USE OF LACTOFERRIN FOR PREVENTION OF NEONATAL SEPSES IN

PREMATURE NEWBORNS

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Field of the art

The present invention relates to the use of lactoferrin for the prevention of neonatal sepses in premature newborns, in particular during intensive neonatal therapy. Background-Introduction

The various micro-organisms (bacteria andmycetes) are causal agents of frequent, serious, and often fatal infections in preterm newborns.

Preterm newborns are patients of a particular type in so far as they manifest - precisely on account of being premature and without efficient immune defences - a propensity to infection that can result in sepses proper, which are dramatic events that frequently lead to rapid death. It is calculated that in preterm newborns with a neonatal body weight of less than 1500 g, hospitalized for two or more months in intensive neonatal therapy (INT) , the estimated incidence of sepses (at least one episode) is about 30-40%, with a tendency to even higher frequencies the lower the body weight at birth. The raw mortality that can be attributed to sepses in premature newborns is 30-75%, with a variability linked to the causal agent.

Apart from the extremely high mortality, neonatal sepses/infections are the major cause of morbidity in premature newborns, with enormous implications of a medical nature (need for costly and potentially toxic protracted antibiotic treatment; difficulties in feeding and impossibility of gaining weight, etc.), of a management

nature (protracted hospitalizations; interruption of the parent-newborn relationship, difficulty of start and maintenance of breast-feeding, etc.) _? and finally of correlated morbidity (sepses are frequently associated to the feared complications of prematurity in surviving preterm newborns, such as necrotizing enterocolitis, retinopathy of prematurity, and bronchodysplasia of preterm newborns) .

Aπiongst the known risk factors, the colonization by infective agents (such as, for example, Candida spp amongst the mycetes) is the most important variable predictive of invasive infective desease. A prior colonization can almost always be identified, if adequately investigated, preliminarily in the majority of sepses.

Of all the colonization sites, the gastro-enteric tract seems to be the one that as a whole has the highest predictivity for a subsequent dissemination in the event of colonization. Bacterial and fungal colonization at various levels of the intestine is a well-known risk factor for a subsequent dissemination and systemic infective desease in preterm newborns.

On the other hand, preterm newborns in INT are at high risk for disorders of the intestinal micro-ecology, with proliferation of a pathogenic microflora for the reason that they undergo protracted treatments with wide-spectrum antibiotics, and often have difficulty in setting up and maintaining oral alimentation. In these very particular patients, the intestine is considered the most important reservoir and site of colonization for all types of pathogens and is therefore the site from which a systemic dissemination may most frequently start.

It is clear that the best strategy for this problem,

which is of extreme seriousness and importance, would be that of setting up an optimal prophylaxis in order to prevent the infective episode rather than curing it.

Unfortunately, in the current state of the art, there do not exist indications on what exactly to do, on how to do it, and in which subgroups of premature newborns. The only truly effective system of prevention, albeit partial, is the hygiene of healthworkers who come into contact with the newborn, and the minimization (as far as possible) of the invasive procedures connected to the medical or surgical assistance. As may be readily appreciated, these are inevitably partial, albeit indispensable, strategies.

In terms of specific prophylaxis, for mycetes there exists the possibility of performing a prophylaxis with systemic antifungal agents (e.g., fluconazole) , even though this even raises concern for the tolerability and potential capacity of selecting resistant strains, a reason for which this is not yet viewed as "standard of care".

As regards bacteria, there currently does not exist any consensus or guideline regarding a specific strategy of prevention. The attempts to identify a preventive strategy based upon the administration of immunoglobulins, or of factors stimulating the growth of neutrophils, have not yielded convincing results, just as do the various strategies based upon the administration of various antibiotics at a reduced dosage or with alternative dosage strategies. Statement of invention

The task of the present invention is to overcome said situation by proposing an innovative approach of a less "invasive" type, based upon the use of natural defensive factors, such as lactoferrin.

Lactoferrin (LF) is a protein with a peculiar biological action, which is present in the milk of humans and of the major mammals. It is secreted in variable amounts during breast-feeding, reaching the maximum concentrations in the colostrum and then progressively decreasing in mature milk. Amongst the various species of mammals, the milk of humans is the one characterized by the maximum concentration of LF (up to 5-7 mg/ml) . Small amounts of LF are, however, detectable in practically all human biological fluids and in exocrine secretions, on mucosal surfaces, and in neutrophils . Minimal amounts are also detectable in the blood, especially during an inflammatory process, owing to the presence in the blood itself of products of degranulation of neutrophils, amongst which precisely LF. From the biochemical standpoint, LF is a bilobed glycoprotein characterized by a typical action of binding of iron and forms part of the family of the transferrins. The gene that encodes it is located in chromosome 3 in a locus common to that of transferrin. Human LF presents a single-chain sequence of 692 amino acids (689 in the case of bovine LF) , with a molecular mass of approximately 80 kDa, and a structure made up of two similar regions, each possessing an iron-binding site of its own. The two sites (one per lobe) are able to bind, reversibly for each, a molecule of ferric iron (Fe+++) , with a very high affinity (even 300 times greater than that of transferrin itself) , and also in conditions of pH that is more acidic than transferrin. Binding with iron is accompanied by a change of structural conformation in the molecular site involved, which passes from an open conformation to a closed one. Another biochemical

prerogative of LF is that of being (unlike transferrin) a highly basic molecule, with a pH of 8-9. This property guarantees for LF the capacity of binding in a pseudo-specific way to many acidic molecules, primarily a wide range of surface proteins of the cell membranes.

In the newborn who physiologically assumes LF with his mother's milk, catabolism of LF occurs, in a first phase, at an enteric level : in the intestinal lumen, with mechanisms that still remain partially obscure, it is partially degraded into peptides (the "lactoferricins") , some of which still have a biological activity that is a surrogate of that of the starting glycoprotein. In a second phase, LF and its by-products then pass into circulation, are captured by the liver and then eliminated via the hepatic emunctory. It should be noted that LF is resistant to digestion by trypsin, and hence to pancreatic digestion, so much so that high levels of LF are found in the faeces of breast-fed newborns.

Research carried out on LF has highlighted a very extensive series of biological activities and functions. Appearing below is a brief list of the major activities of LF:

1. participation in the homeostasis/metabolism of iron

2. participation in the mechanisms of control of inflammation 3. antitumoural activity

4. various enzymatic and pro-enzymatic activities

5. immunomodulating activity

6. antimicrobial (a-antibacterial; b-antiparassitic; c-antiviral; d-antimycotic) activity. At the current state of knowledge, that the role of LF within mechanisms of martial homeostasis would seem to

be only that of intervening in loco on the iron that is produced or released in the course of inflammatory processes. Given that modest amounts of LF can precisely be released by neutrophils during the process of degranulation, the LF present in in-vivo tissues guarantees a function of support in the mechanisms of transport and chelation of iron during an inflammatory process.

Apart from intervention in mechanisms of inflammation by means of modulation of ferric homeostasis in loco, much evidence points to the fact that LF is able to regulate the inflammatory response directly. The anti-inflammatory role of LF in the inflammation site is likely to start with the releaseofLFitself on the partofthe activated neutrophils . The anti-inflammatory function of LF basically expresses itself through three experimentally demonstrated mechanisms. The first and most obvious one is a direct sequestration of the iron present in the inflammation sites in order to prevent any formation of free radicals with an oxidizing and toxic action. The second envisages binding of LF with a lipopolysaccharidic bacterial endotoxin, which in vivo performs a role of mediation of the inflammatory response in the course of bacterial infections. As a result of said bond, the LPS no longer manages to bind to its receptors, and consequently the chain of up-regulation of the release of cytokynes is interrupted. Finally, a third mode of intervention is that of reduction of the cutaneous inflammation by means of inhibition of the migration of the Langerhans cells.

There then exists experimental evidence that points to an antitumoural role of LF, also in this case in a way independent of the action thereof on iron. LF has also been

indicated as being able to regulate the activity of NK cells, modulate the expression of the proteins Gl, inhibit VEGF-mediated angiogenesis, and facilitate the phenomena of apoptosis. In some studies on animal models, LF has inhibited the development of experimental tumours. These results clearly require further studies to confirm a role in this sense of LF in vivo.

LF possesses numerous enzymatic activities, principally of a ribonuclease, protease, and transcriptional-regulation type. The various purified subfractions of LF have proven able to perform even five different types of enzymatic activity: DNase, RNase, ATPase, phosphatase, and malto-oligosaccharidase. Some of these enzymatic activities (in particular, the protease activity) can support the antimicrobial functions of LF.

Undoubtedly, the activities of immunomodulation and the antimicrobial activity (at various levels: antibacterial, antiparassitic, antiviral, andantimycotic) are the most interesting biological functions of LF, and those offering the most intriguing clinical potential

It has been ascertained that LF performs an important role in immune response, by means of direct and indirect mechanisms of immunomodulation. Directly, LF is able to stimulate the release in the acute phase of inflammation by the neutrophils of inflammatory mediators, such as IL8, to stimulate NK activity, and to suppress the expression of other interleukins, such as ILl, IL2, IL6 and TN-alpha by the leukocytes. Indirectly, LF can affect, in a bifidogenous sense, the composition of the enteric flora (see hereinafter) .

As regards antimicrobial action, LF is able to act on

the growth and development of a large number of infective agents, from viruses up to protozoa, and this thanks to the use of multiple and different mechanisms of action.

The capacity of LF to inhibit bacterial growth in vitro was one of the first biological functions experimentally demonstrated for this protein, and for a long time it was deemed to occur exclusively by means of a mechanism of sequestration of free iron on the part of LF itself in such a way as to deprive bacteria and fungi of an indispensable growth factor. More recently, it has, however, been demonstrated that this mechanism of action is not the only one: in fact, N-terminal peptides, with basic pH, of LF (referred to as lactoferricins) are able to destroy or even penetrate the bacterial cell wall, thus exerting a direct cytolytic action. In this function, bovine LF would be characterized by the presence of a series of lactoferricins even more powerful than those of human LF.

However, notwithstanding the large amount of research into these mechanisms of antimicrobial action in vitro, their effective role in vivo has not so far been sufficiently demonstrated.

On the other hand, certain clinical observations indicate a more concrete role of LF in regard to specific infective agents. There has been demonstrated an effect in vitro of selective stimulation of the growth of intestinal bifidogenous flora exerted by LF and by the peptides, which are the product of degradation of the LF itself, and in an animal model there has been demonstrated a capacity for inhibiting growth of colonies of E. coli in the intestine of the rat. LF thus appears able to act at the same time as immunomodulating and antibacterial factor in the

intestinal lumen.

Of considerable interest then is the demonstration in vivo of a protective effect of LF on infection by Helicobacter pylori in the rat, and this could have implications for the prevention of gastric carcinoma, which, as is known, is correlated to chronic infection by Helicobacter pylori.

The antimycotic action of LF can be implemented both with an iron-dependent mechanism and with an iron-independent mechanism. A component of LF, apo-LF, has been shown in vitro to be able to modify the surface permeability of the cell membrane of Candida albicans and Candida krusei, in this way acting in a direct cytopathic sense. There consequently exists also for LF a rationale of use in the prevention of the colonization and pathological condition correlated to fungal dissemination of enteric origin.

The antiviral action of LF expresses itself by means of different mechanisms. In viruses with envelopes (such as CMV, HSVl, HIV, HBV, HCV), it acts by preventing penetration of the virus within the target cell; in nude viruses (such as Rotavirus), instead, LF prevents viral replication by binding directly to the virus itself.

As regards the action of LF on viruses with envelopes, numerous clinical studies have investigated the effect of the supplementation of LF in patients with chronic infection from HCV and the protective effect in regard to primary infection from HIV.

Consequently, on the basis of the considerations outlined above and of the already considerable amount of experimental data available, there hence exists a vast range of contexts and pathological conditions where a clinical

use of LF has so far been proposed.

There do not exist, however, in the literature indications regarding the possible use of lactoferrin for the prevention of neonatal sepses in premature newborns through its oral administration.

Spurred on by the considerable evidence that militates in favour of a potential effectiveness of LF as supporting therapy in bacterial infections, both as regards an antimicrobial "adjuvant" action and as regards an action on inflammation, the inventors have conducted a wide-ranging research that might enable identification of an optimal prophylaxis of neonatal sepses/infections in order to prevent the infective episode, rather than to cure it, by resorting to the use of lactoferrinas nutritional supplement irrespective of the type of alimentation adopted, and moreover verifying whether its capacity to affect, in a bifidogenous sense, development of intestinal bacterial flora might enable a use thereof in premature newborns, preventing dysmicrobism secondary to prematurity and to the therapies conducted.

Another purpose of the invention is to propose the use of bovine lactoferrin, which is particularly interesting for an exogenous use, given its biochemical affinity with human lactoferrin, allied to its non-toxicity and ease of assumption.

On these premises, which have emerged from basic scientific and nutritional research, a double-blind, randomized, multicentre clinical study versus placebo has been conducted, aimed at evaluating the impact of the administration of LF (by itself or in association with a probiotic) in order to cut the costs for neonatal preventive

medicine in premature newborns having a body weight at birth of less than 1500 g.

The study was performed in Italy and involved 15 third-level centres in various regions, as well as numerous neonatologist experimenters in the years 2006, 2007and2008.

The newborns were enrolled on the condition that they were alive at the moment of randomization and with an expectancy of survival of more than 48 hours. Excluded from enlistment were newborns with an infection already in progress in the case where they were born of mothers affected by puerperal sepses or febrile amniotitis.

Bovine lactoferrin was administered in the amount of 100 mg/day to all the newborns enrolled, starting from the third day of life (i.e., in a time lapse of between 48 and 72 hours of life) .

Administration was via oral route, in a single daily dose, by means of addition to the food envisaged in the programme of assistance for that particular child, and was administered both with a feeding bottle and with an oro-gastric probe in the case where the newborn still presented the inability to suck (as in the case of newborns affected by extreme prematurity or by serious concomitant pathological conditions) .

It should be noted that the standard dosage used in this study (100 mg) is equivalent to a dose per kilogram comprised in the 60-to-200-mg/kg range, a range that obviously refers to the particular effective body weight of the newborn to whom LF was administered. Mature maternal milk (i.e. , after 5-6 days from birth) contains amounts of LF in the region of approximately 1 mg/ml. It is to be noted that the range of the dosage of LF administered in the study

corresponds to approximately twice what a newborn of the same weight would assume with the fresh milk of his own mother in the case where he were able to do so and in the case where the mother herself were able to breast-feed (both of which requisites are anything but easy to meet in the case of seriously premature newborns and babies born following upon complicated or pathological pregnancies) . It is in effect extremely problematical, and indeed almost impossible, to calculate precisely the amounts of human lactoferrin that a seriously premature newborn assumes on a daily basis with the milk of his own mother during his stayin hospital. However, calculating that on average at 10 days of life a newborn weighing 1 kg in good conditions of health assumes (if he is able to suck and if his mother is able to breast-feed) approximately 80-100 ml of fresh maternal milk, in theory the same newborn could assume with the milk itself an approximate amount of 80-100 mg of lactoferrin. Unfortunately, this situation arises very rarely because the alimentary instability typical of the seriously preterm newborn frequently does not allow optimal intake of food to be reached and maintained for several weeks . Furthermore, the objective conditions of difficulty in which a mother that has given birth to a seriously premature newborn comes to find herself (pathological conditions of a physical nature, stress, assumption of drugs as a result of pathological pregnancy, etc.) frequently prevent both onset and maintenance of an optimal rise of milk.

Another peculiarity is that bovine LF, on account of the anti-infective and (presumably) immunomodulating effects at an intestinal level that were studied in this trial, is partially more effective for the human newborn

than human lactoferrin itself, as has been demonstrated in recent in-vitro studies (Guarino et al.) .

The importance of integration with LF added to the food is even greater if it is borne in mind that precisely those babies that were in a "worse condition" and for whom a "additional" exogenous supply of LF is markedly indicated

(given that they are the category at highest risk of infections) are the ones that have fewer possibilities of assuming suitable amounts of LF with the milk of their own mothers. If they are in a worse condition, in fact, it is more difficult for them to manage to feed in an adequate way.

From an analysis of the data gathered, the preliminary results on 225 newborns (70% of the patients programmed for enrolment) show that the administration of LF (by itself or in addition to the probiotic LGG - Lactobacillus GG - with a single daily dose added to the meals via oral route) produces a clear reduction in the incidence of neonatal sepses documented microbiologically via positivity for any pathogenic micro-organism in an examination on a culture usingblood, urine, liquor, or peritoneal liquid. Insubjects treated with bovine LF, in fact, one episode of sepsis in the course of hospitalization occurred in 8.3% of babies, as compared to 28.4% of non-treated subjects (0 0.22; 95% C.I. 0.10-0.49; p<0.001) . There was therefore a reduction to approximately one third of the infective episodes in newborns who assumed LF.

It should be emphasized that a reduction of such an amount in the incidence of neonatal infections has so far not been reported in preterm newborns with any preventive agent or measure studied, either of a medical type, alimentary type, or hygienic type.

It should be noted that there have not been reported or documented side effects of any sort, and no toxicity. LF was always tolerated, and in no case was it at all necessary to suspend administration thereof on account of presumed untoward effects . Furthermore, this nutritional supplement was tolerated at all relative dosages administered in the case where they were analysed according to body weight.

The effect was achieved irrespective of the type of alimentation adopted (fresh maternal milk, milk from a milk bank, formulated milk) , and of the amount of milk assumed. And not only this, but the effect was equally direct for all the types of pathogens in the sense that both infections from Gram-negative bacteria and infections from Gram-positive bacteria were prevented, as well as those from mycetes. Also this datum is peculiar, because there does not exist any antibiotic substance that is able to act simultaneously and with the same degree of effectiveness on micro-organisms that are so different from one another.

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