WO2012084459A1

WO2012084459A1 - Prevention and therapy methods for preterm deliveries and births

Info

Publication number: WO2012084459A1
Application number: PCT/EP2011/071649
Authority: WO
Inventors: Piera Valenti; Rosalba Paesano
Original assignee: Microbo S.R.L.
Priority date: 2010-12-23
Filing date: 2011-12-02
Publication date: 2012-06-28
Also published as: IT1406153B1; ITMI20102380A1

Abstract

Therapy and/or prevention of pathological conditions that induce preterm deliveries and births.

Description

PREVENTION AND THERAPY METHODS FOR PRETERM DELIVERIES AND BIRTHS

Technical Field

The present invention relates to therapy and/or prevention of pathological conditions inducing preterm deliveries and births.

Background art

Deliveries occurring before the thirty- seventh week of pregnancy are defined as preterm. Among preterm deliveries (PTDs), the ones with the most clinical relevance occur before the thirty-second week of pregnancy. PTD affects approximately 10% of deliveries, but this percentage is increasing, mainly because of assisted reproduction and multiple pregnancies.

Preterm delivery, in general, is not a danger (risk) for the health of the mother. On the other hand, preterm births (PTBs) are the cause of the main neonatal pathologies and are correlated with 70% of neonatal deaths. This percentage is expected to decrease because of progress in hospital management of premature infants, although the consequences of a PTB, especially at the neurological level, must always be evaluated in the long term among survivors.

Spontaneous or iatrogenic PTD may be induced by infective processes, nutritional deficiencies, alcoholism, smoking, cervical ripening and dilatation, uterine malformations, polyamnios, multiple pregnancies, placenta previa, placental detachment, premature rupture of membranes, iron deficiency, anemia, diabetes, pre-eclampsia, hypertension and hereditary thrombophilia. Although the causes of PTD are various and multifactorial, the inflammatory state in the maternal-fetal interface is currently considered as the crossroads of the factors known to be associated with PTB. The inflammatory state is mediated by pro-inflammatory cytokines.

Although international literature shows clearly that there is a strong correlation between PTB and inflammation ( ef. Christiaens Inge, Dean B. Zaragoza, Larry Guilbert, Sarah A. Robertson, Bryan F. Mitchell, David M. Olson "Inflammatory processes in preterm and term parturition", Journal of Reproductive Immunology 79, 50-57, 2008; Debra Lyon, Ching-Yu Cheng, Lois Howland, Debra Rattican, Nancy Jallo, Rita Pickler, Lisa Brown and Jacqueline McGrath "Integrated Review of Cytokines in Maternal, Cord, and Newborn Blood: Part I— Associations With Preterm births" Biological Research for Nursing 11(4), 371-376, 2010), clinical trials, performed in order to determine the level and type of cytokines and designed by testing the concentration of pro-inflammatory cytokines in different biological samples such as maternal blood, fetal blood or umbilical cord blood, placenta, amniotic liquid, cervico-vaginal secretions, have yielded contradictory results. Indeed it appears unclear whether the predictability of PTD and the induction thereof can be linked to pro-inflammatory cytokines detected in the blood or in cervico-vaginal secretions.

The generic term cytokine designates small proteins which, once they have been secreted, mediate and regulate immunity, inflammation and hematopoiesis. They are divided into "pro" inflammatory and "anti" inflammatory. Pro-inflammatory cytokines generally include interleukins (IL)-lbeta, IL-6, IL-11, IL-12, IL-17, IL-18, IL-8, tumor necrosis factor alpha (TNF-alpha), and interferon-gamma (IFN-gamma). Anti-inflammatory cytokines include IL-4, IL-10, IL-13, IL-16, IFN-a and tumor growth factor b (TGF-b). Another class of cytokines, hematopoietic growth factors, also known as colony-stimulating factors (CSFs), regulates the production of red blood cells, leukocytes and platelets and is an important marker of maternal and fetal status.

The ratio between pro-inflammatory cytokines and anti-inflammatory cytokines is an important element in the immune response of the mother and the fetus. The different ratio between the cytokines is necessary, moreover, in conception, pregnancy, delivery and in the first phase of fetal life. In pregnancy there is in fact a shift between the levels of T helper 1 (Thl) and T helper 2 (Th2), with an increase in the production of Th2 cells. The Thl cells synthesize IL-2, IFN-gamma, and T F-beta and induce cell immunity. The Th2 cells synthesize IL-4, IL-5, IL-6, and IL-10 and induce the production of antibodies.

Although any classification of cytokines cannot be exhaustive, because of the complexity of their multiple roles, cytokines are certainly associated with labor and delivery.

The beginning of labor appears like an inflammatory reaction that involves not only an afflux of leukocytes in the cervical and endometrial tissues, but also an increase in cytokine and prostaglandin synthesis. In fact, during labor, the synthesized cytokines induce prostaglandin synthase-2, which increases the production of prostaglandins (PGs), with consequent activation of cervical maturation and of uterine contractions. In particular, IL-Ιβ increases the production of matrix metal proteases (MMPs), which induce cervical remodeling and the possible rupture of the membranes, while IL-6 and TNF-a activate leukocytes to invade the uterine tissue, therefore stimulating the expression of oxytocin receptors and increasing prostaglandins. IL-8, which is produced by the placenta, also cooperates to the modification of the cervix by inducing the afflux of leukocytes. In addition, these cytokines affect each other: IL-Ιβ and TNF-a activate IL-8, which then promotes synthesis of IL-Ιβ and IL-6.

The inflammatory process during pregnancy can be activated both by infective events and by non-infective events.

Infections of the genital upper tract or systemic infections, since they cause the release of pro-inflammatory cytokines such as IL-Ιβ, IL-6, and TNF-a, must be treated promptly with antimicrobial drugs in order to avoid or reduce the likelihood of triggering preterm uterine contractions and labor.

Treatment of infections instead appears to be more complex if contractions and preterm labor arise because of inflammatory processes that involve high levels of pro-inflammatory cytokines in the blood as in the case of infections localized in regions other than the cervico-vaginal one or as in the case of inflammations unrelated to the infections.

It has been already reported that increase in body mass, lifestyle (smoking, alcohol and drugs), nutritional status, heart rate, age, states of hypertension, iron deficiency, anemia, diabetes, pre-eclampsia and hereditary thrombophilia cause inflammatory phenomena, especially during pregnancy. Recently, relaxin, a collagenolytic hormone that increases the production of matrix metal proteinase, also has been associated with PTD. Indeed the expression of relaxin, regardless of infections, induces the synthesis of IL-6 and IL-8.

Notwithstanding the significance of an inflammatory event during pregnancy, there is a lack of reliable clinical studies on the relation between inflammation unrelated to the infections and PTBs.

From what has been summarized, the best understanding of the relation between cytokines at the systemic level and at the cervico-vaginal secretions level and PTB certainly can be fundamental in the search for interventions which, in a non-invasive manner and without side effects, may decrease inflammation and synthesis of prostaglandins, prolonging the gestation period and preventing thus both the onset of contractions and PTD.

There are numerous subjective and objective diagnostic methods capable of predicting a PTD, but none of the ones listed below is considered fully reliable.

Because of the lack of a reliable method for predicting and defining the uterine modifications that might lead to a PTD, the subjective evaluation of the woman is taken into serious consideration: i) onset of uterine contractility, perceived by the woman as painful or not painful; ii) onset of steady or intermittent cramps of the menstrual type in the suprapubic region; iii) lumbosacral pain; iv) pelvic pressure. However, the woman's subjective evaluation must be necessarily complemented by objective diagnostics, keeping in mind that in a large percentage of PTD the symptoms that precede it may be extremely scarce.

Considering that performing a digital cervical exam to identify the risk of PTD has shown its limitations because a minimum cervical dilatation (< 3 centimeters) occurs normally in multiparous women and it is not necessarily an indication of an imminent PTD, other diagnostic methods must be performed.

Objective evaluation of a PTD can be performed through ultrasound scanning, cardiotocography, electrohysterogram, measurement of vaginal pH, changes in vaginal secretions, any vaginal hemorrhage, Nugent scores, hematological parameters such as red cells, hemoglobin, total serum iron, serum ferritin, level of pro-inflammatory cytokines such as IL-Ι β, IL-6, IL-8 and TNF-a, level of fetal fibronectin and of PGs in the cervico-vaginal fluid.

Among the methods for evaluating uterine activity, ultrasound scanning, cardiotocography and electrohysterogram appear to be particularly useful.

Ultrasound scanning in fact provides information on the length of the neck of uterus and on the presence of Funneling, i.e., a funnel-like flaring of the internal uterine orifice. A cervix < 30 mm and the presence of Funneling are an indicator of high risk of PTD, while a cervix > 30 mm and the absence of funneling are an indicator of low risk of PTD.

Cardiotocography, which is widespread in obstetrics, is useful for evaluating both fetal well-being (heartbeat and movements) and the presence, frequency and intensity of uterine contractions.

The electrohysterogram discerns, in a more specific way than tocography, the physiological uterine activity from the increased uterine contractility that leads to PTD, but it is not easy and inexpensive to use.

Measurement of vaginal pH, changes in vaginal secretions, any signs of hemorrhage, Nugent scores, and hematological parameters are inexpensive diagnostic tests that are always performed. Expensive specific assays to define the level of pro-inflammatory cytokines such as IL-lb, IL-1 and IL-6 and TNF-a in the blood and in the cervico-vaginal fluid, as well as the level of PGs such as PGE₂ and PGF_2a in the cervico-vaginal fluid, are performed far more rarely.

The same can be said for a biochemical test, which has been approved by the Food and Drug Administration: fetal fibronectin. The fetal fibronectin (fFN) is a glycoprotein that acts as a bonding agent between the maternal decidua and the amniotic membranes. In physiological pregnancies it is high in cervico-vaginal secretions during the first 24 weeks of pregnancy, but decreases between the twenty-fourth and thirty-fourth week. The presence of high concentrations of fFN in cervico-vaginal secretions between the twenty-fourth and the thirty-fourth week of pregnancy is, therefore, referred as a parameter that can be associated with a separation of the amniotic membranes from the uterine wall and with a risk of PTD in symptomatic and asymptomatic pregnant women. However, the presence of infective processes, blood traces or lubricants used for the examination may alter the result of this test.

From what has been summarized, it is clear that no analysis can predict, on its own, a PTD and that in order to avoid uncertain and ambiguous diagnoses it is necessary to perform multiple analyses in parallel.

In short, up to now no close connection between the levels, the type and the localization of pro-inflammatory cytokines and PTD has been ascertained in order to reduce the incidence of PTB through more targeted prevention and therapy.

The medical therapy for PTD is still a problem with no easy solution. There are various types of drugs, such as antagonists of the oxytocin receptor, beta agonists and calcium antagonists. Actually, for none of these drugs real effectiveness in stopping labor has been proved. Conversely, the aim of these drugs is mainly to prolong the pregnancy in order to have time to induce fetal pulmonary maturity by means of betamethasone. Tocolytic drugs are the ones mainly used in the attempt to inhibit uterine contractility, although in numerous studies they have not demonstrated significant effects. The use of non-steroidal anti-inflammatory drugs (FANS, prostaglandin inhibitors) is still debated and appears to be questionable.

The only improvement in perinatal outcomes field is associated with the administration of cortisone-based drugs with the sole purpose of inducing fetal pulmonary maturity between the twenty-fourth and the thirty- fourth week of pregnancy. These therapies, together with the great progress in the field of neonatal assistance, have improved remarkably the prognosis of infants born before the term but have not yielded positive results in the prevention of births and PTDs or in the reduction of the incidence of preterm deliveries and births.

The aim of the present invention is to provide a method for preventing and/or treating multifactorial pathological conditions that induce preterm deliveries and births.

The term prevention is understood to reference the use of the present invention in pregnant women who, even in the absence of uterine contractions and other objective data, are at risk of PTD due to previous or concomitant non-infective pathologies.

The term therapy is understood to reference the use of the present invention in pregnant women for whom, at a preterm gestational period, a risk of preterm delivery is diagnosed subjectively and objectively.

Disclosure of the invention

This aim and these and other objects that will become better apparent hereinafter are achieved by means of lactoferrin, or at least one fragment thereof, for use in therapy and/or prevention of pathological conditions inducing preterm deliveries and births, wherein said use comprises the administration of lactoferrin, or at least one fragment thereof, to a pregnant woman in the preterm gestational period.

Within the scope of the present invention, therapy and/or prevention of pathological conditions, particularly multifactorial ones, that induce preterm deliveries and births is correlated to the reduction, disappearance and/or lack of onset of the typical symptoms of preterm deliveries and births reported by the woman, confirmed by means of objective diagnostic methods by comparing the results obtained before and after the therapy with particular reference to the reduction or absence of contractions, to the reduction of the concentration of fetal fibronectin, of pro-inflammatory cytokines in the blood and in cervico-vaginal secretions, and of PGE₂ and PGF_2o[ prostaglandins in the cervico-vaginal secretions, and to the restoration of the physiological hematological values such as the number of red cells and the concentration of hemoglobin, total serum iron and serum ferritin.

Ways of carrying out the invention

The expressions "topical administration" or "topically" are understood to reference a local administration of lactoferrin, preferably in the mucosae of the vaginal cavity.

The expressions "oral administration", "per os" or "orally" are understood to reference an oral administration that allows gastrointestinal absorption of the lactoferrin.

Within the scope of the invention, the human, bovine, or human or bovine recombinant lactoferrin or fragments thereof can be administrated orally by means of capsules, tablets or chewable pills formulated with excipients such as corn starch, microcrystalline cellulose, vegetable magnesium stearate or other excipients that are well-known to a person skilled in the art, including preservatives, antioxidants or coloring agents. Optionally, said formulations for oral use may be in a gastroprotected and/or slow-release form.

Gastroprotection can be obtained by means of a surface coating of said capsules, tablets or chewable pills, that remains stable at a pH lower than 3, which is characteristic of the stomach, and dissolves at a pH higher than 5, which is typical of the small intestine. The materials for surface coating comprise fatty acids, polymers and other materials known to a person skilled in the art.

Slow release can be obtained by microencapsulation or compounds that form a cavity wherein the human, bovine, or human and bovine recombinant lactoferrin or fragments thereof is arranged so as to be able to obtain administrations of greater quantities, which are released over time at concentrations lower than the total content.

Within the scope of the present invention, the human, bovine, or human and bovine recombinant lactoferrin or fragments thereof can be administered for topical use, including intravaginal use, by means of creams, freeze-dried product, freeze-dried product carried by liposomes, nanoparticles, microcapsules, polysaccharides or other carriers with no cytotoxicity, tablets or pessaries with fast or slow release, non-aqueous solutions, anhydrous gels, suspensions, emulsions or ointments formulated with the appropriate excipients and known to a person skilled in the art.

Within the scope of the present invention, the term "fragment" of lactoferrin is understood to reference specific sequences of lactoferrin obtained after digestion with proteolytic enzymes. The specific sequences can be the N-lobe, the C-lobe, and large peptidic fragments that belong to the N-lobe and the C-lobe. The term N-lobe designates the fraction having a molecular weight (MW) of about 33 KDa that corresponds to the fragment from amino acid 1 to 280 and part of the same lobe with MW of approximately 20 KDa that corresponds to the fragment from amino acid 86 to 258 of whole lactoferrin digested enzymatically and subsequently purified (Siciliano , Rega B, Marchetti M, Seganti L, Antonini G, Valenti P. "Bovine lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type 1 infection" Biochem Biophys Res Commun. 1999 Oct 14, 264(1), 19-23).

The term C-lobe designates the fraction with a molecular weight of approximately 38 KDa that corresponds to the fragment from 345 to 692 and another fraction with a MW of 47 KDa that corresponds to the fragment from 285 to 692 of whole lactoferrin digested enzymatically and subsequently purified. The expression "large peptidic fragments" is understood to reference the fragments aa 86-258 MW 20 KDa and aa 285- 692 MW 47 kDa (Siciliano , Rega B, Marchetti M, Seganti L, Antonini G, Valenti P. "Bovine lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type 1 infection" Biochem Biophys Res Commun. 1999, 264(1), 19-23).

The lactoferrin, which can have different origins comprising the human or bovine recombinant one, is a highly cationic glycoprotein capable of chelating with high affinity two ferric ions per molecule. Lactoferrin is important in the homeostasis of iron, since it is capable of preventing and curing disorders of iron metabolism as shown in WO2007065482. Oral administration of lactoferrin also affects the homeostasis of the inflammation, through the inhibition of the synthesis of some proinflammatory cytokines, as shown in WO2007065482, that is herein enclosed by reference.

The unproven correlation between the induction of PTD and proinflammatory cytokines that can be found in the blood or in cervico-vaginal secretions supports strongly the innovation of the present invention, which claims that the prevention and cure of a PTD cannot be based on the exclusive reduction of cytokines at the cervico-vaginal level but entails a simultaneous decrease of the inflammatory state at the systemic level. In support of this assertion, it is sufficient to mention the association between a preterm delivery and a pathology of the oral cavity such as periodontal disease. In fact, in pregnant women affected by periodontal disease and more at risk for PTD, topical intravaginal therapy aimed at decreasing pro- inflammatory cytokines in cervico-vaginal secretions alone has been found to be ineffective, and PTD is induced all the same because the blood levels of pro-inflammatory cytokines remain high.

In one aspect, the present invention relates to lactoferrin, or at least one fragment thereof, for use in therapy and/or prevention of pathological conditions inducing preterm deliveries and births, wherein said use comprises the administration of lactoferrin, or at least one fragment thereof, to a pregnant woman during the preterm period.

In another aspect, the present invention relates to a method for the therapy and/or prevention of pathological conditions that induce preterm deliveries and births, wherein said method comprises the administration of lactoferrin, or at least one fragment thereof, to a pregnant woman during the preterm period.

Infective events supported by mycetes such as Candida spp, or by Gram-positive or Gram-negative bacteria are, if untreated, frequent causes of preterm deliveries and births. Non-infective events that cause preterm deliveries and births include nutritional deficiencies, alcoholism, smoking, cervical ripening and dilatation, uterine malformations, polyamnios, multiple pregnancies, placenta previa, placental detachment, premature rupture of membranes, iron deficiency, anemia, diabetes, pre-eclampsia, hypertension and hereditary thrombophilia.

It has been found surprisingly that, in infective events or severe infections localized in other regions than the cervico-vaginal one, simultaneous oral and topical intravaginal administration prevents and contrasts uterine contractions and the subsequent start of labor and preterm delivery and birth. In other previously listed non-infective events that increase the risk of PTD, including iron deficiency and anemia, the lactoferrin must be administrated orally preventively in the prevention of pathological conditions that induce preterm deliveries and births and, at the onset of PTD symptoms, must be administrated simultaneously topically and orally. In these last cases, the intravaginal lactoferrin and the lactoferrin administrated orally are prepared and formulated separately in two different preparations.

Simultaneous intravaginal and oral administration of lactoferrin is particularly advantageous for women in whom, together with infective episodes including periodontal disease or non-infective episodes, a series of events predisposing for PTD has been observed, such as the onset of subjectively and objectively established uterine contractility, the change in vaginal pH, iron deficiency, anemia, an increase in pro-inflammatory cytokine concentration in the blood and in the cervico- vaginal secretions, prostaglandins and fetal fibronectin.

The effectiveness of simultaneous intravaginal and oral administration of lactoferrin can be indicated by a decrease in uterine contractility, demonstrated subjectively and objectively. Moreover, in infective events it is possible to observe an inhibition of the number of microorganisms in the cervico-vaginal region, a decrease of proinflammatory cytokines in cervico-vaginal secretions and in the blood, of prostaglandins and of fetal fibronectin and in non-infective events or events that are infective at a site that is distal with respect to the cervix, a decrease of pro-inflammatory cytokines in blood, of prostaglandins and of fetal fibronectin.

A new function of lactoferrin (of any origin or human or bovine recombinant) administered intravaginally in combination with oral administration has been found surprisingly which manifests itself with a fast decrease in the frequency and intensity of uterine contractions, as well as of prostaglandins with particular reference to PGE₂ and PGF_2a in cervico- vaginal secretions. This new function also manifests itself with the decrease in fetal fibronectin concentration that indicates that the therapy and/or prevention of PTD by means of simultaneous oral and intravaginal administration of lactoferrin can also be ascribed to stabilization of fetal or maternal-fetal membranes, as demonstrated by the decrease in fetal fibronectin, which is a bonding agent between the maternal decidua and the amniotic membranes.

The effectiveness of intravaginal lactoferrin associated with oral lactoferrin can be associated also with a decrease of pro-inflammatory cytokines in cervico-vaginal secretions and in the blood.

If the therapy with intravaginal lactoferrin associated with the oral lactoferrin in all pathologies, preferably in multifactorial ones, that induce PTD, including hypoferremia and anemia, is performed daily, preterm deliveries and births are reduced considerably in their incidence and delivery occurs at term or in 40% of the subjects between the thirty-sixth and the thirty-seventh week of pregnancy, with an average weight at birth that varies between 2800 grams for the male infant and 2500 for the female infant.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered within 8 hours of the recording of uterine contractility.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered within 8 hours of the recording of the increase in prostaglandins in cervico-vaginal secretions.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered within 8 hours of the recording of an increase in fetal fibronectin in cervico-vaginal secretions in order to stabilize maternal and fetal membranes.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered within 8 hours of the recording of the increase in pro-inflammatory cytokines in cervico-vaginal secretions and in the blood.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered within 8 hours of the recording of the onset of iron deficiency and anemia.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered simultaneously topically intravaginally and orally.

Preferably, for the use of the present invention, the lactoferrin, or at least one fragment thereof, is administered topically intravaginally or, to prevent pathologies inducing preterm deliveries and births, orally.

Preferably, for the use of the present invention, PTD is induced by at least one infective process.

Preferably, for the use of the present invention, PTD is induced by a non-infective process selected among nutritional deficiencies, alcoholism, smoking, cervical ripening and dilatation, uterine malformations, polyamnios, multiple pregnancies, placenta previa, placental detachment, premature rupture of membranes, iron deficiency, anemia, diabetes, pre- eclampsia, hypertension and hereditary thrombophilia.

Preferably, for the use of the present invention, the preterm period is before the thirty-second or between the thirty-second and the thirty- seventh week of pregnancy.

Preferably, for the use of the present invention, the intravaginal daily dose of lactoferrin, or at least one fragment thereof, ranges from 200 mg to 600 mg, the oral daily dose of lactoferrin, or at least one fragment thereof, ranges from 200 mg to 400 mg, and the daily total dose ranges from 400 mg to 1000 mg, preferably 600 mg.

Preferably, for the use of the present invention, the lactoferrin is human, bovine, or human or bovine recombinant.

More preferably, for the use of the present invention, the lactoferrin is bovine.

Preferably, for the use of the present invention, at least one fragment of lactoferrin is a large fragment obtained by enzymatic digestion.

Preferably, for the use of the invention, at least one fragment of lactoferrin is selected from N-lobe (from 1 to 280 aa), C-lobe (from 345 to 692 aa) or peptidic fragments comprising those from 86 to 258 aa and from 285 to 692 aa.

Preferably, for the use of the invention, the lactoferrin is saturated with iron, zinc, copper and/or manganese in different degrees ranging from 1 to 100%.

In particular, in the use according to the present invention, at the first onset of subjective signs reported by the pregnant woman (subjective diagnosis) one proceeds with immediate confirmation by means of objective diagnostic methods.

In case of diagnosis of risk of preterm delivery and birth, intravaginal topical therapy is started within 8 hours with a lactoferrin, preferably a bovine lactoferrin, associated with a lactoferrin, preferably bovine lactoferrin, for oral use aided by an antibiotic therapy if a microbial infection has been diagnosed. After 24 hours and daily, the subjective data reported by the woman under therapy are recorded, while the data obtained with objective diagnostic methods are recorded weekly. If the risk of PTD is associated also with a maternal anemic state, oral therapy with lactoferrin, preferably bovine lactoferrin, must begin within 8 hours. The effectiveness of the lactoferrin is evaluated by the disappearance of the symptoms reported by the woman, confirmed by means of objective diagnostic methods by comparing the results obtained before and after the therapy with particular reference to the decrease or absence of contractions, the decrease of the concentration of fetal fibronectin, of pro-inflammatory cytokines and of prostaglandins PGE₂ and PGF_2a in cervico-vaginal secretions and in the blood, and also to the restoration of the hematological values, such as the number of red cells, hemoglobin, total serum iron, serum ferritin and hematocrit.

Studies have been performed on the effectiveness of lactoferrin in the use according to the present invention by including pregnant women who, at the twentieth week, showed no indication of PTD, together with pregnant women who, despite showing no symptoms at the twentieth week, had pathologies that might affect and increase the risk of PTD, such as women affected by periodontal diseases, iron deficiency, anemia, diabetes, hypertension and thrombophilia. Some of these women, during the pregnancy, contracted infections in the urinary and genitourinary tract and in the amniotic liquid and had febrile episodes.

Pregnant women who had been subjected to cerclage because of cervical ripening and dilatation and the presence of Funneling were instead excluded.

In the absence or in the presence of infective phenomena, Tables 1-6 summarize the average parameters of both the subjective data reported by the woman and the objective data checked with different diagnostic methods in women with risk of PTD in different weeks of the pregnancy, before and after the therapy with intravaginal administration of 100 mg of lactoferrin, preferably bovine lactoferrin, every 4, 6, 8 or 12 hours depending on the frequency of the uterine contractions and of the gestational age, always associated with an oral therapy of 100 mg of lactoferrin, preferably bovine lactoferrin, twice a day.

In the more serious cases, the same intervals of administration were performed with an intravaginal and oral administration of 200 mg of lactoferrin, preferably bovine lactoferrin.

Table 1 : Intravaginal use of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 22^nd and 27^th week of pregnancy not induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Subjective and objective symptoms of Before therapy After 7 days of After 30 days of PTD between 22 and 27 weeks of therapy therapy pregnancy

Onset of uterine contractility* 1 to 5 <l-5 Not every day contractions/day contractions/day Intermittent cramps of the menstrual Yes in 100% of the Frequency decrease Absent type in suprapubic region subjects

Lumbosacral pain Yes in 100% of the Intensity decrease Absent

subjects

Pelvic pressure Yes in 80% of the Absent Absent

subjects

IL-6 (pg/ml) 450 280 120

Average values

Fetal fibronectin (ng/ml) >50 >50 <50

Average values

PGE₂ (pg/ml) 432 250 25

Average values

PGF₂« (pg/ml) 920 740 345

Average values

Notes:

* The criterion "painful or painless contraction" is excluded because it is too subjective. Table 2: Intravaginal application of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 28^th and 34^th week of pregnancy not induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Subjective and objective symptoms of Before therapy After 7 days of After 30 days of PTD between 28 and 34 weeks of therapy therapy pregnancy

Onset of uterine contractility* >4/20 min or >8/h <4/20 min l/2h

Intermittent cramps of the menstrual Yes in 100% of the Frequency decrease Absent type in suprapubic region subjects

Lumbosacral pain Yes in 100% of the Intensity decrease Absent

subjects

Pelvic pressure Yes in 80% of the Absent Absent

subjects

IL-6 (pg/ml) 480 260 130

Average values

Fetal fibronectin (ng/ml) >50 >50 <50

Average values PGE₂ (pg/ml) 480 278 100

Average values

PGF₂„ (pg/ml) 840 670 373

Average values

Notes:

* The criterion "painful or painless contraction" is excluded because it is too subjective. Table 3: Intravaginal application of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 35^th and 37^th week of pregnancy not induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Notes:

* The criterion "painful or painless contraction" is excluded because it is too subjective.

Table 4: Intravaginal use of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 22^nd and 27^th week of pregnancy induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Notes:

Table 5: Intravaginal application of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 28^th and 34^th week of pregnancy induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Onset of uterine contractility* >4/20 min or <4/20 min l/2h

>8/h

Intermittent cramps of the menstrual Yes in 100% of the Frequency Absent type in suprapubic region subjects decrease Lumbosacral pain Yes in 100% of the Intensity decrease Absent

subjects

Pelvic pressure Yes in 80% of the Absent Absent

subjects

IL-6 (pg/ml) 1850 1170 180

Average values

Fetal fibronectin (ng/ml) >50 >50 <50

Average values

PGE₂ (pg/ml) 560 298 120

Average values

PGF₂« (pg/ml) 970 640 425

Average values

Notes:

Table 6: Intravaginal application of lactoferrin (100 mg every 6 hours) in women with risk of PTD between the 35^th and 37^th week of pregnancy induced by infective phenomena combined with 100 mg of lactoferrin per os twice a day.

Subjective and objective symptoms of Before therapy After 7 days of After 15-30 PTD between 35 and 37 weeks of therapy days of pregnancy therapy

Onset of uterine contractility* 1/1-3 min 1/10 min 8/day

Intermittent cramps of the menstrual type Yes in 100% of the Frequency decrease Absent in suprapubic region subjects

Lumbosacral pain Yes in 100% of the Intensity decrease Absent subjects

Pelvic pressure Yes in 80% of the Absent Absent subjects

IL-6 (pg/ml) 2250 1480 320 Average values

Fetal fibronectin (ng/ml) >50 >50 <50 Average values

PGE₂ (pg/ml) 670 450 220 Average values PGF₂« (pg/ml) 1240 970 650

Average values

Notes:

From the results of the study, following the use of lactoferrin both intravaginally and orally according to the invention a considerable decrease in the subjective and objective symptoms was found in all the women with risk of preterm delivery between the twenty-second and the thirty- seventh week of pregnancy, in the absence and in the presence of infective phenomena.

The disclosures in Italian Patent Application No. MI2010A002380 from which this application claims priority are incorporated herein by reference.

Claims

1. Lactoferrin, or at least one fragment thereof, for use in therapy and/or prevention of pathological conditions inducing preterm deliveries and births, wherein said use comprises the administration of lactoferrin or at least one fragment thereof, to a pregnant woman at a preterm gestational age.

2. Lactoferrin, or at least one fragment thereof, for the use according to claim 1 wherein lactoferrin, or at least one fragment thereof, is administered within 8 hours from the recording of uterine contractility.

3. Lactoferrin, or at least one fragment thereof, for the use according to claim 1 or 2 wherein lactoferrin, or at least one fragment thereof, is administered within 8 hours from the recording of an increase of prostaglandins in the cervico-vaginal secretions.

4. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims wherein lactoferrin, or at least one fragment thereof, is administered within 8 hours from the recording of an increase of fetal fibronectin in the cervico-vaginal secretions in order to stabilize maternal and fetal membranes.

5. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims wherein lactoferrin, or at least one fragment thereof, is administered within 8 hours from the recording of an increase of pro-inflammatory cytokines in the cervico-vaginal secretions and in the blood.

6. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims wherein lactoferrin, or at least one fragment thereof, is administered within 8 hours from the recording of the onset of iron deficiency and anemia.

7. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims wherein lactoferrin, or at least one fragment thereof, is administered by simultaneous topical intravaginal and oral administration.

8. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims wherein lactoferrin, or at least one fragment thereof, is orally administered to prevent pathologies inducing preterm deliveries and births.

9. Lactoferrin, or at least one fragment thereof, for the use according to one of claims from 1 to 6 wherein lactoferrin, or at least one fragment thereof, is administrated by topical intravaginal administration.

10. Lactoferrin, or at least one fragment thereof, for the use according to one of preceding claims, wherein preterm delivery is induced by at least one infective process.

11. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims, wherein preterm delivery is induced by a non-infective process selected among nutritional deficiencies, alcoholism, smoking, cervical ripening and dilatation, uterine malformations, polyamnios, multiple pregnancies, placenta previa, placental detachment, premature rupture of membranes, iron deficiency, anemia, diabetes, preeclampsia, hypertension and hereditary thrombophilia.

12. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims, wherein the preterm gestational age is before the thirty-second or between the thirty-second and the thirty- seventh week of pregnancy.

13. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims, wherein the intravaginal daily dose of lactoferrin, or at least one fragment thereof, ranges from 200 mg to 600 mg, the oral daily dose of lactoferrin, or at least one fragment thereof, ranges from 200 mg to 400 mg and the daily total dose of lactoferrin, or at least one fragment thereof, ranges from 400 mg to 1000 mg.

14. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims, wherein lactoferrin is human, bovine, or recombinant human or bovine.

15. Lactoferrin, or at least one fragment thereof, for the use according to claim 14 wherein lactoferrin is bovine lactoferrin.

16. At least one lactoferrin fragment, for the use according to claim 14, wherein the fragment is selected among N-lobe (1-280 aa), C-lobe (345-

692 aa) or peptide fragments including that from 86 to 258 aa and from 285 to 692 aa.

17. Lactoferrin, or at least one fragment thereof, for the use according to one of the preceding claims, wherein the lactoferrin is saturated with iron, zinc, copper and/or manganese in different degrees ranging from 1 to 100%.