EP1991224A2

EP1991224A2 - Use of piperacillin in topical intravaginal preparations for the treatment of vaginal bacterial infections

Info

Publication number: EP1991224A2
Application number: EP07713470A
Authority: EP
Inventors: Enrico Boldrini; Giulia Falcone
Original assignee: Opocrin SpA
Current assignee: Opocrin SpA
Priority date: 2006-02-24
Filing date: 2007-02-23
Publication date: 2008-11-19
Also published as: ITRM20060094A1; WO2007096931A3; WO2007096931A2

Abstract

Piperacillin, a broad-range semi-synthetic β-lactamic antibiotic currently used for parenteral administrations, is used for the production of topical preparations of bactericide activity that are effective on the pathogens responsible for vaginal ecosystem alterations, and that also lend themselves to the treatment and prevention of gynaecological and obstetric bacterial infections by means of topical vaginal application. The piperacillin-based topical preparation has bactericide activity against Gram-positive aerobic micro-organisms, Gram-negative aerobic micro-organisms, Gram-positive anaerobic micro-organisms and Gram-negative anaerobic micro-organisms in the vaginal tract of pregnant and non-pregnant women, and is particularly active in Gram-positive aerobic vaginitis such as from group B streptococci or Streptococcus agalactiae.

Description

USE OF PIPERACILLIN IN TOPICAL INTRAVAGINAL PREPARATIONS FOR THE TREATMENT OF VAGINAL BACTERIAL INFECTIONS

The present invention concerns the use of piperacillin in topical intrav- aginal preparations for the treatment of gynaecological and obstetric bacterial vaginal infections. More specifically, the invention concerns the use of a broad-range semi-synthetic β-lactamic antibiotic, piperacillin, for the production of bactericide preparations effective on pathogens responsible for vaginal ecosystem alterations and which lend themselves to the treatment and pre- vention of gynaecological and obstetric bacterial infections via topical vaginal application.

As is known, normal vaginal flora consists of both aerobic and anaerobic bacteria. The predominating micro-organisms are those of the Lactobacillus genus (facultativeanaerobes) which account for about 95% of all the bacteria present. These micro-organisms provide a defence against infections by maintaining an acidic pH in the vagina, with normal values less than 4.5. The lactobacilli of normal women tend to contain several phenotypes which produce hydrogen peroxide, which damages the pathogen micro-organisms (many of which do not have free radical scavengers) thereby inhibiting their colonisation of the vagina.

Bacterial vaginosis, previously known as "aspecific vaginitis", was recognised at the end of the nineteenth century and then more clearly identified in the 1950s as a bacterial syndrome characterised by the depletion of the micro-organisms in the normal vaginal flora, and particularly the lactobacilli, with a simultaneous increase, up to 100-1,000 times the normal quantities, of the pathogenic vaginal anaerobic bacteria, including Gardnerella vaginalis, Mobiluncus spp., Prevotella spp., Mycoplasma spp. and, in particular, Mycoplasma hominis, and Bactericides spp.. The pathology is accompanied by detectable phenomena contributing to a reliable formulation of the diagnosis, including an increase in vaginal pH to values above 4.5 and homogeneous malodorous vaginal secretions with their characteristic "fishy" (ammine) odour when added with potassium hydroxide. Bacterial vaginosis is considered to be the most common cause of vaginal leakages and, as an infection of the female genital tract, it can be associated with a number of more serious gynaecological complications such as post-surgical infections or inflammatory pathologies of the pelvis. Although it was recently observed that changes in the characteristics of vaginal bacterial flora could have a considerable impact on pregnancy complications, such as preterm birth and preterm foetal membrane rupture or corionamnionites, it was only a few years ago that it resulted clear that "bacterial vaginosis" is not in itself enough to account for all the clinical symptoms, the therapeutic failures and the surprising outcomes of some studies on the connection between bacterial flora and the incidence of adverse effects. After accurate tests, the data often highlighted a different outcome depending on whether they concerned women with anaerobic bacterial flora or women with bacterial flora referred to as "intermediate". Serious complications with preg- nancy loss halfway through the third trimester, corionamnionitis and also abortion in the first trimester have all been connected to the so-called "intermediate" flora.

In this regard, some authors have reported that Escherichia coli and group B streptococci (both aerobics), alongside micro-organisms of bacterial vaginosis, can be responsible for preterm deliveries. These observations found abnormalities of vaginal bacterial flora lying within the definition of "aerobic vaginitis", different from bacterial vaginosis or anaerobic vaginosis.

The term "aerobic vaginitis" was recently proposed by Donders (Donders Gilbert G. G. et al., Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis, Br. J. Obstet. Gynecol., January 2002, 109: 34-43) in order to define a vaginal pathology not classifiable as specific vaginitis or as bacterial vaginosis, characterised by a pH above 5, by yellowish and malodorous secretions (but negative in the "fish odour test" or "whiff test"), inflammatory manifestations and disparheunia. This pathology differs from bacterial vaginosis for the lack of increase in anaerobic organisms and for the presence of aerobes coming from the rectal region (E. coli, Enterococcus spp. and Streptococcus agalactiae) and thus indicative of an alteration of the vaginal homeostasis with the resulting increase in enteric bacterial colonisation.

As already noticed, in both pregnant and non-pregnant women, colonisation with suitable numbers of lactobacilli producing hydrogen peroxide involves a lower acquisition of bacterial vaginosis or aerobic vaginitis compared to women who have low lactobacillic colonisation. Subjects with bacterial vaginosis or aerobic vaginitis lack lactobacilli, however, but the most common micro-organisms seen in patients with bacterial vaginosis are anaerobic pathogens such as Gardenella vaginalis or Mobiluncus. On the other hand, in patients with aerobic vaginitis, group B streptococci are the most frequent component, and namely Streptococcus agalactiae. Other common pathogens include Escherichia coli and Staphilococcus aureus.

As in bacterial vaginosis, the concentration of vaginal lactate is depressed also in aerobic vaginitis, but unlike patients with bacterial vaginosis, the vaginal succinate is not produced in the aerobic pathology. A further difference is that aerobic vaginitis causes an immune response in the host; this mostly leads to a high production of interleukin-6 with interleukin 1-b.

Turning back to bacterial vaginosis, the most common antibiotic treatments consist of oral preparations of metronidazole or clindamycin, to which 80-90% of women show a positive effect. In particular, clindamycin shows a better activity with regard to Mobiluncus spp. and Gardenella vaginalis compared to metronidazole, but the latter has the advantage of being less harmful on lactobacilli. However, 15-30% of the treated patients have a recurrence within three months, and in patients with recurrent bacterial vagi- nosis the initial response to the treatment appears to be even lower. In effect, the heterogeneity of the micro-organisms involved contributes to the therapeutic failure or to increasing the possibility of recurrences.

According to the literature (Barbara A. Majeroni, Bacterial Vaginosis: An Update, American Family Physician, 1998, Vol. 57, No. 6), the treatment regimen for bacterial vaginosis consist of 500 mg of metronidazole or 300 mg of clindamycin for oral therapy twice a day for seven days. These doses turned out to be equivalent to topical treatment with clindamycin in the form of 2% vaginal cream or with metronidazole in vaginal gel, administered daily in 5 g doses, respectively once a day for seven days, or twice a day for five days. However, in the case of bacterial vaginosis during pregnancy, the literature shows that there is no general consensus as regards screening and pharma- cological treatment. It has been reported that, although oral metronidazole and clindamycin have reduced the incidence of vaginal bacterial pathologies in pregnancy, the same efficacy is not found with topical vaginal administration (Joesef M. R. et al., Intravaginal clindamycin treatment for bacterial vaginosis: effects on preterm delivery and low birth weight, Am. J. Obstet Gynecol., 1995, 173: 1527-31 ; McGregor J.A. et al., Bacterial vaginosis is associated with prematurity and vaginal fluid mucinase and sialidase: results of a controlled trial of topical clindamycin cream, Am. J. Obstet Gynecol., 1994, 170: 1048-60; Kurkinen-Raty M. et al., A randomized controlled trial of vaginal clindamycin for the treatment of abnormal genital tract flora in pregnancy, Infect. Dis. Obstet. Gynecol. , 2003, 11 (4): 181-9).

In effect, the treatment regimens recommended by the Centres for Diseases Control and Prevention for pregnant women include 250 mg of metronidazole for oral administration three times a day for seven days or 300 mg of clindamycin for oral administration twice a day for seven days (Centres for Diseases Control and Prevention, Prevention of Perinatal Group B Streptococcal Disease: Revised Guidelines from CDC, Morb. Mortal. WkIy. Rep., 2002, 51 : 1-22).

On the other hand, the antibiotics in use have so far not been seen to appreciably reduce adverse obstetric outcomes such as premature membrane rupture, corionamnionitis, premature delivery, endometritis and neonatal complications. As already mentioned, this is due to the fact that the pathogenic micro-organisms to be controlled and eradicated in pregnancy are not so much the anaerobic ones typical of bacterial vaginosis, but the aerobic ones which have been recognised as etiological agents of anaerobic vaginitis. An important risk factor for pregnancy and for the newborn is genital colonisation by Streptococcus agalactiae or Group B Streptococcus (SGB), a micro-organism belonging to vaginal pathogenic flora of aerobic vaginitis. It is a facultative aerobic Gram-positive coccus of about 2.0 μm, whose colonisation causes complications not just limited to the neonatal period. According to some researchers, SGB colonisation during pregnancy increases the risk of miscarriage and affects the pathogenesis of premature rupture of foetal mem- branes, of preterm delivery and of the low body weight of the newborn at birth. Moreover, the complications due to vaginal colonisation by Streptococcus agalactiae during pregnancy do not only concern pregnancy: it has been reported that this micro-organism is directly responsible for corionamnionitis, endometritis, obsteomielitis and meningitis. Maternal colonisation is in general defined via culture medium research carried out at the 26^th-28^th week of pregnancy. Antibiotic therapy during the prenatal, intrapartum and post-natal periods was used above all to prevent the development of neonatal pathologies. Objectively, prenatal therapy was unable to eradicate the micro-organisms once and for all, perhaps because of often persisting or intermittent colonisation. Another similarly poorly effective strategy is the isolated administration of antibiotic prophylaxis during the postnatal period to colonised mothers and to all their newborns. Therefore, the experience gained by using these strategies has shown that the most effective method currently available is intrapartum prophylaxis in order to prevent mother-foetus transmission of infection from Streptococcus agalactiae (El Beitune P. et al., Colonization by Streptococcus agalactiae during pregnancy: maternal and perinatal prognosis, Braz. J. Infect. Dis., 2005, Vol. 9, No. 4; Decoster L. et al., Antimicrobial susceptibility of group B streptococci collected in two Belgian hospitals, Acta Clin. BeIg., 2005, 60(4): 180-4). In accordance with the guidelines, the protocols envisage the intravenous administration of β-lactamic antibiotics during child delivery. Among the antibiotics widely used against SGB are penicillin G and ampicillin, but penicillin G continues to be the preferred antibiotic for intrapartum prophylaxis of colonised mothers, due to its effective transplacental passage, its low cost, but especially its broad range of action even against Gram-positive cocci, with a lower probability of the emergence of resistant micro-organisms. Erythromycin and clindamycin are considered acceptable alternatives for mothers who are allergic to penicillin, although the resistance to these antibiotics may be significant: the prevalence of the resistance may vary from 7% to 25% for erythromycin and from 3% to 15% for clindamycin.

Other antibiotics that have been experimented for topical vaginal ap- plication in aerobic vaginitis are kanamicin and meclocyclin (Tempera G. et al., Microbiological/clinical characteristics of topical therapy with kanamicin in aerobic vaginitis: a pilot study, Internal J. Antimicr. Ag., 2004, 24(1): 85-88). Clinical trials showed a better performance for kanamicin compared to meclocyclin in terms of action duration. The micro-organisms involved in the study were Gram-negative aerobic bacteria, and in particular E. coli.

To date, therefore, attempts at eradicating Streptococcus agalactiae from the genital tract during pregnancy by means of oral or topical treatments based on commercially available antibiotics have only been partly successful. In view of the above, it would be clinically very important to be able to use a broad-range product that could be effective for topical vaginal treatment, given the capacity of similar products to be well-tolerated without any adverse effects in women during the reproduction period and especially during pregnancy, and also applicable without needing the direct intervention of medical staff. A product of this kind should be able to eradicate vaginal bacterial infections - even recurring ones of the gynaecological and obstetric kind, which are probably correlated to the presence of bacterial biofilms adhering to the epithelium of the vaginal wall (Swidsinski et al., Adherent Biofilms in Bacterial Vaginosis, Obstet. Gynecol., 2005; 106(5): 1013-1023), with particular regard to vaginal colonisation by Streptococcus agalactiae, a micro-organism that causes serious pregnancy and neonatal complications.

Moreover, in order to increase the in-situ effectiveness of the drug and to enhance its safety and compliance, it would be best to be able to use low systemic absorption topical formulations that can maintain suitable concentra- tions of the active ingredient in the vaginal environment over time. At present, no commercially available product containing antibiotics which can be administered vaginally meets these requirements, since: - Topical metronidazole and clindamycin are only active on anaerobic bacteria and are thus targeted solely for bacterial vaginosis therapy;

- Topical kanamicin and meclocyclin are active only on Gram-negative aerobic bacteria and thus effective in the treatment of aerobic vaginitis from E. coli.

According to the present invention, it was found that a known antibiotic molecule, piperacillin, formulated such that it can be used for topical vaginal administration, preferably with protracted release over time, has bacteri- cide activity against Gram-positive aerobic micro-organisms, Gram-negative aerobic micro-organisms, Gram-positive anaerobic micro-organisms and Gram-negative anaerobic micro-organisms present in the vaginal ecosystem of both pregnant and non-pregnant women. As is known, piperacillin is a broad-range semi-synthetic β-lactamic antibiotic derived from D(-)-α- aminobenzylpenicillin, with the brute formula C₂₃H₂₇N5O₇S and molecular weight of 517.56. Its chemical name is (2S,5R,6R)-6-[(R)-2-(4-ethyl-2,3-dioxo- i-piperazinecarboxamido^-phenylacetamidoJ-S.S-dimethyl^-oxo-^tia-i- azabiciclo[3.2.0]heptane-2-carboxylic acid, and it has the following structural formula:

Its sodium salt, 02₃H_2GN₅NaO₇S, which constitutes the normally marketed product, is in the form of a white or whitish powder that is completely soluble in water and in alcohol; the pH of an aqueous solution containing 400 mg/L of sodium piperacillin is between 5.5 and 7.5. Since it was first synthesised in the latter half of the 1970s, piperacillin has been developed mainly for systemic use and is commercially available only as a powder for parenteral administration, specifically via intravenous and intramuscular injection. In effect, this active ingredient is not absorbed when administered orally, but is rapidly absorbed after intramuscular injection, reaching a peak concentration in the bloodstream about thirty minutes after injection. After parenteral administration, piperacillin is largely distributed in body tissues and fluids, including the bones, prostate gland and heart, and reaches high concentration in bile. Sodium piperacillin, in the form for parenteral administration, is mainly used with the following indications:

■ For serious infections, such as septicemia, nosocomial bronchitis, intraabdominal infections, aerobic and anaerobic gynaecological infections, and skin and soft tissue infections, doses of 12-18 g/day, administered intravenously every 4-6 hours, are recommended;

■ For complicated infections of the urinary tract, doses of 8-16 g/day, administered intravenously every 6-8 hours, are recommended;

■ For non-serious infections of the urinary tract and for common bronchitis, doses of 6-8 g/day, administered via intramuscular or intravenous injection every 6-12 hours, are recommended;

■ For simple infections (gonorrhea), a single daily dose of 2 g via intramuscular injection is recommended.

With specific reference to gynaecological infective pathologies that can lead to chorioamnionitis during pregnancy and to preterm foetal mem- brane rupture, piperacillin was also clinically experimented, but always exclusively for intravenous administration (Lockwood CJ. et al., Double-blind, placebo-controlled trial of piperacillin prophylaxis in preterm membrane rupture, 1993, 169(4): 970-6).

The only non-parenteral applications of sodium piperacillin which were studied were in connection with the possible topical use of this antibiotic for direct application on bum wounds, and particularly for the prevention of infections from Pseudomonas aeruginosa. In this case, however, the current litera- ture concerns the association of silver sulfadiazin with sodium piperacillin (Modak S. & Fox CL. Jr., Synergistic action of silver sulfadiazine and sodium piperacillin on resistant Pseudomonas aeruginosa in vitro and in experimental burn wound infections, J. Trauma, 1985, 25(1): 27-31). US patent no. 4535078, by the same authors, has the same object and describes an antibacterial composition useful for the treatment of burn wounds and based on silver sulfadiazin and sodium piperacillin.

According to the present invention, it has been found that piperacillin has considerable bactericidal activity on micro-organisms deriving from vagi- nal fluid in pregnant and non-pregnant women diagnosed with bacterial vaginosis and aerobic vaginitis, and that this activity is advantageously carried out via topical intravaginal administration of the product, without the need to use parenteral administrations. Since piperacillin, even if for solely systemic use, has been known and used for some time, its clinical value and safety have been widely validated all over the world.

Therefore, the present invention specifically provides the use of piperacillin or one of its pharmaceutically acceptable salts for the production of a topical intravaginal preparation for the treatment and prevention of gynaecological and obstetric bacterial vaginal infections. As already noted, this topical preparation is recommended for the treatment and prevention of both bacterial vaginosis and aerobic vaginitis.

Specifically, the intravaginal topical preparation based on piperacillin according to the present invention is recommended for the treatment and prevention of aerobic vaginitis in pregnancy, with particular regard to aerobic vaginitis from Gram-positive aerobic germs such as Group B streptococci or Streptococcus agalactiae.

According to the most convenient embodiments of the present invention, the topical intravaginal preparation contains between 0.005% and 5% weight of piperacillin or of one of its pharmaceutically acceptable salts. In particular, the said pharmaceutically acceptable salt may be sodium salt, and the preparation may contain preferably between 0.03% and 5% weight of sodium piperacillin. The topical formulations reported in the present invention are preferably prepared in order to allow a constant and protracted release of sodium pipercillin over time. In particular, the piperacillin-based topical intravaginal drug may contain - besides the common excipients - also one or more bioad- hesive or mucoadhesive carriers in order to enhance the in-situ resistance time of the active ingredient. More specifically, the bioadhesive or mucoadhesive carrier may be an agent selected from the group consisting of hy- droxypropylcellulose, carbomers, alginates, pectin, xyloglucans, chitosan, xanthan gum and polycarbophil. In some preferred embodiments, sodium piperacillin is conveyed in preparations containing xanthan gum as a bioadhesive or chitosan and/or alginates.

The topical intravaginal drug proposed may be presented in the form of ovules, vaginal tablets, cream, paste, emulsion, unguent, solution or suspension. Advantageously, the preferred pharmaceutical form should enable the protracted release over time for a period up to 4 hours, in order to reduce the frequency of application. This possibility is guaranteed, in particular, by the formulation in vaginal tablets.

According to a preferred embodiment of the present invention, therefore, the said topical intravaginal drug is in the form of a coated vaginal tablet containing from 1 % to 2% weight of sodium piperacillin.

According to another specific alternative, the topical intravaginal drug according to the present invention may be in the form of a gel containing between 0.2 and 2% weight of sodium piperacillin.

As well as the aforesaid bioadhesives and mucoadhesives, the piper- acillin-based topical preparation according to the present invention may include further optional ingredients, such as thickening agents, antioxidants, stabiliser, surfactants or other agents. Moreover, the preparation may contain preservatives or antimicrobial agents such as methyl-, propyl- or ethyl- paraben, benzoic acid, benzyl acid, as well as other substances convention- ally used in the pharmaceutical art such as excipients and carriers for preparations for topical administration. Thθ topical piperacillin-based bactericide product according to the present invention may be applied in doses between 0.3 and 3 mg of active ingredient, with a daily application for 3-10 consecutive days. For the treatment of infectious pathologies during pregnancy and for the prevention of pregnancy complications such as preterm foetal membrane rupture or chorioamnionitis and preterm delivery, the preparation is preferably administered between the 26^th and 28^th week of pregnancy.

The specific features of the present invention, as well as its advantageous aspects, will be more evident with reference to a detailed description presented merely for exemplificative purposes below, along with the results of the experiments conducted on it.

Some examples of piperacillin-based formulations for topical intrav- aginal use according to the present invention are presented merely for exemplificative purposes below. EXAMPLES 1-3

Piperacillin formulations in ovules for topical intravaginal application

Some sodium piperacillin-based preparations in vaginal ovules were produced according to procedures commonly used in the pharmaceutical art, on the basis of the compositions reported below (where the percentages are expressed in weight). Formulation 1

Sodium piperacillin 0.035 % polycarbophil 1.0 % gelatine 21.0 % glycerol 34.30 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 % water 43.605 %

Formulation 2 sodium piperacillin 0.035 % polycarbophil 0.5 % gelatine 27.0 %

PEG 300 30.30 % methylparaben 0.28 % ethylparaben 0.014 % water 42.105 %

Formulation 3 sodium piperacillin 0.035 % xanthan gum 0.3 % gelatine 26.0 % glycerol 34.30 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 % water 39.305 %

EXAMPLES 4-6

Piperacillin formulations in tablets for topical intravaginal application

The coating for the vaginal tablets consisted of a film-type one (ca. 0.5 mg) composed of methylhydroxypropylcellulose (ca. 0.45 mg) and PEG 6000

(ca. 0.05). The sodium piperacillin-based compositions contained inside the coating were as follows.

Formulation 4 sodium piperacillin 1 .04 mg mannitol 17.9 mg pregelatinized com starch 8 .0 mg magnesium stearate 0.4 mg polycarbophil 57.7 mg

Formulazione 5 sodium piperacillin 1.04 mg mannitol 17.9 mg pregelatinized corn starch 8.0 mg magnesium stearatθ 0.4 mg xanthan gum 57.7 mg

Formulation 6 sodium piperacillin 1.04 mg mannitol 17.9 mg pregelatinized corn starch 8.0 mg magnesium stearate 0.4 mg hydroxypropylmethylcellulose 57.7 mg

EXAMPLES 7-10 Piperacillin formulations in gel form for topical intravaginal application

Some sodium piperacillin-based preparations in gel form were produced according to procedures commonly used in the pharmaceutical art, on the basis of the compositions reported below (where the percentages are expressed in weight). Formulation 7 sodium piperacillin 0.416 % polycarbophil 51.619 % vaseline oil 41.0 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 %

PEG 6.909 %

Formulation 8 sodium piperacillin 0.416 % xanthan gum 51.619 % vaseline oil 41.0 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 %

PEG 6.909 %

Formulation 9 sodium piperacillin 0.416 % xanthan gum 60.584 % vaseline oil 35.0 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 %

PEG 3.944 %

Formulation 10 sodium piperacillin 0.416 % polycarbophil 46.584 % vaseline oil 41.0 % methylparaben 0.028 % ethylparaben 0.014 % propylparaben 0.014 % PEG 11.944 %

EXAMPLE 11 Piperacillin formulations in cream form for topical intravaginal application

According to common procedures of the pharmaceutical art, various sodium piperacillin-based preparations were produced in cream form, with tenors of the active ingredient ranging from 1% to 3%. The excipients used were liquid paraffin, propylene glycol, polysorbate 60, cetostearylic alcohol, cethylpalmitate, stearic acid, sorbitan monostearate, benzylic alcohol and water. Preparation and evaluation of mucoadhesive vaginal tablets

As already noted, in order to increase the effectiveness of piperacillin and to enhance safety and compliance, it would be best to have topical formulations of low systemic absorption and which can maintain adequate concentrations of the active ingredient in the vaginal environment over time. There- fore, the formulations described below were arranged and evaluated in order to enable a constant protracted release of the drug.

Cylindrical vaginal tablets of about 6 mm in diameter and 3 mm in height, each weighing 85 mg, were prepared by using a hydraulic press with a compression strength of 1000 kg. The composition of each tablet undergoing the subsequent trials is reported in Table 1 below.

Some formulations were excluded on the basis of preliminary screen- ing. Some of them (CPR1 , CPR9 and CPR10) did not swell and did not show signs of adherence to the walls of the glass vessel, while others (CPR4 and CPR5) dissolved in a few minutes with effervescence.

TABLE 1

85 mg vaginal tablets - Composition (mg) of the formulations under study

Evaluation of mucoadhesive properties

The evaluation of the mucoadhesive properties of the formulations under study was carried out by measuring the work necessary to separate two mucous surfaces between which the formulation under study was placed. The mucous surfaces were composed of portions of rabbit vaginal mucous: the vagina was cut longitudinally and spread out taking care not to touch the mucous layer; then two sections of the vagina were mounted on two supports of the tensile apparatus. The equipment used consisted of a microbalance, a mobile platform and a computerised system that could record the force necessary to separate the two surfaces (sample under study/mucous layer) by stretching.

From the graphs obtained, the area under the curve was calculated, which represents the adhesion work (force x elongation) The results obtained are reported in the attached Figure 1 and in Table 2 below.

TABLE 2 Mucoadhesive properties

As may be noted from the collected data, the best mucoadhesive properties are shown by the formulations containing chitosan, CPR6 and CPR12, although even the other formulations show comparable mucoadhe- sion to the reference substances. The adhesion work of the tablets decreased in the following order: CPR12>CPR6>CPR3>CPR8>CPR2=CPR7=CPR11.

Evaluation of swelling capacity

The tablets were weighed, placed on a wire gauze of known weight and immersed at room temperature in 60 ml of simulated vaginal fluid having the following composition: NaCI (3.51 g), KOH (1.40 g), Ca(OH)2 (0.222 g), bovine serum albumin (0.018 g), lactic acid (2.00 g), acetic acid (1.00 g), glycerol (0.16 g), urea (0.4 g), glucose (5.0 g), enough water to make up 1 litre taking the pH to 4.2. The samples were periodically (30 min) weighed after removing the excess surface water. The hydrating capacity of the tablets was quantified by means of the swelling index according to the following ratio:

(Pt-Pi) Swelling index: Pi

Pt: tablet weight at time t Pi: initial tablet weight

The variation in the swelling index over time, for each formulation es- sayed, is reported in Figure 2. The graph shows the behaviour of the CPR7 formulation - the only one to contain lactose as excipient - which in a very short time starts to break up with virtually no swelling at all.

The excipient which leads to the greatest swelling is chitosan, that is present in the same quantities in formulations CPR6 and CPR12: during the trial, these two formulations considerably increased in volume reaching swelling index values around 14-15. CPR12 presented a more regular swelling, probably due to the presence of crosspovidon that leads to the forming of channels within the polymer matrix and facilitates the input of water.

In the other tablets, the hydration, swelling and erosion processes took place at the same time, and the formulations maintained a constant weight for long periods of time with constant swelling index values: CPR2 showed a diameter of 1.5 cm after 5 hours hydration, even if its swelling index only reached a value of 7.8. Comparing the profiles of CPR2 and CPR3, which present the same composition except for the polymer, we note the greater hydration capacity of xanthan gum compared to HPMC.

Analysis of the drug The analysis of piperacillin (PPC) was carried out by HPLC, by using a Shimadzu LC 10AS system with a UV detector and integration system. The injection valve was a Rheodyne with a 20 μl capacity. The column was a Bondclone one (Phenomenex) 30 cm in length and having an internal diameter of 3.9 mm, packed with a 10 μm C18 phase. The mobile phase was com- posed of NaH₂PO₄ 0.05M pH 7.0 and CH₃CN in a ratio of 80:20, with a flow rate of 1 ml/min; the detection wavelength was 220 nm. Quantitative determination was carried out by comparison with an external standard. Sample analysis was carried out within 6 hours of the preparation or sampling.

Evaluation of the dissolution To evaluate the dissolution profile of the formulations studied, a rotary drum dissolver (FU Xl) was used in the following conditions: Method A

- rotation speed: 74 rpm

- temperature: 37⁰C - dissolution medium: 600 ml of vaginal fluid simulating solution

- volume sampled: 10 m Method B

- rotation speed: manual

- temperature: 25⁰C - dissolution medium: 1.5 ml of vaginal fluid simulating solution

- volume sampled: 0.375 ml

These conditions were chosen in order to simulate "in-vivo" conditions:

- daily vaginal fluid production: 6ml

- amount continuously present: 0.5-0.6ml - turnover: 0.25 ml/hour

Figure 3 shows the dissolution profiles of the studied formulations as a percentage of the active ingredient dissolved in the medium with respect to the theoretical quantity contained in each tablet, equivalent to 100% of PPC released. The results are comparable regardless of the method used. The release parameters, analysed by applying the empirical exponential equation M_t/M» = ktⁿ, where M_t/M» is the fraction of the drug ceded at time t; k is the dissolution velocity constant characteristic of the system; n is the char- acteristic diffusional exponent of the release mechanism, are reported in Table 3 below as Rso%, instantaneous velocity at 50% of release and t.₅₀% is the time necessary to release 50% of the drug.

TABLE 3 Release parameters

The behaviour of formulation CPR7 can be highlighted since it completely releases the drug within 2.5 hours. This behaviour may be due to the presence of lactose, which is only contained in CPR7, that leads to a massive penetration of water inside the matrix, thereby increasing the diffusion velocity of the drug.

Formulations CPR6 and CPR12, both containing chitosan, sodium alginate and CMCNa, do not dissolve but swell a great deal: drug release is therefore much slower due to the long passage through the gelatinous mass that the PPC must cross. Moreover, the drug that is strongly soluble in water tends to stay within the hydrated tablet.

Even the presence of HPMC in the formulation leads to the forming of a hydrogel that hinders the diffusion of the drug. The data obtained shows that passing from HPMC K4M, present in CPR8, to HPMC K100M, in CPR3 and CPR11 , decreases the capacity of PPC to spread and thus its release velocity R_50% falls from 0.42±0.01 for CPR8 to 0.19+0.02 and 0.16±0.03 min^"1 respec- tively for CPR3 and CPR11. This phenomenon may be explained by the increase in molecular weight of the polymer, and thus by the increased tangling of the polymer chains, which reduce the mobility of the macromolecules in the swelled system.

Conclusions

Compared to the other formulations tested, the CPR2 formulation presents the best properties since it is characterised by a good dissolution profile with a prolonged and almost complete PPC release in 6 hours, and a high degree of initial hydration and swelling followed by erosion/dissolution in contact with simulated vaginal fluid.

Straight after CPR2, the formulations CPR6 and CPR8 present a good combination between dissolution profile and hyd ration/swelling profile. Later, formulations such as CPR2, CPR6 and CPR8 were created with in- creasing concentrations of piperacillin, up to 10 mg. The dissolution profiles for the three formulations and for the concentrations of active ingredient in the range between 1 mg and 10 mg turned out to be unchanged. These dissolution profiles appear independent of the measurement conditions used (Method A or Method B) and are comparable.

Experimental results

The results of some in-vitro trials aimed at testing the efficacy of using topical piperacillin in the treatment of vaginal infective pathologies from Gram- positive and Gram-negative aerobic and anaerobic germs are illustrated be- low. The trials aimed to assess the in-vitro strength of the active ingredient on vaginal pathogens of both collection and recent isolation kinds.

The vaginal tampons brought to the laboratory were placed in specific culture mediums for growing various bacterial species identified and assayed in vitro with regard to sodium piperacillin. a) Anaerobic bacteria

As regards the bacteria prevalently responsible for bacterial vaginosis, piperacillin activity was tested on 30 strains of anaerobic Gram-positive and Gram-negative bacteria, both standard strains and those isolated through clinical samples: Gardnerella vaginalis ATCC, Fusobacterium spp. (1 ATCC and 6 clinically isolated ones), Porphyromonas spp. (1 NCTC and 3 clinically isolated ones), Prevotella spp. (7 clinically isolated strains), Eubacterium len- turn (2 clinically isolated strains) and Peptosterococcus spp. (9 clinically isolated strains).

MIC determination

The piperacillin was assayed at concentrations from 0.06 mg/L to 256 mg/L. MIC evaluation was determined by means of the broth dilution method in microslides, according to the NCCLS (1997), using Wilkins-Chalgren broth added with hemin and vitamin K1. The microslide wells were inoculated with a bacterial suspension of 5x10⁵ CFU/mL, and the slides were then placed in an anaerobic atmosphere and read after 48 hours. MIC/MBC ratio determination

After determining the MIC of piperacillin, the subcultures were arranged on ASA (agar blood for anaerobic bacteria) slides, by taking the inco- culum from the microslide wells which did not show any visible growth to the naked eye. The slides were incubated at 37⁰C for 48h in an anaerobic atmos- phere. MBC is defined as the lowest concentration determining the absence of visible growth. b) Aerobic bacteria

As regards the bacteria mainly responsible for aerobic vaginitis, the culture mediums used were: Mac Conkey agar (OXOID) for E. coli, Bile Escu- line agar (OXOID) for enterococcus spp. and agar blood CNA (OXOID) for S. agalactiae.

When possible, the lactobacilli (the main ones responsible for vaginal flora equilibrium in fertile-age women) were also isolated by using MRS agar (OXOID). After inoculation, the slides were incubated at 37°C for 24h; the Streptococcus agalactiae and lactobacilli were incubated in the presence of CO₂.

A total of 40 strains were identified and purified, and both MIC (Mini- mum Inhibiting Concentration) and MBC (Minimum Bactericide Concentration) determination was carried out on them with regard to piperacillin.

MIC determination

E. coli and Enterococcus spp.: The inoculum was prepared by diluting 1-2 colonies of the examined bacteria strains in 5 ml of Muller Hinton broth (OXOID) in order to obtain a turbidity of 0.5 McFarland. The tubes containing the inoculum were incubated at 37⁰C for 24 h.

After this time, MIC was determined by means of the solid medium technique. Scalar dilutions of the antibiotic from 1/128 to 1/0.125 were prepared in sterile tubes.

1 ml of each dilution was poured into Petri dishes to which was added 19 ml of Muller Hinton agar (OXOID). In this way, each slide corresponded to a specific dilution. The inoculum was placed onto the slides which were then incubated at 37°C for 24h.

In some strains of E. coli the inoculum effect on the MIC value was also assessed at concentrations of 10³, 10⁵ and 10⁷ CFU/ml.

Lactobacilli:

The procedure was identical to the previous one. However, MRS broth (OXOID) was used in order to obtain the inoculum and MRS agar containing the various dilutions for the antibiotic was used for sowing. Moreover, incubation was carried out in the presence of CO₂.

S. aaalactiae:

For Streptococcus agalactiae, the inoculum was obtained in Muller Hinton broth, but the MIC was instead determined by using the microdilution method in a liquid medium (Muller Hinton broth). For incubation, the mi- crowells were sealed with parafilm in order to obtain an oxygen-free atmosphere.

MIC/MBC ratio determination After determining the MIC of piperacillin, the subcultures were arranged on ISO sensitest agar slides, taking the inoculum by means of a calibrated handle/loop starting from those samples which did not show any visible growth to the naked eye. The slides were incubated at 37°C for 24h (for the lactobacilli, in the presence of CO₂). The MBC is defined as the lowest concentration determining the absence of visible growth.

The results are summarised in the three tables below, showing piperacillin activity (assessed as MIC μg/ml) on bacteria isolated from clinical samples (vaginal fluid) of pregnant women (Table 4), from clinical samples of nonpregnant women (Table 5) and on standard bacteria strains (Table 6)

TABLE 4 Piperacillin activity on samples taken from pregnant women

Micro-organism MIC

Fusobacterium spp. (anaerobic Gram-) 0.125

Prevotella melaninogenica (anaerobic Gram-) 0.5

Prevotella intermedia (anaerobic Gram-) 4

Prevotella spp. (anaerobic Gram-) 4

Eubacterium lentum (anaerobic Gram+) <0.06

Peptostreptococcus spp. (anaerobic Gram+) <0.06

Peptostreptococcus micros (anaerobic Gram+) 1

Peptostreptococcus micros (anaerobic Gram+) 4

Peptostreptococcus micros (anaerobic Gram+) 0.125

E. coli 16

E. coli 32

Enterococcus spp. (aerobic Gram +) 32

Enterococcus spp. (aerobic Gram +) 4

Enterococcus spp. (aerobic Gram +) 8

Enterococcus spp. (aerobic Gram +) 1

Enterococcus spp. (aerobic Gram +) 32

S. agalactiae (aerobic Gram +) <0.125

S. agalactiae (aerobic Gram +) 2

S. agalactiae (aerobic Gram +) <0.125

S. agalactiae (aerobic Gram +) <0.125 TABLE 5 Piperacillin activity on samples taken from non-pregnant women

Micro-organism MIC

Fusobacterium nucleatum (anaerobic Gram-) <0.06

Fusobacterium varium (anaerobic Gram-) 0.06

Fusobacterium spp. (anaerobic Gram-) 2

Fusobacterium spp. (anaerobic Gram-) 1

Prevotella melaninogenica (anaerobic Gram-) 1

Prevotella intermedia (anaerobic Gram-) 2

Prevotella spp. (anaerobic Gram-) 2

Eubacterium lentum (anaerobic Gram+) <0.06

Peptostreptococcus spp. (anaerobic Gram+) 0.5

Peptostreptococcus micros (anaerobic Gram+) 2

Peptostreptococcus micros (anaerobic Gram+) 1

Porphyromonas spp. (anaerobic Gram-) 0.5

Porphyromonas spp. (anaerobic Gram-) 1

E. coli 8

E. coli 16

E. coli 1

E. coli 16

E. coli 8

E. coli 1

Enterococcus spp. (aerobic Gram +) 32

Enterococcus spp. (aerobic Gram +) 2

Enterococcus spp. (aerobic Gram +) 32

S. agalactiae . (aerobic Gram +) <0.125

S. agalactiae (aerobic Gram +) 2

S. agalactiae (aerobic Gram +) <0.125

S. agalactiae (aerobic Gram +) 1

S. agalactiae (aerobic Gram +) <0.125 TABLE 6 Piperacillin activity on standard strains

Micro-organism MIC

Gardnerella vaginalis ATCC (anaerobic optionally Gram-

<0.06 or Gram variable)

Fusobactθrium nucleatum (anaerobic Gram-) 0.5

Porphyromonas gingivalis (anaerobic Gram-) 4

As it may be seen from the above tables, piperacillin activity on the anaerobic pathogens involved in bacterial vaginosis is good and is of the bactericide type. In particular for Gardnerella vaginalis, 100% of the assayed strains were sensitive to the antibiotic.

Piperacillin activity on aerobic pathogens involved in aerobic vaginitis showed: - a good result as regards Escherichia coli and Enterococcus, with MIC values showing high percentages of sensibility according to NCCLS (2003) break-points;

- an excellent result with Streptococcus agalactiae, with 100% sensibility of the assayed strains at very low MIC values (easily reached with an in-situ formulation).

Also in this case the activity turned out to be of the bactericide type.

In view of the above, piperacillin can be the preferred antibiotic in infections of women's lower genital tract, such as bacterial vaginitis (vaginosis and aerobic vaginitis), where the therapeutic decision is difficult owing to the polymicrobic nature of these pathologies and the little availability of antibiotics having a broad range of activity against both Gram-positive and Gram- negative anaerobic/aerobic bacteria.

The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifica- tions and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.

Claims

1. Use of piperacillin or of a pharmaceutically acceptable salt thereof for the production of a topical intravaginal preparation for the treatment and prevention of gynaecological and obstetric bacterial vaginal infections.

2. Use according to claim 1 , wherein the said topical preparation is recommended for the treatment and prevention of bacterial vaginosis and aerobic vaginitis.

3. Use according to claim 2, wherein the said topical preparation is recommended for the treatment and prevention of aerobic vaginitis during pregnancy.

4. Use according to claims 2 or 3, wherein the said topical preparation is recommended for the treatment and prevention of aerobic vaginitis due to group B streptococci or Streptococcus agalactiae.

5. Use according to any one of claims 1-4, wherein the said topical intravaginal preparation contains from 0.005% to 5% weight of piperacillin or of a pharmaceutically acceptable salt thereof.

6. Use according to any one of claims 1-5, wherein the said pharmaceutically acceptable salt is sodium salt.

7. Use according to claim 6, wherein the said topical intravaginal preparation contains from 0.03% to 5% weight of sodium piperacillin.

8. Use according to any one of claims 1-7, wherein the said topical intravaginal drug also contains one or more bioadhesive or mucoadhesive carriers.

9. Use according to claim 8, wherein the said bioadesive or mucoadhesive carrier is chosen from the group consisting of hydroxypropylcellulose, carbomers, alginates, pectin, xyloglucans, chitosan, xanthan gum and poly- carbophil.

10. Use according to claim 9, wherein the said bioadhesive or muco- adhesive carrier is xanthan gum.

11. Use according to claim 9, wherein the said bioadhesive or mucoadhesive carrier is chosen from chitosan, alginates and/or their mixtures.

12. Use according to any one of claims 1-11 , wherein the said topical intravaginal drug is in the form of ovules, vaginal tablets, cream, paste, emulsion, unguent, solution or suspension.

13. Use according to any one of claims 1-11, wherein the said topical intravaginal drug is in the form of a coated vaginal tablet containing from 1% to 2% weight of sodium piperacillin.

14. Use according to any one of claims 1-11 , wherein the said topical intravaginal drug is in the form of a gel containing from 0.2 to 2% weight of sodium piperacillin.