Evaluation of the effects of a new intravaginal gel, containing purified bovine colostrum, on vaginal blood flow and vaginal atrophy in ovariectomized rat.

ORIGINAL RESEARCH—PHARMACOTHERAPY Evaluation of the Effects of a New Intravaginal Gel, Containing Purified Bovine Colostrum, on Vaginal Blood Flow and Vaginal Atrophy in Ovariectomized Rat Silvia Vailati, MSc,* Elsa Melloni, MSc,* Ermanno Riscassi, MSc,† Delphine Behr Roussel, PhD,‡§ and Marco Sardina, MD, PhD* *Zambon S.p.A., Bresso (Milan), Italy; †Pharma Research Consulting s.r.l, Milan, Italy; ‡Pelvipharm, MontignyLe-Bretonneux, France; §EA 4501 Université Versailles Saint Quentin en Yvelines, Montigny-Le-Bretonneux, France DOI: 10.1002/sm2.8

ABSTRACT

Introduction. Vaginal dryness due to vaginal atrophy is a common complaint of postmenopausal women, interfering with sexual function and quality of life. Hormone replacement therapy is the only effective therapy but with known risks that leave unmet medical needs. A new product, ZP-025 vaginal gel, containing puriﬁed (dialyzed lyophilized) bovine colostrum, has been developed for the treatment of vaginal dryness secondary to vaginal atrophy. Aim. The study aims to investigate the effects of intravaginal application of ZP-025 on vaginal atrophy using an animal model. Methods. Ovariectomized female Sprague-Dawley rats were used. Three weeks after surgery, rats were divided into four groups and treated for 4 weeks (twice a day) with placebo or ZP-025 at low (0.5%) or high (2.3%) concentrations of colostrum; in the control group, rats did not receive any treatment. Changes in vaginal blood ﬂow due to pelvic nerve stimulation were assessed by laser Doppler ﬂowmetry and vaginal tissue was collected for histological assay. Main Outcome Measures. The main outcome measures were vaginal blood ﬂow before and after pelvic nerve stimulation and histology of vaginal epithelium. Results. Treatment with ZP-025 to ovariectomized rats induced an increase of vaginal blood ﬂow parameters (vascular capacitance, amplitude and area under the curve of the response) in response to pelvic nerve stimulation compared with control group, statistically signiﬁcant at 2.3%. Vaginal epithelium showed a physiological estrous cycle aspect in treated animals, with at least ﬁve cell layers vs. one or two cell layers in control rats. As expected from a topical formulation, systemic effects on body weights and uterine wet weights were not observed with application of ZP-025. Conclusions. In this study, the new product ZP-025, containing puriﬁed colostrum, was shown to have beneﬁcial effects on vaginal atrophy in ovariectomized rats, improving vaginal hemodynamics and thickness of vaginal epithelium. Vailati S, Melloni E, Riscassi E, Behr Roussel D, and Sardina M. Evaluation of the effects of a new intravaginal gel, containing purified bovine colostrum, on vaginal blood flow and vaginal atrophy in ovariectomized rat. Sex Med 2013;1:35–43. Key Words. Vaginal Atrophy; Bovine Colostrum; Ovariectomized Rat; Vaginal Blood Flow

Introduction

aginal atrophy is caused by a decrease in oestrogen production and is a common

complaint of postmenopausal women, interfering with sexual function and quality of life. In about 45% of menopausal women, vaginal atrophy can be clinically manifest as a syndrome of vaginal

Sex Med 2013;1:35–43 © 2013 The Authors. Sexual Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Sexual Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Vailati et al.

dryness, itching, irritation, and dyspareunia [1,2]. The vaginal atrophy becomes clinically apparent 4–5 years after menopause and its hallmarks are thinning of vaginal epithelia layers, increased vaginal pH, decrease in the local blood ﬂow, and diminished vaginal secretion. Clinical studies have shown that these signs signiﬁcantly correlate with the decline in circulating ovarian hormones [3–6]. While systemic and local estrogen-based hormonal therapy is effective in treating symptoms of vaginal atrophy in postmenopausal women [3,7– 9], these medications are contraindicated in some populations (women with unknown vaginal/ uterine bleeding or those with a known or suspected ovary, endometrial cancer, and breast cancer) and other women choose not to take them for fear about their safety [10,11]. Because of these issues, recent work has focused on developing more speciﬁc, and in many cases nonhormonal, alternatives to traditional postmenopausal hormonal therapy to treat vaginal dryness in presence or in absence of vaginal atrophy [12]. Zambon SpA (Bresso, Milan, Italy) has developed a gel product (ZP-025 vaginal gel— Monurelle Biogel), containing puriﬁed bovine colostrum to be administrated topically, for the treatment of vaginal dryness also in presence of vaginal atrophy. Colostrum is a substance produced from female mammary gland during the ﬁrst few hours postpartum, and numerous investigators have reported that the colostrum imparts many advantages for the development of the infants [13]. Thanks to its constituents, colostrum is able to help the local defense (immunoglobulin A); modulate the immunitary response (immunoglobulin E, M, G); promote antibacteric and antiviral action (lactoferrin and transferring); modulate the inﬂammation factors (cytokine, interleukine); promote normal cell growth, normal cell activities, cell migration and proliferation, and tissue repair (transforming growth factor alpha and beta; epidermal growth factor); stimulate the mucosal restore; and accelerate wound healing (insulin-like growth factors—IGF-1 and -2). In particular, IGF-1 is involved in the regulatory feedback of growth hormone [14] and interferes with insulin-like growth factor binding proteins [15]. To date, based on the best scientiﬁc knowledge available to us, no sexual hormones are directly present in bovine colostrum. Because vaginal mucosa, because of its anatomic and physiologic features, is exposed to epithelium damage and to vaginal ecosystem and local defense Sex Med 2013;1:35–43

factors alterations, colostrum can help in promoting the mucosal trophic restoration and preserving from bacteric and viral aggression. Our hypothesis is that topical application of colostrum could have a beneﬁcial effect speciﬁcally on vaginal atrophy. Besides puriﬁed colostrum, this vaginal gel contains other natural substances with humectants, hydrating, re-epithelizing, and antioxidant properties at concentrations 0.05) nor at the time of starting treatments (day 21). Likewise, twice-a-day treatments with placebo, ZP-025 0.5%, and ZP-025 2.3% for 4 weeks did not modify the body weight change between the beginning (day 21) and the end of application period (day 49) (Figure 1B) compared with control. Determination of Peripheral Sexual Response: Vaginal Blood Engorgement by LDPM The intravaginal application of placebo for 4 weeks, 3 weeks after ovariectomy, did not signiﬁcantly change baseline mean BP and baseline LDPM compared with control group (Figure 2).

Likewise, neither ZP-025 0.5% nor ZP-025 2.3% modiﬁed either BP (Figure 2A) or baseline LDPM (Figure 2B) compared with placebo. Following PN stimulation, the parameters of vaginal blood engorgement (i.e., maximal amplitude of the response, AUC of the response, and vascular capacitance) increased in each group in parallel with the frequency of PN electrical stimulation. No statistically signiﬁcant difference was shown in each parameter between placebo and control group (Figure 3). The efﬁcacy of the product was evaluated by statistical comparison between the two different doses of colostrum and placebo in order to establish a possible superiority of the product due to the presence of colostrum. After 4 weeks of intravaginal twice-daily treatment with ZP-025 0.5%, no statistical difference in the amplitude, the AUC, and the vascular capacitance was present when compared with placebo group (Figure 4). On the contrary, the treatment with ZP-025 2.3% signiﬁcantly increased all the parameters of vaginal blood engorgement following PN stimulation, that is, maximal amplitude of the

A Mean body weight (g)

450 400

Control

350

Placebo ZP-025 0.5%

300

ZP-025 2.3% 250

Treatment 200 0

Surgery

Days of study

Body weight change (g) (day 21-49)

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Control Placebo ZP-025 0.5% ZP-025 2.3%

Figure 1 Evolution of (A) body weight starting from surgery until the end of the treatment and (B) body weight change during the 4 weeks of intravaginal treatment in either placebo or ZP-025 0.5% or ZP-025 2.3% groups compared with control group in ovariectomized rats (oneway ANOVA, nonsignificant).

Effects of a New Gel on Vaginal Atrophy in Rats

response, AUC of the response, and vascular capacitance when compared with placebo group (***P < 0.001 for each parameter, two-way anova analysis) (Figure 4).

BP (mmHg)

Baseline mean BP

Control

Placebo

ZP-025 0.5% ZP-025 2.3%

LDPM (au)

Baseline vaginal LDPM

Control

Placebo

ZP-025 0.5% ZP-025 2.3%

Figure 2 (A) Baseline mean blood pressure (BP) and (B) baseline vaginal laser Doppler perfusion measurement (LDPM) measured in anesthetized ovariectomized rats (one-way ANOVA, nonsignificant). au = arbitrary unit.

Vagina and Uterus Weights Intravaginal treatment with placebo, ZP-025 0.5%, or ZP-025 2.3% for 4 weeks in OVX rats did not induce any modiﬁcation of the vagina and uterus macroscopic aspect. Indeed, in all groups of treatment (control, placebo, ZP-025 0.5%, and ZP-025 2.3%), the appearance of vagina and uterus was normal and thin uterine horns were observed. Moreover, after 4 weeks of twice daily intravaginal treatment with placebo, ZP-025 0.5% and ZP-025 2.3%, vagina and uterus tissue wet weight normalized to body weight were not modiﬁed when compared with control (Table 1). Histopathological Evaluation of Vagina and Uterus Seven weeks after ovariectomy, at the end of the treatment period, uterus and cervix atrophy was noted in all animals of all experimental groups. The atrophy involved endometrium and myometrium. B

AUC % baseline.s

% baseline

Amplitude

Frequency (Hz)

Frequency (Hz) Control Placebo

Control Placebo

C Figure 3 Effect of twice-a-day intravaginal treatment with placebo or control in anesthetized ovariectomized rats on vaginal blood engorgement: amplitude of the response (A); AUC of the response (B); and vascular capacitance (C) elicited by pelvic nerve electrical stimulations at increasing frequencies (two-way ANOVA analysis). AUC = area under the curve; ns = nonsignificant.

% baseline.mmHg-1

Vascular capacitance

Frequency (Hz)

Control Placebo

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Vailati et al. B

AUC

% baseline

% baseline.s

Amplitude

Frequency (Hz)

Frequency (Hz) Placebo ZP-025 0.5% ZP-025 2.3%

Placebo ZP-025 0.5% ZP-025 2.3%

% baseline.mmHg-1

C Vascular capacitance

Figure 4 Effect of twice-a-day intravaginal treatment with placebo or ZP-025 0.5% or ZP-025 2.3% during 4 weeks in anesthetized ovariectomized rats on vaginal blood engorgement: amplitude of the response (A); AUC of the response (B); and vascular capacitance (C) elicited by pelvic nerve electrical stimulations at increasing frequencies. (*P < 0.05, ***P < 0.001, 2-way ANOVA). AUC = area under the curve; ns = nonsignificant.

Frequency (Hz) Placebo ZP-025 0.5% ZP-025 2.3%

Atrophy was also noted in vaginal epithelium in all control animals (Figure 5A). In particular, a signiﬁcant thinning was present in the proximal and central part of the vagina epithelium, which was reduced to one or two cell layers and reaching at maximum three to four cell layers in the distal part of some control animals. On the contrary, the vaginal epithelium showed a physiological estrous cycle morphological aspect in animals receiving placebo or ZP-025 0.5% or ZP-025 2.3% (Figure 5B). The epithelium consisted of one to two cell layers with ﬂattened superﬁcial cells only in the proximal part with progressive increase in cell layers (at least 5) in the central and distal part. In the central portion, the superﬁcial layers of epithelial cells were in some instances cuboidal with minimal muciﬁcation, while in the distal portion, the epithelium of at Table 1

least ﬁve cell layers was squamous with presence of stratum corneum in the majority of animals receiving placebo or ZP-025 0.5% or ZP-025 2.3%. Such changes are characteristic of estrous cycle [22]. Discussion

Our study shows that the application of a new vaginal gel containing puriﬁed colostrum for 4 weeks provided beneﬁcial effects in an animal model of vaginal atrophy. Frequency-depending increases of vaginal blood ﬂow elicited by PN stimulation is a widely used experimental paradigm to mimic female sexual response in intact animals [16,23–25]. Ovariectomy, reducing estrogen levels, causes a marked decrease in vaginal hemodynamics, also in

Vagina and uterus wet tissue–body weight ratios at the end of the 4-week treatment in OVX rats Wet weight–body weight ratio (g/100 g) (mean ± SEM)

Vagina Uterus

Control

Placebo

ZP-025 0.5%

ZP-025 2.3%

0.030 ± 0.001 0.027 ± 0.001

0.032 ± 0.002 0.026 ± 0.001

0.032 ± 0.001 0.029 ± 0.001

0.034 ± 0.01 0.029 ± 0.001

ns = nonsignificant, one-way ANOVA

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Effects of a New Gel on Vaginal Atrophy in Rats

Control

Epithelium (1-2 cell layers) in the proximal and central part of the vagina. Atrophy.

ZP-025 2.3% Epithelium (at least 5 cell layers with stratum corneum) in the central part of the vagina. Estrous.

Figure 5 Hematoxylin and eosin staining of rat vaginal epithelium at ×100 magnification. Representative tissue sections are shown. (A) Vaginal epithelium of a rat from control group; (B) vaginal epithelium of a rat receiving twice-a-day intravaginal application of ZP-025 2.3% for 4 weeks. Tissue from control animal shows an atrophic vaginal epithelium, with a lower number of cell layers (A). The treatment with ZP-025 2.3% is able to restore an estrous condition in ovariectomized rat (B).

response to PN stimulation [16]; however, estrogen replacement in OVX rats ameliorates vaginal blood ﬂow and relating parameters, following PN stimulation [16]. In this study, after PN stimulation at different frequencies, only the treatment with ZP-025 containing 2.3% of colostrum increases all the parameters of vaginal blood engorgement, that is, maximal amplitude of the response, AUC of the response, and vascular capacitance in a statistically signiﬁcant manner. On the contrary, for the same parameters no differences between control and placebo were noted and only a trend with the low dose of colostrum was present when compared with placebo. Previous studies demonstrate that ovariectomy produces a signiﬁcant decrease in animal uterus and vaginal tissue wet weight and epithelial thickness [16,19,26]. In fact, the stratiﬁed squamous epithelium, consisting of approximately six to eight layers of cells in intact rats, is reduced to one or two cell layers after ovariectomy. Estrogens, when delivered subcutaneously, restore the uterus

and vaginal wet weight as well as epithelial thickness, with the same number of layers seen in the non-OVX rats [16,26]. Our study shows that a twice-a-day application of placebo, ZP-025 at 0.5% and 2.3% of colostrum to OVX rats for 4 weeks increases the thickness of vaginal epithelium compared with untreated OVX rats and a physiological estrous cycle morphological aspect of the vaginal epithelium is observed. Despite the fact that the product is not able to restore completely the epithelial thickness to the same condition as before the ovariectomy or with estrogens [26], nor it is able to signiﬁcantly enhance tissue wet weight, an evident effect is present in treated animals, with an epithelium of at least ﬁve cell layers, compared with one or two cell layers in untreated animals. This effect of ZP-025 at all concentrations of colostrum could be due not only to the well-known growth factors included in the colostrum per se, but also to the other re-epithelizing components present in the formula, such as betaine, sericin, and panthenol [27,28]. Moreover, the antioxidant compound (Vitamin E) included in the product could retard biologically destructive chemical reactions in living organisms through their ability to scavenge oxidants and free radicals [29]. As a consequence, the placebo cannot be considered totally devoid of any effect, as shown in the histology analysis of vaginal epithelium, where the response to placebo indicates that it is not completely inert; however, a more complete response was obtained with the gel containing colostrum on other parameters. Finally, the fact that uterus atrophy remains histologically unchanged in all experimental groups after a 4-week treatment indicates that the effects of the product are speciﬁcally limited to the vagina, as expected for a topical preparation. This is in contrast with estrogen preparations for topical use, that anyway lead to increased systemic estrogen levels, thus increasing the well-known risk of systemic actions [30–32]. Although many women report that they gained weight at or near the time of their menopause [33], the amplitude and even the reality of the menopause-related gain in weight is not consistent between studies [34,35]. In rats, ovariectomy is associated to body weight increase [23,36], mainly due to hyperphagia and hypoactivity [37,38]. It has been shown that 17β-estradiol hormonal replacement is able to reduce body weight gain in OVX rats [36,38], while in the present study with ZP-025 there is no signiﬁcant difference between experimental

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groups in the animal body weight, both measured at the start of treatments and at the end (after 4 weeks of treatment). Conclusion

The new ZP-025 vaginal gel containing colostrum improves vaginal hemodynamics and thickness of vaginal epithelium in rats with OVX-induced vaginal atrophy. The marked changes in the vaginal hemodynamics and histology resulting from estrogen deprivation are improved by a treatment with ZP-025 containing 2.3% concentration of puriﬁed colostrum. Clinical trials are necessary to determine if these beneﬁts could be translated to menopausal women. Corresponding Author: Silvia Vailati, MSc, Zambon S.p.A., Via Lillo del Duca 10, 20091 Bresso (Milan), Italy. E-mail: [emailprotected] Conﬂict of Interest: S. Vailati, E. Melloni and M. Sardina are employees of Zambon S.p.A. and E. Riscassi is an independent consultant supporting Zambon S.p.A. in the development projects. References 1 Iosif CS, Bekassy Z. Prevalence of genito-urinary symptoms in the late menopause. Acta Obstet Gynecol Scand 1984;63:257– 60. 2 Bygdeman M, Swahn ML. Replens versus dienoestrol cream in the symptomatic treatment of vaginal atrophy in postmenopausal women. Maturitas 1996;23:259–63. 3 Labrie F, Diamond P, Cusan L, Gomez JL, Bélanger A. Effect of 12-month DHEA replacement therapy on bone, vagina, and endometrium in postmenopausal women. J Clin Endocrinol Metab 1997;82:3498–505. 4 Sarrel PM. Ovarian hormones and vaginal blood ﬂow using Laser Doppler velocimetry to measure effects in a clinical trial in post-menopausal women. Int J Impot Res 1998;10:S91–3. 5 Myers LS, Morokoff PJ. Physiological and subjective sexual arousal in pre- and postmenopausal women taking replacement therapy. Psychophysiology 1986;23:283–92. 6 Semmens JP, Wagner G. Estrogens deprivation and vaginal function in postmenopausal women. JAMA 1982;248:445–8. 7 Bercovici B, Uretzki G, Palti Y. The effects of estrogens on cytology and vascularization of the vaginal epithelium in climacteric women. Am J Obstet Gynecol 1972;113:98– 103. 8 Abrams RM, Stanley H, Carter R, Notelovitz M. Effect of conjugated estrogens on vaginal blood ﬂow in surgically menopausal women. Am J Obstet Gynecol 1982;143:375–8. 9 Campbell S, Whitehead M. Oestrogens therapy and the menopausal syndrome. Clin Obstet Gynaecol 1977;4:31–47. 10 Notelovitz M. Urogenital aging: Solutions in clinical practice. Int J Gynaecol Obstet 1997;59(suppl 1):S35–S39. 11 The Society of Obstetricians and Gynaecologists of Canada. The detection and management of vaginal atrophy. Int J Gynaecol Obstet 2005;88:222–8. 12 Ibe C, Simon JA. Vulvovaginal atrophy: Current and future therapies. J Sex Med 2010;7:1042–50.

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Evaluation of the effects of a new intravaginal gel, containing purified bovine colostrum, on vaginal blood flow and vaginal atrophy in ovariectomized rat. - PDF Download Free (2025)