WO2015144219A1 - Method of treatment - Google Patents

Abstract

The present invention relates to recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant either risking growth restriction or neurodevelopmental delay. The treatment comprises enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth of said infant. Moreover, rhBSSL for use in treatment of human infants being small for their gestational age is also disclosed.

Description

METHODS FOR TREATMENT

Field of the invention

The present invention relates to recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of human infants for at least around four weeks, wherein such treatment may be advantageous for example in reducing the risk of growth restriction. The present invention further relates to methods wherein such rhBSSL is administered to human infants.

Background

Low birth weight and preterm infants

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 have a weight appropriate for its birth; 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.

In early infancy, and especially in the preterm infants, pancreatic exocrine functions are not fully developed (Manson and Weaver, Arch Dis Child Fetal Neonatal Ed 76(3):F206-1 1 (1997)). The expression of pancreatic lipases involved in lipolysis of dietary lipids is low in the preterm pancreas compared with in the adult pancreas (Lombardo, Biochim Biophys Acta 1533(1 ):1 -28 ( 2001 )). This is compensated for by expression of bile salt- stimulated lipase (BSSL) in the lactating mammary gland and secretion of the enzyme with the milk.

Many low birth (LBW) babies require specialized care in Newborn Intensive Care Units (NICUs) as they are especially susceptible to health problems such as respiratory distress syndrome (RDS), cyanotic attacks, bleeding in the brain (intraventricular hemorrhage, IVH), cerebral palsy, heart problems such as patent ductus artiousus (PDA), hypocalcemia,

hypoglycaemia, intestinal problems such as necrotizing enterocolitis (NEC), jaundice and retinal-development problems such as retinopathy of prematurity (ROP). Beyond such acute care, being born underweight has been found to be associated with a number of mid-term health problems, including poor weight gain and head growth in infancy; developmental delay and later language problems in early childhood; neurological abnormalities, and 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.

Bile salt-stimulated lipase

Bile-salt-stimulated lipase accounts for about 1 % of the total protein in human breast milk and is present at concentrations from 0.1 to 0.2 g/L (Stromqvist et al, Arch Biochem Biophys 347(1 ):30-6 (1997)). The levels of BSSL are similar throughout the day (Freed et al, J Pediatr Gastroenterol Nutr. 5(6):938-42 (1986)) and BSSL production is maintained for at least 3 months (Hernell et al, Am J Clin Nutr 30(4):508-1 1 (1977)). Triglycerides comprise about 98 % or more of all lipids in human breast milk or infant formula and account for about 50 % of the energy content. Therefore, the lipases that hydrolyze the TGs, making the resulting hydrolysis products available for absorption, are of great importance for efficient energy utilization for the rapidly growing newborn infant. However, BSSL is inactivated during pasteurization of human breast milk and is not present in any infant 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 with pasteurized breast milk (PBM) (Andersson et al, Acta Paediatr 96(10):1445-9 (2007);Williamson et al, Arch Dis Child 53(7):555-63 (1978)).

Bile-salt-stimulated lipase has a broader substrate specificity than most lipases. Not only is the enzyme capable of completely hydrolyzing TGs but also vitamin A and cholesteryl esters. Thus, BSSL drives the intraluminal lipolysis toward completion and results in the formation of glycerol and free fatty acids, including long-chain polyunsaturated fatty acids, the latter being indispensable building blocks for the developing central nervous system. Growth in low birth weight and preterm infants

Growth in preterm infants, in terms of both weight gain and longitudinal growth, is often inadequate despite efforts to optimize parenteral nutrition and enteral feeding (Bloom et al, Pediatrics 1 12(1 Pt 1 ):8-14 (2003); Dusick et al, Semin Perinatol 27(4):302-10 (2003)). In a growth observational study in extremely low-birth-weight infants, it was found that, at discharge from the NICU at 36 weeks postmenstrual age, most were below the 10th percentile weight for completed weeks of gestation (Ehrenkranz et al, Pediatrics 104(2 Pt 1 ):280-9 (1999)). It has also been concluded that growth velocity during NICU hospitalization exerts a significant effect on growth and

neurodevelopment at 18 to 22 months corrected age (Ehrenkranz et al, Pediatrics 1 17(4):1253-61 (2006)). In yet another observational study in a NICU, it was found that preterm infants could rarely meet the recommended dietary intakes and accrued a nutrient deficit that could not be regained before hospital discharge (Embleton et al, Pediatrics 107(2):270-3 (2001 )). In general, the LBW preterm infants, whether appropriate for gestational age (AGA) or SGA, who show catch-up growth toward normal in the first year of life are more likely to have more optimal outcomes in health, growth, and developmental status through childhood than children who do not catch up (Casey, Semin Perinatol 32(1 ):20-7. (2008)).

Treatment of preterm infants with BSSL

Two Phase 2 studies in 63 preterm infants (BVT.BSSL-020 and

BVT.BSSL-021 ), the first clinical trials with rhBSSL in this patient population, have been completed. RhBSSL was well tolerated with a safety profile similar to that of placebo. In these double-blind crossover studies, patients fed with infant formula (BVT.BSSL-020) or PBM (BVT.BSSL-021 ) were randomly assigned to have rhBSSL 0.15 g/L or placebo added to their food for the first 7 days. After a washout period of 2 days, the patients crossed over to the other treatment regimen and received an additional 7 days of treatment. At enrollment, the feeding volume was within the range of 150 to 180 mL/kg/day. The volume was kept constant for each individual throughout the study. The primary efficacy assessment (coefficient of fat absorption) was made by measuring the fat in the food and corresponding stool during the last 3 days (72 hours) of each treatment period.

A prospectively defined meta-analysis of the 2 studies showed that growth velocity was statistically significantly greater with rhBSSL than with placebo (P<0.001 ) and increased by 2.93 g/kg/day more on rhBSSL compared with placebo. This is considered clinically relevant and corresponds to a 20% improvement. Analysis of coefficient of fat absorption showed no statistically significant improvement with rhBSSL compared with placebo (P=0.069). There was no difference between rhBSSL and placebo in change in knee-to-heel length. An exploratory analysis of the coefficient of absorption for arachidonic acid (AA) and docosahexaenoic acid (DHA) showed increased absorption of both these fatty acids with rhBSSL compared with placebo.

The above mentioned clinical studies are also referred to in

WO 2012/052059 and WO 2012/052060, which relate to methods to increase the absorption of unsaturated fatty acids in human infants, and to increase the growth velocity of human infants.

For estimating nutrient requirements in the neonatal period, the

American Academy of Pediatrics (AAP) recommends that "postnatal growth should approximate the in utero growth of a normal fetus of the same postconceptional age" (Kleinman; Pediatric nutrition handbook, Ch 4:

Nutritional needs of the preterm infant, 6th ed. 79-1 12.). The optimal weight gain to aim for should be similar to that in the third trimester of 17 g/kg/day, i.e. within the range of 15 to 20 g/kg/day. Although awareness and

understanding of the importance of optimized nutrition exists, there is room for considerable improvement in the nutritional regimens of preterm newborn infants. Disclosure of the invention

It is an object of the present invention to provide novel methods and

compositions that are beneficial for infants, in particular for underweight, small and preterm infants. It is moreover an object of the present invention to provide novel dosage regimes for treatment of preterm infants being small for gestational age and thus risking short- and long-term consequences.

In one aspect, there is provided a recombinant human bile-salt- stimulated lipase (rhBSSL) for use in treatment of a human infant risking growth restriction, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth of said infant.

Growth restriction is here defined as whether or not a human infant's weight is below the 10th percentile after said period of at least four weeks of treatment. The 10th percentile at a certain gestational age is defined from the intrauterine growth curves previously established by Olsen et al (Pediatrics 125:214-224 (2010)). By reducing the risk for growth restriction, an infant may grow in accordance with normal growth curves. Preterm infants may thus be of particular interest for such a treatment, but also full term infants that for some reason may risk growth restriction, for example due to a low birth weight or underweight in the early post-natal period or early infancy. The therapeutic use of rhBSSL as disclosed herein may consequently benefit such infants by improving growth. In one embodiment thereof, said treatment prevents growth restriction.

An "infant" as used herein should primarily be understood as a child in the first year of life. In some instances, however, infant refers to a child in the first two years of life.

The preterm period, during which growth impairment may occur, corresponds not only to the early post-natal period, but also represents the late-fetal period had the infant been carried to term. This is a period during which significant phases of organ development occur, including brain development and organ differentiation, and for many organs this period represents a very narrow chronological window for their appropriate completion. Important phases of development can be seriously compromised if such growth windows are missed. The inventors have found, as

demonstrated in a clinical study reproduced herein as Example 3, that enteral administration of rhBSSL to preterm infants for a time period of at least around four weeks advantageously reduces the risk for growth restriction. In particular, it has been demonstrated that such treatment reduces the risk for a preterm infant of becoming growth restricted. Thus, in one embodiment, said human infant is a preterm infant.

For preterm infants, the optimal weight gain to aim for is a postnatal growth that approximates the in utero growth of a normal fetus of the same post-conceptional age. Thus in some embodiments of the present invention, the growth should preferably be at least 15 g/kg/day, such as in the range of from 15 g/kg/day to 23 g/kg/day. The term sub-optimal growth which is also used herein may be understood as a growth velocity of less than 15 g per kilogram bodyweight per day during a period of treatment of at least four weeks. As demonstrated in Example 3, treatment with rhBSSL

advantageously reduces the risk of growth restriction in terms of sub-optimal growth. The treatment with rhBSSL as disclosed herein for a period of at least four weeks may moreover positively influence long-term growth and development. For some embodiments, improved growth, e.g. in terms of reduced risk of growth restriction and/or prevention of growth restriction, is achieved in the treatment period of at least four weeks. In other

embodiments, improved growth is achieved in the neonate period, herein primarily defined as the first 28 days after birth, or in the first 3 months after birth, in the first 6 months after birth, in the first year after birth, or in the first two years after birth. A period of at least four weeks of early dietary manipulation with rhBSSL in human infants, such as preterm infants, may thus have major beneficial consequences for later growth and development, which suggests that improved nutrition in postnatal early life may be important.

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. For example, the growth 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 e.g. growth velocity; i.e. an amount of growth in a unit period (such as per day). In certain embodiments of the present invention, an improvement in growth 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. 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, a growth as 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 during which the infant is being administered rhBSSL or receiving medical care such as within a NICU.

As already accounted for, many LBW infants are susceptible to health problems both in an acute phase e.g. while staying NICU, as well as in a midterm and/or long-term perspective. It might therefore be important to find ways to reduce the short- and long-term complications, such as for example Cerebral Palsy, impairment in hearing and vision, and delays or deficiencies in neurocognitive development, that may result from a low birth weight.

In one aspect, there is provided recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant risking

neurodevelopmental delay, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve neurodevelopment of said infant.

The inventors have unexpectedly found that rhBSSL treatment for a period of four weeks reduces the risk of neurodevelopmental delay and/or developmental disability. The mechanism behind the improvement is not yet fully understood, but it is hypothesized that improved absorption of important fatty acids may play a role in preventing neurodevelopmental delay. In one particular embodiment of this aspect, said treatment prevents

neurodevelopmental delay, in particular in preterm infants and/or SGA infants.

Neurodevelopment and in particular incidence of neurodevelopmental delay may suitably be assessed by the Bayley III scales for infant and toddler development. In Examples 3 and 4, Bayley III scales were used at 12 months of corrected age and a neurodevelopment composite is used at 24 months of corrected age for assessing an infant's neurodevelopment. The term

"corrected age" as used herein should be understood as the age of the infant calculated from the expected date of birth.

The neurodevelopment disability composite is defined as presence of any one of the following: • A composite score of less than 85 on any of the cognitive, language or motor domains of Bayley-lll

• Bilateral deafness, defined as need for bilateral amplification

• Bilateral blindness, defined as corrected visual acuity of less than

20/200 (or equivalent) in the better eye

• Cerebral palsy (CP), defined as hypotonia, spastic diplegia, hemiplegia or quadriplegia causing functional deficits that require rehabilitation services

The Bayley-lll is an individually administered instrument that assesses the developmental functioning of infants and young children between 1 month and 42 months of age, across five domains: cognitive, motor, language, social-emotional, and adaptive behavior. Assessments of the cognitive, motor and language domains are conducted using items administered to the child; assessment of the social-emotional and adaptive behavior domains are conducted using parent/primary caregiver response to a questionnaire.

Bayley-lll is primarily used to identify young children with neurodevelopmental delay and to assist health care providers in the intervention planning. A score below 85 on the Bayley scale is considered a neurodevelopmental delay while a score below 70 is considered a severe neurodevelopmental delay. A score of 95-100 roughly represents a normal development. The Bayley

Scales of Infant and Toddler Development (2^nd ed) has been used extensively in research to track the effects of intervention on children's development. The psychometric properties of the Bayley Scales of Infant and Toddler

Development 2^nd ed, have been maintained in Bayley-lll ("Administration Manual for the Bayley Scales of Infant and Toddler Development" 3^rd ed, San Antonio, TX: Pearson (2006)).

An infant risking neurodevelopmental delay may be an infant already considered as suffering from delayed neurodevelopment or an infant at risk of becoming neurodevelopmentally delayed. An infant facing the risk of delayed neurodevelopment may for example be a preterm infant, for reasons already accounted for elsewhere herein, or an infant being SGA.

In one embodiment, said neurodevelopment, such as at an corrected age of 12 months or 24 months, is assessed based on a score from Bayley Scales of Infant and Toddler Development (Bayley III). Thus, at e.g. 12 months corrected age, neurodevelopment of an infant may be assessed and any neurodevelopmental delay may be detected. In one embodiment, said Bayley III score is based on assessment(s) of one or more of cognitive, motor, and language function(s) of the infant. It should be understood that neurodevelopmental delay can exist in one of said functions only, or in two of said functions or in all of said functions.

In an alternative embodiment, neurodevelopment is assessed based on one or more of cognitive, motor, and language function(s) of the infant.

Neurodevelopment may thus be assessed using other methods but Bayley III. Such methods are considered known to the skilled person.

In one embodiment, said neurodevelopmental delay corresponds to a Bayley III score of less than 85. The infant thus risks neurodevelopmental delay corresponding to a Bayley III score of less than 85. In some instances, the neurodevelopmental delay is considered severe and in such cases said Bayley III score is less than 70. The infant thus risks severe

neurodevelopmental delay corresponding to a Bayley III score of less than 70.

In one embodiment, said neurodevelopment is assessed at a corrected age of the infant of 12 months, such as 24 months.

In one aspect, there is provided recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant being small for its gestational age (SGA), said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth velocity of said infant.

As has been demonstrated by the inventors (see Example 3), in particular SGA infants being small at birth benefit from treatment with rhBSSL for a period of four weeks. It has been found that said rhBSSL treatment improves growth by improving growth velocity in this particularly vulnerable patient group. In addition, said rhBSSL treatment has been found to reduce the risk of growth restriction for a human infant being SGA. This can e.g. be defined as reducing the risk of having a daily growth velocity of less than 15 g/kg weight during the treatment period. Thus in one embodiment, said use of rhBSSL reduces the risk of growth restriction, in particular when the infant is SGA and preterm.

The following embodiments specifically relate to all aspects of the present invention.

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).

In particular embodiments of the present invention, the human infant is small for its gestational age (SGA) at birth. SGA should be understood as defined elsewhere herein and primarily refers to an infant's weight at birth. An infant considered as SGA is thus small compared to an expected normal weight at a certain gestational age. This may for example be due to intrauterine growth retardation (IUGR), i.e. an insufficient intra-uterine growth. SGA infants may be preterm but can also be full-term.

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 have not fully developed their pancreas and liver functions, and can often not thrive as well as full-term babies. In alternative embodiments, said preterm infant may be AGA, i.e. appropriate for

gestational age, or SGA, i.e. small for gestational age, at birth.

Accordingly, in other particular embodiments 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. 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. In particular embodiments, said preterm human infant is born before week 35 of gestation, such as before week 34 of gestation, such as before week 33 of gestation, such as before week 32 of gestation, such as before week 31 of gestation, such as before week 30 of gestation, such as before week 29 of gestation, such as before week 28 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.

As will be appreciated by the person of ordinary skill, a human infant is thus (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, in certain embodiments of the invention the rhBSSL 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 with said infant receiving 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 minutes, 15 minutes or 5 minutes, 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.

In certain embodiments of the invention the human infant is not 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 exclusively breastfed or not exclusively fed from expressed (fresh) breast milk from its own mother will receive nutrition 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, and/or 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.

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 rhBSSL 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 rhBSSL 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 thus comprising rhBSSL 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 rhBSSL 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. In one embodiment of the present invention, said treatment moreover improves feeding utilization. Administration of rhBSSL added to e.g. infant formula or PBM has been found to aid utilization of the food. This has been demonstrated in Example 3, wherein preterm infants receiving rhBSSL for a time period of four weeks were found to utilize their food better than the preterm infants not receiving rhBSSL.

In certain embodiments of the invention, the rhBSSL is added to non- fresh breast milk prior to administration to the infant. Thus, before each administration of rhBSSL to the infant, rhBSSL can be added to non-fresh breast milk such that a non-fresh breast milk comprising rhBSSL is formed. In particular, the non-fresh breast milk to which the rhBSSL is added is 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. 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.

In yet another alternative embodiment of the present invention, the recombinant human bile-salt-stimulated lipase is added to an infant formula prior to administration to the infant. RhBSSL is preferably added to infant formula before each enteral administration of rhBSSL such that a infant formula comprising rhBSSL is formed. In one embodiment, said infant is during said time period of at least around four weeks fed infant formula. The skilled person will be aware of the many infant formulae that are commercially available, which include: Enfamil™, Pregestimil™, Nutramigen™, and

Nutramigen AA™ (all marketed or made by Mead Johnson); Similac™, Isomil™, Alimentum™, and EleCare™ (all marketed or made by Abbott Laboratories, Ross division); Nestle: 12 %, the largest producer of formula in the world, makes GoodStart™ (marketed or made by Nestle/Gerber Products Company); Farexl™ and Farex2™ (marketed or made by Wockhardt

Nutrition). For preterm infants, other infant formulae such as Similac Neosure, Entramil Premature, Similac Special Care, Cow & Gate Nutriprem 2 and Entramil Enfacare 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.

A suitable ratio between the amounts of rhBSSL and the other components in the infant feed for the present invention can be obtained if the rhBSSL is present in a concentration of 0.03-0.5 g/L formula or milk. Thus, rhBSSL may be added to infant formula or (previously) pasteurized and/or frozen breast milk to a final concentration of 0.03-0.5 g/L. In particular embodiments the rhBSSL 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. 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. The enzymatic activity as given here has been determined by use of the PNPB assay as described in the attached Example 1 .

In certain embodiments of the present invention, wherein rhBSSL is added to or (co)administered with infant formula, said infant formula

comprises protein in an amount of 2.8 to 4.1 g/100 kcal, carbohydrates in an amount of 9.5 to 12.0 g/100 kcal; and lipids in an amount of 4.4 to

6.0 g/100 kcal. 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 eicosapentaenonic acid (C20:5 n-3) is added its concentration does not exceed the content of DHA.

In the present invention, the amount of rhBSSL 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 reduce the risk of growth restriction, to reduce the risk of neurodevelopmental delay, or to improve growth velocity of the human (e.g. preterm and/or SGA) infant when said lipase is administered to the infant according to aspects and

embodiments described herein. Suitable amounts of rhBSSL that may be administered to the infant in any given day may range from an amount per day of between 1 and 100 mg per Kg weight of infant. In particular

embodiments between 5 and 50 mg per Kg weight of infant or between 15 and 40 mg per Kg weight of infant may be administered over a day, such as between about 22.5 and 27 mg of rhBSSL 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 in Example 1 ), 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 rhBSSL 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.

In one embodiment of the present invention, the rhBSSL is administered at least once per day (such as with at least one feed), over said time period of least around four weeks. In particular such embodiments, the rhBSSL 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 rhBSSL. In another non-limiting embodiment, the rhBSSL is co-administered with the feed to the infant every fifth hour, such as every fourth hour, such as every third or every second hour . 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.

In one embodiment of the present invention, said treatment comprises enteral administration of rhBSSL for a time period of around four weeks. The beneficial effects of such treatment have been demonstrated in e.g. Example 3.

Various embodiments of rhBSSL, useful in the aspects of the invention as defined herein, will now be described. For example, rhBSSL as used herein should be understood as including 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.

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" (www.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 BSSL 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; WO 91/15234; WO 91/18923; 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_001798.2; Swiss-Prot ID:

P19835). In further examples, other shorter isoforms of human bile-salt- stimulated lipase are described in Venter et al (Science 291 :1304-1351

(2001 )); GenBnk ID: AAC71012.1 ; Pasqualini et al (J Biol Chem 273:28208- 28218 (1998)); GenBank ID: EAW88031 .1 ; WO 94/20610 and Blackberg et al (Eur J Biochem 228: 817-821 (1995)).

In particular embodiments of the present invention, the human BSSL 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, supra; WO 91/15234, WO 91/18923; RefSeq ID: NP_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 BSSL 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 ).

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 BSSL 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 be useful in the present invention, and hence are also encompassed by the term "human bile- salt-stimulated lipase". In certain such embodiments, the term encompasses 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, 71 1 , 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.

In other embodiments of the present invention, the human BSSL 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 (supra). 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 3rd 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 BSSL 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).

In a particular embodiment of the present invention, the rhBSSL is produced by expression from a nucleic acid described, defined or referred to herein.

A rhBSSL defined or referred to herein, in the context of the present invention 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, i.e. 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.

In a particular embodiment of the present invention, the rhBSSL 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 Examples appended herein.

The recombinant human bile-salt-stimulated lipase may be enterally administered according to the present invention by various means, including oral administration. 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 weak infants such tube-based forms of administration may be more practical, or may be necessary, to administer the rhBSSL according to the instant invention.

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.

In one embodiment of the present invention, rhBSSL is comprised in a composition suitable for enteral administration. Such a composition may, apart from an effective amount of rhBSSL, also comprise one or more of the following constituents: a buffer, such as sodium phosphate, a solubility agent, such as sodium chloride, a bulking agent, such as mannitol, and a stabilizing agent, such as glycine. Such a composition, also denoted rhBSSL

composition, may advantageously be used in any one of the embodiments or aspects as disclosed herein.

RhBSSL may preferably be provided as a lyophilized powder for oral solution. In certain embodiments, the lyophilized powder is sterile. In yet an embodiment, the lyophilized rhBSSL powder comprises a rhBSSL

composition as previously defined herein. The lyophilized powder may be reconstituted and added to infant formula or PBM, preferably in a

concentration representing the physiological concentration found in breast milk. As the compound is most likely not absorbed when administered orally, distribution will remain localized to the gastrointestinal tract. It is hypothesized that the compound will be deactivated by proteases to smaller peptides and amino acids. Any intact BSSL is thus expected to be excreted unchanged in the feces. Amino acids resulting from the enzymatic degradation of BSSL may be absorbed and handled as other dietary amino acids.

Numerous analyses, studies and reviews have previously discussed the link between unsaturated fats, especially long-chain polyunsaturated fatty acids (LCPUFAs) derived from linoleic acid (LA) and alpha-linolenic acid (LNA), and visual and/or cognitive development or function, for example as summarized by McCann & Ames in 2005 (Am J Clin Nutr, 82: 281 -295).

Indeed, on the basis of all available evidence, it has been recommended that infant formulas be supplemented with the LCPUFAs docosahexaenoic acid (DHA) and arachidonic acid (AA), and for pregnant and lactating women to include some food sources of DHA in the diet in view of their assumed increase in LCPUFA demand and the relationship between maternal and fetal/infant DHA status (Koletzko et al, J Perinat Med 36: 5-14 (2008)).

In one aspect, there is provided recombinant human bile-salt-stimulated lipase (rhBSSL) for use in increasing the absorption of at least one

unsaturated fatty acid in a human infant risking fat malabsorption, said use comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks.

It is hypothesized that the rhBSSL treatment for four weeks improves absorption of at least one unsaturated fatty acid, and that this improvement may be related to the reduced occurrence of neurodevelopmental delay observed in preterm infants treated with rhBSSL for four weeks. Thus, the present aspect may in some instances also constitute an embodiment to the previous aspects, in particular the aspect providing rhBSSL for use in reducing the risk of neurodevelopmental delay.

The term "unsaturated fatty acid" will be readily identified by the person of ordinary skill in the art, and for example encompasses any carboxylic acid with an un-branched aliphatic tail (chain) that has at least one double bond between two adjacent carbon atoms in the chain. The two carbon atoms in the chain that are bound next to either side of the double bond can occur in a cis or trans configuration. In certain embodiments of the present invention at least one double bond in said unsaturated fatty acids is in the cis

configuration. In further embodiments of the present invention, the

unsaturated fatty acids may be further characterized as described 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.

In one embodiment, said unsaturated fatty acid is selected from the group of: an essential fatty acid; a polyunsaturated fatty acid; an unsaturated fatty acid that has an aliphatic chain of 20 or more carbon atoms; and/or a polyunsaturated fatty acid that has an aliphatic chain of 20 or more carbon atoms (long chain polyunsaturated fatty acid - LCPUFA). RhBSSL may also improve uptake of LCPUFAs such as AA and DHA. These LCPUFAs are required for normal growth and maturation of numerous organ systems, most importantly the brain and eye. As outlined above, it is hypothesized that absorption of fatty acids may play a role in preventing neurodevelopmental delay.

In one embodiment, said at least one unsaturated fatty acid is one selected from the group consisting of: eicosadienoic acid (C20:2 n-6), dihomo-gamma-linolenic acid (C20:3 n-6), eicosatrienoic acid (C20:3 n-3), arachidonic acid (C20:4 n-6) and docosahexaenoic acid (C22:6 n-3), linoleic acid (C18:2 n-6) and alpha-linolenic acid (C18:3 n-3), preferably wherein said unsaturated fatty acid is arachidonic acid (C20:4 n-6) and/or

docosahexaenoic acid (C22:6 n-3).

In one embodiment common to the aspects disclosed herein, said rhBSSL for use further improves quality of growth by reducing the risk of impaired growth quality. Quality of growth may herein refer to a growth not only resulting in a weight increase but also in e.g. growth of the brain (as measured e.g. by head circumference) and body length. Alternative methods for measuring quality of growth are known to the skilled person, e.g.

measurements of body composition using Caliper, measurement of ratio of body fat/muscle/skeleton. Alternatively, an increased absorption of fatty acids may in some instances indicate an improved quality of growth.

In one aspect, there is provided a method of treatment of a human infant risking growth restriction, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth of said infant. The advantages and benefits of the method aspects are the similar to the advantages and benefits disclosed in relation to the different aspects and embodiments above directed to rhBSSL for use in different methods of treatment. The advantages and benefits are thus not repeated here, but reference is instead made to the advantages and benefits mentioned above in relation to the corresponding rhBSSL aspect/embodiment.

In one embodiment, growth restriction corresponds to a daily growth of less than 15 g/kg weight of the infant. In one aspect, there is provided a method of treatment of a human infant risking neurodevelopmental delay, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve neurodevelopment of said infant.

In one particular embodiment, neurodevelopment is assessed based on a score from Bayley Scales of Infant and Toddler Development (Bayley III).

In one particular embodiment, said Bayley III score is based on assessment(s) of one or more of cognitive, motor, and language function(s) of the infant.

In an alternative embodiment, neurodevelopment is assessed based on one or more of cognitive, motor, and language function(s) of the infant.

Neurodevelopment may thus be assessed using other methods but Bayley III. Such methods are considered known to the skilled person.

In one embodiment, said neurodevelopmental delay corresponds to a Bayley III score of less than 85.

In one particular embodiment, said neurodevelopmental delay is assessed at a corrected age of the infant of 12 months, such as 24 months.

In one embodiment, said treatment prevents neurodevelopmental delay.

In one particular embodiment, said infant suffers from underweight, such as low birth weight.

In one particular embodiment, said infant is small for gestational age at birth.

In one aspect, there is provided a method of treatment of a human infant human being small for its gestational age, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth velocity of said infant. In one particular embodiment of the method aspects, said infant suffers from preterm birth.

Other embodiments of the method aspects correspond to the

embodiments disclosed for the aspects providing rhBSSL for use.

In one aspect, there is provided a method to increase the absorption of at least one unsaturated fatty acid in a human infant risking fat malabsorption, comprising enteral administration of recombinant human bile-salt-stimulated lipase to said infant for a time period of at least around four weeks. In embodiments of this aspect, the unsaturated fatty acid is as defined in related aspects above.

In yet a related aspect, there is provided a modified infant formula comprising recombinant rhBSSL in an amount effective for use in treatment to reduce the risk of growth restriction, to reduce the risk of neurodevelopmental delay, or to improve growth velocity of a human infant, such as a preterm or underweight infant. Said modified infant formula is fed to said infant over an administration regimen for at least four weeks as described or defined elsewhere herein.

In one embodiment of such an 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 °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. In particular embodiments, the infant formula may have a protein, carbohydrate, and/or lipid content as defined herein.

In a related aspect, there is provided a modified pasteurized breast milk comprising rhBSSL in an amount effective for use in treatment to reduce the risk of growth restriction, to reduce the risk of neurodevelopmental delay, or to improve growth velocity of a human infant, such as a preterm or underweight infant. Said modified pasteurized breast milk is for example fed to said infant over an administration regimen for at least four weeks as described or defined elsewhere herein. In one embodiment of such an 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 °C.

In one related aspect, there is provided a pharmaceutical composition comprising between 0.1 and 100 mg of rhBSSL, wherein said lipase is preferably not isolated from the milk or transgenic sheep, and wherein said pharmaceutical composition is for use in treatment to reduce the risk of growth restriction, to reduce the risk of neurodevelopmental delay, or to improve growth velocity of a human infant, such as a preterm or underweight infant. Said pharmaceutical composition is preferably intended for treatment of said infant for a time period of at least four weeks.

Embodiments of the infant formula, PBM, and pharmaceutical composition aspects correspond to the embodiments disclosed for the aspects providing rhBSSL for use.

Brief description of the drawings

The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawing which is given by way of illustration only, and thus are not limitative of the present invention, and wherein:

Figure 1 shows a schematic plan of the clinical studies of rhBSSL added to infant formula or to pasteurized breast milk.

Examples

The following examples, 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. Example 1 :

Preparation and characterization of rhBSSL

Material and methods:

An exemplary drug substance, i.e. 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. Cells from the master cell bank (MCB) were thawed, expanded in shaker flasks using EX-CELL 302 medium without glutamine and glucose (SAFC) and later supplemented with glutamine and glucose, followed by growth in 15 and 100 L bioreactors, before inoculating the 700 L production bioreactor where BSSL was constitutively expressed and produced in a fed-batch process. The culture was harvested as a single batch and the mature rhBSSL polypeptide (i.e. without the leader sequence) was purified from cells, cell debris and other contaminates via a number of downstream steps, including an anion exchange chromatography step.

Contaminating viruses was inactivated by low pH treatment and a dry heat treatment step. The rhBSSL Drug Substance (DS) bulk was diafiltered and concentrated to the appropriate formulation. After formulation, the material was divided in one to three batches for lyophilization and heat treatment, generating one to three DS batches.

Specific activity was determined using a 4-nitrophenyl ester butyric acid (PNPB) assay. Each sample was specifically purified by HA-HPLC and SE-HPLC before determination of specific activity. Specific activity was determined using 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) was prepared in PBS with 0.1 % BSA. 200 μΙ of these rhBSSL solutions is added to 25 μΙ of an activation solution containing 20 mM sodium cholate (as bile-salt activator) in PBS with 0.1 % BSA. These solutions were preincubated in a spectrophotometer at 27°C for 5 minutes. Just before measuring, 25 μΙ of a well-mixed substrate solution containing 5 mM PNPB in PBS-Tween was added. The formation of 4-nitrophenol was detected by its absorbance at 400 nm and the increase in absorbance was measured during 90 seconds. The active amount of BSSL was determined using a standard curve of an rhBSSL reference standard.

Results:

Production of rhBSSL in this mammalian-cell expression system produced rhBSSL having a predicted amino acid sequence as shown in SEQ ID NO:1 . A predicted structure has been previously disclosed in Fig. 1 .1 of

WO 2012/052060, wherein potential glycosylation sites also are marked.

This form of rhBSSL appeared to exhibit glycosylation that is different to native hBSSL found in human milk (BSSL-MAM) and also to rhBSSL-OVI (produced from transgenic sheep) (results not shown). It was also found that by C-terminal amino acid sequence analysis that a large proportion of the lipase molecules are shortened by one (occasionally two) amino acids compared to the (predicted) full length polypeptide molecules. Differences in functional properties have also been 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.

Example 2:

Preparation of investigational drug product

Material and methods:

The investigational medicinal product was prepared from lyophilized drug substance, i.e. rhBSSL, for example produced as described in Example 1 . The drug substance was dissolved in water for injection, the resulting solution was pre-filtered (10 μηη), and adjusted to the final (active) concentration with water for injection. The product was filtered through a 0.22 μηη filter and filled into pre-sterilized 10 ml_ glass vials. The vials were stoppered with sterilized stoppers and sealed with aluminium caps.

Example 3:

Data from a Phase III study with rhBSSL

(Protocol number: BVT.BSSL-030, EudraCT Number: 2010-023909-35) A phase III study has been performed with rhBSSL during four weeks of treatment in preterm infants. The purpose of this prospective, randomized, double-blind phase III study was to determine the efficacy (improved growth) of rhBSSL and to compare the safety and tolerability of rhBSSL treatment with that of placebo treatment after oral administration by addition to the food, i.e. to infant formula and/or pasteurized breast milk (PBM). In addition, long- term safety and effect (neurodevelopment and anthropometrics) of rhBSSL up to 12 months corrected age of the infants were assessed. The study design is outlined in Figure 1 . Objectives: Objectives of this study was to demonstrate that rhBSSL improves growth in preterm infants as compared with placebo when administered in infant formula or PBM. Other objectives were to determine the effect of rhBSSL treatment in decreasing risk of growth restriction, to determine the effect of rhBSSL treatment in increasing the levels of DHA and AA, and to determine the effect of rhBSSL treatment on neurodevelopment.

Exploratory objectives were e.g. to assess the effect of rhBSSL treatment on fatty acid levels; to assess the effect of rhBSSL on the body composition, and to assess the effect of rhBSSL treatment on the fecal calprotectin and microbiota contents.

Study design and treatments: 410 evaluable patients were randomly assigned to receive either treatment with rhBSSL or placebo. Patients were randomly assigned to study drug only if they met all of the inclusion and none of the exclusion criteria as outlined in the sections below. The randomization was stratified by feeding regimen (PBM or infant formula) and by size for gestational age category (SGA or AGA). An infant having a birth weight that lies above the 10th percentile for the gestational age on the gender-specific intrauterine growth curves presented by Olsen et al (Pediatrics 125(2):e214- 24 (2010)) is defined as AGA. An infant with a birth weight at or below the 10th percentile is defined as SGA.

Patients having reached a level of enteral feeding of at least

100 mL/kg/day, were randomly assigned to receive rhBSSL 8700 U or placebo added to 100 mL of the food. The feeding volumes were decided according to each unit's feeding plan, and the patient's body weight.

The addition of study drug to the food started as soon as possible after randomization, either on the day of randomization or the day after. The predose assessments on day 1 (the day the first dose of study drug is administered) constituted the patient's baseline values.

RhBSSL, which can be prepared as described in Example 2, was delivered as a sterile powder for oral solution, in a single-dose glass vial containing 8700 U (corresponding to 15 mg active rhBSSL). The content of 1 vial was intended for reconstruction in 1 mL of sterile water before addition to 100 mL of food. The matching placebo was also delivered as a sterile powder for oral solution in an identical single-dose container.

Study Endpoints: Apart from growth velocity in grams per kilogram per day during 4 weeks of treatment, the study moreover comprised the following efficacy endpoints:

• Change from Baseline in body weight (g) at 3 months

• Body weight (g) at 12 and 24 months corrected age

• Growth restriction, defined as growth velocity <15 g per kilogram

bodyweight per day during 4 weeks of treatment

• Levels of DHA and AA at 4 weeks

One of the secondary safety endpoints was Bayley Scales of Infant and Toddler Development (3rd edition, Bayley-lll) scores at 12 and 24 months of corrected age, consisting of:

■ Cognitive domain composite scores.

^■ Language domain composite scores.

^■ Motor domain composite scores.

^■ Scaled scores for each subtest and for the cognitive domain (in total 5 scaled scores).

Lastly, exploratory endpoints were levels of fatty acids, percent body fat, fecal calprotectin concentration, and fecal microbiota. Inclusion criteria: Each patient had to meet all of the following criteria to be enrolled in this study:

• Preterm infant born before week 32 of gestation and who is <33 weeks postmenstrual age at the time of randomization.

· Preterm infant who is AGA or SGA at birth.

• Preterm infant who is receiving food enterally (bottle or gavage tube) at a level of at least 100 mL/kg/day at randomization.

• Preterm infant whose enteral feeding consists of only infant formula or only PBM at the time of inclusion, and who are expected to remain on only infant formula for 4 weeks, or only PBM for at least 2 weeks following treatment initiation.

• Preterm infant who is expected not to receive any fresh breast milk for 4 weeks following treatment initiation. Exclusion criteria: Patients meeting any of the following criteria were not eligible for enrollment:

• Expected stay at the hospital is less than 4 weeks from the first dose of study drug.

• Currently receiving mechanical ventilation via endotracheal tube

(continuous positive airway pressure, CPAP, or high-flow nasal cannula are not criteria for exclusion).

• Require >30% oxygen (if on CPAP or in head box) or >0.5 L/min oxygen.

• Evidence of severe brain disease or damage, including grade III or IV peri- or intraventricular hemorrhage, meningitis or hydrocephalus, grade III or IV intracranial hemorrhage, or periventricular leukomalacia.

• Presence of major dysmorphology or congenital abnormalities that are likely to affect growth and/or development.

• Current clinical evidence of hemodynamically significant persistent ductus arteriosus.

· Clinical evidence of sepsis (including low or high white blood cell count and/or low platelet count and bacteriologically proven evidence of systemic infection). This should be based on the investigator's opinion and available local laboratory reference ranges.

• Systemic anti-infective treatment within 48 hours prior to randomization, other than prophylactic treatment (eg, antifungal prophylaxis with fluconazole) as per local clinical practice.

• Evidence of congenital infection (eg, cytomegalovirus). • Previous or current diagnosis of necrotizing enterocolitis (Bell's stage 2 or greater).

• Prior or current treatment with corticosteroids, except hydrocortisone.

• Presence of any condition that in the opinion of the investigator makes the patient unsuitable for inclusion.

• Enrolled in another concurrent clinical intervention study.

Food requirements: Before inclusion of the first patient, each study center had to determine a target food volume, to be used for all infants in the study. This volume was in the range of 150 to 180 mL/kg/day. Once an infant reached the target volume, the food volume was not to be changed during the treatment period.

The daily volume (ml_) of food enterally fed to the patient was recorded every day during the treatment period. The volume recorded took into account the food that remained in the bottle at the end of feeding.

The total daily volume (ml_) of parenterally administered nutrition received by the patient during the treatment period was recorded. The product name and volume of any parenterally administered nutrition containing a fat emulsion were also recorded.

Each study center selected one infant formula that all formula fed patients at that study center was fed. They were to remain on the same infant formula throughout the treatment period, unless medically contraindicated.

The range of protein, carbohydrate, and lipid content for the infant formulas allowed for use in this study are indicated below in Table 2.

Table 2. Infant formula content

In addition, all infant formulas to be used in the study had to contain arachidonic acid (AA) and docosahexaenoic acid (DHA). The infant formula had to contain less than or equal to 40 % of medium-chain triglycerides (TGs).

There were no special requirements with regard to the source or quality of the PBM. The milk could be the mother's own or come from a milk bank (donor milk). The composition of the milk (fat, protein, and lactose) was recorded when available. Fortification of the milk was done according to a predefined study center-specific schedule. Preferably, one fortifier was used at a study center, and the amounts added was fixed, i.e., the same

concentration added to all PBM. Fortification could not be individualized based on body weight. All use of fortifiers, including lipid emulsions, was to be recorded.

The availability of PBM varied between the neonatal intensive care units (NICUs), and thus the time period during which an infant was given only PBM before switching to infant formula varied. In this study, the time of switching to infant formula was not based on body weight. Instead, each study center using PBM in this study had to, before the first infant was randomized, declare which of the following alternatives was to be followed for the infants in the study: 1 ) PBM only for 4 weeks, or 2) PBM only for 2 weeks followed by infant formula for 2 weeks.

No fresh breast milk were to be used during the 29 days of treatment. However, for ethical reasons, breast feeding could not be prohibited.

Efficacy assessments: Body weight at birth and the lowest measured body weight were retrospectively recorded. The patient's body weight in grams was, as a minimum, measured at Baseline (Day 1 , before start of study drug administration), on Days 8, 15, 22, and 29, and on at least 2 other time points per week. Thereafter, body weight was recorded at least weekly until discharge, and at each scheduled follow-up visit.

Fatty acids, including DHA and AA, were determined in either serum or plasma at Baseline (Day 1 ) and at the end of treatment (Day 29).

Determination of fatty acids other than DHA and AA were considered exploratory assessments. Safety assessments: In this study, Bayley-lll was used to assess

neurodevelopment. The Bayley Scales of Infant and Toddler Development, third edition (Bayley-lll; Bayley, Administration Manual for the Bayley Scales of Infant and Toddler Development, Third Edition. San Antonio, TX: Pearson; 2006) is an individually administered instrument that assesses the

developmental functioning of infants and young children between 1 month and 42 months of age, across five domains: cognitive, motor, language, social-emotional, and adaptive behavior. Assessments of the cognitive, motor and language domains are conducted using items administered to the child; assessment of the social-emotional and adaptive behavior domains are conducted using parent/primary caregiver response to a questionnaire.

Bayley-lll is primarily used to identify young children with neurodevelopmental delay and to assist health care providers in the intervention.

Bayley III was used to assess neurodevelopment. The cognitive, language and motor domains was assessed at 12 months corrected age. Bayley III is also assessed at 24 months corrected age, at which point the social-emotional and adaptive behavior domains also are assessed.

There are 91 items in the cognitive domain scale. The language domain scale is comprised of two subtests, receptive communication, which is comprised of 49 items and expressive communication, which is comprised of 48 items. The motor scale is composed of two subtests, fine motor, which is comprised of 66 items and gross motor, which is comprised of 72 items.

Raw scores were determined for each child. Four types of non- referenced scores can also be obtained: scaled scores; composite scores, percentile ranks and growth scores. For the purposes of the analysis at 12 months corrected age visit, composite score was calculated for each of the domains, and scaled scores were calculated for each subtest as well as for the cognitive domain. A score of less than 70 was considered as an indication of severe neurodevelopmental delay.

At 24 months corrected age, the Bayley-lll scores are evaluated as secondary efficacy endpoints.

In addition, a neurodevelopment disability composite is used to assess neurodevelopment, This composite was defined as presence of any one of the following:

• A composite score of less than 85 on any of the cognitive, language or motor domains of Bayley-lll

• Bilateral deafness, defined as need for bilateral amplification

· Bilateral blindness, defined as corrected visual acuity of less than

20/200 (or equivalent) in the better eye

• Cerebral palsy (CP), defined as hypotonia, spastic diplegia, hemiplegia or quadriplegia causing functional deficits that require rehabilitation services Exploratory assessments: Fatty acids (Phosphatidylcholine (PC), Total Fatty Acid (TFA) and Triglyceride (TG)) are determined in serum at Baseline (TFA only) and end of treatment (4 weeks, PC and TG only).

Body composition (percent fat mass) is determined by air displacement plethysmography at study centers with access to the necessary equipment (Pea Pod®). Measurements were performed at Baseline (Day 1 ), at the end of treatment (Day 29), at 40 weeks postmenstrual age, and 3 months corrected age, with the 2 latter visits scheduled only for patients with body composition assessments during the treatment period.

A fecal sample was collected at Baseline (Day 1 ) and at the end of treatment (Day 29). The samples are analyzed with respect to calprotectin and bacterial contents.

Demography: The following was recorded at Baseline: body weight, total body length, head circumference, sex, race, actual birth date, expected birth date, body weight at birth, and information related to multiple birth.

Results

The full-analysis set (FAS) was the primary analysis set and consisted of all 410 patients randomly assigned to treatment who had a baseline and at least 1 post-baseline assessment of body weight. All analyses using the FAS grouped patients according to randomized treatment.

Demography: The demography and disposition of the patient group in the FAS is accounted for below in Table 3:

Table 3: Patient demo ra h FAS

Since the SGA group of infants proved to be of particular interest,

demographic variables were studied also for this patient group, see Table 4 below.

Table 4. Demography in SGA group

rhBSSL Placebo Total (N=62) Total (N=32) (N=30) SGA pts (N=410) SGA pts SGA pts All patients

Gender Male 15 (46.9%) 17 (56.7%) 32 (51.6%) 189 (46.1 %)

Female 17 (53.1 %) 13 (43.3%) 30 (48.4%) 221 (53.9%)

Gestational Mean (SD) 28.9 (1.6) 29.3 (1.6) 29.1 (1.6) 28.8 (1.7) age at birth Median (Min- 29.0 (25.0- 29.5 (25.9- 29.3 (25.0- 29.0 (24.0- (weeks) Max) 31.3) 31.6) 31.6) 31.9)

PMA at Mean (SD) 32.0 (0.9) 32.2 (0.9) 32.1 (0.9) 31 .9 (1.0) randomization Median (Min- 32.4 (29.6- 32.5 (29.0- 32.4 (29.0- 32.3 (26.9-

(weeks) Max) 32.9) 32.9) 32.9) 32.9)

Age at Mean (SD) 3.2 (1.6) 3.0 (1.3) 3.1 (1.4) 3.2 (1.5) randomization Median (Min- 2.8 (1.0-7.9) 2.8 (1.0-6.4) 2.8 (1.0-7.9) 2.9 (1.0-8.4) (weeks) Max)

Baseline Mean (SD) 1091 (202) 1 106 (193) 1098 (196) 1387 (284) weight Median (Min- 1 160 (690- 1088 (740- 1 105 (690- 1387 (690- Max) 1455) 1500) 1500) 2130)

Size for GA SGA 32 (100%) 30 (100%) 62 (100%) 62 (15%) category AGA 0 0 0 348 (85%)

Feeding Formula 20 (62%) 18 (60%) 38 (61 %) 255 (62%) regimen PBM 12 (38%) 12 (40%) 24 (39%) 155(38%) The demographics of the SGA group corresponded well with the

demographics for the FAS. Differences were as expected however found in the baseline weight; SGA infants on average having a lower birth weight (1098 g) compared to other infants in the FAS.

Efficacy endpoints: The primary efficacy measurement (growth velocity) was made by frequent (at least 3 times per week) measurements of the infants' weight during treatment. Although no significant effect of rhBSSL treatment on growth velocity (results not shown) compared to placebo could be seen as such in the full population of the study group, other effects of rhBSSL was surprisingly observed.

Notably, in the SGA group, consisting in total of 62 patients of which 32 received rhBSSL and 30 received placebo, a statistically significant improvement in growth velocity was observed. The least squares (LS) mean in the rhBSSL group was 17.1 g/kg/day, whereas the LS mean in the placebo group was 15.1 g/kg/day. The difference in growth velocity between the group was thus 1 .95 g/kg/day. RhBSSL administered with infant formula or PBM over a time period of four weeks was thus found to improve growth velocity for infants being SGA at birth.

Moreover, rhBSSL was found to reduce the risk of growth restriction.

The number of patients in the rhBSSL group (50 patients, corresponding to 24.3 %, see Table 4 below) having a growth velocity below 15 g/kg/day during the four week treatment period were fewer compared to the number of patients in the corresponding placebo group (58 patients, corresponding to 28.4 %). In addition, the number of patients (68, corresponding to 33.3 %) in the group that received rhBSSL that had a weight below the 10^th percentile at 4 weeks were fewer than the number of corresponding patients (74, 36.3 %) in the group that received placebo. Thus, despite the fact that the patients in the placebo group on average received a larger daily feeding volume than the patients in the rhBSSL group (the difference was 3-4 mL/kg/day), a reduced incidence of growth restriction could thus be observed in the rhBSSL group. Table 5. Growth restriction, FAS and SGA infants

In addition, rhBSSL treatment for a period of four weeks was found to reduce growth restriction in SGA infants. A numerical (but not statistically significant) improvement was observed in favor of rhBSSL compared to placebo in the proportion of patients with a growth velocity < 15 g/kg/day, or a weight below the 10th percentile.

Feeding utilization, i.e. the ability to utilize administered food, was found to be significantly improved in the infant population that received rhBSSL. Feeding utilization was found to be 1 1 1 .1 g/L in the rhBSSL group and 106.8 g/L in the placebo group. Thus, although the infants in the rhBSSL group on average received less food, they could utilize the food better. For an infant receiving a feeding volume of 150 ml/kg/day, the observed difference (4.3 g/L) in feeding utilization corresponds to a weight increase of 0.65 g/kg/day. See Table 6 below.

Table 6. Feeding and feeding utilization

rhBSSL (N=206) Placebo (N=204)

Average feeding (ml/kg/day)

Mean (SD) 145.6 (17.8) 149.6 (16.2)

Median (Min - Max) 148.9 (56.0 - 184.4) 151 .6 (49.5 - 197.6)

Feeding Utilization (g/L)

Mean (SD) 1 13.9 (23.6) 109.7 (23.3)

Median (Min - Max) 1 13.7 (46.8 - 210.8) 109.4 (63.8 - 233.0)

Least Squares Mean 1 1 1 .1 106.8

95% CI (107.3, 1 14.9) (102.9, 1 10.6)

Least Squares Mean difference 4.3

95% CI (0.120, 8.574) p-value 0.044 It was further demonstrated that feeding utilization in favor of rhBSSL was even better in the SGA group. In this group, average feeding volume was 1 -2 ml/kg/day lower for the infants that received rhBSSL compared to the infants that received placebo. A statistical significant advantage for rhBSSL was observed in feeding utilization. The SGA infants that received rhBSSL had a feeding utilization of 120.4 g/L, whereas the infants that received placebo had a feeding utilization of 107.7 g/L (LS mean values). The observed difference of 12.7 g/L corresponds to a weight increase of 1 .9 g/kg/day in an infant receiving a feeding volume of 150 ml/kg/day.

Safety endpoints: A higher incidence of adverse events was observed during the four weeks treatment period in the rhBSSL treated infants, with signs of gastrointestinal intolerance and potential cases of necrotising enterocolitis (NEC). Further investigation is required to better understand these findings.

Although the Bayley Scales were included in the study as safety endpoints, it was surprisingly demonstrated that administration of rhBSSL added to infant formula or PBM for a time period of four weeks actually improved neurodevelopment.

Neurodevelopment was assessed for 201 infants at 12 months corrected age across the three domains as indicated in the Table above. Scores were obtained for each domain. Although the mean and median scores were quite similar for the rhBSSL group (99 infants) and the placebo group (102 infants), the number of infants considered as suffering from severe

neurodevelopmental delay (a score less than 70) was smaller in the rhBSSL group compared to in the placebo group across all three domains. The number of infants considered as suffering from neurodevelopmental delay (a score less than 85) based on an assessment of the language domain was smaller in the rhBSSL group compared to in the placebo group.

Improvements in neurodevelopment were also observed in the SGA group (29 infants). Among the SGA infants that received rhBSSL (13 infants), no severe neurodevelopmental delay could be observed in any of the studied domains (cognitive, language, and motor domain). In comparison, a number of infants in the placebo group showed signs of severe neurodevelopmental delay (a score less than 70) in the language (12 %) and motor (6 %) domains. No clear tendency could however be observed between the groups and no conclusion could thus be made for neurodevelopmental delay (a score less than 85).

Results from blood sample analysis of fatty acids are expected to partly explain some of the above findings, for example the observed improvements in reducing incidence of neurodevelopmental delay.

Conclusions: The conducted clinical study demonstrated that rhBSSL added to infant formula or PBM and administered during a time period of four weeks compared to placebo surprisingly:

• improved growth velocity of infants being small for gestational age

(SGA; below the 10^th percentile) at birth. The growth velocity for SGA infants that received rhBSSL were 17.1 g/kg/day while the growth velocity for SGA infants that received placebo was 15.1 g/kg/day.

• reduced the risk of growth restriction for preterm infants. Growth

restriction is defined as a weight below the 10^th percentile at a certain gestational age, or as sub-optimal growth, i.e. a growth velocity below 15 g/kg/day. RhBSSL moreover reduces the risk for SGA infants of becoming growth restricted.

• reduced the risk of (severe) neurodevelopmental delay in preterm

infants. Neurodevelopment was assessed using Bayley III Scores. · improved feeding utilization. Example 4:

Assessment of long-term effects of rhBSSL treatment on neurodevelopment

The study described in Example 3 included the assessments of long-term effect of rhBSSL treatment. The study moreover includes assessments of neurodevelopment at 24 months corrected age. Thus, the assessments in accordance with Bayley-lll are once more performed at 24 months of corrected age. Bayley-lll assessments: Bayley-lll is used to assess neurodevelopment as described in Example 3. The cognitive, motor, and language domains are assessed at 24 months corrected age. At 24 months corrected age, the social-emotional and adaptive behavior domains are also assessed.

A composite score is calculated for each of the 5 domains, and scaled scores are calculated for each subtest as well as for the cognitive and social- emotional domains.

At 24 months corrected age, the Bayley-lll scores are evaluated as secondary efficacy endpoints. Child behavior checklist: The child behavior checklist (CBCL) for 1 .5 to 5 years of age is a parent-rated scale that is used to evaluate mental health and behavioral development of children at 24 months corrected age. It consists of 99 problem items and one open-ended item for recording other problems not listed on the form. Each item will be rated by the parent/primary caregiver on a 0-2 point scale, where 0=not true, 1 = somewhat or sometimes true and 2=very true or often true.

There are 7 syndrome subscales (emotionally reactive,

anxious/depressed, somatic complaints, withdrawn, sleep problems, attention problems and aggressive behavior). These subscales are evaluated by summarizing the ratings for the items comprising each syndrome (Rescorla, Ment Retard Dev D R 11 :226-37 (2005)). The subscales are also summarized as internalizing problems (emotionally reactive, anxious/depressed, somatic complaints and withdrawn items) and externalizing problems (attention problems and aggressive behavior items), and a total problem score is calculated. Neurodevelopment disability composite: A neurodevelopment disability composite is assessed at 24 months corrected age and is defined as presence of any one of the following:

• A composite score of less than 85 on any of the cognitive, language or motor domains of Bayley-lll

• Bilateral deafness, defined as need for bilateral amplification

• Bilateral blindness, defined as corrected visual acuity of less than

20/200 (or equivalent) in the better eye

• Cerebral palsy (CP), defined as hypotonia, spastic diplegia, hemiplegia or quadriplegia causing functional deficits that require rehabilitation services

Assessment of confounders of neurodevelopment: Even with randomization, confounding of intervention groups may occur when assessing developmental outcomes. Numerous factors related to child developmental outcome have been identified as confounding variables which need to be considered in randomized, controlled, trials of infant nutritional interventions. These variables include socioeconomic status, parental education, race, income, maternal IQ, age, psychological status, parity, marital status, child gender, number of children in the home and exposure to teratogenic substances (Singer, Semin Neonatol 6:393-401 (2001 )). In this study, confounders deemed to have a possible influence on the neurodevelopmental outcome are selected.

The following confounders of neurodevelopment were recorded as soon as informed consent was provided (earliest at the 12-months-corrected-age visit): medical and social factors of the biological mother, namely maternal height, weight and age at time of conception; presence of diabetes and/or preeclampsia during this pregnancy; nicotine use, alcohol intake and/or drug abuse during this pregnancy, and number of previous pregnancies (all pregnancies that lasted beyond week 16 of gestation to be included).

The following confounders of neurodevelopment are recorded at the 24- months-corrected-age visit: factors of the household the patient currently lives in; namely number of children <18 years of age in the household, number of parents/primary caregivers in the household, socio economic status of parents/primary caregivers (educational level, employment status and income level). Results:

It is hypothesized that rhBSSL treatment would reduce the risk of

(neuro)developmental delay as assessed also when the infant is 24 months (corrected age).

Claims

Claims

1 . Recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant risking growth restriction, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth of said infant.

2. RhBSSL for use according to claim 1 , wherein growth restriction corresponds to a daily growth of less than 15 g/kg weight of the infant.

3. Recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant risking neurodevelopmental delay, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve neurodevelopment of said infant.

4. RhBSSL for use according to claim 3, wherein neurodevelopment is assessed based on a score from Bayley Scales of Infant and Toddler

Development (Bayley III).

5. RhBSSL for use according to claim 4, wherein said Bayley III score is based on assessment(s) of one or more of cognitive, motor, and language function(s) of the infant.

6. RhBSSL for use according to any one of claims 4-5, wherein said neurodevelopmental delay corresponds to a Bayley III score of less than 85.

7. RhBSSL for use according to any one of claims 3-6, wherein said neurodevelopment is assessed at a corrected age of the infant of 12 months, such as 24 months.

8. RhBSSL for use according to any one of claims 3-7, wherein said treatment prevents neurodevelopmental delay.

9. RhBSSL for use according to any one of the preceding claims, wherein said infant suffers from underweight, such as low birth weight.

10. RhBSSL for use according to any one of the preceding claims, wherein said infant is small for its gestational age at birth.

1 1 . Recombinant human bile-salt-stimulated lipase (rhBSSL) for use in treatment of a human infant being small for its gestational age, said treatment comprising enteral administration of rhBSSL to said infant for a time period of at least around four weeks to improve growth velocity of said infant.

12. RhBSSL for use according to any one of the preceding claims, wherein said infant suffers from preterm birth.

13. RhBSSL for use according to claim 12, wherein said human infant is born before week 37 of gestation, or is born between about week 37 and about week 32 of gestation, between about week 32 and about week 25 of gestation, or between about week 25 and about week 22 of gestation.

14. RhBSSL for use according to any preceding claim, wherein said infant during said time period of at least around four weeks is not fed fresh mother's milk.

15. RhBSSL for use according to any preceding claim, wherein said rhBSSL is comprised in an infant formula and/or pasteurized breast milk.

16. RhBSSL for use according to any preceding claim, wherein said infant during said time period of at least around four weeks is fed infant formula.

17. RhBSSL for use according to any one of the preceding claims, wherein said infant during said time period of at least around four weeks is fed pasteurized breast milk.

18. RhBSSL for use according to any preceding claim, wherein said rhBSSL is administered in an amount per day of between 1 and 100 mg per Kg weight of said infant, between 5 and 50 mg per Kg weight, between 15 and 40 mg per Kg weight, or between about 22.5 and 27 mg per Kg weight.

19. RhBSSL for use according to any preceding claim, wherein said rhBSSL is administered with at least one feed per day over said time period of at least around four weeks.

20. RhBSSL for use according to claim 19, wherein said rhBSSL is administered with most of or with all feeds per day.

21 . RhBSSL for use according to any preceding claim, wherein said treatment comprises enteral administration of rhBSSL for a time period of four weeks.

22. Method of treatment of a human infant risking growth restriction, said treatment comprising enteral administration of recombinant human bile-salt- stimulated lipase (rhBSSL) to said infant for a time period of at least around four weeks to improve growth of said infant.

23. Method according to claim 22, wherein growth restriction corresponds to a daily growth of less than 15 g/kg weight of the infant.

24. Method of treatment of a human infant risking neurodevelopmental delay, said treatment comprising enteral administration of recombinant human bile-salt-stimulated lipase (rhBSSL) to said infant for a time period of at least around four weeks to improve neurodevelopment of said infant.

25. Method according to claim 24, wherein neurodevelopment is assessed based on a score from Bayley Scales of Infant and Toddler Development (Bayley III).

26. Method according to claim 25, wherein said Bayley III score is based on assessment(s) of one or more of cognitive, motor, and language

function(s) of the infant.

27. Method according to claim 25 or 26, wherein said neurodevelopmental delay corresponds to a Bayley III score of less than 85.

28. Method according to any one of claims 24-27, wherein said

neurodevelopment is assessed at a corrected age of the infant of 12 months, such as 24 months.

29. Method according to any one of claims 24-28, wherein said treatment prevents neurodevelopmental delay.

30. Method according to any one of claims 22-30, wherein said infant suffers from underweight, such as low birth weight.

31 . Method according to any one of claims 22-30, wherein said infant is small for gestational age at birth.

32. Method of treatment of a human infant human being small for its gestational age, said treatment comprising enteral administration of recombinant human bile-salt-stimulated lipase (rhBSSL) to said infant for a time period of at least around four weeks to improve growth velocity of said infant.

33. Method according to any one of claims 22-32, wherein said infant suffers from preterm birth.

34. Method according to claim 33, wherein said human infant is born before week 37, or is born between about week 37 and about week 32 of gestation, between about week 32 and about week 25 of gestation, or between about week 25 and about week 22 of gestation.

35. Method according to any one of claims 22-34, wherein said infant during said time period of at least around four weeks is not fed fresh mother's milk.

36. Method according to any one of claims 22-35, wherein said rhBSSL is comprised in an infant formula and/or pasteurized breast milk.

37. Method according to any one of claims 22-36, wherein said infant during said time period of at least around four weeks is fed infant formula.

38. Method according to any one of claims 22-37, wherein said infant during said time period of at least around four weeks is fed pasteurized breast milk.

39. Method according to any one of claims 22-38, wherein said rhBSSL is administered in an amount per day of between 1 and 100 mg per Kg weight of said infant, between 5 and 50 mg per Kg weight, between 15 and 40 mg per Kg weight, or between about 22.5 and 27 mg per Kg weight.

40. Method according to any one of claims 22-39, wherein said rhBSSL is administered with at least one feed per day over said time period of at least around four weeks.

41 . Method according to claim 40, wherein said rhBSSL is administered with most of or with all feeds per day.

42. Method according to any one of claims 22-41 , wherein said treatment comprises enteral administration of rhBSSL for a time period of four weeks.