A aplicação da espectroscopia Raman confocal (ECR) para o interrogatório biofísico da pele humana in vivo foi lançada no final de 1990 por Caspers et al. (1998, 2000, 2001). A Espectroscopia Raman pode ser aplicada a amostras de qualquer formato (gases, líquidos, soluções, suspensões, sólidos).
Os espectros de Raman são ferramentas poderosas para a classificação e caracterização de células e tecidos, incluindo a quantificação da sua composição molecular . Uma vez que a técnica não é destrutivo , e não necessita de preparação de amostra , reagentes, corantes , etiquetas ou outros agentes de contraste de reforço , é perfeitamente adequado para a aplicação in vivo . Análise de dados em tempo real é possível, por meio de algoritmos de análise de dados sofisticados.
Esta técnica é aceita como o "padrão ouro" para a medição da espessura do estrato córneo (SC), in vivo. Recentemente, a caracterização da permeação in vivo de retinol, também foi relatada (PUDNEY et al., 2007).
As medições são realizadas utilizando um analisador de pele invertido (Skin Analyzer River Diagnostics CRS modelo 3510). O instrumento é equipado com dois lasers de diodo bombeado juntamente com fibras com dois diferentes comprimentos de onda: 671 nm e 785 nm. O laser de 671 nm, coleta dados na alta região de comprimento de onda alta (2500- 4000 cm-1) e o laser de 785 nm, corresponde à região denominada de impressão digital do espectro (400-2000 cm-1). (MELOT et al., 2009).
Os componentes básicos de um instrumento Raman são um laser, um compartimento de amostra, onde a luz laser é focada na amostra em que a luz dispersa e recolhida, e um espectrômetro de um detector para medir o espectro da luz dispersa para a análise de dados (CASPERS et al., 2001).
Diversas aplicações biológicas compostas de estudos de soluções altamente purificadas e concentradas de biomoléculas, tais como ácidos nucleicos e proteínas, foram iniciados na década de 60 e 70 do século passado. Naquele tempo que levou várias horas, com poderosos lasers de alta precisão para gravar um único espectro. Entretanto, a tecnologia tem chegado a um ponto em que as medições in vivo de amostras biológicas complexas, tais como células e tecidos pode ser levada a cabo em uma questão de segundos ou menos (CASPERS et al., 2000).
O compartimento da amostra de um microespectrômetro de Raman é um microscópio óptico (a objetiva do microscópio é utilizada para focar a luz de laser na amostra
e para recolher a luz dispersa a partir da mesma). Isto permite que os espectros de Raman sejam obtidos com a mesma resolução espacial sub-mícron, como é comum em microscópios ópticos. As fibras ópticas podem ser usadas para guiar a luz do laser para a amostra e para orientar o espectro Raman de volta para o espectrômetro. Deste modo, as sondas de fibra óptica Raman permitem o espectro de laser localizado de forma remota na amostra do tecido
in vivo (CASPERS et al., 2001).
A luz do laser é focada na pele através de uma objetiva de microscópio de imersão em óleo, a uma profundidade bem definida usando uma unidade alta precisão piezoelétrica. A luz difundida é focada no núcleo do tecido rejeitando sinais de fora das regiões de foco da pele. O sinal de espalhamento é detectado com uma sensibilidade elevada através de uma câmera CCD arrefecida com ar retro iluminado (CASPERS et al., 2008).
Até a concepção do desenho de estudo da presente tese, não existiam relatos na literatura avaliando a permeação de hormônios pela via transdérmica utilizando a ECR.
REFERÊNCIAS
AMIGONI, S.; MORELLI, P.; PARAZZINI, F.; CHATENOUD, L. Determinants of elevated blood pressure in women around menopause: results from a cross-sectional study in Italy. Maturitas, v. 34, n. 1, p. 25–32, 2000.
ANDERSON, G. L.; LIMACHER, M.; ASSAF, A. R.; BASSFORD, T.; BERESFORD, S. A.; PRETO, H. et al. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA, v. 291, n. 14, p. 1701–1712, 2004.
BEST, P. J.; BERGER, P. B.; MILLER, V. M.; LERMAN, A. The effect of estrogen replacement therapy on plasma nitric oxide and endothelin-1 levels in postmenopausal women. Ann. Intern. Med., v. 128, n. 4, p. 285–288, 1998.
BOTELHO, M. A.; QUEIROZ, D. B.; BARROS, G.; GUERREIRO, S.; FECHINE, P. et al. Nanostructured transdermal hormone replacement therapy for relieving menopausal symptoms: a confocal Raman spectroscopy study. Clinics (São Paulo), v. 69, n. 2, p. 75- 82, 2014a.
BOTELHO, M. A.; GUERREIRO, S. J.; QUEIROZ, D. B.; BARROS, G.; CAVALCANTE, M.; SOUZA, J. M. O. et al. Depth-scanning confocal Raman for rapid in vivo determination of testosterone concentration profiles in human skin. Medical Express, v. 1, n. 1, p 31-35, 2014b.
BOTELHO, M. A.; QUEIROZ, D. B.; FREITAS, A.; GUERREIRO, S.; UMBELINO, S.; BARROS, G. et al. Effects of a new testosterone transdermal delivery system, Biolipid B2®- testosterone in healthy middle aged men: a confocal raman spectroscopy study. J. Pharm. Sci. Innov., v. 2, p. 1-7, 2013.
BUSH, D. E.; JONES, C. E.; BASS, K. M.; WALTERS, G. K.; BRUZA, J. M.; OUYANG, P. Estrogen replacement reverses endothelial dysfunction in postmenopausal women. Am. J. Med., v. 104, n. 6, p. 552–558, 1998.
CACCIATORE, B.; PAAKKARI, I.; HASSELBLATT, R.; NIEMINEN, M. S.; TOIVONEN, J.; TIKKANEN, M. I. et al. Randomized comparison between orally and transdermally administered hormone replacement therapy regimens of long-term effects on 24-hour ambulatory blood pressure in postmenopausal women. Am. J. Obstet. Gynecol., v. 184, n. 5, p. 904–909, 2001.
CAGNACCI, A.; CANNOLETTA, M.; PALMA, F.; ZANIN, R.; XHOLLI, A.; VOLPE, A. Menopausal symptoms and risk factors for cardiovascular disease in postmenopause. Climacteric, v. 15, n. 2, p. 157–162, 2012.
CAGNACCI, A.; MODENA, M. G.; MALMUSI, S.; MUIA, N.; VOLPE, A. Effect of prolonged administration of transdermal estradiol on flow-mediated endothelium-dependent and endothelium-independent vasodilation in healthy postmenopausal women. Am. J. Cardiol., v. 84, n. 3, p. 367–370, 1999.
CAGNACCI, A.; ZANIN, R.; CANNOLETTA, M.; GENERALI, M.; CARETTO, S.; VOLPE, A. Menopause, estrogens, progestins, or their combination on body weight and anthropometric measures. Fertil. Steril., v. 88, n. 6, p. 1603–1608, 2007.
CASPERS, P. J., LUCASSEN, G. W.; WOLTHUIS, R.; BRUINING, H. A.; PUPPELS, G. J. In vitro and in vivo Raman spectroscopy of human skin. Biospectroscopy, v. 4, n. 5 Suppl., p. S31–S39, 1998.
CASPERS, P. J.; LUCASSEN, G. W.; BRUINING, H. A.; PUPPELS, G. J. Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin. J. Raman Spectrosc., v. 31, p. 813–818, 2000.
CASPERS, P. J.; LUCASSEN, G. W.; CARTER, E. A.; BRUINING, H. A.; PUPPELS, G. J. In vivoconfocal Raman microspectroscopy of the skin: non-invasive determination of molecular concentration profiles. J. Invest. Dermatol., v. 116, n. 3, p. 434–442, 2001.
CHAKRABARTI, S.; MORTON, J. S.; DAVIDGE, S. T. Mechanisms of estrogen effects on the endothelium: an overview. Can. J. Cardiol., v. 30, n. 7, p. 705–712, 2014.
CIFKOVA, R.; PITHA, J.; LEJSKOVA, M.; LANSKA, V.; ZECOVA, S. Blood pressure around the menopause: a population study. J. Hypertens., v. 26, n. 10, p. 1976–1982, 2008. CRUZ, M. N.; AGEWALL, S.; SCHENCK-GUSTAFSSON, K.; KUBLICKIENE, K. Acute dilatation to phytoestrogens and estrogen receptor subtypes expression in small arteries from women with coronary heart disease. Atherosclerosis, v. 196, n. 1, p. 49–58, 2008.
DAVIS, S. R.; VAN DER MOOREN, M. J.; VAN LUNSEN, R. H.; LOPES, P.; RIBOT, C.; REES, M.; MOUFAREGE, A.; RODENBERG, C.; BUCH, A.; PURDIE, D. W. Efficacy and safety of a testosterone patch for the treatment of hypoactive sexual desire disorder in surgically menopausal women: a randomized, placebo-controlled trial. Menopause, v. 13, n. 3, p. 387-396, 2006.
DUBEY, R. K.; OPARIL, S.; IMTHURN, B.; JACKSON, E. K. Sex hormones and hypertension. Cardiovasc. Res., v. 53, n. 3, p. 688–708, 2002.
FRANKLIN, S. S.; LARSON, M. G.; KHAN, S. A.; WONG, N. D.; LEIP, E. P.; KANNEL, W. B.; LEVY, D. Does the relation of blood pressure to coronary heart disease risk change with aging? The Framingham Heart Study. Circulation, v. 103, p. 1245–1249, 2001.
FUNG, M. M.; PODDAR, S.; BETTENCOURT, R.; JASSAL, S. K.; BARRETT-CONNOR, E. A cross-sectional and 10-year prospective study of postmenopausal estrogen therapy and blood pressure, renal function, and albuminuria: the Rancho Bernardo Study. Menopause, v. 18, n. 6, p. 629–637, 2011.
GERHARD, M.; WALSH, B. W.; TAWAKOL, A.; HALEY, E. A.; CREAGER, S. J.; SEELY, E. W.; GANZ, P.; CREAGER, M. A. Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women. Circulation, v. 98, n. 12, p.1158–1163, 1998.
GILLIGAN, D. M.; BADAR, D. M.; PANZA, J. A.; QUYYUMI, A. A.; CANNON, R. O. Acute vascular effects of estrogen in postmenopausal women. Circulation, v. 90, n. 2, p. 786–791, 1994.
GONZAGA, L. W.; BOTELHO, M. A.; QUEIROZ, D. B.; FECHINE, P. B.; FREIRE, R.; AZEVEDO, E. et al. Nanotechnology in Hormone Replacement Therapy: Safe and Efficacy of Transdermal Estriol and Estradiol Nanoparticles after 5 Years Follow-Up Study. Latin Am. J. Pharm., v. 3, n. 31, p. 442-450, 2012.
GUPTA, B. B. P.; LALCHHANDAMA, K. Molecular mechanisms of glucocorticoid action. Curr. Sci., v. 83, n. 9, p. 1103–1111, 2002.
HARVEY, P. J.; MOLLOY, D.; UPTON, J.; WING, L. M. Dose response effect of conjugated equine oestrogen on blood pressure in postmenopausal women with hypertension. Blood Press, v. 9, n. 5, p. 275–282, 2000.
KEARNEY, P. M.; WHELTON, M.; REYNOLDS, K.; MUNTNER, P.; WHELTON, P. K.; HE, J. Global burden of hypertension: analysis of worldwide data. Lancet, v. 365, n. 9455, p. 217–223, 2005.
LIEBERMAN, E. H.; GERHARD, M. D.; UEHATA, A.; WALSH, B. W.; SELWYN, A. P.; GANZ, P.; YEUNG, A. C.; CREAGER, M. A. Estrogen improves endothelium-dependent, flow-mediated vasodilation in postmenopausal women. Ann. Intern. Med., v. 121, n. 12, p. 936–941, 1994.
MANITOBA HEALTH. Routes for Drug Administration. Emergency Treatment Guidelines Appendix. 2003. Retrieved April 2, 2013.
MANWARING, P.; MORFIS, L.; DIAMOND, T.; HOWES, L. G. Effects of hormone replacement therapy on ambulatory blood pressure and vascular responses in normotensive women. Blood Press, v. 9, n. 1, p. 22–27, 2000.
MARCINKOWSKA, E.; WIEDŁOCHA, A. Steroid signal transduction activated at the cell membrane: from plants to animals. Acta Biochim. Pol., v. 43, n. 9, p. 735–745, 2002.
MÉLOT, P.; PUDNEY, P. D.; WILLIAMSON, A. M.; CASPERS, P. J.; VAN DER POL, A.; PUPPELS, G. J. Studying the effectiveness of penetration enhancers to deliver retinol through the stratum cornum by in vivo confocal Raman spectroscopy. J. Control. Release, v.138, p. 32–39, 2009.
MOORE, F. L.; EVANS, S. J. Steroid hormones use non-genomic mechanisms to control brain functions and behaviors: a review of evidence. Brain Behav. Evol., v. 51, n. 4, p. 41– 50, 1995.
MOREY, A. K.; RAZANDI, M.; PEDRAM, A.; HU, R. M.; PRINS, B. A.; LEVIN, E. R. Oestrogen and progesterone inhibit the stimulated production of endothelin-1. Biochem. J., v. 330, Pt. 3, p. 1097–1105, 1998.
MUECK, A. O.; SEEGER, H.; LÜDTKE, R.; GRÄSER, T.; WALLWIENER, D. Effect on biochemical vasoactive markers during postmenopausal hormone replacement therapy: estradiol vs estradiol/dienogest. Maturitas, v. 38, n. 3, p. 305–313, 2001.
NAHAR, L.; SARKER, S. D.; TURNER, A. B. A review on synthetic and natural steroid dimers: 1997-2006. Curr. Med. Chem., v. 14, n. 12, p. 1349–1370, 2007
ONEDA, B.; CARDOSO JR, C. G.; FORJAZ, C. L.; ARAUJO, T. G.; BERNARDO, F. R.; DE GUSMÃO, J. L. et al. Effects of estrogen therapy and aerobic training on sympathetic activity and hemodynamics in healthy postmenopausal women: a double-blind randomized trial. Menopause, v. 21, n. 4, p. 369–375, 2014.
PAAKARI, I.; HASSELBLATT, R.; NIEMINEN, M. S.; CACCIATORE, B.; TOIVONEN, J.; TIKKANEN, M. J.; YLIKORKALA, O. Comparison of the effect of transdermal and oral HRT on ambulatory blood pressure in postmenopausal women. Maturitas, v. 27, n. 1001, p. 98, 1997.
PINES, A.; FISMAN, E. Z.; DRORY, Y.; SHAPIRA, I.; AVERBUCH, M.; ECKSTEIN, N.; MOTRO, M.; LEVO, Y.; AYALON, D. The effects of sublingual estradiol on left ventricular function at rest and exercise in postmenopausal women: an echocardiographic assessment. Menopause, v. 5, n. 2, p. 79–85, 1998.
POTTS, P. O.; GUY, R. H. Predicting skin permeability. Pharm. Res., v. 9, p. 663–669, 1992.
PRESTON, R. A. Comparative effects of conventional vs novel hormone replacement therapy on blood pressure in postmenopausal women. Climacteric, v. 12, Suppl. 1, p. 66–70, 2009. PUDNEY, P. D. A. M. E.; MÉLOT, P. J.; CASPERS, A.; VAN DER POL, G. J. Puppels, In- vivo confocal Raman study of the delivery of the skin active retinol to the skin. Appl. Spectroscopy, v. 61, p. 804–811, 2007.
RECKELHOFF, J. F.; FORTEPIANI, L. A. Novel mechanisms responsible for postmenopausal hypertension. Hypertension, v. 43, n. 5, p. 918–923, 2004.
ROUSSEAU, G. G. Fifty years ago: the quest for steroid hormone receptors. Mol. Cell. Endocrinol., v. 375, p. ½, p. 10–13, 2013.
RYLANCE, P. B.; BRINCAT, M.; LAFFERTY, K.; De TRAFFORD, J. C.; BRINCAT, S.; PARSONS, V.; STUDD, J. W. Natural progesterone and antihypertensive action. Br. Med. J., v. 290, n. 6461, p. 13–14, 1985.
STAHL, S.M. Stahl's Essential Psychopharmacology: Neuroscientific basis and practical applications, New York: Cambridge University Press, 2008.
SMELTZER, S.C.; Bare, B.G. Textbook of Medical-Surgical Nursing, 9th ed, Philadelphia: Lippincott, 2000.
SCHUNKERT, H.; DANSER, A. H.; HENSE, H. W.; DERKX, F. H.; KURZINGER, S.; RIEGGER, G. A. Effects of estrogen replacement therapy on the renin–angiotensin system in postmenopausal women. Circulation, v. 95, n. 1, p. 39–45, 1997.
SCUTERI, A.; BOS, A. J.; BRANT, L. J.; TALBOT, L.; LAKATTA, E. G.; FLEG, J. L. Hormone replacement therapy and longitudinal changes in blood pressure in postmenopausal women. Ann. Intern. Med., v. 135, n. 4, p. 229–238, 2001.
SEELY, E. W.; WALSH, B. W.; GERHARD, M. D.; WILLIAMS, G. H. Estradiol with or without progesterone and ambulatory blood pressure in postmenopausal women. Hypertension, v. 33, n. 5, p. 1190–1194, 1999.
SHAH, S.; BELL, R. J.; SAVAGE, G.; GOLDSTAT, R.; PAPALIA, M. A.; KULKARNI, J.; DONATH, S.; DAVIS, S. R. Testosterone aromatization and cognition in women: a randomized, placebo-controlled trial. Menopause, v. 13, n. 4, p. 600-608, 2006.
STAESSEN, J.; BULPITT, C. J.; FAGARD, R.; LIJNEN, P.; AMERY, A. The influence of menopause on blood pressure. J. Hum. Hypertens., v. 3, n. 6, p. 427–433, 1989.
SUMINO, H.; ICHIKAWA, S.; KUMAKURA, H.; TAKAYAMA, Y.; KANDA, T.; SAKAMAKI, T.; KURABAYASHI, M. Effects of hormone replacement therapies on office and ambulatory blood pressure in Japanese hypertensive postmenopausal women. Hypertens. Res., v. 26, n. 5, p. 369–376, 2003.
TOTH, M. J.; TCHERNOF, A.; SITES, C. K.; POEHLMAN, E. T. Effect of menopausal status on body composition and abdominal fat distribution. Int. J. Obes. Relat. Metab. Disord., v. 24, n. 2, p. 226–231, 2000.
VESTERGAARD, P.; HERMANN, A. P.; STILGREN, L.; TOFTENG, C. L.; SORENSEN, O. H.; EIKEN, P.; NIELSEN, S. P.; MOSEKILDE, L. Effects of 5 years of hormonal replacement therapy on menopausal symptoms and blood pressure – a randomised controlled study. Maturitas, v. 46, n. 2, p. 123–132, 2003.
VIINIKKA, L.; ORPANA, A.; PUOLAKKA, J.; PYÖRÄLÄ, T.; YLIKORKALA, O. Different effects of oral and transdermal hormonal replacement on prostacyclin and thromboxane A2. Obstet. Gynecol., v. 89, n. 1, p. 104–107, 1997.
VOLTERRANI, M.; ROSANO, G.; COATS, A.; BEALE, C.; COLLINS, P. Estrogen acutely increases peripheral blood flow in postmenopausal women. Am. J. Med., v. 99, n. 2, p. 119– 122, 1995.
VONGPATANASIN, W.; TUNCEL, M.; MANSOUR, Y.; ARBIQUE, D.; VICTOR, R. G. Transdermal estrogen replacement therapy decreases sympathetic activity in postmenopausal women. Circulation, v. 103, n. 24, p. 2903–2908, 2001.
WASSMANN, K.; WASSMANN, S.; NICKENIG, G. Progesterone antagonizes the vasoprotective effect of estrogen on antioxidant enzyme expression and function. Circ. Res., v. 97, n. 10, p. 1046–1054, 2005.
WENNER, M. M.; STACHENFELD, N. S. Blood pressure and water regulation: understanding sex hormone effects within and between men and women. J. Physiol., v. 590, Pt. 23, p. 5949–5961, 2012.
WREN, B. G.; ROUTLEDGE, A. D. The effect of type and dose of oestrogen on the blood pressure of post-menopausal women. Maturitas, v. 5, n. 2, p. 135–142, 1983.
APÊNDICE A – ARTIGOS DERIVADOS DA TESE
ARTIGO SUBMETIDO AO INTERNATIONAL JOURNAL OF NANOMEDICINE
ORIGINAL RESEARCH
Short running header: Transdermal progesterone relieving menopausal symptoms Queiroz et al
TRANSDERMAL BIOIDENTICAL PROGESTERONE NANOPARTICLES RELIEVING MENOPAUSAL SYMPTOMS: A 5 YEARS FOLLOW-UP STUDY
Dinalva Brito QUEIROZ3, Pierre B. FECHINE2, Luiz Wagner GONZAGA3, Rafael FREIRE2, Lucindo QUINTANS JR1, Marco Antonio BOTELHO1,3*
1. Department of Physiology, Federal University of Sergipe, São Cristóvão-SE- BRAZIL.
2. Department of Analytical Chemistry, Federal University of Ceara Group of Advanced Biomaterial in Chemistry (GQMAT) Fortaleza – CE-BRAZIL.
3. Post graduation Program in Biotechnology, Laboratory of Nanotechnology, Potiguar University, Natal-RN-BRAZIL
Corresponding Author: Marco Antonio Botelho, MSc, PhD.
University Potiguar, School of Health, Av. Salgado Filho, 1610 Natal, Rio Grande do Norte, BRAZIL. Phone: ++ 55 84 3215.1234 / Fax: ++ 55 84 3215.1275 Coordinator of the Post Graduation Program in Biotechnology Email: [email protected]
Abstract:
Purpose: To determine the safety and efficacy of a bioidentical progesterone (10%) transdermal delivery system (lipophilic emulsion-type base) in restoring the FSH levels and relieving menopausal symptoms.
Patients and methods: A novel protocol on hormonal replacement was administered daily to 122 menopausal Brazilian women with climacteric symptoms during a period of 6o months to mimic the normal ovary secretory pattern. The transdermal bioidentical nanoemulsion was applied in the right forearm. Clinical parameters including weight, blood pressure, degree of satisfaction with symptomatic relief, serum concentrations of FSH and bilateral mammography BI-RADS were compared between the baseline and five years after treatment.
Results: Improvement in climacteric symptoms was reported in 95% of women evaluated before and after 60 months of treatment. Serum concentrations of follicle stimulating hormone (FSH) changed significantly (p<0,05) after treatment, the values of serum FSH decreased after 60 months from 79.29 ± 4.9 IU/mL to 59.27 ± 4.3 IU/mL. Bilateral mammography assessment of the breasts found normal results in all women. No adverse health-related events were attributed to this bioidentical hormone replacement therapy.
Conclusion: The nanostructured formulation is safe and effective in reestablishing FSH optimal serum levels and relieving the symptoms of the menopause.
Keywords: Nanoparticles, Transdermal delivery, Menopause, symptoms, hormone replacement therapy.
Introduction
There is a dilemma and a permanent debate about side effects on drugs administered orally. The first-pass metabolism is related with many side effects, since the metabolites of these compounds affect the course of human physiology.1-3
The menopause, marks the end of reproductive status, this condition is related with many symptoms such as hot flushes, insomnia, mood disturbances and vaginal dryness.3
The recent approaches on hormone therapy has been trying to mimic the normal ovary secretory pattern to minimize menopause symptoms, however, there is very few long term studies focused on Transdermal Bioidentical Hormone Therapy (TBHRT) as an alternative strategy for treating and controlling menopause symptomatology. 4-6
The stratum cornum is the least permeable skin layer. Thus, in order to improve the transdermal absorption of hormones, enhancement strategies have been developed, specially regarding to allow the controlled released system on bioidentical molecules through the skin.7
Progesterone can be given by several routes including oral, vaginal and intramuscular injection. The dosage of progesterone has been widely studied, for different ways of administration such as transdermal creams, intranasal solutions and vaginal gel. A recent long term study has provided strong evidences about the safety and efficacy of an enhancer capable of delivery nanoparticles of estriol and estradiol through the skin. 8,3
The technology of controlled release of drugs represents one of the frontiers on the medical science; it involves multidisciplinary aspects and may provide
important contribution to improve human health. The confocal Raman spectroscopy is the elected method to describe in real time important information about the drugs concentration in each layer of the skin.9,10
The Nanotechnology have been proving to become a potent and effective tool, bringing new perspectives in the medicine field such as transdermal absorption, since, recently new evidences suggests that this kind of drugs have interesting and unique properties.11-14
The present study was designed to determine the long term efficacy and safety of a nanostructured formulation of Progesterone (10%) for treating signs and symptoms related to menopause and evaluate its effects on clinical laboratorial and radiographic parameters after 5 years follow-up.
Material and methods
Ethics
Iinitially, a written informed consent was provided for individuals willing to participate in a protocol approved by the Institutional Review Board of the Paulista University, Brazil. Eligible patients were then assigned were then enrolled to the study.
This was a long-term clinical trial study of female patients aged 45-72 years old treated for menopause-related hormone imbalances from January 1, 2003 to April 30, 2008. The Paulista University Health Science Center at Sao Paulo Institutional Review Board reviewed and approved this study; protocol #533/2009. Other results of this study are published elsewhere.
Study Design
Volunteers were recruited from a referenced Gynecology Medical Center in Fortaleza City, the Capital of Ceara state, located in Northeast Brazil.
A total of 122 menopausal and post-menopausal women (aged 45-72 years) with climacteric symptoms were included in this study. “menopausal” was defined as amenorrhea that had persisted for ≥ 6 months. All patients had undergone natural menopause for at least 3 to 6 months and had not received TBHRT prior to study entry.
Inclusion criteria included patients with typical climacteric complaints such as hot flushes, sweating, insomnia, depressive disturbance, genital itching and dyspareunia.
Exclusion criteria included patients unwilling to complete the treatment protocol, subjects with any disease that would impede the use of the substances used in the study (e.g, mental diseases) as well as subjects that had used any type of HRT within six months prior to the study.
Clinical Evaluation
The TBHRT consultation consists of an educational lecture about TBHRT. The patients menopausal symptoms were classified as “absent=0," "mild=1," "moderate=2" or "severe=3". During initial evaluation and follow-up visits, it was used a standardized form to monitor symptoms resolution and any adverse effects.
The patients also were educated on several components of this kind of therapy such as: hormonal changes in menopause, associated factors, symptoms, risks and benefits of TBHRT dosage forms and the importance of the follow-up visits. Blood samples were collected from the subjects early in the morning after an overnight fast. To minimize variation, each subject sample was analyzed at the same time of the day.
After serum testing, the identification hormone deficiencies, was determined by the gynecologist, and then, if necessary, additional transdermal bioidentical progesterone was recommended. The patients were evaluated each 6 months after TBHRT initiation. All the patients were instructed about how to use the pump for transdermal application (performed in the presence of an experienced gynecologist) that performed all clinical examinations to guarantee standardization and correct use of the TBHRT. Compliance was defined as completing seventy percent or more of the transdermal applications. Satisfaction with the hormone therapy was also evaluated.
Compliance with the regimen was checked by personal interviews. Furthermore, serum concentrations of FSH, blood pressure, blood lipids, biochemical inflammatory markers were measured (data not shown).
During the 5 years of trial, Patients were instructed to complete evaluation forms each six months after the first consultation to monitor menopausal symptoms and side effects. In addition, demographic data including age, yearly family income and level of education was also assessed.
Transdermal Hormone Replacement Therapy
Patients received a transdermal dose on the right forearm a bioidentical nanoparticulated formulation of progesterone (10%); (Progesterone/ Biolipid B2®; Evidence Pharm. Group, Fortaleza,CE Brazil) given daily for 60 months.
The severity of the menopausal symptoms was evaluated according to the answers in the form full filled each six months of the study. Satisfaction with the hormone therapy was also evaluated at those times. Compliance with the regimen was checked by personal interviews. Furthermore, serum concentrations of FSH and blood pressure were measured.
During the 5 years of trial, Patients were instructed to complete evaluation forms each six months after the first consultation to monitor menopausal symptoms and side effects.
The subjects continued to use their regular non-supervised, self-performed transdermal bioidentical hormone measures. The clinical exam with the same parameters was repeated at 3 and after six months during five years.
Nanostructured Bioidentical Hormone Emulsion Preparation
The Nanostructured Bioidentical Hormones formulation was prepared at the Department of Nanotechnology, Institute of Applied Biotechnology (Patent Pending