increase the growth velocity
of a human infant, or relates to a medical method of one of the
protective (or accumulation-limiting) methods described above. In
such aspect, the infant may be in medical need of increased growth
velocity, such as body weight, in need or protection from damage to
the small bowel mucosa or in need of limiting the accumulation of
incompletely digested or excess fat/lipid, and the amount of
recombinant human bile-salt-stimulated lipase may be a
therapeutically effective amount.
[0124] In a further aspect related to that above, the present
invention therefore also relates to a therapeutic method to treat a
human infant suffering from underweight or premature birth, said
method comprising the step of enteral administration of recombinant
human bile-salt-stimulated lipase to an infant in medical need
thereof. Infants in particular need of such medical intervention
may be small for gestational age (SGA), Low Birth Weight (LBW)
infants, those suffering from a failure to thrive (FTT) and/or
infants born before about week 37 of gestation; in each case as
described or defined elsewhere herein. Alternatively, a further
aspect of the present invention also relates to one or more of the
therapeutic methods to: (X) protect the small bowel mucosa of a
human infant from damage; to (Y) protect an immature intestinal
epithelium of a human infant from the deleterious effects of
incompletely digested and/or excess fat and/or lipid; and/or to (Z)
limit accumulation of incompletely digested and/or excess fat
and/or lipid in the ileum of a human infant; in each case said
methods comprising the step of enteral administration of
recombinant human bile salt stimulated lipase to an infant in
medical need thereof.
[0125] To practice certain embodiments of the present invention, it
may be useful to first prepare an infant feed that contains
recombinant human bile-salt-stimulated lipase. Accordingly, in
another aspect the present invention relates to a method of
preparing a modified infant formula or modified pasteurized breast
milk comprising rhBSSL, said modified infant formula or modified
pasteurized breast milk useful for increasing the growth velocity
of a human infant. In particular embodiments such method comprises
the steps of:
[0126] i. providing a first quantity of recombinant human
bile-salt-stimulated lipase and a second quantity of an unmodified
infant formula or unmodified pasteurized breast milk; and
[0127] ii. adding an amount of said lipase to said unmodified
infant formula or unmodified pasteurized breast milk so as to form
a modified infant formula or modified pasteurized breast milk,
[0128] so as to form a modified infant formula or modified
pasteurized breast milk that includes an amount of lipase effective
to increase the growth velocity of a human infant when said
modified infant formula or modified pasteurized breast milk is fed
to said infant over an administration regimen as described or
defined elsewhere herein.
[0129] In an alternative aspect, the present invention also relates
to analogous methods to the method above, where the modified infant
formula or modified breast milk is useful to, and the amount of
rhBSSL therein is effective to, respectively: (X) protect the small
bowel mucosa of a human infant from damage; to (Y) protect an
immature intestinal epithelium of a human infant from the
deleterious effects of incompletely digested and/or excess fat
and/or lipid; and/or to (Z) limit accumulation of incompletely
digested and/or excess fat and/or lipid in the ileum of a human
infant.
[0130] In certain embodiments of the present invention, the
recombinant human bile-salt-stimulated lipase is provided in a form
that is suitable for storage, distribution and/or incorporation
into the modified infant formula or modified milk of the present
invention. For example, in certain embodiments said lipase is
provided as a lyophilized formulation. Typically, the lyophilized
formulation of said lipase will be provided in a conveniently sized
container such as in a vial, and may comprise an appropriate
quantity of recombinant human bile-salt-stimulated lipase. In
certain such embodiments the container is a sterile container,
including being a sterile vial. When provided as a lyophilized
formulation, the rhBSSL may be solubilized, such as with sterile
water, prior to addition to the infant formula or milk, or
alternatively the lyophilized formulation of rhBSSL may be
solubilized directly in said infant formula or milk.
[0131] For convenience or other reasons, such as for sterility or
safety, in certain embodiments of the present invention the
recombinant human bile-salt-stimulated lipase is provided as a unit
dose. A unit dose may provide sufficient (or slightly more) rhBSSL
as is required for a single administration in a discrete unit or
container. Alternatively, a small number of such discrete units or
containers together, such as between 2 and 5 such discrete units or
containers, provides sufficient (or slightly more) rhBSSL as is
required for a single administration. In certain such embodiments,
the unit dose form comprises an amount of recombinant human
bile-salt-stimulated lipase that is between 1.5 and 75 mg lipase.
In particular such embodiments the amount of rhBSSL is between 5
and 45 mg, or about 20 mg of said lipase.
[0132] In another embodiment, the recombinant human
bile-salt-stimulated lipase is provided as a solution. The
concentration of rhBSSL in such solution may be between 1.5 and 150
mg/mL, and in certain such embodiments may be at a concentration of
between 7.5 and 30 mg/mL, such as at a concentration of about 15
mg/mL.
[0133] In particular embodiments of the present invention, the
recombinant human bile-salt-stimulated lipase is provided as a
composition or as a pharmaceutical formulation, such as a
lyophilized or solution composition, that includes one or more
pharmaceutically acceptable carriers as well as the rhBSSL.
Suitable pharmaceutically acceptable carriers, if required, will be
known the person of ordinary skill and include those described
elsewhere herein.
[0134] In particular embodiments of this aspect of the invention,
this method of preparing a modified infant formula or modified
pasteurized breast milk comprising rhBSSL includes the further step
of feeding said modified infant formula or modified pasteurized
breast milk (so prepared by the method) to a human infant, thereby
administering the rhBSSL to said infant by enteral administration.
In certain such embodiments said infant is fed the modified infant
formula or modified pasteurized breast milk over an administration
regimen as described or defined elsewhere herein.
[0135] The inventors have disclosed herein that an infant feed
(formula or pasteurized breast milk) including rhBSSL shows, under
controlled clinical trial conditions, the effect of increasing the
growth velocity of a human infant. Accordingly, in another aspect
the instant invention relates to a modified infant formula that
includes recombinant human bile-salt-stimulated lipase in an amount
effective to increase the growth velocity of a human infant; for
example, when said modified infant formula is fed to said infant
over an administration regimen described or defined elsewhere
herein.
[0136] In certain embodiments of such aspect, the modified infant
formula is already prepared for feeding. In other embodiments, the
modified infant formula is subjected to processing before being fed
to said infant. For example, the formula may be dissolved in water
and/or warmed to an appropriate temperature for feeding such as
37.degree. C. In particular such embodiments the modified infant
formula is provided as a power or granules, or as a ready-to-use
liquid or as a concentrated suspension or solution.
[0137] In an analogous aspect, the instant invention also relates
to a modified pasteurized breast milk that includes recombinant
human bile-salt-stimulated lipase in an amount effective to
increase the growth velocity of a human infant; for example when
said modified pasteurized breast milk is fed to said infant for at
least one feed per day over at least around 4 days, for at least
one feed per day over an administration regimen as described or
defined elsewhere herein.
[0138] In certain embodiments of such aspect, the modified breast
milk is already prepared for feeding. In other embodiments, the
modified breast milk is subjected to processing before being fed to
said infant. For example, the modified breast milk may be thawed
from a frozen state and/or warmed to an appropriate temperature for
feeding such as 37.degree. C.
[0139] In other aspects, the present invention also relates to: a
modified pasteurized breast milk that includes recombinant human
bile-salt-stimulated lipase; a modified infant formula that
includes recombinant human bile-salt-stimulated lipase; a
pharmaceutical composition that includes recombinant human
bile-salt-stimulated lipase; or relates to recombinant human
bile-salt-stimulated lipase per-se; in each case for use in: (X)
protection of the small bowel mucosa of a human infant from damage;
in (Y) protection of an immature intestinal epithelium of a human
infant from the deleterious effects of incompletely digested and/or
excess fat and/or lipid; and/or for use in (Z) limitation of
accumulation of incompletely digested and/or excess fat and/or
lipid in the ileum of a human infant. Particular embodiments of
such aspects are in each case for use in: (V) protection of the
small bowel mucosa of said infant from mucosal and/or transmucosal
necrosis and/or inflammation; and/or for use in (W) protection of a
human infant from necrotizing enterocolitis and/or ileal
perforation, and/or prevention of or reduction of the likelihood,
severity and/or prevalence of necrotizing enterocolitis and/or
ileal perforation in said infant. In certain embodiments of such
aspects, the recombinant human bile-salt-stimulated lipase is
adapted for enteral administration to said infant. In further
certain embodiments of all such aspects, the human infant is fed by
enteral (or gastric) tube and/or is born premature.
[0140] In another aspect, the present invention also relates to a
use of recombinant human bile-salt-stimulated lipase for the
manufacture of a medicament for: (X) protection of the small bowel
mucosa of a human infant from damage; for (Y) protection of an
immature intestinal epithelium of a human infant from the
deleterious effects of incompletely digested and/or excess fat
and/or lipid; and/or for (Z) limitation of accumulation of
incompletely digested and/or excess fat and/or lipid in the ileum
of a human infant. Particular embodiments of such aspects are in
each case for: (V) protection of the small bowel mucosa of said
infant from mucosal and/or transmucosal necrosis and/or
inflammation; and/or for (W) protection of a human infant from
necrotizing enterocolitis and/or ileal perforation, and/or
prevention of or reduction of the likelihood, severity and/or
prevalence of necrotizing enterocolitis and/or ileal perforation in
said infant. In a certain embodiment of such use aspect, the
recombinant human bile-salt-stimulated lipase is adapted for
enteral administration to said infant. In further certain
embodiments of all such aspect, the human infant is fed by enteral
(or gastric) tube and/or is born premature.
[0141] As will now be readily apparent to the person of ordinary
skill from the disclosure above, one or more of any of the
embodiments described earlier--for example those describing the
various recombinant human bile-salt-stimulated lipase, dosage
amounts, administration modes and/or regimens, infant
sub-populations, and also that administration with rhBSSL can
result in an increase of growth velocity and a limited increase in
overall CFA--may also further characterize these composition and/or
use aspects of the present invention. For example, such composition
and/or use may use a rhBSSL isolated from an expression product of
a recombinant hamster ovary cell, and/or may be administered in an
amount per day of between 1 and 100 mg of said lipase per Kg weight
of infant, such as administered in an infant formula to a preterm
infant born before about week 37 of gestation.
[0142] A particularly practical aspect of the instant invention
relates to a kit for the preparation of a modified infant formula
or modified breast milk that comprises rhBSSL, said modified infant
formula or modified pasteurized breast milk useful for increasing
the growth velocity of a human infant. In certain embodiments said
kit comprises the components:
[0143] a. at least one first container that includes a first amount
of recombinant human bile-salt-stimulated lipase, such as in a
lyophilized or solution formulation; and
[0144] b. at least one second container, which is distinct from the
first container, that includes a second amount of unmodified infant
formula or unmodified pasteurized breast milk,
[0145] where said lipase and said unmodified infant formula, or
unmodified pasteurized breast milk, are each in an amount
sufficient to prepare a modified infant formula or modified
pasteurized breast milk, respectively, that includes an amount of
said lipase effective to increase the growth velocity of a human
infant; for example when said modified infant formula or modified
pasteurized breast milk is fed to said infant over an
administration regimen as described or defined elsewhere
herein.
[0146] In an alternative aspect, the present invention also relates
to analogous kits to the above kit, where the modified infant
formula or modified breast milk is useful to, and the amount of
rhBSSL therein is effective to, respectively: (X) protect the small
bowel mucosa of a human infant from damage; to (Y) protect an
immature intestinal epithelium of a human infant from the
deleterious effects of incompletely digested and/or excess fat
and/or lipid; and/or to (Z) limit accumulation of incompletely
digested and/or excess fat and/or lipid in the ileum of a human
infant.
[0147] In certain embodiments, the kit further comprises
instructions. Such instructions may describe how to use the kit or
particular components of said kit. For example, said instructions
may describe the steps of:
[0148] i. preparing a modified infant formula or modified
pasteurized breast milk, such as by adding an amount of recombinant
human bile-salt-stimulated lipase to an unmodified infant formula
or unmodified pasteurized breast milk so as to form a modified
infant formula or modified pasteurized breast milk,
respectively;
[0149] ii. feeding said modified infant formula or modified
pasteurized breast milk to a human infant; for example over an
administration regimen as described or defined elsewhere
herein.
[0150] In other embodiments of this aspect, said instructions
further describe that the human infant to be administered
recombinant human bile-salt-stimulated lipase is an underweight or
LBW infant, one that was born premature and/or one that is fed by
enteral (or gastric) tube. For example, an infant may be one that
falls under any of the underweight or premature classes described
or defined elsewhere herein.
[0151] In another aspect, the instant invention relates to a method
to increase the growth velocity of a human infant, said method
comprising the steps of:
[0152] i. preparing or otherwise providing a modified infant
formula or a modified pasteurized breast milk in each case
comprising rhBSSL or as prepared by the method or by using the kit
above;
[0153] ii. feeding the modified infant formula or modified
pasteurized breast milk so prepared or otherwise provided to said
infant; and
[0154] iii. repeating the preceding steps over an administration
regimen as described or defined herein.
[0155] In an alternative aspect, the present invention also relates
to a method to: (X) protect the small bowel mucosa of a human
infant from damage; to (Y) protect an immature intestinal
epithelium of a human infant from the deleterious effects of
incompletely digested and/or excess fat and/or lipid; and/or to (Z)
limit accumulation of incompletely digested and/or excess fat
and/or lipid in the ileum of a human infant; in each case said
method comprising the three steps set out in the preceding
method.
[0156] Of particular utility for the medical or therapeutic
applications provided herein by the present invention is a yet
further aspect that relates to a pharmaceutical composition in a
unit dose that includes between 0.1 and 100 mg of recombinant human
bile-salt-stimulated lipase. A unit dose will be readily understood
by the person skilled in the art, and includes for example, those
described or defined elsewhere herein. In certain embodiments of
such aspect, the unit dose includes between 1.5 and 75 mg of
rhBSSL. In particular such embodiments, the unit dose includes
between 5 and 45 mg of said rhBSSL, such as about 10, 15, 20 or 25
mg of said lipase.
[0157] As will be appreciated from the discussion on enzyme amounts
above, in certain embodiments of the invention, the unit dose of
recombinant human bile-salt-stimulated lipase may be expressed in
various ways, including in terms of the absolute mass of rhBSSL, or
in terms of the mass of active rhBSSL. Alternatively (or in
addition), the amount of rhBSSL may be expressed in terms of units
(U) of enzyme. Accordingly, in particular embodiments the unit dose
includes an amount of between about 2,000 and 20,000 units of
rhBSSL (U), between about 5,000 and about 15,000, such as between
about 7,000 and 10,000 units of rhBSSL.
[0158] In certain embodiments of the present invention, the
pharmaceutical composition is adapted for enteral administration,
and/or for administration to a human infant, such as wherein said
unit dose is specifically adapted for enteral administration to a
human infant. For example, said unit dose is a lyophilized,
solubilized or frozen amount of recombinant human
bile-salt-stimulated lipase in an amount and/or formulation
suitable for addition to or preparation as an infant formula or
breast milk feed. In other embodiments, the unit dose may be
provided in a form, container or amount of rhBSSL as described or
defined elsewhere herein.
[0159] In another particular aspect, the invention also relates to
a pharmaceutical composition that includes between 0.1 and 100 mg
of recombinant human bile-salt-stimulated lipase, where said lipase
is not isolated from the milk or transgenic sheep.
[0160] As will now be appreciated by the person of ordinary skill,
the recombinant human bile-salt-stimulated lipase that comprises
the any of the kits or pharmaceutical compositions, or used in any
of the methods, may be any of the recombinant human
bile-salt-stimulated lipases described or defined elsewhere
herein.
[0161] In a related aspect, the instant invention additionally
relates to a packaged-pharmaceutical-product comprising a
pharmaceutical composition that includes an amount of recombinant
human bile-salt-stimulated lipase, wherein said
packaged-pharmaceutical-product further comprises instructions that
describe the steps of:
[0162] i. preparing a modified infant formula or modified
pasteurized breast milk that contains rhBSSL in an amount effective
to increase the growth velocity of a human infant when said
modified infant formula or modified pasteurized breast milk is fed
to said infant for at least one feed per day over at least around 4
days, for at least one feed per day over at least around 5 days, or
for at least one feed per day over at least around 7 days; and
[0163] ii. enteral administration of said amount of rhBSSL by
feeding said modified infant formula or modified pasteurized breast
milk to a human infant over an administration regimen as described
or defined herein.
[0164] In an alternative aspect, the present invention also relates
to analogous packaged-pharmaceutical-products, wherein the
instructions therein describe the step (i.) as preparing a modified
infant formula or modified pasteurized breast milk that contains
rhBSSL in an amount effective to: (X) protect the small bowel
mucosa of a human infant from damage; to (Y) protect an immature
intestinal epithelium of a human infant from the deleterious
effects of incompletely digested and/or excess fat and/or lipid;
and/or to (Z) limit accumulation of incompletely digested and/or
excess fat and/or lipid in the ileum of a human infant; in each
case when said modified infant formula or modified pasteurized
breast milk is fed to said infant for at least one feed per day
over at least around 4 days, for at least one feed per day over at
least around 5 days, or for at least one feed per day over at least
around 7 days
[0165] In other embodiments of the present invention, a
packaged-pharmaceutical-product further comprises an infant formula
or pasteurized breast milk. Said infant formula or pasteurized
breast milk may be included in the packaged-pharmaceutical-product
as a separate component to the recombinant human
bile-salt-stimulated lipase; i.e., it may be an unmodified infant
formula or unmodified pasteurized breast milk. In an alternative
such embodiment, the packaged-pharmaceutical-product may include
the infant formula or pasteurized breast milk already comprising
the recombinant human bile-salt-stimulated lipase; i.e., it may be
a modified infant formula or unmodified pasteurized breast milk. In
either of such embodiments, the infant formula may be provided as
dried granulate or powder for solubilizing, or may be provided as a
liquid (either at an appropriate concentration or as a concentrate)
in a suitable container or as a frozen sample.
[0166] In certain embodiments of these
packaged-pharmaceutical-products, the pharmaceutical composition is
one described or defined elsewhere herein.
[0167] In other certain embodiments, the instructions of a
packaged-pharmaceutical-product describe that the human infant to
be administered recombinant human bile-salt-stimulated lipase does
suffer from, or should suffer from, being underweight, being of
LBW, being born premature birth and/or fed by enteral (or gastric)
tube. For example, said infant may suffer from one or more of the
weight or premature aliments or indications as described elsewhere
herein.
[0168] In particular embodiments of the invention, the instructions
of the packaged-pharmaceutical-products or the kits describe that
the modified infant formula or modified pasteurized breast milk, or
the recombinant human bile-salt-stimulated lipase, is for, is
effective for, or has been shown/demonstrated to be efficacious and
safe in a clinical trial and for: (A) increasing the growth
velocity of a human infant; and/or (B) for: (X) protection of the
small bowel mucosa of a human infant from damage; for (Y)
protection of an immature intestinal epithelium of a human infant
from the deleterious effects of incompletely digested and/or excess
fat and/or lipid; and/or for (Z) limitation of accumulation of
incompletely digested and/or excess fat and/or lipid in the ileum
of a human infant.
[0169] With regards to the present invention, in any of its methods
requiring the preparation or provision of a modified infant formula
or modified pasteurized breast milk, or its kits or
packaged-pharmaceutical-product including instructions that
describe such a preparation or provision, in certain embodiments of
such aspects it may be required that and the recombinant human
bile-salt-stimulated lipase and/or the unmodified infant formula or
unmodified pasteurized breast milk is to be thawed and/or
solubilized before the modified infant formula or modified
pasteurized breast milk is prepared. Such preparation or provision
may include that the recombinant human bile-salt-stimulated lipase
is added to an unmodified infant formula (for example, provided as
a dried-premix) or unmodified pasteurized frozen breast milk. In
other embodiments, such preparation or provision may include that
that a modified infant formula or modified pasteurized breast milk
is first thawed and/or warmed to an appropriate temperature for
feeding to a human infant, for example to 37.degree. C. In other
embodiments, an unmodified frozen breast milk is first thawed, the
rhBSSL is then added, and then for example solubilized if said
lipase is provided as an lyophilized power or granulate form.
[0170] As will be appreciated by the person of ordinary skill upon
the disclosure of the present invention herein, the modified infant
formula or modified pasteurized breast milk of the invention, or
the kit, packaged-pharmaceutical-product, rhBSSL or pharmaceutical
composition do not have to be in a quantity, size or amount to
fulfill the needs of an entire treatment regimen. For example, a
fresh quantity of modified infant formula or modified pasteurized
breast milk may be prepared, such as from a kit, or pharmaceutical
compositions of the present invention for each administration to
the human infant, such that multiple kits or pharmaceutical
compositions are utilized during the course of the treatment
regimen.
[0171] It is to be understood that application of the teachings of
the present invention to a specific problem or environment will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein. Examples of the products,
compositions, packages or kits of the present invention and
representative methods or processes for their preparation or use
appear in the following.
[0172] All references, patents and publications cited herein are
hereby incorporated by reference in their entirety.
EXEMPLIFICATION
[0173] The following exemplification, including the experiments
conducted and results achieved, also illustrate various presently
particular embodiments of the present invention, and are provided
for illustrative purposes only and are not to be construed as
limiting the present invention.
[0174] Section 1: Drug Substance, its Characterization and
Preparation of Investigational Drug Product.
[0175] The drug substance, human bile-salt-stimulated lipase,
having a predicted amino acid sequence as shown in SEQ ID NO:1, was
produced by expression from recombinant Chinese hamster ovary (CHO)
cells containing a nucleic acid expression system comprising the
nucleotide sequence encoding human BSSL according to standard
procedures. Briefly, the 2.3 Kb cDNA sequence encoding full-length
hBSSL including the leader sequence (as described by Nilsson et al,
1990; Eur J Biochem, 192: 543-550) was obtained from pS146 (Hansson
et al, 1993; J Biol Chem, 268: 26692-26698) and cloned into the
expression vector pAD-CMV 1 (Boehringer Ingelheim)--a pBR-based
plasmid that includes CMV promoter/SV40 polyA signal for gene
expression and the dhfr gene for selection/amplification--to form
pAD-CMV-BSSL. pAD-CMV-BSSL was then used for transfection of
DHFR-negative CHOss cells (Boehringer Ingelheim)--together with
co-transfection of plasmid pBR3127 SV/Neo pA coding for neomycin
resistance to select for geneticin (G418) resistance--to generate
DHFR-positive BSSL producing CHO cells. The resulting CHO cells
were cultured under conditions and scale to express larger
quantities of rhBSSL. For example, cells from the master cell bank
(MCB) are thawed, expanded in shaker flasks using Ex-Cell 302
medium without glutamine and glucose (SAFC) later supplemented with
glutamine and glucose, followed by growth in 15 and 100 L
bioreactors, before inoculating the 700 L production bioreactor
where BSSL is constitutively expressed and produced in a fed-batch
process. The culture is harvested as a single batch and the mature
rhBSSL polypeptide (i.e., without the leader sequence) is purified
from cells, cell debris and other contaminates via a number of
downstream steps, including an anion exchange chromatography step.
Contaminating viruses may be inactivated by low pH treatment and a
dry heat treatment step. The rhBSSL Drug Substance (DS) bulk is
diafiltered and concentrated to the appropriate formulation. After
formulation, the material is divided in one to three batches for
lyophilization and heat treatment, generating one to three DS
batches.
[0176] Production of rhBSSL in this mammalian-cell expression
system produces rhBSSL having a predicted amino acid sequence as
shown in SEQ ID NO:1 and a structure as schematically represented
in FIG. 1.1, also marking the potential glycosylation sites.
[0177] This form of rhBSSL appears to exhibit glycosylation that is
different to native hBSSL found in human milk (BSSL-MAM) and also
to rhBSSL-OVI (produced from transgenic sheep). For example, using
high pH anion exchange chromatography with pulsed amperiometric
detection (HPAEC-PAD), the monosaccharide and sialic acid
glycosylation level was determined for the CHO-derived rhBSSL
produced and used for the clinical trials described herein
(rhBSSL-CHO), and is found to have a total glycosylation level that
is lower than BSSL-MAM, but higher than rhBSSL-OVI (see Table 1.1).
These overall levels of glycosylation correlated to the overall
molecular masses of each form of BSSL which, determined by MALDI-MS
are found to be about 85 KDa for rhBSSL-CHO compared to 100 KDa and
78 KDa for BSSL-MAM and rhBSSL-OVI, respectively. As shown in Table
1.1, the pattern or profile of glycosylation (monosaccharide and
sialic acid) on the possible glycosylation sites, particularly that
of O-glycans, differs for rhBSSL-CHO compared to rhBSSL-MAM and to
rhBSSL-OVI (detection using capillary electrophoresis with laser
induced fluorescence detection [CE-LIF] and/or HPAEC-PAD).
TABLE-US-00001 TABLE 1.1 Monosaccharide and Sialic Acid content
[mole/(mole BSSL)] for rhBSSL-CHO, rhBSSL-OVI and hBSSL-MAM rhBSSL-
hBSSL- rhBSSL- CHO MAM OVI Monosaccharide content Fucose 2.0 30.6
1.3 Galactosamine 16.6 15.8 3.0 Glucosamine 2.1 37.6 0.0* Galactose
17.5 51.8 3.4 Glucose 0.0 0.0 0.0 Mannose 5.0 9.8 2.5 Total 43.2
145.6 10.2** Sialic acid content N-Acetyl neuraminic acid 27.9 16.4
0.5 N-Glycosyl neuraminic acid 0.0 0.0 5.0 Total 27.9 16.4 5.5
*When analyzing for glucosamine in the rhBSSL-OVI material, a small
peak in the chromatogram was seen. However no value was reported
since such low amount was calculated as a negative value due to a
greater intersection point of the calibration curve, which was
subtracted. An estimated absolute/uncorrected value was 1.8 mole
glucoseamine/mole BSSL. **The total sum including
(absolute/uncorrected) glucosamine was 12 mole/mole BSSL.
[0178] Not only is the degree and distribution of glycosylation for
rhBSSL-CHO different to that of BSSL-MAM and to that of rhBSSL-OVI,
but it is found that by C-terminal amino acid sequence (determined
for example, by endoprotein Glu-C peptide mapping and sequence
identification using liquid chromatography in combination with
electrospray ionization mass spectrometry [LC-ESI-MS-MS]) that a
large proportion of the lipase molecules are shortened by one
(occasionally two) amino acids compared to the (predicted) full
length polypeptide molecules. For every molecule with a full-length
C-terminus sequenced, there are detected about three molecules
having a C-terminus truncated by the last amino acid. A small
proportion of C-terminal sequences are detected that were truncated
by the last 2 amino acids. For example, of this population of (near
full-length) lipase molecules, about 25% are full length, around
75% are shorter by one amino and less than 1% are shorter by two
amino acids.
[0179] Differences in functional properties are observed between
rhBSSL-CHO and BSSL-MAM and from rhBSSL-OVI. The specific activity
of rhBSSL-CHO is observed to be higher than that of the other forms
of BSSL. The specific activities of BSSL-MAM and rhBSSL-OVI are
only 80% of that of rhBSSL-CHO based on mass. Each sample is
specifically purified by HA-HPLC and SE-HPLC before determination
of specific activity. Specific activity is determined using
4-nitrophenyl ester butyric acid (PNPB) as a substrate for BSSL,
and detection of the release of 4-nitrophenol. Briefly, a dilution
series of rhBSSL (for example, from 20 to 160 ng activity/mL) is
prepared in PBS with 0.1% BSA. 200 .mu.l of these rhBSSL solutions
is added to 25 .mu.l of an activation solution containing 20 mM
sodium cholate (as bile-salt activator) in PBS with 0.1% BSA. These
solutions are preincubated in a spectrophotometer at 27.degree. C.
for 5 minutes. Just before measuring, 25 .mu.l of a well-mixed
substrate solution containing 5 mM PNPB in PBS-Tween is added. The
formation of 4-nitrophenol can be detected by its absorbance at 400
nm and the increase in absorbance is measured during 90 seconds.
The active amount of BSSL is determined using a standard curve of
an rhBSSL reference standard.
[0180] The investigational medicinal product was prepared from
lyophilized Drug Substance that is dissolved in water for
injection. The solution is pre-filtered (10 .mu.m), and adjusted to
the final (active) concentration with water for injection. The
product is filtered through a 0.22 .mu.m filter and filled into
pre-sterilized 10 mL glass vials. The vials are stoppered with
sterilized stoppers and sealed with aluminium caps.
[0181] Section 2: Abbreviated Report on Combined Data from Two
Phase II Studies with rhBSSL
[0182] Protocol Number: BVT.BSSL-020
[0183] EUDRACT Number: 2007-002423-33
[0184] Clinicaltrials.gov identifier: NCT00658905
[0185] A prospective, randomized, double-blind crossover study
comparing 0.15 g/L rhBSSL added to infant formula versus placebo
during one week of treatment in preterm infants born before week 32
of gestational age
[0186] And
[0187] Protocol Number: BVT.BSSL-021
[0188] EUDRACT Number: 2007-002434-10
[0189] Clinicaltrials.gov identifier: NCT00659243
[0190] A prospective, randomized, double-blind crossover study
comparing 0.15 g/L rhBSSL added to pasteurized breast milk versus
placebo during one week of treatment in preterm infants born before
week 32 of gestational age
LIST OF ABBREVIATIONS
[0191] AA Arachidonic Acid [0192] AE Adverse Event [0193] ANCOVA
Analysis of Covariance [0194] ANOVA Analysis of Variance [0195]
BSSL Bile-salt-stimulated Lipase [0196] CFA Coefficient of Fat
Absorption [0197] CRF Case Report Form [0198] DHA Docosahexaenoic
Acid [0199] FA Fatty acid [0200] FAS Full Analysis Set [0201] g
Gram [0202] ICH International Conference on Harmonization [0203] kg
Kilogram [0204] MedDRA Medical Dictionary for Regulatory Activities
[0205] mm Millimeter [0206] N/A Not Applicable [0207] PP
Per-Protocol [0208] PT Preferred Term [0209] rhBSSL Recombinant
human bile-salt-stimulated lipase [0210] SAE Serious Adverse Event
[0211] SAP Statistical Analysis Plan [0212] SAS.RTM. Statistical
Analysis Software [0213] SD Standard Deviation [0214] SOC System
Organ Class [0215] TEAE Treatment-Emergent Adverse Event [0216]
TLFs Tables, Data Listings, and Figures
1. INTRODUCTION
[0217] Two phase II studies have been performed with rhBSSL in
preterm infants, studies BVT.BSSL-020 and -021. The primary
objective in both studies was to compare the fat absorption
(coefficient of fat absorption, CFA) in preterm infants following
treatment with rhBSSL to that with placebo when administered in
formula (study-020) or pasteurized breast milk (study-021).
Secondary objectives were to compare the length and body weight in
preterm infants following treatment with rhBSSL to that in placebo
when administered in infant formula/pasteurized breast milk, and to
study the safety of rhBSSL when administered in infant formula
pasteurized breast milk.
[0218] The sample size estimation in each study was based on an
estimated 10% difference in CFA units between treatment periods and
a standard deviation of 15%, with a power of 90% and a significance
level of 5%. It was anticipated that a 10% difference in CFA would
result in a 2 g/kg/day difference in growth velocity. However, none
of the studies was expected to have a sufficient power to
demonstrate an improvement in growth, due to the small number of
patients (32) in each study and the short duration of treatment (1
week). Therefore, a pre-defined combined analysis of the two
studies, with the primary objective to demonstrate improved growth
following treatment with rhBSSL as compared to placebo when
administered in infant formula or pasteurized breast milk was
described in a separate statistical analysis plan (SAP). The SAP
for the combined data was developed and finalized prior to database
lock and unblinding of the clinical database in either of the two
studies.
[0219] In addition, some post hoc analyses, not described in any
SAP, have also been performed and are reported here.
[0220] The present report is a summary of the design and results
from the two studies, focusing on the combined analysis but also in
many cases presenting results by study. It is based on information
given in the individual study reports, the statistical report of
the combined analysis, and on a statistical report of the post hoc
analysis.
[0221] Both studies were conducted according to ICH GCP guidelines
and the Declaration of Helsinki. Both trials were approved by the
appropriate Independent Ethics Committees and informed consent was
signed by the guardians of all included patients.
2. ANALYSIS OBJECTIVES OF THE COMBINED ANALYSIS
2.1 Primary Objective
[0222] The primary objective of the combined analysis was to
demonstrate improved growth following treatment with recombinant
human bile-salt-stimulated lipase (rhBSSL) as compared to placebo
when administered in infant formula or pasteurized breast milk.
2.2 Secondary Objectives
[0223] The secondary objectives were as follows: [0224] To
demonstrate improved fat absorption in preterm infants following
treatment with rhBSSL as compared to placebo when administered in
infant formula or pasteurized breast milk. [0225] To compare the
knee-to-heel length in preterm infants following treatment with
rhBSSL to that in placebo when administered in infant formula or
pasteurized breast milk. [0226] To evaluate safety and tolerability
of rhBSSL in preterm infants when administered in infant formula or
pasteurized breast milk.
3. STUDY DESIGN
[0227] The study designs and procedures of the two studies were the
same with the exception of the feeding regimen (formula was used in
study BVT.BSSL-020 and pasteurized milk in study BVT.BSSL-021),
thus the combining of the data from the two studies is appropriate.
Each study planned to enroll 32 patients in order to obtain 26
evaluable patients.
[0228] Patients were randomized to infant formula/pasteurized
breast milk supplemented with rhBSSL at a final concentration of
0.15 g/L, or to infant formula/pasteurized breast milk
"supplemented" with sterile water for injection (as placebo) for
the first 7 days. After a washout period of 2 days, the patient was
"crossed over" to the other treatment regimen during a second 7-day
treatment period. Collection of feces samples for CFA assessment
were performed during the last 3 days (72 hours) of each treatment
period.
[0229] Patients were enrolled and randomized into these studies at
the neonatal intensive care unit, after fulfilling all of the
inclusion and none of the exclusion criteria. Infants who were
receiving other infant formula prior to enrollment in the 020 study
were to be switched from their current formula to the study formula
on the day of enrollment. For patients in the 021 study who were
receiving milk fortifiers other than Eoprotin, it was required to
discontinue the milk fortification and/or switch to Eoprotin at
least 2 days before the first dose.
[0230] The study design is presented in FIG. 2.1.
[0231] The schedule of study assessments is provided below in Table
2.1.
TABLE-US-00002 TABLE 2.1 Schedule of Study Assessments (in two
parts to fit on the page) Visit 1* 2* Screening Baseline 3 4 5 6 7
8 (up through the 8th day) Day -7 to -1 1 2 3 4 5 6 7 Informed
consent x Medical history x Inclusion/Exclusion x Demographic data
x Routine Laboratory, if available # .sup. x.sup.# .sup. x.sup.#
.sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup.
x.sup.# .sup. x.sup.# Physical examination x x Randomization x Body
weight (gram) x x x x x x x x Growth-knee-to-heel (millimeter) x x
x x x x x x Body temperature x x x x x x x x Blood Pressure/Heart
rate x x x x x x x x ECG, if available # .sup. x.sup.# .sup.
x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.#
.sup. x.sup.# .sup. x.sup.# Check for nappy rash x x x x x x x
Concomitant medication x x x x x x x x Administration of study drug
x x x x x x x Documentation of food intake x x x x x x x Weighing
of vomiting x x x x Tracer dye x x Stool collection** x x x x
Tolerability assessments (stool x x x x x x x consistency/color,
regurgitation) Adverse Event x x x x x x x Visit 18 9 WO 10 WO 11
12 13 14 15 16 17 F-Up (from the 9th-18th day) Day 8 9 10 11 12 13
14 15 16.sup.## 23 .+-. 3 Informed consent Medical history
Inclusion/Exclusion Demographic data Routine Laboratory, if
available # .sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.#
.sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup.
x.sup.# .sup. x.sup.# Physical examination x x x Randomization Body
weight (gram) x x x x x x x x x x Growth-knee-to-heel (millimeter)
x x x x x x x x x x Body temperature x x x x x x x x .sup.
x.sup.### x Blood Pressure/Heart rate x x x x x x x x .sup.
x.sup.### x ECG, if available # .sup. x.sup.# .sup. x.sup.# .sup.
x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.# .sup. x.sup.#
.sup. x.sup.# .sup. x.sup.# .sup. x.sup.# Check for nappy rash x x
x x x x x x x x Concomitant medication x x x x x x x x x x
Administration of study drug x x x x x x x Documentation of food
intake x x x x x x x Weighing of vomiting x x x x Tracer dye x x
Stool collection** x x x x Tolerability assessments (stool x x x x
x x x x x consistency/color, regurgitation) Adverse Event x x x x x
x x x x x *Visit 1 and visit 2 could take place at the same time.
All baseline assessments were to be performed and documented in the
CRF prior to study drug administration **Collection of stool began
with the appearance of the first dye and continued until the second
dye appeared. The stool containing the second marker was not
collected. ***Study formula or milk intake continued until the
second dye appeared in stool. .sup.#Only recorded when available
within routine care. No extra blood samples or ECG taken for the
study. .sup.##Visit 17 extended beyond Day 16 if necessary, until
second tracer dye appears. .sup.###Vital signs collected daily
until second dye appeared
4. PATIENT SELECTION
[0232] Patients selected for these studies were infants born before
week 32 of gestational age and who were .ltoreq.32 weeks and 6 days
of gestation (extrapolated age) at the time of enrollment. Infants
enrolled in these studies did not receive parenteral nutrition
(except glucose).
4.1 Inclusion Criteria
[0233] A patient must fulfill the following criteria in order to be
included in the study:
[0234] 1. Preterm infants born before week 32 of gestation and who
were .ltoreq.32 weeks and 6 days of gestation (extrapolated age) at
the time of enrollment
[0235] 2. Preterm infants appropriate for gestational age (each
site should use its own growth curves or procedures and keep a copy
of those used in the investigator's file. The same growth curve
should be used for all patients at one site)
[0236] 3. Preterm infants receiving infant formula whose mothers
are not intending to provide breast milk
[0237] 4. Preterm infants receiving oral or enteral nutrition
(bottle or nasal tube)
4.2 Exclusion Criteria
[0238] The presence of any of the following will exclude a patient
from inclusion in the study:
[0239] 1. Infants receiving parenteral nutrition (except
glucose)
[0240] 2. For BVT.BSSL-020: Infants receiving milk fortifiers
(e.g., Enfamil.RTM., Nutriprem.RTM., Milupa.RTM. Eoprotin.RTM.)
[0241] Otherwise eligible infants who are receiving milk fortifiers
may be enrolled if the use of fortifiers is discontinued 2 days
before the first dose [0242] For BVT.BSSL-021: Infants receiving
milk fortifiers other than Eoprotin.RTM. (e.g., Enfamil.RTM.,
Nutriprem.RTM.). [0243] Otherwise eligible infants who received
milk fortifiers than Eoprotin.RTM. could be enrolled if the use of
fortifiers was discontinued 2 days before the first dose;
[0244] 3. Infants requiring mechanical ventilation
[0245] 4. Infants small for their gestational age (SGA)
[0246] 5. Infants requiring .gtoreq.30% O.sub.2
[0247] 6. Infants receiving phototherapy (babies who have completed
phototherapy and otherwise qualify for the study may be
admitted)
[0248] 7. Infants with severe brain disease including grade III or
IV periventricular or intra ventricular hemorrhage, meningitis or
hydrocephalus, intracranial hemorrhage of grade III or IV,
periventricular leukomalacia
[0249] 8. Major dysmorphology or congenital abnormalities that can
affect growth and development
[0250] 9. Infants with hemodynamically significant persistent
ductus arteriosus (PDA)
[0251] 10. Clinical evidence of sepsis (including low or high white
cell count and/or low platelet count, and bacteriologically proven
evidence of systemic infection)
[0252] 11. Documented congenital infection (e.g. CMV)
[0253] 12. Presence of necrotizing enterocolitis
[0254] 13. Hemorrhagic pulmonary events
[0255] 14. Prior or concomitant treatment with corticosteroids,
except hydrocortisone
[0256] 15. Any condition which in the opinion of the investigator
makes the patient unsuitable for inclusion
[0257] 16. Enrollment in another concurrent clinical study within 2
days of the screening visit through the completion of the follow-up
visit
4.3 Removal of Subjects from Therapy or Assessment
[0258] A patient was to be withdrawn from the study drug if, in the
opinion of the investigator, it was medically necessary, or if it
were the wish of the patient's parents or legal guardian. Other
reasons for withdrawal from treatment could include the following:
[0259] incorrect entry in the study [0260] major protocol violation
[0261] adverse event
5. TREATMENTS
5.1 Treatments Administered
[0262] The amount of formula or milk given was based on the
patient's body weight as recorded on the CRF each morning. The
concentration of rhBSSL in the formula or pasteurized breast milk
remained constant at 0.15 g/L. Patients received formula (study
020) or pasteurized breast milk (study 021) with or without rhBSSL
for 7 days depending on the randomization schedule. A matching
amount of sterile water for injection (WFI) was added to the
pasteurized breast milk without rhBSSL when the patient was
assigned to placebo. The amount of formula/milk given each day was
recorded on the CRF.
TABLE-US-00003 rhBSSL dose in formula/ Feeding Treatment Drug
Dosage Form Route milk regimen A BSSL rhBSSL Liquid Oral 0.15 g/L*
According solution to body weight* B Placebo Sterile Liquid Oral
Volume to According water for solution match to body injection
rhBSSL weight* *Infants were to receive approximately 150 to 180 mL
milk/kg body weight per day. The feeding amount on a mL/kg basis
for a particular infant was to remain constant for both treatment
periods.
5.2 Identity of Investigational Product
[0263] Recombinant human BSSL drug substance and the
investigational medicinal product (IMP) was prepared as described
in Section 1 of the Exemplification (above).
[0264] Recombinant human BSSL was delivered as a frozen oral
solution in a 10 mL glass vial. The strength was 15 mg/mL and the
fill volume 1.3 mL. The study drug had to be stored frozen
(-25.degree. C. to -15.degree. C.) at the study centre in a place
inaccessible to unauthorized persons.
[0265] Before administration, the frozen solution was thawed and a
0.9 mL aliquot of the rhBSSL solution was transferred to 90 mL of
formula (study 020) or pasteurized breast milk (study 021) to give
a final concentration in the feed of 0.15 g/L. The placebo
formula/milk was prepared in the same way, where 0.9 mL of sterile
water was substituted for rhBSSL solution.
[0266] Two lots of IMP were used in both these studies.
[0267] The addition of the fortifier Eoprotin.RTM. as supplement
was only allowed throughout study 021 (breast milk); however, the
amount of Eoprotin.RTM. had to remain constant during the treatment
phase.
5.3 Selection of Concentration
[0268] The concentration of rhBSSL to be added to pasteurized milk
and formula has been selected based on the levels normally present
in breast milk which is in the range of 0.1-0.2 g/L.
5.4 Blinding
[0269] The randomization schedules were maintained in a secure,
locked location by Biovitrum's designee and were not revealed to
any hospital personnel, investigators, Biovitrum personnel, or
parents until after the database locks had been achieved. The
addition of rhBSSL/placebo to formula or pasteurized breast milk
was performed by a pharmacist or designee who was unblinded to the
treatment assignment and was not involved in the evaluation of the
patients.
5.5 Prior and Concomitant Therapy
[0270] Other therapy considered necessary for the patient's welfare
could be given at the discretion of the Investigator. All such
therapies were to be recorded on the CRF. The concomitant
administration of parenteral nutrition (except glucose), milk
fortifiers (with the exception of Eoprotin.RTM. in study 021, as
described above) within 2 days of the first dose of study
medication through 2 days following the last dose, and
corticosteroids, except hydrocortisone, was prohibited during the
study. No other drug under investigation was to be used
concomitantly with the study drug. The patients were not allowed to
participate concurrently in another clinical study.
[0271] Preterm infants often experience complications that need
therapeutic intervention. This was acceptable as long as the
medication did not interfere with feeding. If concomitant
medication resulted in the need for parenteral feeding, the patient
was to be withdrawn from the study. Similarly, the development of
complications that affect the absorption of enteral nutrition, such
as necrotizing enterocolitis or abdominal obstruction, required
that the patient discontinue participation in the study.
[0272] The use of ointments for the treatment of skin irritation
was prohibited during the 72-hour fecal collection period. Diapers
were to be changed frequently during the 72-hour collection period
to keep the skin dry. Patients with skin rash severity leading to
discontinuation of the stool collection were to be withdrawn from
the study.
6. STUDY ASSESSMENTS FOR ANALYSIS ON COMBINED DATA
6.1 Efficacy Assessments in Each Study
6.1.1 Body Weight
[0273] The patient's weight in grams was recorded each day using a
scale with an accuracy of at least +/-5 grams and entered on the
CRF. To the extent possible, body weight was measured at
approximately the same time each day.
6.1.2 Sample Collection
[0274] In study BVT.BSSL-021, aliquots of the breast milk were
taken prior to addition of rhBSSL or placebo on Days 4-7 and Days
13-16.
[0275] The collection of feces for the determination of CFA was
performed over a period corresponding to the fat, i.e., formula or
milk, ingestion during 72 hours toward the end of each treatment
period. Diapers supplied to each site were used for feces
collection. During the two treatment periods, a carmine red tracer
dye was given as a marker together with a meal (approximately at
noon) on Day 4 and Day 13, respectively, and collection of stool
commenced with the appearance of the first carmine red marker in
the stool. The stool containing the first marker was collected and
the date and time of the first stool collected was recorded on the
CRF. At 72 hours following administration of the first red marker,
the second carmine marker was given, and stool collection continued
until the second carmine marker appeared. The stool containing the
second marker was not collected, but the date and time of the
appearance of the second marker was recorded on the CRF. Diapers
were weighed before placement and after removal and the difference
in weight was recorded on the CRF. The times of each collection and
the elapsed duration of the entire collection period was also
recorded on the CRF. The use of ointments for the treatment of skin
irritation was prohibited during the stool collection period.
Diapers were to be changed frequently during the collection period
to keep the skin dry. Patients with skin rash severity leading to
discontinuation of the stool collection were to be withdrawn from
the study. Specific collection methods were provided in a separate
laboratory manual. If applicable, vomit from the stool collection
periods of both treatment periods was weighed. A small cloth/linen
was weighed and placed under the head of each infant. When the
cloth/linen was soiled with vomit, it was removed and re-weighed.
If an additional cloth was used to remove vomit from the infant,
that cloth was also weighed before and after use. The weight of
vomit (total weight minus the weight of the cloth/linen) was
recorded on the CRF. All other feed losses, e.g., formula or milk
left in bottle, were measured and the amount accounted for in the
calculation of the volume of formula consumed at each feeding.
[0276] All diapers and paper napkins used during each collection
period were collected. They were placed in a sealed bag, labeled
with patient ID and date and time and stored at -20.degree. C.
until shipment to the analytical laboratory.
6.1.3 Sample Analysis
[0277] Feces samples, the formula (study 020) and the milk aliquots
(study 021) were analyzed by a central laboratory. Individual fatty
acids, including the long-chain polyunsaturated fatty acids DHA and
AA, were quantified in feces and feed by a gas chromatographic
method following extraction by the Folch method. In both studies,
the Omegawax.RTM. 250 column (Supelco) was used for separation of
the fatty acids. However, due to co-elution of DHA with nervonic
acid (C24:1), which was only present in the breast milk, samples
from patients of the per-protocol analysis set from study 021 were
also analyzed using a SP-2380 column (Supelco) in order to quantify
DHA for those samples from study 021. This column provides good
separation of DHA and C24:1, but is less suitable for overall
separation of other fatty acids in the formula and milk; hence
individual fatty acids from these samples from study 021 (breast
milk) were separated and analyzed using (separately) the SP-2380
column (for DHA) and the Omegawax.RTM. 250 column (for all other
fatty acids). Total lipids were calculated as the sum of the
individual fatty acids. (See Section 7.5.1). The same analytical
principle was used to determine lipids in each of the batches of
formula and aliquots of breast milk used in the study.
6.1.4 Knee-to-Heel Length
[0278] The length of the patient's leg was measured from the knee
to the heel using a knemometer provided to the sites. Knee-to-heel
length was recorded in millimeters on the CRF. To the extent
possible, length was measured at approximately the same time each
day and by the same person. Three measurements were made and the
mean value was entered on the CRF.
6.2 Safety Assessments: Adverse Events
[0279] The adverse event (AE) reporting period in each study began
upon administration of the first dose (Day 1) of investigational
medication and ended at the Follow-up Visit (1 week.+-.3 days after
the last dose of study drug intake). All AEs that occurred in a
patient during the adverse event reporting period were to be
reported, whether or not the event was considered
medication/product related. In addition, any known untoward event
that occurred subsequent to the AE reporting period that the
investigator assessed as possibly, probably, or definitely related
to the investigational product were also to be reported as an
AE.
7. STATISTICAL METHODOLOGY
7.1 Analysis Populations
[0280] Safety Analysis Set: All randomized patients who received at
least one dose of randomized study medication (rhBSSL or placebo).
The analysis of safety variables was performed using the safety
analysis set.
[0281] Full Analysis Set (FAS): All randomized patients who
received at least one dose of randomized study medication, and had
a baseline and at least one post-baseline weight assessment in both
treatment periods.
[0282] Per-Protocol Analysis Set (PP): All patients included in FAS
who had reasonable compliance and no other major protocol
violations.
[0283] The assessment of patients who qualified for the PP analysis
set within each study was performed prior to database lock and
unblinding of the respective study. For both FAS and PP, the
combined datasets included exactly the same patients as in the
individual studies.
7.2 Statistical Objective of the Combined Analysis
7.2.1 Primary Efficacy Objective and Hypothesis
[0284] The primary objective of the analysis on the combined data
from the two studies was to demonstrate improved growth following
treatment with rhBSSL as compared to placebo when administered in
infant formula or pasteurized breast milk.
[0285] The null hypothesis presupposed no difference between the
treatments with respect to growth velocity. The alternative
hypothesis was as follows: rhBSSL improves growth velocity as
compared to placebo when administered in infant formula or
pasteurized breast milk.
7.2.2 Secondary Efficacy Objectives
[0286] The secondary efficacy objectives of the analysis on the
combined data from the two studies were as follows: [0287] To
demonstrate improved fat absorption in preterm infants following
treatment with rhBSSL as compared to placebo when administered in
infant formula or pasteurized breast milk. [0288] To compare the
knee-to-heel length in preterm infants following treatment with
rhBSSL to that in placebo when administered in infant formula or
pasteurized breast milk.
[0289] With respect to CFA, the null hypothesis presupposed no
difference between the treatments. The alternative hypothesis was
as follows: rhBSSL improves fat absorption as compared to placebo
when administered in infant formula or pasteurized breast milk.
[0290] No statistical hypothesis test has been performed with
respect to the knee-to-heel length.
7.2.3 Safety Objective
[0291] Safety objectives of the analysis on the combined data from
the two studies were to evaluate safety and tolerability of rhBSSL
in preterm infants when administered in infant formula or
pasteurized breast milk.
7.3 Patient Disposition
[0292] Patient disposition was summarized by treatment sequence and
was based on all patients randomized in both studies. The summary
table included the number of patients randomized, the number (%) of
patients who completed each study, the number (%) of patients who
discontinued from each study, and the number (%) of patients for
each reason for discontinuation. The summary table also reported
the number (%) of patients included in the safety, FAS, and PP
analysis sets, and the number (%) of patients who completed each
treatment period.
7.4 Patient Demographic and Baseline Characteristics
[0293] Demographic characteristics included actual age and
extrapolated gestational age on the day of first dose of study
medication, gestational age at birth, gender, race, and ethnicity.
Baseline characteristics included knee-to-heel length and body
weight. Two summary tables were provided for demographic and
baseline characteristics. The first table provided a summary of
combined data by treatment sequence, and the second table provided
summaries of demographic and baseline characteristics by study.
Continuous variables were summarized by the number of patients,
mean, standard deviation (SD), median, minimum, and maximum values.
Categorical variables were summarized by the number and percentage
of patients in each category.
7.5 Analysis of Efficacy
[0294] All efficacy data collected in these two studies were
summarized for each study and for the combined analysis using
descriptive statistics. Efficacy analyses for the individual trials
were conducted in accordance with their efficacy objectives, as
described in the Introduction (results of these analyses are not
presented in this Report).
[0295] The primary analysis of the analysis on the combined data
from the two studies was based on a 2-sided test using an alpha
level of significance of 0.05. A stepwise sequential testing
procedure was used to ensure a multiple level of significance of
0.05. [0296] 1.sup.st step: The null hypothesis of no difference
between the treatments with respect to growth velocity was tested
using an alpha level of significance of 0.05. If the null
hypothesis was rejected, then the 2.sup.nd step of the sequential
testing procedure was to be performed. [0297] 2.sup.nd step: The
null hypothesis of no difference between the treatments with
respect to CFA was tested using an alpha level of significance of
0.05. If the null hypothesis was rejected, then a confirmatory
claim was also to be made with respect to CFA.
[0298] This multiple comparison procedure controls that the
multiple level of significance is no more than 5%.
[0299] Primary and secondary efficacy analyses reported point
estimates and 95% confidence intervals around the estimates for
each treatment and the estimated difference between treatments
accompanied with the corresponding 95% confidence interval. No
hypothesis testing was performed for variables other than growth
velocity and CFA as stated above.
[0300] Continuous variables were summarized using n, mean, SD,
median, minimum, and maximum values. Categorical variables were
summarized using the number and percentage of patients in each
category.
[0301] If a final assessment was not available when calculating the
growth velocity during a period, the growth velocity was calculated
at the last available assessment and carried forward to the final
day. Otherwise, no imputation of missing data was performed.
7.5.1 Efficacy Variables for Analysis
[0302] The Primary Efficacy Variable was:
[0303] Growth velocity (g/kg/day): Growth velocity was defined as,
for the first period, (the weight at the last assessment in the
first period minus the weight at Day 1) divided by [the weight at
Day 1 and (the day of the last assessment in the first period minus
1)], and for the second period, (the weight at the last assessment
in the second period minus the weight at Day 10) divided by [the
weight at Day 10 and (the day of the last assessment in the second
period minus 10)].
[0304] The Secondary Efficacy Variables were:
[0305] CFA measured in food and feces samples collected between the
tracer markers during the final 3 days (72 hours) of each treatment
period.
[0306] CFA was calculated as [Fat (g/period) in food-Fat (g/period)
in stool]/[Fat (g/period) in food)]*100.
[0307] Fat in food was calculated as ([Food (mL)-Vomit (mL)]*[Fat
Content in Food (g/100 mL)]/100. This formula was based on the
following assumptions: (a) fat content in vomit is the same as the
fat content in food; (b) density of vomit is the same as density of
food.
[0308] Fat content of food (formula or pasteurized breast milk) was
determined using the same method as for the stool analysis and was
performed by the same lab. Food (mL) and Vomit (mL) were calculated
as the total amount of food or vomit recorded on or after the first
tracer ingestion and prior to the second tracer ingestion. Vomit
was recorded in grams on the CRF. Therefore, Vomit (mL) was
calculated as Vomit (g)/Density.
[0309] There was one difference in the calculation of fat
(g/period) in stool and fat content in food (g/100 mL) between the
two studies. That difference relates to different contents of fatty
acids in milk and formula, as described below:
[0310] BVT.BSSL-020:
[0311] Fat (g/period) in stool was calculated as a sum of the
following fatty acids divided by 1000, since each fatty acid was
provided in mg by the lab: C12:0, C14:0, C16:0, C18:0, C18:1, C18:2
n-6, C18:3 n-3, C20:4 n-6, and C22:6 n-3.
[0312] Each fatty acid in food was provided in g/100 mL. Fat
content in food (g/100 mL) was calculated as the sum of the same
fatty acids as in the stool.
[0313] BVT.BSSL-021:
[0314] Fat (g/period) in stool was calculated as a sum of the
following fatty acids divided by 1000, since each fatty acid was
provided in mg by the lab: C12:0, C14:0, C16:0, C16:1, C18:0,
C18:1, C18:2 n-6, C18:3 n-3, C18:3 n-6, C20:1, C20:2 n-3, C20:3
n-6, C20:4 n-6, C22:6 n-3 and C24:1.
[0315] Each fatty acid in food was provided in g/100 mL. Fat
content in food (g/100 mL) was calculated as the sum of the same
fatty acids as in the stool.
[0316] Combined Analysis:
[0317] The combined statistical analysis of CFA data used the
overall CFA values as calculated for each infant/treatment-period
from each of the two individual studies.
[0318] Change in length (mm): Change in length was defined as the
change in length from knee to heel from Day 1 to Day 7 in the first
period and Day 10 to Day 16 in the second period.
7.5.2 Efficacy Analysis Methodology
[0319] The primary and secondary efficacy analyses were based on
the FAS of the combined data from the two studies. Supportive
efficacy analyses were based on the PP analysis set of combined
data from the two studies. In addition, analyses of each efficacy
variable were provided by study for the FAS and for the PP analysis
set.
[0320] The primary efficacy outcome, growth velocity, was analyzed
by an analysis of variance (ANOVA) with treatment, regimen
(pasteurized breast milk or infant formula), period, sequence, and
patient nested within regimen and sequence as factors. All main
effects were tested against the residual mean square from the ANOVA
model.
[0321] The normality assumption of growth velocity distribution
based on the combined data was tested using the Shapiro-Wilk test.
If the normality assumption was not met, then the ranked values
were to be used for the ANOVA.
[0322] The secondary efficacy outcome, CFA from the last three days
of each treatment period, was analyzed in the same way as growth
velocity by an analysis of variance (ANOVA) with treatment, regimen
(pasteurized breast milk or infant formula), period, sequence, and
patient nested within regimen and sequence as factors.
[0323] Descriptive statistics for the total amount of fat in food
and the total amount of fat in stool were provided by
treatment.
[0324] Another secondary efficacy outcome, change in knee-to-heel
length, was analyzed by an analysis of covariance (ANCOVA) with
treatment, regimen, period, sequence, and patient nested within
regimen and sequence as factors using the baseline value as a
covariate.
7.6 Analysis of Safety: Adverse Events
[0325] All adverse events (AE) analyses were based on the safety
analysis set of the combined data from both studies. Results were
presented using descriptive statistics. No hypothesis testing was
performed.
[0326] MedDRA dictionary version 10.0 was used to classify all AEs
reported during either study by system organ class (SOC) and
preferred term (PT). All summary tables included counts of patients
with treatment-emergent adverse events (TEAEs). The assessment of
TEAEs was made in each individual study. TEAEs were defined as
those AEs that either had an onset on or after the start of study
drug and no more than 14 days (30 days for serious AEs) after the
last dose of study drug, or were ongoing at the time of study drug
initiation and increased in severity or became closer in
relationship to study drug during the treatment period. All TEAEs,
treatment related TEAEs (definite, probable, and possible), SAEs,
and TEAEs leading to withdrawal of study drug were summarized by
MedDRA SOC, PT, and treatment. Both the incidence (proportion of
patients) and number of each TEAE were summarized. Additionally,
TEAEs were summarized by maximum severity (mild, moderate, or
severe). An overall summary of TEAEs was presented by treatment
sequence and total and presented the number (%) of patients with
TEAEs for each treatment sequence allocated to (1) BSSL only; (2)
Placebo only; (3) Both BSSL and placebo; and (4) Neither
Treatment.
8. RESULTS
8.1 Disposition of Patients
[0327] A summary of disposition of patients in the two studies by
treatment sequence is shown in Table 2.2. Patient disposition by
study was also collected and summarized (not shown in this
Report).
TABLE-US-00004 TABLE 2.2 Patient Disposition rhBSSL/Placebo
Placebo/rhBSSL Total Number of Patients Randomized 32 33 65 Safety
Analysis Set.sup.a 31 (100.0%) 32 (100.0%) 63 (100.0%) Full
Analysis Set (FAS).sup.b 30 (96.8%) 30 (93.8%) 60 (95.2%)
Per-Protocol Analysis Set (PP).sup.c 24 (77.4%) 22 (68.8%) 46
(73.0%) Completed Period 1.sup.d 30 (96.8%) 31 (96.9%) 61 (96.8%)
Completed Period 2.sup.d 29 (93.5%) 30 (93.8%) 59 (93.7%) Completed
the study 29 (93.5%) 30 (93.8%) 59 (93.7%) Discontinued the Study 2
(6.5%) 2 (6.3%) 4 (6.3%) Adverse Event(s) 2 (6.5%) 2 (6.5%) 4
(6.3%) Protocol Violation(s) 0 0 0 Withdrew Consent 0 0 0 Lost to
Follow-up 0 0 0 Sponsor's Request 0 0 0 Principal Investigator
Decision 0 0 0 Other 0 0 0 .sup.aThe safety analysis set includes
all patients who received at least one dose of randomized study
medication. .sup.bThe full analysis set includes all randomized
patients who received at least one dose of randomized study
medication and had a baseline and at least one post-baseline weight
assessment in both treatment periods. .sup.cThe per-protocol
analysis set includes patients in the FAS who had reasonable
compliance and no other major protocol violations. .sup.dCompleted
period defined as patients who received study medication for 7 days
in the treatment period.
[0328] A total of 65 patients were randomized across both studies:
33 patients in BVT.BSSL-020 and 32 patients in BVT.BSSL-021. A
total of 63 patients received at least one dose of randomized study
medication and were included in the safety analysis set: 33
patients in BVT.BSSL-020 and 30 in BVT.BSSL-021. The FAS included a
total of 60 patients who were in the safety analysis set and who
had a baseline and at least one post-baseline weight assessment in
both treatment periods: 33 patients in BVT.BSSL-020 and 27 patients
in BVT. BSSL-021. A total of 46 patients were included in the PP
analysis set: 26 patients in BVT.BSSL-020 and 20 patients in
BVT.BSSL-021. There were 14 patients who were not included in the
PP analysis set due to incomplete or incorrect stool
collection.
[0329] Of the 63 patients in the safety analysis set, 31 patients
were randomized to the rhBSSL/Placebo treatment sequence and 32
patients to Placebo/rhBSSL. A total of 61 patients completed Period
1, and a total of 59 patients completed Period 2. All but four
patients completed the studies; these four patients discontinued
due to AEs.
8.2 Demographic and Baseline Characteristics
[0330] Demographic and baseline characteristics for the combined
data in the two studies by treatment sequence are shown below in
Table 2.3. Demographic and baseline characteristics by study were
also collected and summarized (not shown in this Report).
TABLE-US-00005 TABLE 2.3 Demographics and Baseline Characteristics
rhBSSL/Placebo Placebo/rhBSSL Total Characteristic (N = 31) (N =
32) (N = 63) Age (Weeks).sup.a N 31 32 63 Mean (SD) 4.14 (1.553)
3.60 (1.393) 3.87 (1.487) Gestational Age at Birth (Weeks) N 31 32
63 Mean (SD) 28.39 (1.575) 28.96 (1.542) 28.68 (1.572) Extrapolated
Gestational Age (Weeks).sup.a N 31 32 63 Mean (SD) 32.53 (.447)
32.58 (.541) 32.56 (.494) Gender Male 15 (48.4%) 18 (56.3%) 33
(52.4%) Female 16 (51.6%) 14 (43.8%) 30 (47.6%) Ethnicity Hispanic
or Latino 13 (41.9%) 13 (40.6%) 26 (41.3%) Not Hispanic or Latino
18 (58.1%) 19 (59.4%) 37 (58.7%) Race White 25 (80.6%) 27 (84.4%)
52 (82.5%) Black 1 (3.2%) 2 (6.3%) 3 (4.8%) Asian 1 (3.2%) 1 (3.1%)
2 (3.2%) Native Hawaiian or Other 1 (3.2%) 0 1 (1.6%) Pacific
Islander Other 3 (9.7%) 2 (6.3%) 5 (7.9%) Knee-to-heel Length
(mm).sup.b N 31 32 63 Mean (SD) 100.09 (5.490) 99.78 (6.573) 99.93
(6.017) Weight (g) N 31 32 63 Mean (SD) 1463.4 (169.28) 1469.6
(216.25) 1466.6 (193.02) .sup.aAge on the day of first dose.
.sup.bMeasured with a knemometer.
[0331] In the combined analysis, the mean age on the day of first
dose was higher for patients randomized to rhBSSL/Placebo (4.14
weeks) compared to the mean age for patients randomized to
Placebo/rhBSSL (3.60 weeks). Other demographic and baseline
characteristics were comparable between treatment sequences.
[0332] A difference in mean age on the day of first dose was also
noticeable between the two studies: the mean age was lower for
patients in BVT.BSSL-020 (3.39 weeks) compared to the mean age in
BVT.BSSL-021 (4.39 weeks). Mean gestational age at birth was about
one week higher in BVT.BSSL-020 (29.18 weeks) versus BVT.BSSL-021
(28.13 weeks). However, the gestational age on the day of first
dose was similar in the two studies. A difference in ethnicity was
also observed between the two studies: the percentage of Hispanic
or Latino patients was higher in BVT.BSSL-020 (63.6%) compared to
BVT.BSSL-021 (16.7%). Other demographic and baseline
characteristics were comparable between the studies.
8.3 Treatment Compliance
[0333] Treatment compliance in study BVT.BSSL-020 is summarized
below in Table 2.4 and for study BVT.BSSL-021 in Table 2.5.
TABLE-US-00006 TABLE 2.4 Treatment Compliance by Treatment - Study
BVT.BSSL.020 Variable rhBSSL Placebo Statistics (N = 33) (N = 33) n
33 33 Treatment Compliance (%) <60 0 0 .gtoreq.60, <70 0 0
.gtoreq.70, <80 0 1 (3.0%) .gtoreq.80, <90 0 1 (3.0%)
.gtoreq.90, <100 28 (84.8%) 25 (75.8%) .gtoreq.100 5 (15.2%) 6
(18.2%) Mean 98.79 97.24 Std Dev 1.639 4.967 Median 99.34 98.56
Minimum 92.6 73.0 Maximum 100.7 101.8
TABLE-US-00007 TABLE 2.5 Treatment Compliance by Treatment - Study
BVT.BSSL.021 Variable rhBSSL Placebo Statistics (N = 28) (N = 29) n
28 29 Treatment Compliance (%) <60 0 0 .gtoreq.60, <70 1
(3.6%) 0 .gtoreq.70, <80 0 0 .gtoreq.80, <90 2 (7.1%) 1
(3.4%) .gtoreq.90, <100 16 (57.1%) 19 (65.5%) .gtoreq.100 9
(32.1%) 9 (31.0%) Mean 95.97 97.52 Std Dev 7.033 3.820 Median 98.17
97.75 Minimum 66.7 87.2 Maximum 101.8 103.7
8.4 Efficacy Analysis
8.4.1 Primary Efficacy Variable
[0334] The primary efficacy variable in the combined analysis was
the growth velocity. Combined results for growth velocity based on
the combined analyses of the two clinical studies in the FAS and PP
analysis sets are shown in Table 2.6. Growth velocity analysis
results by study based on the FAS and PP analysis sets are shown in
Tables 2.7a and 2.7b respectively.
TABLE-US-00008 TABLE 2.6 Analysis of Growth Velocity (g/kg/day) in
the FAS and PP Analysis Sets FAS Analysis Set PP Analysis Set Study
Analysis Set and Placebo rhBSSL Statistics rhBSSL (N = 60) (N = 60)
(N = 46) Placebo (N = 46) n 60 60 46 46 Mean (SD) 16.92 (4.540)
14.00 (5.942) 17.08 (4.424) 15.04 (5.048) Median 16.84 14.95 16.84
15.09 Minimum 7.5 -4.5 8.3 0.0 Maximum 26.5 26.4 26.5 26.4 LS Mean
16.86 13.93 17.15 15.06 95% CI (15.73, 17.98) (12.80, 15.05)
(15.92, 18.38) (13.83, 16.29) LS Mean Difference (rhBSSL - 2.93
2.08 Placebo) 95% CI of LS Mean Difference (1.35, 4.51) (0.36,
3.81) p-value for LS Mean Difference <0.001 0.019
[0335] The combined results of the two clinical studies showed a
significant increase in growth velocity during rhBSSL treatment
compared to during placebo treatment in both the FAS and PP
analysis sets. In the FAS, the growth velocity LS means were 16.86
g/kg/day with rhBSSL and 13.93 g/kg/day with placebo. The
difference in growth velocity between rhBSSL and placebo was
statistically significant: LS mean difference (rhBSSL-Placebo) was
2.93 g/kg/day (p<0.001). In the PP analysis set, the LS mean
difference (rhBSSL-Placebo) of 2.08 g/kg/day was also statistically
significant (p=0.019).
[0336] Table 2.7a below displays the growth velocity results in the
FAS analysis set for each of the two clinical studies, and Table
2.7b displays the same for the PP analysis set.
TABLE-US-00009 TABLE 2.7a Analysis of Growth Velocity (g/kg/day) by
Study in the FAS Analysis Set BVT.BSSL-020 BVT.BSSL-021 Statistics
rhBSSL (N = 33) Placebo (N = 33) rhBSSL (N = 27) Placebo (N = 27) n
33 33 27 27 Mean (SD) 18.06 (3.964) 14.29 (6.493) 15.54 (4.880)
13.63 (5.292) Median 18.39 15.51 15.95 13.98 Minimum 9.2 -4.5 7.5
-3.1 Maximum 25.5 23.3 26.5 26.4 LS Mean 18.05 14.31 15.58 13.63
95% CI (16.52, 19.58) (12.78, 15.84) (13.82, 17.33) (11.87, 15.39)
LS Mean Difference 3.74 1.95 (rhBSSL - Placebo) 95% CI of LS Mean
(1.58, 5.90) (-0.54, 4.43) Difference p-value for LS Mean 0.001
0.119 Difference
[0337] The improvement in growth velocity during rhBSSL treatment
compared to placebo was more pronounced in study BVT.BSSL-020 than
in study BVT.BSSL-021. Based on the FAS, in the BVT.BSSL-020 study,
the LS mean difference (rhBSSL-Placebo) was 3.74 g/kg/day (p=0.001)
whereas in the BVT.BSSL-021 study, it was 1.95 g/kg/day (p=0.119).
Similar results by study were observed in the PP analysis set (see
Table 2.7b).
[0338] Another observation from Table 2.7a was that patients on
formula gained weight more rapidly than patients on pasteurized
breast milk. In the FAS, the mean growth velocity during rhBSSL
treatment was 18.06 and 15.54 g/kg/day in BVT.BSSL-020 and
BVT.BSSL-021 respectively, and during placebo treatment it was
14.29 and 13.63 g/kg/day in the respective studies. Similar results
were observed in the PP analysis set (see Table 2.7b).
[0339] The normality assumption of growth velocity distribution
based on the combined data was tested using the Shapiro-Wilk test.
The test for normality was significant in the FAS (p-value
<0.001), indicating that the normality assumption was not met.
(A similar result was seen for the PP analysis set.) Therefore, an
analysis of growth velocity using the ranked values was also
performed. The result of this sensitivity analysis was consistent
with the primary analysis with a resulting p-value of <0.001,
demonstrating a significant improvement in growth during rhBSSL
treatment as compared to placebo.
TABLE-US-00010 TABLE 2.7b Analysis of Growth Velocity (g/kg/day) by
Study in the PP Analysis Set BVT.BSSL-020 BVT.BSSL-021 Statistics
rhBSSL (N = 26) Placebo (N = 26) rhBSSL (N = 20) Placebo (N = 20) n
26 26 20 20 Mean (SD) 17.79 (4.013) 15.39 (5.412) 16.16 (4.856)
14.59 (4.630) Median 17.98 16.08 16.80 14.95 Minimum 9.2 0.0 8.3
3.4 Maximum 24.0 23.3 26.5 24.6 LS Mean 17.75 15.45 16.47 14.76 95%
CI (16.22, 19.28) (13.92, 16.98) (14.23, 18.71) (12.51, 17.00) LS
Mean Difference (rhBSSL - 2.30 1.71 Placebo) 95% CI of LS Mean
Difference (0.14, 4.47) (-1.46, 4.88) p-value for LS Mean
Difference 0.038 0.271
8.4.2 Secondary Efficacy Variables
[0340] The secondary efficacy variables were CFA, and change in
knee-to-heel length between the start and end of each treatment
period.
CFA
[0341] Only patients in the PP analysis set had complete/correct
stool collection, essential for the determination of CFA.
Therefore, the presentation in the present report is limited to
data for the PP analysis set, with the exception of Table 2.8a
below that shows CFA results of the combined analysis of the two
clinical studies for both the FAS and the PP analysis set. The CFA
analysis results by study based on the PP analysis sets are
provided in Table 2.8b.
TABLE-US-00011 TABLE 2.8a Analysis of CFA (%) in the FAS and PP
Analysis Sets Study Analysis Set and FAS Analysis Set PP Analysis
Set Statistics rhBSSL (N = 60) Placebo (N = 60) rhBSSL (N = 46)
Placebo (N = 46) n 59* 59* 46 46 Mean (SD) 67.80 (16.663) 64.06
(16.319) 69.08 (14.683) 65.66 (16.126) Median 71.09 66.50 71.83
67.15 Minimum 11.7 25.7 31.2 25.7 Maximum 93.2 93.0 93.2 93.0 LS
Mean 67.78 64.08 69.06 65.50 95% CI (64.73, 70.83) (61.03, 67.13)
(66.31, 71.80) (62.75, 68.25) LS Mean Difference (rhBSSL - 3.70
3.56 Placebo) 95% CI of LS Mean Difference (-0.60, 8.00) (-0.29,
7.40) p-value for LS Mean Difference 0.090 0.069 *One patient in
study 020 withdrawn before stool collection period.
[0342] The combined results of the two clinical studies showed a
numerical increase in CFA in rhBSSL compared to placebo in both the
FAS and PP analysis sets. In the PP analysis set, the LS mean CFA
were 69.1% during rhBSSL treatment and 65.5% for placebo; the LS
mean difference (rhBSSL-Placebo) was 3.56% (p=0.069).
[0343] The improvement in CFA during rhBSSL treatment compared to
placebo was higher in BVT.BSSL-021 compared to BVT.BSSL-020. In the
PP, the LS mean difference (rhBSSL-Placebo) was 4.86% (p=0.073) in
BVT.BSSL-021 and 2.08% (p=0.462) in BVT.BSSL-020. Similar results
were observed in the FAS analysis set by study (see Table
2.8b).
TABLE-US-00012 TABLE 2.8b Analysis of CFA (%) by Study in the PP
Analysis Set BVT.BSSL-020 BVT.BSSL-021 Statistics rhBSSL (N = 26)
Placebo (N = 26) rhBSSL (N = 20) Placebo (N = 20) n 26 26 20 20
Mean (SD) 69.55 (14.452) 67.07 (14.849) 68.46 (15.333) 63.82
(17.875) Median 70.99 67.15 75.41 67.09 Minimum 36.8 25.7 31.2 35.9
Maximum 89.0 93.0 93.2 91.3 LS Mean 69.46 67.38 68.56 63.70 95% CI
(65.40, 73.53) (63.31, 71.45) (64.78, 72.35) (59.92, 67.49) LS Mean
Difference (rhBSSL - 2.08 4.86 Placebo) 95% CI of LS Mean
Difference (-3.67, 7.84) (-0.50, 10.22) p-value for LS Mean
Difference 0.462 0.073
[0344] Table 2.9a provides the total amount of fat in food consumed
between food tracer markers and the total amount of fat in stool
from stool samples collected between tracer markers in stools by
study in the combined analysis (PP analysis set). Data by treatment
for the combined analysis are provided in Table 2.9b.
TABLE-US-00013 TABLE 2.9a Total Amount of Fat in Food and Total
Amount of Fat in Stool by Study in the PP Analysis Set.
BVT.BSSL-020 BVT.BSSL-021 Statistics rhBSSL (N = 26) Placebo (N =
26) rhBSSL (N = 20) Placebo (N = 20) Total Amount of Fat in Food
(g) n 26 26 20 20 Mean (SD) 29.12 (5.037) 28.50 (5.047) 19.00
(5.110) 20.51 (6.718) Median 29.11 27.98 18.27 18.70 Minimum 21.1
17.7 12.1 13.3 Maximum 44.0 39.3 29.1 43.7 Total Amount of Fat in
Stool (g) n 26 26 20 20 Mean (SD) 8.53 (3.416) 8.97 (3.278) 6.16
(3.550) 7.56 (4.785) Median 8.63 9.06 4.99 6.09 Minimum 3.2 2.0 0.9
2.0 Maximum 15.0 14.7 13.3 18.0
[0345] Patients on formula consumed more fat from food than
patients on pasteurized breast milk. In the PP, the mean total
amount of fat in food consumed during rhBSSL treatment (72-hour
collection period) was 29.12 g and 19.00 g in BVT.BSSL-020 and
BVT.BSSL-021 respectively, and during placebo treatment it was
28.50 g and 20.51 g in the respective studies. Patients on formula
also excreted more fat in stool than patient on pasteurized breast
milk. The mean total amount of fat excreted in stool during rhBSSL
treatment was 8.53 g and 6.16 g in BVT.BSSL-020 and BVT.BSSL-021
respectively, and during placebo it was 8.97 g and 7.56 g in the
respective studies.
[0346] Table 2.9b summarizes the total amount of fat in food and
the total amount of fat in stool, during the 72-hour collection
interval, for the combined results in the PP and analysis set. Fat
intake and fat excretion were comparable for the two treatments. In
the combined data from the two studies, in the PP, the mean amount
of fat in food consumed during rhBSSL treatment was 24.72 g, and
the mean amount consumed during placebo was 25.03 g. The amount of
fat excreted in stool was 7.50 g and 8.36 g, respectively.
TABLE-US-00014 TABLE 2.9b Total Amount of Fat in Food and Total
Amount of Fat in Stool, combined data, in the PP Analysis Set. PP
Analysis Set Statistics rhBSSL (N = 46) rhBSSL (N = 46) Total
Amount of Fat in Food (g) n 46 46 Mean (SD) 24.72 (7.133) 25.03
(7.018) Median 25.28 24.35 Minimum 12.1 13.3 Maximum 44.0 43.7
Total Amount of Fat in Stool (g) n 46 46 Mean (SD) 7.50 (3.635)
8.36 (4.018) Median 7.05 8.34 Minimum 0.9 2.0 Maximum 15.0 18.0
[0347] There was little difference between the mean volume of
infant formula or breast milk ingested between the different
studies, or the volume ingested between treatment periods with
rhBSSL or with placebo.
Correlation Between Growth Velocity and Fat Absorption
[0348] FIG. 2.2 presents the difference in growth velocity
(rhBSSL-placebo) vs. the difference in CFA (rhBSSL-placebo) in the
combined analysis of data from the PP analysis sets from the two
studies.
[0349] As seen in this graph, there was no statistically
significant correlation (p-value 0.177) between the effect of
rhBSSL on growth velocity and fat absorption (CFA).
Change in Knee-to-Heel Length
[0350] The results of change in knee-to-heel length for the
combined analysis from the two studies in the FAS and PP analysis
were collected and summarized (not shown in this Report).
[0351] No noticeable differences were observed between treatments
with respect to mean change in knee-to-heel length measurements in
either the FAS or PP analysis sets in the combined data from both
studies, or by study.
8.5 Analysis of Safety: Adverse Events
8.5.1 Extent of Exposure
[0352] A summary of treatment exposure, as number of days on
treatment, is provided in Table 2.19 below.
TABLE-US-00015 TABLE 2.19 Extent of Treatment Exposure - Safety
Analysis Set Variable rhBSSL Placebo Statistic (N = 61) (N = 62)
Number of Days on Treatment [1] 1 0 0 2 0 0 3 1 (1.6%) 1 (1.6%) 4 0
0 5 0 0 6 0 1 (1.6%) 7 60 (98.4%) 60 (96.8%) n 61 62 Mean 6.9 6.9
Std Dev 0.51 0.52 Median 7.0 7.0 Minimum 3 3 Maximum 7 7 [1] Number
of days on treatment = Last day of treatment period - First day of
treatment period + 1.
[0353] The extent of treatment exposure was comparable between
treatments. 98.4% of patients had 7 days of rhBSSL treatment and
96.8% of patients had 7 days of placebo treatment. One patient
discontinued from BVT.BSSL-020 after 3 days of placebo treatment
during the second period. Three (3) patients in BVT.BSSL-021
discontinued during the first treatment period: 2 patients
discontinued after 6 and 7 days of placebo treatment, respectively,
and one patient discontinued after 3 days of rhBSSL treatment.
[0354] A summary exposure to of rhBSSL is provided in Table 2.20
below.
TABLE-US-00016 TABLE 2.20 Extent of Exposure to rhBSSL - Safety
Analysis Set Variable Total Statistic (N = 63) Total amount of
rhBSSL (g) [1] n 61 Mean 0.2717 Std Dev 0.05172 Median 0.2682
Minimum 0.063 Maximum 0.397 [1] Total amount of rhBSSL (g) = 0.15
g/L * (Total amount of food (L) ingested during rhBSSL treatment
period - Total amount of vomit (L) during rhBSSL treatment period).
Vomit was not collected on Days 1, 2, 3, 10, 11, and 12. Note:
Concentration of rhBSSL in food is 0.15 g/L according to the
protocols.
[0355] In the combined analysis results, the mean (SD) amount of
rhBSSL consumed was 0.27 g (0.052 g).
8.5.2 Brief Summary of Adverse Events
[0356] The overall incidence rate of TEAEs is shown below in Table
2.21.
TABLE-US-00017 TABLE 2.21 Overall Summary of Treatment-emergent
Adverse Events-Safety Analysis Set rhBSSL Placebo Total (N = 61) (N
= 62) (N = 63) n (%) of Total AEs n (%) of Total AEs n (%) of Total
AEs Patients (n) Patients (n) Patients (n) Patients with any TEAE
29 (47.5%) 56 32 (51.6%) 78 45 (71.4%) 134 Patients with any
serious 0 0 2 (3.2%) 2 2 (3.2%) 2 TEAE Patients with any TEAE 1
(1.6%) 1 3 (4.8%) 3 4 (6.3%) 4 leading to discontinuation from the
study Patients with any related 5 (8.2%) 6 4 (6.5%) 6 8 (12.7%) 12
TEAE Patients with any severe 1 (1.6%) 1 6 (9.7%) 16 6 (9.5%) 17
TEAE Patients who died 0 0 1 (1.6%) 1 1 (1.6%) 1 Related includes
definitely, probably, or possibly study medication related.
[0357] A total of 134 treatment-emergent adverse events (TEAEs)
were experienced by 45 of 63 (71.4%) patients in these two studies.
There was no noticeable difference observed in the proportion of
patients with TEAEs between treatments. The proportions of patients
with TEAEs were comparable between the studies: 23 (69.7%) patients
experienced AEs in BVT.BSSL-020 and 22 (73.3%) in BVT.BSSL-021.
However, the total number of TEAEs were higher in BVT.BSSL.021 (81
events) compared to BVT.BSSL.020 (53 events). (Tabulated data by
study not shown in this report.)
[0358] Across the two studies, 2 (3.2%) patients reported one
serious TEAE during placebo treatment, 4 (6.3%) patients reported
one TEAE leading to discontinuation from the study (1 patient
during rhBSSL treatment and 3 patients during placebo treatment), 8
(12.7%) patients reported at least one TEAE considered treatment
related (5 patients had during rhBSSL treatment, 4 patients during
placebo treatment, where one of these patients had a related TEAE
during both periods), and one patient died during placebo
treatment.
8.5.3 Display of Adverse Events
[0359] A summary of the most commonly reported TEAEs (reported in
>=4% of the patients) is provided below in Table 2.22. A summary
of all reported TEAEs was collected and summarized (not shown in
this Report).
TABLE-US-00018 TABLE 2.22 Most Commonly Reported Treatment-Emergent
Adverse Events-Safety Analysis Set rhBSSL Placebo Total (N = 61) (N
= 62) (N = 63) n (%) of Total AEs n (%) of Total AEs n (%) of Total
AEs Preferred Term Patients (n) Patients (n) Patients (n)
Dermatitis diaper 13 (21.3%) 20 13 (21.0%) 15 21 (33.3%) 35 Anemia
3 (4.9%) 3 6 (9.7%) 6 8 (12.7%) 9 Cardiac murmur 4 (6.6%) 4 2
(3.2%) 2 6 (9.5%) 6 Bradycardia 1 (1.6%) 2 5 (8.1%) 15 5 (7.9%) 17
Hypokalemia 3 (4.9%) 3 2 (3.2%) 2 5 (7.9%) 5 Anemia neonatal 2
(3.3%) 2 1 (1.6%) 1 3 (4.8%) 3 Thrombocythemia 0 0 3 (4.8%) 3 3
(4.8%) 3 Urinary tract infection 1 (1.6%) 1 2 (3.2%) 2 3 (4.8%) 3
Note: This table includes AEs reported in >=4% of patients. If a
patient had more than one count for a particular preferred term,
the patient was counted once for that preferred term.
[0360] The most common TEAE in the combined results for the two
studies was dermatitis diaper reported by 21 (33.3%) patients. The
incidence of this event was comparable between treatments. Other
most commonly reported TEAEs were anemia in 8 (12.7%) patients,
cardiac murmur in 6 (9.5%) patients, bradycardia and hypokalemia
each reported by 5 (7.9%) patients, and anemia neonatal,
thrombocythemia, and urinary tract infection each reported by 3
(4.8%) patients. All of the most common TEAEs were reported in both
treatments, with the exception of thrombocythemia which was
reported in placebo only. In addition, all of the most common TEAEs
were reported in both studies, with the exception of urinary tract
infection which was reported in BVT.BSSL-020 only and hypokalemia
which was reported in BVS.BSSL-021 only.
9. CONCLUSIONS
[0361] The results of the combined analysis are consistent with the
results of the individual studies supporting the following
conclusions: [0362] rhBSSL significantly improves growth as
compared to placebo in preterm infants receiving pasteurized breast
milk or infant formula. [0363] There is a numerical but not
significant improvement in fat absorption following rhBSSL
treatment as compared to placebo. [0364] No difference with respect
to the change in knee-to-heel length was observed after one week of
rhBSSL treatment as compared to placebo. [0365] rhBSSL added to
infant formula or pasteurized breast milk was well tolerated.
[0366] No apparent difference in the safety profile during rhBSSL
treatment as compared to placebo was observed. [0367] Patients on
formula consumed more fat and gained more weight than patients on
pasteurized breast milk.
EXHIBIT A
[0368] Proposed compositional requirements of infant formula--
ESPGHAN recommended standards (adapted from Koletzko et al
2005):
TABLE-US-00019 Component Unit Minimum Maximum Energy kcal/100 ml 60
70 Proteins Cows' milk protein g/100 kcal 1.8* 3 Soy protein
isolates g/100 kcal 2.25 3 Hydrolyzed cows' milk protein g/100 kcal
1.8.dagger. 3 Lipids Total fat g/100 kcal 4.4 6 Linoleic acid g/100
kcal 0.3 1.2 a-linolenic acid g/100 kcal 50 NS Ratio
linoleic/a-linolenic acids mg/100 kcal 5:1 15:1 Lauric + myristic
acids % of fat NS 20 Trans fatty acids % of fat NS 3 Erucic acid %
of fat NS 1 Carbohydrates Total carbohydrates.dagger-dbl. g/100
kcal 9 14 Vitamins Vitamin A ug RE/100 kcal.sctn. 60 180 Vitamin D3
ug/100 kcal 1 2.5 Vitamin E mg a-TE/100 kcal& 0.5{ 5 Vitamin K
ug/100 kcal 4 25 Thiamin ug/100 kcal 60 300 Riboflavin ug/100 kcal
80 400 Niacin# ug/100 kcal 300 1500 Vitamin B6 ug/100 kcal 35 175
Vitamin B12 ug/100 kcal 0.1 0.5 Pantothenic acid ug/100 kcal 400
2000 Folic acid ug/100 kcal 10 50 Vitamin C mg/100 kcal 8 30 Biotin
ug/100 kcal 1.5 7.5 Minerals and trace elements Iron (formula based
on cows' milk protein and protein mg/100 kcal 0.3** 1.3
hydrolysate) Iron (formula based on soy protein isolate) mg/100
kcal 0.45 2 Calcium mg/100 kcal 50 140 Phosphorus (formula based on
cows' milk protein and mg/100 kcal 25 90 protein hydrolysate)
Phosphorus (formula based on soy protein isolate) mg/100 kcal 30
100 Ratio calcium/phosphorus mg/mg 1:1 2:1 Magnesium mg/100 kcal 5
15 Sodium mg/100 kcal 20 60 Chloride mg/100 kcal 50 160 Potassium
mg/100 kcal 60 160 Manganese ug/100 kcal 1 50 Fluoride ug/100 kcal
NS 60 Iodine ug/100 kcal 10 50 Selenium ug/100 kcal 1 9 Copper
ug/100 kcal 35 80 Zinc mg/100 kcal 0.5 1.5 Other substances Choline
mg/100 kcal 7 50 Myo-inositol mg/100 kcal 4 40 L-carnitine mg/100
kcal 1.2 NS *The determination of the protein content of formulae
based on non-hydrolyzed cows' milk protein with a protein should be
based on measurement of true protein content between 1.8 and 2.0
g/100 kcal ([total N minus NPN] .times. 6.25) .dagger.Formula based
on hydrolyzed milk protein with a protein content less than 2.25
g/100 kcal should be clinically tested. .dagger-dbl.Sucrose
(saccharose) and fructose should not be added to infant formula.
.sctn.1 mg RE (retinol equivalent) = 1 mg all-trans retinol = 3.33
IU vitamin A. Retinol contents shall be provided by preformed
retinol, while any contents of carotenoids should not be included
in the calculation and declaration of vitamin A activity. &1 mg
a-TE (a-tocopherol equivalent) = 1 mg d-a-tocopherol. {Vitamin E
content shall be at least 0.5 mg a-TE per g PUFA, using the
following factors of equivalence to adapt the minimal vitamin E
content to the number of fatty acid double bonds in the formula:
0.5 mg a-TE/g linoleic acid (18:2n-6); 0.75 mg a-TE/g a-linolenic
acid (18:3n-3); 1.0 mg a-TE/g arachidonic acid (20:4n-6); 1.25 mg
a-TE/g eicosapentaenoic acid (20:5n-3); 1.5 mg a-TE/g
docosahexaenoic acid (22:6n-3). #Niacin refers to preformed niacin.
**In populations where infants are at risk of iron deficiency, iron
contents higher than the minimum level of 0.3 mg/100 kcal may be
appropriate and recommended at a national level. NS, not
specified.
EXHIBIT B
[0369] Proposed levels of optional ingredients, if added-- ESPGHAN
recommended standards (adapted from Koletzko et al 2005):
TABLE-US-00020 Optional ingredients Unit Minimum Maximum Taurine
mg/100 kcal 0 12 Total added nucleotides mg/100 kcal 0 5 Cytidine
5#-monophosphate (CTP) mg/100 kcal 0 1.75 Uridine 5#-monophosphate
(UMO) mg/100 kcal 0 1.5 Adenosine 5#-monophosphate mg/100 kcal 0
1.5 (AMP) Guanosine 5#-monophosphate mg/100 kcal 0 0.5 (GMP)
Inosine 5#-monophosphate (IMP) mg/100 kcal 0 1 Phospholipids mg/100
kcal 0 300 Docosahexaenoic acid* % of fat 0 0.5 *If docosahexaenoic
acid (22:6n-3) is added to infant formula, arachidonic acid
(20:4n-6) contents should reach at least the same concentration as
DHA. The content of eicosapentaenoic acid (20:5n-3) should not
exceed the content of docosahexaenoic acid.
SEQUENCE LISTING
TABLE-US-00021 [0370] SEQ ID NO:1: AKLGAVYTEG GFVEGVNKKL GLLGDSVDIF
KGIPFAAPTK ALENPQPHPG 50 WQGTLKAKNF KKRCLQATIT QDSTYGDEDC
LYLNIWVPQG RKQVSRDLPV 100 MIWIYGGAFL MGSGHGANFL NNYLYDGEEI
ATRGNVIVVT FNYRVGPLGF 150 LSTGDANLPG NYGLRDQHMA IAWVKRNIAA
FGGDPNNITL FGESAGGASV 200 SLQTLSPYNK GLIRRAISQS GVALSPWVIQ
KNPLFWAKKV AEKVGCPVGD 250 AARMAQCLKV TDPRALTLAY KVPLAGLEYP
MLHYVGFVPV IDGDFIPADP 300 INLYANAADI DYIAGTNNMD GHIFASIDMP
AINKGNKKVT EEDFYKLVSE 350 FTITKGLRGA KTTFDVYTES WAQDPSQENK
KKTVVDFETD VLFLVPTEIA 400 LAQHRANAKS AKTYAYLFSH PSRMPVYPKW
VGADHADDIQ YVFGKPFATP 450 TGYRPQDRTV SKAMIAYWTN FAKTGDPNMG
DSAVPTHWEP YTTENSGYLE 500 ITKKMGSSSM KRSLRTNFLR YWTLTYLALP
TVTDQEATPV PPTGDSEATP 550 VPPTGDSETA PVPPTGDSGA PPVPPTGDSG
APPVPPTGDS GAPPVPPTGD 600 SGAPPVPPTG DSGAPPVPPT GDSGAPPVPP
TGDSGAPPVP PTGDAGPPPV 650 PPTGDSGAPP VPPTGDSGAP PVTPTGDSET
APVPPTGDSG APPVPPTGDS 700 EAAPVPPTDD SKEAQMPAVI RF 722 SEQ ID NO:2:
1 accttctgta tcagttaagt gtcaagatgg aaggaacagc agtctcaaga taatgcaaag
61 agtttattca tccagaggct gatgctcacc atggggcgcc tgcaactggt
tgtgttgggc *** 121 ctcacctgct gctgggcagt ggcgagtgcc gcgaagctgg
gcgccgtgta cacagaaggt 181 gggttcgtgg aaggcgtcaa taagaagctc
ggcctcctgg gtgactctgt ggacatcttc 241 aagggcatcc ccttcgcagc
tcccaccaag gccctggaaa atcctcagcc acatcctggc 301 tggcaaggga
ccctgaaggc caagaacttc aagaagagat gcctgcaggc caccatcacc 361
caggacagca cctacgggga tgaagactgc ctgtacctca acatttgggt gccccagggc
421 aggaagcaag tctcccggga cctgcccgtt atgatctgga tctatggagg
cgccttcctc 481 atggggtccg gccatggggc caacttcctc aacaactacc
tgtatgacgg cgaggagatc 541 gccacacgcg gaaacgtcat cgtggtcacc
ttcaactacc gtgtcggccc ccttgggttc 601 ctcagcactg gggacgccaa
tctgccaggt aactatggcc ttcgggatca gcacatggcc 661 attgcttggg
tgaagaggaa tatcgcggcc ttcggggggg accccaacaa catcacgctc 721
ttcggggagt ctgctggagg tgccagcgtc tctctgcaga ccctctcccc ctacaacaag
781 ggcctcatcc ggcgagccat cagccagagc ggcgtggccc tgagtccctg
ggtcatccag 841 aaaaacccac tcttctgggc caaaaaggtg gctgagaagg
tgggttgccc tgtgggtgat 901 gccgccagga tggcccagtg tctgaaggtt
actgatcccc gagccctgac gctggcctat 961 aaggtgccgc tggcaggcct
ggagtacccc atgctgcact atgtgggctt cgtccctgtc 1021 attgatggag
acttcatccc cgctgacccg atcaacctgt acgccaacgc cgccgacatc 1081
gactatatag caggcaccaa caacatggac ggccacatct tcgccagcat cgacatgcct
1141 gccatcaaca agggcaacaa gaaagtcacg gaggaggact tctacaagct
ggtcagtgag 1201 ttcacaatca ccaaggggct cagaggcgcc aagacgacct
ttgatgtcta caccgagtcc 1261 tgggcccagg acccatccca ggagaataag
aagaagactg tggtggactt tgagaccgat 1321 gtcctcttcc tggtgcccac
cgagattgcc ctagcccagc acagagccaa tgccaagagt 1381 gccaagacct
acgcctacct gttttcccat ccctctcgga tgcccgtcta ccccaaatgg 1441
gtgggggccg accatgcaga tgacattcag tacgttttcg ggaagccctt cgccaccccc
1501 acgggctacc ggccccaaga caggacagtc tctaaggcca tgatcgccta
ctggaccaac 1561 tttgccaaaa caggggaccc caacatgggc gactcggctg
tgcccacaca ctgggaaccc 1621 tacactacgg aaaacagcgg ctacctggag
atcaccaaga agatgggcag cagctccatg 1681 aagcggagcc tgagaaccaa
cttcctgcgc tactggaccc tcacctatct ggcgctgccc 1741 acagtgaccg
accaggaggc cacccctgtg ccccccacag gggactccga ggccactccc 1801
gtgcccccca cgggtgactc cgagaccgcc cccgtgccgc ccacgggtga ctccggggcc
1861 ccccccgtgc cgcccacggg tgactccggg gccccccccg tgccgcccac
gggtgactcc 1921 ggggcccccc ccgtgccgcc cacgggtgac tccggggccc
cccccgtgcc gcccacgggt 1981 gactccgggg ccccccccgt gccgcccacg
ggtgactccg gggccccccc cgtgccgccc 2041 acgggtgact ccggcgcccc
ccccgtgccg cccacgggtg acgccgggcc cccccccgtg 2101 ccgcccacgg
gtgactccgg cgcccccccc gtgccgccca cgggtgactc cggggccccc 2161
cccgtgaccc ccacgggtga ctccgagacc gcccccgtgc cgcccacggg tgactccggg
2221 gccccccctg tgccccccac gggtgactct gaggctgccc ctgtgccccc
cacagatgac 2281 tccaaggaag ctcagatgcc tgcagtcatt aggttttagc
gtcccatgag ccttggtatc .sctn..sctn..sctn. 2341 aagaggccac aagagtggga
ccccaggggc tcccctccca tcttgagctc ttcctgaata 2401 aagcctcata
cccctaaaaa aaaaaaaa
[0371] The start and stop codons are marked (underneath) with "***"
and ".sctn..sctn..sctn." respectively. The leader sequence is
underlined.
Sequence CWU 1
1
21722PRTHomo sapiens 1Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly
Phe Val Glu Gly Val1 5 10 15 Asn Lys Lys Leu Gly Leu Leu Gly Asp
Ser Val Asp Ile Phe Lys Gly 20 25 30 Ile Pro Phe Ala Ala Pro Thr
Lys Ala Leu Glu Asn Pro Gln Pro His 35 40 45 Pro Gly Trp Gln Gly
Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys 50 55 60 Leu Gln Ala
Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80 Leu
Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg 85 90
95 Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly
100 105 110 Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp
Gly Glu 115 120 125 Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr
Phe Asn Tyr Arg 130 135 140 Val Gly Pro Leu Gly Phe Leu Ser Thr Gly
Asp Ala Asn Leu Pro Gly145 150 155 160 Asn Tyr Gly Leu Arg Asp Gln
His Met Ala Ile Ala Trp Val Lys Arg 165 170 175 Asn Ile Ala Ala Phe
Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly 180 185 190 Glu Ser Ala
Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr 195 200 205 Asn
Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu 210 215
220 Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys
Val225 230 235 240 Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala
Arg Met Ala Gln 245 250 255 Cys Leu Lys Val Thr Asp Pro Arg Ala Leu
Thr Leu Ala Tyr Lys Val 260 265 270 Pro Leu Ala Gly Leu Glu Tyr Pro
Met Leu His Tyr Val Gly Phe Val 275 280 285 Pro Val Ile Asp Gly Asp
Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr 290 295 300 Ala Asn Ala Ala
Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320 Gly
His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn 325 330
335 Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350 Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val
Tyr Thr 355 360 365 Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys
Lys Lys Thr Val 370 375 380 Val Asp Phe Glu Thr Asp Val Leu Phe Leu
Val Pro Thr Glu Ile Ala385 390 395 400 Leu Ala Gln His Arg Ala Asn
Ala Lys Ser Ala Lys Thr Tyr Ala Tyr 405 410 415 Leu Phe Ser His Pro
Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly 420 425 430 Ala Asp His
Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala 435 440 445 Thr
Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met 450 455
460 Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met
Gly465 470 475 480 Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr
Thr Glu Asn Ser 485 490 495 Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly
Ser Ser Ser Met Lys Arg 500 505 510 Ser Leu Arg Thr Asn Phe Leu Arg
Tyr Trp Thr Leu Thr Tyr Leu Ala 515 520 525 Leu Pro Thr Val Thr Asp
Gln Glu Ala Thr Pro Val Pro Pro Thr Gly 530 535 540 Asp Ser Glu Ala
Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala545 550 555 560 Pro
Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr 565 570
575 Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala
580 585 590 Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val
Pro Pro 595 600 605 Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr
Gly Asp Ser Gly 610 615 620 Ala Pro Pro Val Pro Pro Thr Gly Asp Ser
Gly Ala Pro Pro Val Pro625 630 635 640 Pro Thr Gly Asp Ala Gly Pro
Pro Pro Val Pro Pro Thr Gly Asp Ser 645 650 655 Gly Ala Pro Pro Val
Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 660 665 670 Thr Pro Thr
Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp 675 680 685 Ser
Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro 690 695
700 Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val
Ile705 710 715 720 Arg Phe22428DNAHomo sapiens 2accttctgta
tcagttaagt gtcaagatgg aaggaacagc agtctcaaga taatgcaaag 60agtttattca
tccagaggct gatgctcacc atggggcgcc tgcaactggt tgtgttgggc
120ctcacctgct gctgggcagt ggcgagtgcc gcgaagctgg gcgccgtgta
cacagaaggt 180gggttcgtgg aaggcgtcaa taagaagctc ggcctcctgg
gtgactctgt ggacatcttc 240aagggcatcc ccttcgcagc tcccaccaag
gccctggaaa atcctcagcc acatcctggc 300tggcaaggga ccctgaaggc
caagaacttc aagaagagat gcctgcaggc caccatcacc 360caggacagca
cctacgggga tgaagactgc ctgtacctca acatttgggt gccccagggc
420aggaagcaag tctcccggga cctgcccgtt atgatctgga tctatggagg
cgccttcctc 480atggggtccg gccatggggc caacttcctc aacaactacc
tgtatgacgg cgaggagatc 540gccacacgcg gaaacgtcat cgtggtcacc
ttcaactacc gtgtcggccc ccttgggttc 600ctcagcactg gggacgccaa
tctgccaggt aactatggcc ttcgggatca gcacatggcc 660attgcttggg
tgaagaggaa tatcgcggcc ttcggggggg accccaacaa catcacgctc
720ttcggggagt ctgctggagg tgccagcgtc tctctgcaga ccctctcccc
ctacaacaag 780ggcctcatcc ggcgagccat cagccagagc ggcgtggccc
tgagtccctg ggtcatccag 840aaaaacccac tcttctgggc caaaaaggtg
gctgagaagg tgggttgccc tgtgggtgat 900gccgccagga tggcccagtg
tctgaaggtt actgatcccc gagccctgac gctggcctat 960aaggtgccgc
tggcaggcct ggagtacccc atgctgcact atgtgggctt cgtccctgtc
1020attgatggag acttcatccc cgctgacccg atcaacctgt acgccaacgc
cgccgacatc 1080gactatatag caggcaccaa caacatggac ggccacatct
tcgccagcat cgacatgcct 1140gccatcaaca agggcaacaa gaaagtcacg
gaggaggact tctacaagct ggtcagtgag 1200ttcacaatca ccaaggggct
cagaggcgcc aagacgacct ttgatgtcta caccgagtcc 1260tgggcccagg
acccatccca ggagaataag aagaagactg tggtggactt tgagaccgat
1320gtcctcttcc tggtgcccac cgagattgcc ctagcccagc acagagccaa
tgccaagagt 1380gccaagacct acgcctacct gttttcccat ccctctcgga
tgcccgtcta ccccaaatgg 1440gtgggggccg accatgcaga tgacattcag
tacgttttcg ggaagccctt cgccaccccc 1500acgggctacc ggccccaaga
caggacagtc tctaaggcca tgatcgccta ctggaccaac 1560tttgccaaaa
caggggaccc caacatgggc gactcggctg tgcccacaca ctgggaaccc
1620tacactacgg aaaacagcgg ctacctggag atcaccaaga agatgggcag
cagctccatg 1680aagcggagcc tgagaaccaa cttcctgcgc tactggaccc
tcacctatct ggcgctgccc 1740acagtgaccg accaggaggc cacccctgtg
ccccccacag gggactccga ggccactccc 1800gtgcccccca cgggtgactc
cgagaccgcc cccgtgccgc ccacgggtga ctccggggcc 1860ccccccgtgc
cgcccacggg tgactccggg gccccccccg tgccgcccac gggtgactcc
1920ggggcccccc ccgtgccgcc cacgggtgac tccggggccc cccccgtgcc
gcccacgggt 1980gactccgggg ccccccccgt gccgcccacg ggtgactccg
gggccccccc cgtgccgccc 2040acgggtgact ccggcgcccc ccccgtgccg
cccacgggtg acgccgggcc cccccccgtg 2100ccgcccacgg gtgactccgg
cgcccccccc gtgccgccca cgggtgactc cggggccccc 2160cccgtgaccc
ccacgggtga ctccgagacc gcccccgtgc cgcccacggg tgactccggg
2220gccccccctg tgccccccac gggtgactct gaggctgccc ctgtgccccc
cacagatgac 2280tccaaggaag ctcagatgcc tgcagtcatt aggttttagc
gtcccatgag ccttggtatc 2340aagaggccac aagagtggga ccccaggggc
tcccctccca tcttgagctc ttcctgaata 2400aagcctcata cccctaaaaa aaaaaaaa
2428
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