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Evaluation of efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness A multicenter, randomized, open, positive and parallel controlled clinical trial Clinical Study Final Report Study centers: □ Peking University First Hospital □ Peking University Third Hospital □ Beijing Chao-Yang Hospital Attached to Capital Medical University □ Beijing Shijitan Hospital Sponsor: Zhaoke Pharmaceutical (Hefei) Co., Ltd Report completion: July 20, 2010 Hyaluronic acid vaginal gel (Hyalofemme) Clinical Study Final Report Study device: Hyaluronic acid vaginal gel (Hyalofemme) Study objective: to evaluate the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness Study type: clinical verification of medical device type III Study design: a multicenter, randomized, open, positive and parallel controlled clinical trial Product standard: standard for registration Inspection report: SFDA, Jinan Quality Supervision and Inspection Center for Medical Devices, Report no.: Y2008022105 Sponsor: Zhaoke Pharmaceutical (Hefei) Co., Ltd Study centers and investigators: Peking University First Hospital Peking University Third Hospital Beijing Chao-Yang Hospital Attached to Capital Medical University Beijing Shijintan Hospital Professor Professor Liao Qinping Geng Li Professor Professor Song Xuehong Li Hongxia Statistic Department: Institute of Basic Medical Sciences Chinese Academy of Medical Sciences Professor Han Shaomei Study principal investigator: Professor Study period: May, 2009-May, 2010 Liao Qinping TABLE OF CONTENTS ABBREVIATIONS.......................................................................................................4 Abstract of Hyaluronic acid vaginal gel (Hyalofemme) .......................................5 Clinical Study Final Report .......................................................................................5 Study General Information ..........................................................................................12 1 Clinical general information.................................................................................13 1.1 Inclusion criteria..........................................................................................15 1.2 Exclusion criteria.........................................................................................15 1.3 Withdrawal criteria.....................................................................................16 1.4 Elimination criteria .....................................................................................16 1.5 Withdrawal procedures .............................................................................16 2 Clinical trial method (including setup of control group if necessary) ...........17 2.1 Randomization ............................................................................................17 2.2 Study control................................................................................................17 2.3 Study products ............................................................................................17 2.4 Packaging of study products.....................................................................18 2.5 Dispensation of study products................................................................18 2.6 Concomitant therapy..................................................................................19 2.7 Study procedures ........................................................................................19 2.8 Safety evaluation.........................................................................................21 2.9 Statistical analysis .......................................................................................22 3 Statistical method and evaluation method.........................................................22 4 Clinical evaluation standards...............................................................................23 4.1 Efficacy evaluation......................................................................................23 4.2 Safety evaluation.........................................................................................24 5 Clinical trial results ................................................................................................24 5.1 Allocation, dropout and elimination of subjects ....................................24 5.2 Baseline information and comparability analysis ..................................26 5.3 Analysis of concomitant medications ......................................................28 5.4 Efficacy analysis ..........................................................................................30 5.5 Safety analysis .............................................................................................35 5.4 Endometrial thickness ................................................................................40 6 Adverse events and side effects and the treatment during the clinical trial .41 7 Analysis of clinical trial results ............................................................................44 8 Conclusion of the clinical trial..............................................................................47 9 Indications, applicable range, contraindication and attentions ......................47 10 References .............................................................................................................48 11 Participants of clinical trial .....................................................................................49 12 Declaration and signatures of investigators.....................................................50 13 Opinions of clinical trial management department of medical institution .51 ABBREVIATIONS AE Adverse Event ADR Adverse Drug Reacion BP Blood Pressure VSH Vagina Health Score CRF Case Report Form EC Ethics Committee GCP Good Clinical Practice ICH International Conference on Harmonization SAE Serious Adverse Event SFDA State Food and Drug Administration SOP Standard Operating Procedure FAS Full Analysis Set PPS Per Protocol Set SAS Safety Analysis Set Abstract of Hyaluronic acid vaginal gel (Hyalofemme) Clinical Study Final Report Sponsor Zhaoke Pharmaceutical (Hefei) Co., Ltd Clinical Zhaoke Pharmaceutical (Hefei) Co., Ltd Monitoring Evaluation of efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness Study title A multicenter, randomized, open, positive and parallel controlled clinical trial Study product Hyaluronic acid vaginal gel(Hyalofemme) Inspection SFDA, Jinan Quality Supervision and Inspection Center for Medical certificate Devices, Report no.: Y2008022105 Clinical Peking University First Hospital, Obstetrics-Gynecology study Liao Qinping, director, professor responsible center and Principal investigator Starting time May, 2009 Completion May, 2010 time Report July, 2010 completion time Study To evaluate the efficacy and safety of Hyaluronic acid vaginal gel objective (Hyalofemme) for improvement of vaginal dryness Study method The study is a multicenter, randomized, open, positive and parallel controlled study, evaluating the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness. According to the protocol design, all subjects will be randomized into test group and control group. Each subject will be treated continuously by the device for 10 times, once every 3 days and the period will last for about one month (30 days). The investigator has responsibility for observation and evaluation of the efficacy and safety of the study device throughout the course and after finishing. If any serious adverse event related to test group occurs during the trial, it should be discontinued and the investigator should discuss the relationship between the event and test product. If the adverse event is closely related to the test product, the trial will discontinue, otherwise it will go on. The investigator will judge the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness after that. Sample size 144 cases, test group:72 cases, control group: 72 cases Study The vaginal mucosa tunica consists of a non cornified, stratified squamous epithelium rich in glycogen and of basal lamina rich in elastic fibers. The characteristics of the vaginal epithelium change depending on the variations in the concentration of estrogenic and progestational hormones in which women undergo during their life span. During the fertile period it is stratified squamous epithelium and rich in glycogen, moistening and elastic, while during menopause, due to a decrease in estrogenic hormones, the epithelium atrophies. The cells residing in the intermediate levels of the stratified squamous epithelium are mainly involved in this process thus leading to thinning of the vaginal mucosa. The number of stratification diminishes and only basal and parabasal cells, which however are dystrophic and flake easily leaving the chorion unprotected, remain. The vagina mucosa in these conditions is more susceptible directly to germs and is more permeable to their toxic metabolic products. As a consequence, it is not unusual to observe small lacerations that may easily become infected. background The vagina is soft and elastic during the fertile period due to the balance mechanism of the vaginal epithelium that maintains the appropriate hydration and lubrication. During some situations, such as post partum, lactation and menopause, oral contraceptives or stress, the balance may be disturbed. In all of these situations, signs of vaginal dryness, burning sensation or itching may be observed. Because of the insufficient vaginal lubrication, the epithelium is injured thus leading to symptoms of vaginal dryness, burning sensation, dyspareunia and itching. The physiological lubricating film that characterizes the fertile period of a woman’s life plays an important role in maintaining the integrity of the vaginal mucosa. This film is no longer present during menopause thus leading to fragility of the vagina mucosa. The therapeutic resolutions in these situations are represented by topically applied products that produce a hydrating effect to the vagina mucosa and help to regain elasticity and softness. Hyaluronic acid vaginal gel (Hyalofemme) is a colorless gel with high hydrating properties. The gel contains Hydeal-D®, a derivative of hyaluronic acid, which maintains the biocompatibility and interactivity of hyaluronic acid, and also adequate hydration of mucosa. Topical administration of the gel has a good adhesiveness to the vaginal mucosa and long acting hydration thus improving the dry state of the vagina. It also improves the spontaneous recovery of small lesions that caused by friction of vagina. Hyaluronic acid vaginal gel (Hyalofemme), a colorless gel with high hydrating properties, is manufactured by Fidia Advanced Biopolymers, srl, Italy and has been marketed in Italy for years. Hyaluronic acid is a natural molecule that can maintain a right level of hydration and plays an important role in tissue repair. The product has no pharmacological effect for it is not a drug but a type Ⅲ medical device. The gel’s hydrating properties are attributed to the characteristics of the Hydeal-D® which releases water molecules to the tissue, thus improving the dry state of the vagina. Therefore, the gel is applicable to supplementary treatment of 【 】 vaginal dryness symptoms induced by any reason 1 . The vaginal ecosystem is composed of a complex microflora, constituted of different microorganisms. The presence of these microorganisms is subject to qualitative and quantitative modifications some of which can result in pathological conditions. Thus it is of utmost importance to understand the influence of Hyalofemme gel in vaginal ecosystem. However, due to the extreme complexity of the factors which influence this ecosystem, there is no single theory capable of explaining the mechanisms which govern the colonization and the homeostasis of this organ. The hormone dependent dynamic nature of the vaginal physiology has been demonstrated. The hormonal state of the subject determines directly or indirectly the qualitative and quantitative variations of the vaginal microbial components, mainly influencing the lactobacilli. This microorganism is responsible for the vaginal homeostasis by: ·producing lactic acid which maintains an acidic vaginal ambient, ·competing for bacterial adherence, ·producing hydrogen peroxide, ·producing bacteriocins, ·stimulating the local immune system. For these reasons the vaginal homeostasis is assessed through vagina PH and vaginal smears (variations of the vaginal microbial components), so as to evaluate the influence of Hyalofemme gel on vaginal ecosystem and the safety. Major z Primary efficacy endpoint: percentage of improvement of vaginal dryness symptoms evaluation criteria z Secondary efficacy endpoint: percentage of improvement of itching, dyspareunia and burning sensation z Safety evaluation: vital signs, laboratory examinations of vaginal microecosystem, vaginal PH value, vaginal B ultrasound and incidence of adverse event. Statistical Institute of Basic Medical Sciences Chinese Academy of Medical Sciences method undertook the overall process from statistical analysis and data management to analysis summary. Statistical analysis report was provided after completion of data analysis. Interim analysis was not done in the study. Statistical analysis: use SAS 9.1 statistic analysis software for calculation. All statistical tests used double-sided tests, and the tested difference was considered of statistical significance when P-value≤0.05. Measurement data of each visit in the two groups would apply sample size, mean, standard deviation, median, maximum and minimum for statistical description. Comparison of differences before and after within group would be carried out with paired t-test. Changes of the two groups prior and post treatment would use t-test and Wilcoxon rank-sum test for comparison between groups. Numeration data of each visit in different groups would use frequency (constituent ratio) for descriptive statistics. Comparison between two groups would be carried out with χ2 test or Fisher’s exact test. Main contents of statistical analysis: ·Efficacy analysis: The rate of disappearance between two groups used χ2 test. Multicenter effect analysis used Cochran-Mantel-Haenszel χ2 test. The differences of vagina health score between two groups were compared by Wilcoxon rank-sum test. ·Safety analysis Comparison for incidence of adverse events between two groups used χ2 test and all adverse events occur in the study were described in a table. Normal/abnormal changes of laboratory examination results before and after study and relationship to the study products. Results and conclusions The study was a multicenter, randomized, open, positive and parallel controlled clinical trial, to evaluate the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness. The results showed that applying Hyaluronic acid vaginal gel and Estriol cream in test group and control group according to the protocol, could remarkably improve the clinical symptoms of vaginal dryness. The final (after administration for 10 times) improvement rate were 84.44% and 89.42%, respectively, without significant statistical differences between them (P>0.05). Mean of rate difference was -3.33% and 95%CI was [-9.40, 2.74], the lower limit of which did not exceed the boundary value of non-inferiority evaluation (15%). Thus we could think that the efficacy of the Hyaluronic acid vaginal gel is not inferior to that of Estriol cream, with no significant difference between them. At the first visit, the improvement rate of vaginal dryness in Hyaluronic acid vaginal gel group and Estriol cream group were 49.17% and 53.53%, and had no statistical significant differences. That meant the two products had a short onset time and they could release the clinical symptoms of vaginal dryness after three times of administration of the products. That the improvement rate on first visit was lower than that on final visit showed the continuous release effect on the symptoms as time of medication increased. The final improvement rate of the secondary efficacy endpoints (itching, dyspareunia and burning sensation) in Hyaluronic acid vaginal gel group and Estriol cream group were 86.23% and 81.97%, 56.96% and 62.33%, 85.83% and 87.87%, respectively. The differences between the two groups had no statistical significance (P>0.05), and FAS results were in line with PPS. See Table 4.1.1 ~ Table 4.2.3. Totally 13 adverse events occurred throughout the clinical study, seven in the test group (incidence: 9.7%, 7/72) and six in the control group (incidence: 8.3%, 6/72). The differences between the two groups had no statistical significance (P>0.05). No serious adverse event occurred, and the AE that were related to the test product or suspicious were in mild level. See Table 5.1 and Table 6. No changes of vaginal micro-ecological environment appeared after treatment in 80.6% (54/67) of subjects in Hyaluronic acid vaginal gel group, while 77.27% (51/66) in Estriol cream group. It indicated that applying Hyaluronic acid vaginal gel would not alter the internal environment of vagina and it was able to maintain the homeostasis, with a high safety. See Table 5.2. Mean of vaginal PH value in Hyaluronic acid vaginal gel group was 5.63 prior to treatment and 5.30 after treatment, with no statistical difference. Vaginal PH value after treatment (5.30) in Hyaluronic acid vaginal gel was higher than that in Estriol cream group and the difference between them had statistical significance (P<0.05). It suggested that the hyaluronic acid vaginal gel was not as good as the estriol cream on reducing vaginal PH value. It was mainly because the estrin in the estriol cream could help the growth of lactobacilli in vagina, promoting lactose broken down into lactic acid, to reduce PH value. While the hyaluronic acid vaginal gel had no pharmacological effect. Its biological effect was acted through Hydeal-D®, the derivative of hyaluronic acid, without mechanism to reduce PH value. See Table 5.3. The endometrial thickness prior to treatment in test group and control group were 3.19mm and 3.21mm, respectively. While 3.43mm and 3.52mm after treatment. The difference between the two group had no statistical difference (P>0.05). However, the endometrial thickness of the No. 003 subject (applying estriol cream) in Peking University First Hospital increased from 3mm to 9mm. On follow-up after drug withdrawal, the endometrial thickness of the subject reduced gradually and the symptoms were relieved. It indicated that estriol cream had influence on the endometrial thickness. The doctors should apply it strictly in accordance with the indications. Because of its narrow applicability, the doctors should be cautious when using it. The hyaluronic acid vaginal gel did not have hormonelike effect, thus it had no influence on endometrium or hormones-endocrine system and had a high safety. See Table 5.4. Hyaluronic acid vaginal gel (Hyalofemme), a colorless gel with high hydrating properties, is manufactured by Fidia Advanced Biopolymers, srl, Italy and has been marketed in numerous foreign countries for years. Hyaluronic acid is a natural molecule that can maintain a right level of hydration and plays an important role in tissue repair. The product has no pharmacological effect for it is not a drug but a type Ⅲ medical device. The gel’s hydrating properties are attributed to the characteristics of the hyaluronic acid based biopolymer which releases water molecules to the tissue, thus improving the dry state of the vagina without any irritation of vaginal mucosa. Therefore, the gel is applicable to supplementary treatment of vaginal dryness symptoms induced by any reason. Compared with estriol cream, hyaluronic acid vaginal gel has the characteristics of wider applicable range, no hormonelike effect, higher safety and better acceptability of patients. The clinical trial showed that the hyaluronic acid vaginal gel played a part in repair of vaginal epithelial cells. It could improve the epithelium condition, making the subjects feel better after application. The mechanism need further study. Though the results would be inevitably influenced by the investigator because of the open-label trial, they still had reference value. The certain evaluation of the efficacy and safety of hyaluronic acid vaginal gel depended on further blind trial. To sum up, the hydration and adhesion of hyaluronic acid vaginal gel can improve vaginal dryness throughout the spontaneous recovery of small lesions that caused by friction of vagina. Therefore, the gel is applicable to supplementary treatment of vaginal dryness symptoms induced by any reason. Study General Information Clinical study responsible center: Peking University First Hospital Address: No.8, Xishiku Avenue, Xicheng District, Postcode: 100034 Beijing Email: [email protected] Investigator: Professor Liao Qinping Tel.:010-85299246 Study center: Peking University Third Hospital Address: No.49, Huayuanbei Road, Haidian District, Postcode: 100083 Beijing Email: [email protected] Investigator: Professor Geng Li Tel.: 010-62017691 Study center: Beijing Chao-Yang Hospital Attached to Capital Medical University Address: No.8, Baijiazhuang, Chaoyang District, Postcode: 100020 Beijing Email: [email protected] Investigator: Professor Song Xuehong Tel.:010-85231715 Study center: Beijing Shijitan Hospital Address: No.10, Yangfangdian Tie Hospital Road, Postcode: 100038 Haidian District, Beijing Email:[email protected] Investigator: Professor Li Hongxia Tel.: 010-63926375 Statistic Department: Institute of Basic Medical Sciences Chinese Academy of Medical Sciences Address: No.1, Wangfujing Shuaifuyuan, Dongcheng Postcode: 100730 District, Beijing Email: [email protected] Principal: Professor Han Shaomei Tel.:010-65296408 Report for serious adverse events Department: Ethics Committee of Peking University First Hospital Contact person: Zhao Xia Address:No.6,Dahongluochang Street, Xicheng District, Beijing Postcode: 200025 Email: Tel.:010-66119025 Sponsor: Zhaoke Pharmaceutical (Hefei) Co., Ltd. Address: New and High-tech Industrial Development Zone, No.669, Changjiangxi Road, Hefei, Anhui Province Project leader: Jiang Su 010-65262690-801/13901063831 Monitor: Jiang Su, Yu Jinhuan Email: [email protected] Tel.: 010-65262690-804/ 13911909335 Fax: 010-65262290 Hyaluronic acid vaginal gel (Hyalofemme) Clinical Study Final Report 1 Clinical general information The vaginal mucosa tunica consists of a non cornified, stratified squamous epithelium rich in glycogen and of basal lamina rich in elastic fibers. The characteristics of the vaginal epithelium change depending on the variations in the concentration of estrogenic and progestational hormones in which women undergo during their life span. During the fertile period it is stratified squamous epithelium and rich in glycogen, moistening and elastic, while during menopause, due to a decrease in estrogenic hormones, the epithelium atrophies. The cells residing in the intermediate levels of the stratified squamous epithelium are mainly involved in this process thus leading to thinning of the vaginal mucosa. The number of stratification diminishes and only basal and parabasal cells, which however are dystrophic and flake easily leaving the chorion unprotected, remain. The vagina mucosa in these conditions is more susceptible directly to germs and is more permeable to their toxic metabolic products. As a consequence, it is not unusual to observe small lacerations that may easily become infected. The vagina is soft and elastic during the fertile period due to the balance mechanism of the vaginal epithelium that maintains the appropriate hydration and lubrication. During some situations, such as post partum, lactation and menopause, oral contraceptives or stress, the balance may be disturbed. In all of these situations, signs of vaginal dryness, burning sensation or itching may be observed. Because of the insufficient vaginal lubrication, the epithelium is injured thus leading to symptoms of vaginal dryness, burning sensation, dyspareunia and itching. The physiological lubricating film that characterizes the fertile period of a woman’s life plays an important role in maintaining the integrity of the vaginal mucosa. This film is no longer present during menopause thus leading to fragility of the vagina mucosa. The therapeutic resolutions in these situations are represented by topically applied products that produce a hydrating effect to the vagina mucosa and help to regain elasticity and softness. Hyaluronic acid vaginal gel (Hyalofemme) is a colorless gel with high hydrating properties. The gel contains Hydeal-D®, a derivative of hyaluronic acid, which maintains the biocompatibility and interactivity of hyaluronic acid, and also adequate hydration of mucosa. Topical administration of the gel has a good adhesiveness to the vaginal mucosa and long acting hydration thus improving the dry state of the vagina. It also improves the spontaneous recovery of small lesions that caused by friction of vagina. Hyaluronic acid vaginal gel (Hyalofemme), a colorless gel with high hydrating properties, is manufactured by Fidia Advanced Biopolymers, srl, Italy and has been marketed in Italy for years. Hyaluronic acid is a natural molecule that can maintain a right level of hydration and plays an important role in tissue repair. The product has no pharmacological effect for it is not a drug but a type Ⅲ medical device. The gel’s hydrating properties are attributed to the characteristics of the Hydeal-D® which releases water molecules to the tissue, thus improving the dry state of the vagina. Therefore, the gel is applicable to supplementary treatment of vaginal dryness symptoms induced by any reason【1】. The vaginal ecosystem is composed of a complex microflora, constituted of different microorganisms. The presence of these microorganisms is subject to qualitative and quantitative modifications some of which can result in pathological conditions. Thus it is of utmost importance to understand the influence of Hyalofemme gel in vaginal ecosystem. However, due to the extreme complexity of the factors which influence this ecosystem, there is no single theory capable of explaining the mechanisms which govern the colonization and the homeostasis of this organ【1】. The hormone dependent dynamic nature of the vaginal physiology has been demonstrated. The hormonal state of the subject determines directly or indirectly the qualitative and quantitative variations of the vaginal microbial components, mainly influencing the lactobacilli. This microorganism is responsible for the vaginal homeostasis by: ·producing lactic acid which maintains an acidic vaginal ambient, ·competing for bacterial adherence, ·producing hydrogen peroxide, ·producing bacteriocins, ·stimulating the local immune system. For these reasons the vaginal homeostasis is assessed through vagina PH and vaginal smears (variations of the vaginal microbial components), so as to evaluate the influence of Hyalofemme gel on vaginal ecosystem and the safety. 144 subjects were enrolled in the clinical study to evaluate the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness. Peking University First Hospital: 36 subjects, Peking University Third Hospital: 40 subjects, Beijing Chao-Yang Hospital Attached to Capital Medical University: 32 subjects and Beijing Shijitan Hospital: 36 subjects. Selection criteria of subjects: 1.1 Inclusion criteria ⑴ Women under 70 years old, with natural menopause or surgery-induced menopause for more than 6 months; ⑵ Patients with vaginal dryness symptoms induced by various reasons; ⑶ Patients without contraindications of local application with estrogen; ⑷ Patients with good compliance who are voluntary to written informed consent. 1.2 Exclusion criteria ⑴ Unmarried women; ⑵ Women during pregnancy and lactation; ⑶ Patients who are allergic to hyaluronic acid gel; ⑷ Patients using other topically applied vaginal products within one week ⑸ Patients with vaginal infections such as trichomonas, candida infection and bacterial vaginosis; ⑹ Patients with breast cancer, uterine cancer and estrogen hormone dependent tumors, genital bleeding of unknown origin; and patients with acute hepatopathy, embolic disorders, severe primary disease of kidney and hematopoietic system and malignant tumor recently; ⑺ Patients using estrogens within one month; ⑻ Patients who are attending or have attended other clinical trials within 2 weeks; ⑼ Patients with mental disorder and no insight, who are unable to express themselves exactly; ⑽ Patients who do not give informed consent; ⑾ Patients who is not suitable to participate in the clinical trial for it may aggravate patients’ condition in the investigator’s opinion. 1.3 Withdrawal criteria ⑴ All subjects, meeting inclusion criteria and signing informed consent forms, who have been randomized and received study products, but do not complete observation period as the protocol stated; ⑵ Protocol violation; ⑶ Patient who is unwilling to continue the study and requests to drop out ⑷ Patient with poor compliance and unable to adhere to treatment though he/she does not request to drop out; ⑸ A severe infection or allergic response occurs thus not able to continue application of the study product; ⑹ A serious adverse event or a serious adverse reaction occurs thus the investigator believes that drop-out is best for the subject; Patient, who drops out because of adverse event, should be recorded and the investigator should inform the monitor; ⑺ Severe complications occurs to the patient during the study, unsuitable to continue; ⑻ Patient’s condition gets worse and needs emergency treatments. 1.4 Elimination criteria ⑴ Patients do not meet selection of study population, including inclusion criteria and exclusion criteria. ⑵ Patients using other medications that can promote lesion healing ⑶ Patients, who have been randomized, haven’t received any treatment. 1.5 Withdrawal procedures Patient can drop out of the study freely at any time, without any influence on the following treatment and follow-up visit. The investigator should ask the patient about the drop-out reason and whether any AE occurs. If possible, the last follow-up visit should be completed. The patient should receive the laboratory examinations and the investigator should complete AE record and follow up the adverse event. 2 Clinical trial method (including setup of control group if necessary) There were no similar products that treat vaginal dryness in China, thus according to suggestion of the investigators (choosing the product with definite efficacy verified to sell in China’s market by SFDA), estriol cream (Ovestin) was used as control article. Because the packages, size and dosage of hyaluronic acid vaginal gel and Ovestin were not identical, blinding method was not suitable and the study would be a multicenter, randomized, open, positive and parallel controlled study. Subjects received hyaluronic acid vaginal gel(Hyalofemme)or Ovestin for evaluation of efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness【2】. 144 patients with symptoms of vaginal dryness were anticipated to be included in the study. There were four study centers, 36 cases in each center while the case number could be regulated on the basis of enrolling conditions. Using hyaluronic acid vaginal gel(Hyalofemme)or Ovestin should be randomized. 2.1 Randomization: the trial used segmented balance randomization. Subjects were randomized to test group and control group in a ratio of 1:1. The random allocation form was produced through SAS program by the staff of Institute of Basic Medical Sciences Chinese Academy of Medical Sciences and at the same time the sealed random envelopes of corresponding sequence 【 】 numbers were created 4 . 2.2 Study control: the trial applied positive control, Ovestin as control group. 2.3 Study products: provided by Zhaoke Pharmaceutical (Hefei) Co., Ltd. 2.3.1 Test group: Name: Hyaluronic acid vaginal gel (Hyalofemme) Size: 30g/aluminum tube Product standard: registered standard Manufacture batch no.:017000 Expiry date: to June, 2010 Manufacturer: Italy FIDIA FARMACEUTICI S.P.A. Instructions 1. Revolve the vaginal applicator into the tube. 2. Squeeze the tube to fill the applicator to half of it (about 5g). 3. Release the applicator from the tube and plug it in patient’s vagina. The patient should keep crouching or lying position. 4. Impel the applicator until all of the gel is discharged. 5. Clean the applicator after administration. Wash out the remaining gel with water and disinfectants, then rinsing it thoroughly. Finally remove the water from the applicator. 6. Put the applicator into the box for use next time. 7. In case of severe dryness, it is suggested that eject a little gel so as to make enough lubricating before plugging the applicator into vagina. Application frequency Application frequency depends on the dry state of the vaginal mucosa. Patients in the study applied the gel every 3 days, totally 10 times continuously except other prescription. 2.3.2 Control group: Name: Estriol cream (Ovestin cream) Size: 15mg/15g/vial Product standard: registered standard Manufacture batch no.:661514 Expiry date: to August, 2011 Manufacturer: provided by Zhaoke Pharmaceutical (Hefei) Company Limited and manufactured by Organon (Ireland) Ltd. Patients in the study applied 0.5g of the cream every 3 days, totally 10 times continuously except other prescription. 2.4 Packaging of study products Test products and control products were packaged respectively in large white boxes. On the boxes should be marked the name of the products, quantity, usage, site number, storage condition, expiry date, sponsor and ‘For Clinical Verification’. 2.5 Dispensation of study products Registration: All products of each site were stored in one place and managed by a specially-assigned person. The dispensation and reclaiming should be registered. Sequence: Random envelope numbers of Peking University First Hospital were 001-036, Peking University Third Hospital 037-072 and Beijing Chaoyang Hospital Attached to Capital Medical Hospital 073-108, Beijing Shijitan Hospital 109-144. 145-192 were reserved random envelope numbers. Dispensation: The investigator opened the corresponding random envelopes from small number to large number according to the sequence of each patient’s visit, confirming the subject received hyaluronic acid vaginal gel (Hyalofemme) or Ovestin cream on the basis of the content. The random envelope number was the unique number of the subject. The product should be given to each subject according to her sequence and random envelope content, and should not be chosen. The subject serial number remained unchanged during the study. The investigator should fill in the Drug Dispensation Form exactly in time and write down related records on the envelope (signature and date). If the patient lost the product, she could get new product from the investigator. The investigator should check the product to ensure it was the same as that of patient’s random number. And should record the reason, quantity and date, then sign his name in the dispensation form. Storage: The study products were conducted unified preservation, management and distribution by each center. The products should be stored in the shade place. Remaining Products: The products remaining in case of patients not using them on time, dropping out midway and not using up or the treatment protocol changing, the products should be reclaimed and recorded in detail. 2.6 Concomitant therapy In order to eliminate any affect of other drugs, all subjects were not allowed to apply other drugs or products that may improve the dry state of vagina, or Chinese patent medicines and drugs for external use with similar efficiency. Subject, who met inclusion criteria but need continued medication for other diseases during the study or addition of other drugs or therapies, should be recorded on the CRF (name of the drugs or therapies, dosage, application frequency and time, etc.) 2.7 Study procedures 144 patients with vaginal dryness were anticipated to be treated in the study. 2.7.1 Screening: patients, who met inclusion criteria but did not accord with exclusion criteria, become subjects after they gave informed consent. Then they were randomized into treatment group according to the random envelope. 2.7.2 Treatment period Day 0: after the selection of qualified patient, the investigator recorded subject’s demographic information and medical history. Physical examinations should be done and subject’s evaluation of vaginal dryness and other symptoms (itching, dyspareunia and burning) by visual analogue scale (VAS) should be recorded. The vaginal microecosystem laboratory examinations were evaluated by taking a vaginal smear (the method is attached below) and measuring the PH (measuring method is attached below), and the endometrial thickness was measured by vaginal B ultrasound. The administration of test group and control group were identical. The investigator dispensed the study products and filled in the dispensation form. 2.7.3 First visit (visit by phone): the investigator should telephone the subjects between the 3rd and the 4th administration, and should record the evaluation of vaginal dryness and other symptoms. 2.7.4 Final visit: the subjects should come to the study centers for the final visit on the 3±1st day after the 10th administration. The investigator should ask the subjects to have physical examinations (weight, breath, heart rate, pulse and blood pressure), vaginal microecosystem laboratory examinations, PH value and endometrial thickness measured by vaginal B ultrasound. Subjects should give the evaluation of their symptoms. Measuring method of vaginal PH value: The PH was measured using strips of “accurate PH indicators〞(produced by Merck &Co., Inc., Germany), the range of PH was from 2.0 to 9.0, with intervals of 0.5 units. It should be used within expiry date and stored in dry and dark place. The patient took lithotomy position and the investigator got some secretions from the 1/3 part of vaginal lateral wall after having exposed the zone with one-off vaginal dilator. The PH value was obtained by confronting the color of the strip, after the secretions touching it for 5 seconds, with the given color scale. The vaginal smear: The vaginal smear was taken from the posterior fornix of the vagina. The microscope slide was then fixed with heat and colored using the Gram method and then observed with an optimal microscope. The following observations were made: intensity and variety of vaginal bacterial flora and predominant bacteria. Throughout the study the investigator should record adverse events and concomitant therapy. Study schedule Items Periods Screening First visit Final visit Treatment (by phone) (on-site) Day 0 between the 3rd and the 3±1st day after the the Informed consent × Inclusion/Exclusion Criteria × 4th administration 10th administration Demographic information and × medical history Physical examinations × *Evaluation of vaginal symptoms × Vaginal PH value × × × × Endometrial thickness × × Dispensation of study products × × × × * Vaginal microecosystem laboratory examinations Reclaiming of study products × Concomitant therapy × × × Adverse reaction/event × × × *The disappearance rate of vaginal symptoms included that of dryness, itching, dyspareunia and burning. * Vaginal microecosystem laboratory examinations included intensity and variety of vaginal bacterial flora and predominant bacteria. 2.8 Safety evaluation 2.8.1 Physical examinations Routine examinations of weight, temperature, breath, blood pressure and pulse. 2.8.2 Vaginal microecosystem laboratory examinations To evaluate whether the vaginal bacterial flora was normal by the results of intensity and variety of vaginal bacterial flora and predominant bacteria. Standard of vaginal microecosystem laboratory examinations: The intensity grading standard of vaginal bacterial flora includes 4 grades, with oil immersion lens (10×100 amplification). Grade 1 (marked ‘+’): average bacterial number 1-9 per visual field, about 105~6/ml bacteria in the sample; grade 2 (++): average bacterial number 10-99 per visual field, about 107~8/ml bacteria in the sample; grade 3 (+++): average bacterial number, more than 100, the visual field is full of bacteria, about 109~10/ml bacteria in the sample; grade 4 (++++): the bacteria aggregate or cover the epithelium, more than 1010/ml bacteria in the sample. The variety of vaginal bacterial flora includes 4 grades: grade 1(marked ‘+’): 1-3 kinds of bacteria are distinguished; grade 2(++): 4-6 kinds of bacteria are distinguished; grade 3 (+++):7-10 kinds of bacteria are distinguished; grade 4 (++++): more than 11 kinds of bacteria are distinguished. Definition of normal value of vaginal microecosystem laboratory examinations: intensity (grade 2-3), variety (grade 2-3) and the predominant bacteria (Large Gram positive bacillus, G+b(L)). 2.8.3 Vaginal PH value 2.8.4 Examination of endometrial thickness 2.8.5 Incidence of adverse events 2.9 Statistical analysis: The statistical analysis applied SAS 9.1 statistic analysis software for calculation. All statistical tests used double-sided tests, and the tested difference was considered of statistical significance when P-value≤0.05. 3 Statistical method and evaluation method 3.1 Selection of statistical analysis data 3.1.1 Full Analysis Set (FAS) According to ITT, eliminate the subject with the minimal and reasonable method. This data set includes all patients except who never used the study products or who had no efficacy data. The missing data of primary endpoint should apply last observation carry forward (LOCF). 3.1.2 Per-Protocol Set (PPS) Composed of all patients who conformed to the protocol and the following terms: ·patients with compliance of 80%-120% ·patients do not use the forbidden medication during the study ·patients who match all inclusion criteria with none of exclusion criteria ·patients in whom there are serious violations to the protocol. 3.1.3 Safety Analysis Set (SAS) Composed of all subjects who received the treatment at least once and whose safety information were collected. 3.2 Statistical analysis plan The statistical analysis applied SAS 9.1 statistic analysis software for calculation. All statistical tests used double-sided tests, and the tested difference was considered of statistical significance when P-value≤0.05. Measurement data of each visit in the two groups applied sample size, mean, standard deviation, median, maximum and minimum for statistical description. Comparison of differences before and after within group were carried out with paired t-test. Changes of the two groups prior and post treatment used t-test and Wilcoxon rank-sum test for comparison between groups. Numeration data of each visit in different groups used frequency (constituent ratio) for descriptive statistics. Comparison between two groups was carried out with χ2 test or Fisher’s exact test. 4 Clinical evaluation standards 4.1 Efficacy evaluation【5、6】 4.1.1 Primary efficacy variable: percentage of improvement of vaginal dryness symptoms Before receiving treatment, after the 3rd and the 10th administration, the patient should give the evaluation of the vaginal dryness with visual analogue scale (VAS, 0-10, 0= absent, 10= intolerable). The patient should report her subjective perception truly and independently. Scoring method: 0___________________________10 Absent intolerable Computing method: Percentage of improvement of vaginal dryness symptoms Mean score after treatment —Mean score before treatment = ---------------------------------------------------------- ×100% Mean score before treatment 4.1.2 Secondary efficacy variable: percentage of other vaginal symptoms Before receiving treatment, after the 3rd and the 10th administration, the patient should give the evaluation of itching, dyspareunia and burning sensation with visual analogue scale (VAS, 0-10, 0= absent, 10= intolerable). The patient should report her subjective perception truly and independently. 4.2 Safety evaluation 4.2.1 Physical examinations: recorded subject’s general conditions including weight, temperature, breath, blood pressure and pulse. 4.2.2 Laboratory examinations of vaginal microecosystem: did the examinations before and after treatment. To evaluate whether the vaginal bacterial flora was normal by the results of intensity and variety of vaginal bacterial flora and predominant bacteria. 4.2.3 Vaginal PH value: measured before and after treatment. 4.2.4 Examination of endometrial thickness: through vaginal B ultrasound before and after treatment, the increased endometrial thickness <5mm. 4.2.5 Incidence of adverse event: regardless of whether the subjects were voluntary, the investigator should collect and record all adverse events observed by physical examinations or other methods. Then the investigator should assess the relationship between the adverse events and the products. In case of serious adverse event the investigator should take emergency measures and report it, recording it in CRF. 5 Clinical trial results 5.1 Allocation, dropout and elimination of subjects 144 subjects were planned to enroll in the clinical trial (36 subjects for each center). In fact, 144 subjects participated in the study and 4 cases were transferred from Beijing Chao-Yang Hospital Attached to Capital Medical University to Peking University Third Hospital. There were 11 subjects dropping out of the study (dropout rate 7.6%, lower than 20%). Five subjects in Group A and six subjects in Group B dropped out. Four subjects among them dropped out because of AE and four subjects for lost of follow-up. Two subjects required to quit the study and one dropped out because of too long suspension of administration. On the basis of completion and dropout distribution analysis, the study data could be considered reliable and the difference of dropout rate between the two groups had no statistical significance. Statistical results were the followings (Table 1.1, Table 1.2, Table 1.3) Group A: Hyaluronic acid vaginal gel (Hyalofemme); Group B: Estriol cream (Ovestin cream) Table 1.1 Subject distribution of each center Group A Cent er Enrolled FAS PPS Group B SS Dropout/ Enrolled FAS PPS Total SS Elimination Dropout/ Enrolled FAS PPS SS Elimination Dropout/ Elimination 1 18 18 16 18 2 18 18 18 18 0 36 36 34 36 2 2 20 20 20 20 0 20 20 17 20 3 40 40 37 40 3 3 16 16 15 16 1 16 16 15 16 1 32 32 30 32 2 4 18 18 16 18 2 18 18 16 18 2 36 36 32 36 4 Total 72 72 67 72 5 72 72 66 72 6 144 144 133 144 11 * Center 1: Peking University First Hospital Center 2: Peking University Third Hospital Center 3: Beijing Chao-Yang Hospital Attached to Capital Medical University Center 4: Beijing Shijitan Hospital Table 1.2 Dropout/Elimination analysis Group Completion N Dropout/Elimination % N % χ2 P 0.0984 0.7537 Total Group A Group B 67 66 93.1 91.7 5 6 6.9 8.3 72 72 Total 133 92.4 11 7.6 144 *χ2 test. Enrollment number: 144, qualified completion number: dropout/elimination number: 11 and dropout/elimination rate: 7.6%. 133, Table 1.3 List of the subjects unqualified to enter PPS Center No. Name Group 1 1 2 2 2 3 3 4 16 29 43 55 67 89 104 116 LSJU LYLI SDME ZXLA ZHYA LSRO ZRHU XXYU A A B B B A B A 4 117 YXLI B Dropout/discontinuation reason Discontinuation time Lost to follow-up First visit Lost to follow-up Final visit Subject asked to quit Final visit AE Final visit AE Final visit Lost to follow-up Final visit AE Final visit Lost to follow-up Final visit The patient gave informed consent on Jan.22 and discontinued administration for business trip. Then restarted receiving administration on Feb.11. FAS PPS SS √ √ √ √ √ √ √ √ √ × × × × × × × × × √ √ √ √ √ √ √ √ √ 4 4 140 144 AIYA ZPKU B A Subject asked to quit AE Final visit Final visit √ √ × × 5.2 Baseline information and comparability analysis Statistical analysis of baseline data prior to the study showed no statistical difference on every parameter between the two groups (age, nationality, weight, systolic pressure, diastolic pressure, body temperature, pulse, breath, menstrual cycle, menopause time and way, diagnosis and treatment history of gynecologic disease)(P>0.05). Based on the statistical analysis of vaginal microecology laboratory examinations, vaginal PH value, endometrial thickness prior to treatment, each parameter between the two groups on baseline had no statistical difference (P>0.05). There were no statistical differences of vital signs (weight, body temperature, breath, systolic pressure, diastolic pressure and heart rate) before and after administration (P>0.05). The followings are statistical results (Table 2.1, Table 2.2) Table 2.1 Demographics of the subjects ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------Nationality n (missing) 72 (0) 72 (0) Fisher 1.0000 The Han People 71 (98.6%) 72 (100.0%) The minority groups 1 (1.4%) 0 (0.0%) Age (years old) n (missing) Mean±SD Median Q1; Q3 Min; Max 72 (0) 54.05 ±4.27 54.07 51.75 ;55.79 39.39; 65.71 72 (0) -0.4860 54.41 ±4.60 54.22 52.14 ;56.75 44.49; 67.61 0.6277 Systolic pressure n (missing) mmHg Mean±SD Median Q1; Q3 Min; Max 72 (0) 72 (0) -0.0926 118.14 ±12.59 118.35 ±14.36 120.00 120.00 110.00; 120.00 110.00 ;124.00 90.00; 150.00 90.00; 160.00 0.9264 Diastolic pressure n (missing) mmHg Mean±SD Median Q1; Q3 Min; Max 72 (0) 77.33 ±7.64 80.00 70.00; 80.00 60.00; 98.00 0.3870 72 (0) 0.8678 76.19 ±8.11 79.00 70.00; 80.00 60.00; 100.00 √ √ Heart rate n (missing) Times/min Mean±SD Median Q1; Q3 Min; Max 72 (0) 75.86 ±7.45 76.00 72.00; 80.00 52.00; 88.00 72 (0) -0.4160 76.40 ±8.16 79.00 70.00; 82.50 56.00; 90.00 n (missing) Mean±SD Median Q1; Q3 Min; Max 72 (0) 36.34 ±0.17 36.30 36.20; 36.50 35.80; 36.70 72 (0) 0.4331 36.33 ±0.18 36.30 36.20; 36.50 35.80; 36.80 0.6656 Breath n (missing) Times/min Mean±SD Median Q1; Q3 Min; Max 72 (0) 20.90 ±4.52 20.00 18.00; 26.00 14.00; 28.00 72 (0) -0.6183 21.36 ±4.38 20.00 18.00; 25.00 14.00; 30.00 0.5374 Weight Kg 72 (0) 60.99 ±7.34 60.00 55.00; 65.50 47.00; 82.00 72 (0) 60.61 ±8.66 60.00 55.00; 65.50 41.00; 82.50 0.2854 0.7757 72 (0) 29.01 ±3.05 28.00 28.00; 30.00 23.00; 40.00 72 (0) 28.39 ±3.63 28.00 28.00; 30.00 20.00; 45.00 0.8923 0.3722 71 (1) 4.44 ±3.71 3.50 1.50; 6.67 0.50; 19.67 72 (0) 5.58 ±5.45 4.08 2.13; 7.00 0.50; 30.92 -1.0824 0.2791 72 (0) 62 (86.1%) 10 (13.9%) 0.055 0.8133 Body temperature ℃ n (missing) Mean±SD Median Q1; Q3 Min; Max Menstrual cycle n (missing) days Mean±SD Median Q1; Q3 Min; Max Menopause time n (missing) years Mean±SD Median Q1; Q3 Min; Max Menopause Way n (missing) 72 (0) Natural menopause 61 (84.7%) Surgical menopause 11 (15.3%) 0.6781 Diagnosis and n (missing) treatment history No of gynecologic Yes disease 72 (0) 46 (63.9%) 26 (36.1%) 72 (0) 52 (72.2%) 20 (27.8%) 1.152 0.2830 Note: 1. Q1: 25% quantile, 2. Q3: 75% quantile, 3. comparison of classification indexes used χ2 test and χ2 were statistics, 4. Group comparison of quantitative indexes used t-test and t was statistic, 5. Comparison of menstrual cycle and menopause time used Wilcoxon rank sum test and statistic was Z. Table 2.2 Vaginal microecology laboratory examination on baseline ------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ------------------------------------------------------------------------------Vaginal microecology n (missing) 72 (0) 72 (0) 0.747 0.3873 laboratory Normal flora 29 (40.3%) 24 (33.3%) examination Abnormal flora Vaginal PH value n (missing) Mean±SD Median Q1; Q3 Min; Max Endometrial thickness mm n (missing) Mean±SD Median Q1; Q3 Min; Max 43 (59.7%) 48 (66.7%) 72 (0) 71 (1) 0.1223 5.63 ±1.04 5.61 ±0.98 5.50 5.45 5.00; 6.50 5.00; 6.50 3.80; 8.00 3.80; 7.50 62 (10) 3.19 ±1.20 3.00 2.40; 4.00 1.00; 8.30 62 (10) 3.21 ±1.80 3.00 2.10; 4.00 1.00; 12.00 -0.0704 0.9028 0.9440 ------------------------------------------------------------------------------Note: 1. Q1: 25% quantile, 2. Q3: 75% quantile, 3. comparison of classification indexes used χ2 test and χ2 were statistics, 4. Group comparison of quantitative indexes used t-test and t was statistic. 5.3 Analysis of concomitant medications Several concomitant medications were applied in the test group and control group during the clinical study. They were mainly antihypertensive drugs. Concomitant medications were appended to nine subjects, two receiving nutrition and health-care medicine and seven because of AE. According to statistical analysis, the difference of concomitant medications between the two groups had no statistical significance (P>0.05). See Table 3.1 and Table 3.2. Table 3.1 Comparison of concomitant medications ------------------------------------------------------------------------------Index Statistic Group A Group B Statistic p-value ------------------------------------------------------------------------------Concomitant n (missing) 72 (0) 72 (0) 2.118 0.1455 medications no 59 (81.9%) 65 (90.3%) yes 13 (18.1%) 7 (9.7%) -----------------------------------------------------------------------------Note: comparison of concomitant medication rate used χ2 test and statistic was χ2. Table 3.2 Concomitant medication list Center Random No. Name Group Medication Reason Starting Date 1 1 LIBI B Lotensin 2001 2 2 2 2 2 2 2 37 39 41 42 50 50 54 ZGFA DXYI HZHU YLHO LXFE LXFE ZHJI B A A B A A A 2 2 55 55 ZXLA ZXLA B B 2 2 55 55 ZXLA ZXLA B B 2 2 2 2 2 2 58 61 62 62 62 62 WZYU ZJPI JSLA JSLA JSLA JSLA A A A A A A Fosinopril Captopril Valsartan Nifedipine Fosinopril Nifedipine Losartan potassium Irbesartan Levothyroxine sodium Levofloxacin Chinese decoction Betaloc Fosinopril Aspirin Metoprolol Amlodipine Shuanghuanglian Oral liquid High blood pressure Hypertension Hypertension Hypertension Hypertension Hypertension Hypertension Hypertension 2 62 JSLA A 2 62 JSLA A Ending Date 20010430 19990901 20081101 19891001 2005 2005 200601 Hypertension 20060330 Hypokalemia 20060330 Cystitis Cystitis Hypokalemia Hypokalemia Hypokalemia Hypokalemia Hypokalemia Upper Respiratory infection Soluble granules Upper For cold Respiratory infection Acetylspiramycin Upper Respiratory infection 20100108 201002 20100114 20041201 200711 2007 2007 2007 20100218 20100224 20100218 20100224 20100218 20100224 2 2 2 66 67 67 WYLI ZHYA ZHYA B B B 2 3 70 80 WSHU HXLI A A 3 4 93 111 CXME LFLA A B 4 111 LFLA B 4 4 111 111 LFLA LFLA B B 4 113 WCRO A 4 131 ZHHO A 4 4 131 142 ZHHO DJHE A A Levoamlodipine Metformin Metronidazole Suppositories Fosinopril Shenshitong (traditional Chinese medicine) Caltrate-D Bixie Fenqing Wan Hypertension 2007 Diabetes Vaginal 201002 itching Hypertension Kidney stone 20100101 Osteoporosis Urinary infection Osteoporosis Alendronate Sodium Tablets Calcitriol Osteoporosis Valsartan capsules Hypertensive disease Yinhua Miyanling Urinary tablets infection Chinese Dragon’s Appendagitis Blood Gongyanping Appendagitis Sporanox Mycotic vaginitis 201003 20100115 20100210 20091223 20091227 20091218 20100117 20091218 20091218 20100117 20100117 20091228 20100104 20100223 20100223 20100331 20100402 5.4 Efficacy analysis 5.4.1 Primary efficacy endpoint: percentage of improvement of vaginal dryness symptoms Non-inferiority test was used for primary efficacy endpoint, thus analysis would apply PPS and the results of FAS were the same as PPS. Prior to administration, the average VAS scores of vaginal dryness symptoms were 5.76 in test group and 5.26 in control group. On the first visit, the scores were 3.01 and 2.55, respectively. On the final visit, the scores were 0.90 and 0.62, respectively. From all the above, both the test group (applying Hyaluronic acid vaginal gel) and the control group (applying Estriol cream) could effectively reduce the scores of vaginal dryness symptoms. And as the administration time prolonged, the symptoms could be almost entirely relieved and even disappeared. The primary efficacy endpoint was percentages of improvement of vaginal dryness symptoms in the two groups. On the first visit, the mean percentages of vaginal dryness were 49.17% in test group and 53.53% in control group. While on the final visit, the percentages were 84.44% and 89.42, respectively. The differences between the two groups had no statistical significance (P> 0.05). On the first visit, the mean difference of the percentages between test group and control group was -3.26% and 95%CI was [-11.23%,4.72%]. On the final visit, the mean difference of the percentages between test group and control group was -3.33% and 95%CI was [-9.40%,2.74%]. Though data showed that the improvement percentages of Estriol cream group were higher than that of Hyaluronic acid vaginal gel group on the two visits, the 95%CI lower limit of mean difference did not exceed the 15% non-inferiority margin specified by protocol design. It proved that the efficacy of Hyaluronic acid vaginal gel was not inferior to that of Estriol cream. The difference between them had no clinical significance. See Table 4.1.1-4.1.4. Table 4.1.1 Primary efficacy endpoint- improvement rate of vaginal dryness (PPS) --------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value --------------------------------------------------------------------------------------------------------------------Vaginal dryness n (missing) 67 (0) 66 (0) -1.6182 0.1056 Score Mean±SD 5.76 ±1.88 5.26 ±1.82 Prior to administration Vaginal dryness n (missing) 67 (0) 66 (0) -1.6658 0.0957 Score Mean±SD 3.01 ±1.74 2.55 ±1.82 First visit Vaginal dryness n (missing) Score Mean±SD Final visit Vaginal dryness n (missing) Improvement Mean±SD Rate First visit 67 (0) 0.90 ±1.18 67 (0) 49.17 ±23.90 66 (0) 0.62 ±1.06 66 (0) 53.53 ±27.67 -1.4993 1.0190 0.1338 0.3082 Vaginal dryness n (missing) 67 (0) 66 (0) 1.5093 0.1312 Improvement Mean±SD 84.44 ±20.60 89.42 ±17.21 Rate Final visit ------------------------------------------------------------------------------------------------------------------Note: t-test was used for comparison within groups and statistic was t. Wilcoxon test was used for group comparison and statistic was Z. Table 4.1.2 Non-inferiority CI of vaginal dryness improvement rate between the two groups with center effects (PPS) ------------------------------------------------------------------------------Indexes Statistics Test group Control group ------------------------------------------------------------------------------53.54 Change values (First visit-Baseline) 50.29 Standard error 2.82 2.85 95%CI [44.70, 55.87] [47.91, 59.17] -----------------------------------Group comparison 1-2mean difference (95%CI) -3.26 [-11.23, Change values (Final visit-Baseline) 85.63 Standard error 2.15 95%CI [81.38, 89.88] 4.72] 88.96 2.17 [84.67, 93.24] -----------------------------------Group comparison 1-2 mean difference (95%CI) -3.33 [ -9.40, 2.74] Note: 1 the covariance analysis used multi-center effects analysis to control the center effects and baseline effects. Mean and the 95% CI were modified. Table 4.1.3 Primary efficacy endpoint- improvement rate of vaginal dryness (FAS) ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------Vaginal dryness n (missing) 72 (0) 72 (0) 1.5014 0.1333 Score Mean±SD 5.71 ±1.89 5.26 ±1.89 Prior to administration Vaginal dryness n (missing) 72 (0) 72 (0) 1.4837 0.1379 Score Mean±SD 3.04 ±1.78 2.64 ±1.88 First visit Vaginal dryness n (missing) Score Mean±SD Final visit Vaginal dryness n (missing) Improvement Mean±SD Rate First visit 72 (0) 1.06 ±1.44 72 (0) 48.19 ±25.00 72 (0) 0.86 ±1.45 72 (0) 51.89 ±27.48 1.0926 0.2746 -0.8746 0.3818 Vaginal dryness n (missing) 72 (0) 72 (0) -1.0146 0.3103 Improvement Mean±SD 81.36 ±25.30 85.03 ±22.84 Rate Final visit ---------------------------------------------------------------------------------------------------------------------Note: t-test was used for comparison within groups and statistic was t. Wilcoxon test was used for group comparison and statistic was Z. Table 4.1.4 Non-inferiority CI of vaginal dryness improvement rate between the two groups with center effects (FAS) ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Test group Control group ---------------------------------------------------------------------------------------------------------------------Change values (First visit-Baseline) 49.40 52.03 Standard error 2.76 2.76 95%CI [43.95, 54.86] [46.58, 57.49] ------------------------------------------------Group comparison 1-2mean difference (95%CI) -2.63 [-10.37, 5.11] Change values (Final visit-Baseline) 82.25 85.21 Standard error 2.71 2.71 95%CI [76.90, 87.61] [79.85, 90.56] --------------------------------------------------Group comparison 1-2mean difference (95%CI) -2.95 [-10.55, 4.64] Note: 1 the covariance analysis used multi-center effects analysis to control the center effects and baseline effects. Mean and the 95% CI were modified. 5.4.2 Secondary efficacy endpoints Evaluating the secondary efficacy endpoints: improvement rate of vaginal itching, dyspareunia and burning sensation. On the first visit, improvement rate of itching were 63.66% in test group and 67.29% in control group; that of dyspareunia were 24.33% and 26.64%, respectively; improvement rate of burning sensation were 53.68% and 65.06%, respectively. On the final visit, improvement rate of itching were 86.23% in test group and 81.97% in control group; that of dyspareunia were 56.96% and 62.33%, respectively; improvement rate of burning sensation were 85.83% and 87.87%, respectively. The differences between the two groups of each secondary endpoints had no statistical significance (P>0.05). And the results of FAS were consistent with that of PPS. See Table 4.2.1-Table 4.2.3 Table 4.2.1 Secondary efficacy endpoint-improvement rate of vaginal itching (PPS) --------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------Prior to n (missing) 67 (0) 66 (0) 0.9546 0.3398 Administration First visit n (missing) Improvement Mean±SD Rate (%) Final visit Rate (%) n (missing) Mean±SD 27 (40) 63.66 ±38.25 27 (40) 86.23 ±26.11 35 (31) 67.29 ±37.79 35 (31) 81.97 ±29.05 -0.3713 0.4381 0.7104 0.6613 Table 4.2.2 Secondary efficacy endpoint- improvement rate of dyspareunia (PPS) -------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value -------------------------------------------------------------------------------------------------------------------Prior to n (missing) 64 (3) 64 (2) 1.1380 0.2551 Administration First visit n (missing) Improvement Mean±SD Rate (%) 56 (11) 24.33 ±31.78 54 (12) 26.64 ±35.62 0.1502 0.8806 Final visit n (missing) 56 (11) 54 (12) 1.0542 0.2918 Improvement Mean±SD 56.96 ±41.47 62.33 ±43.80 Rate (%) -------------------------------------------------------------------------------------------------------------------- Table 4.2.3 Secondary efficacy endpoint- improvement rate of burning sensation (PPS) -------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value --------------------------------------------------------------------------------------------------------------------Prior to n (missing) 67 (0) 65 (1) -1.8758 0.0607 Administration First visit n (missing) Improvement Mean±SD Rate 48 (19) 53.68 ±35.28 36 (30) 65.06 ±33.71 1.3826 0.1668 Final visit n (missing) Improvement Mean±SD Rate 48 (19) 85.83 ±24.63 36 (30) 87.87 ±36.66 1.6111 0.1072 5.5 Safety analysis 5.5.1 Adverse event Thirteen adverse events occurred throughout the clinical trial, seven in test group (9.7%) and six in control group (8.3%). The difference between the two groups had no statistical significance. See Table 5.1. Table 5.1 Comparison of adverse event incidence of the two groups ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------Adverse events n (missing) 72 (0) 72 (0) 0.084 0.7711 no 65 ( 90.3%) 66 ( 91.7%) yes 7 (9.7%) 6 (8.3%) Note: comparison of adverse events used χ2 test and statistic was χ2 5.5.2 Laboratory examinations of vaginal microecosystem The vaginal microenvironment is a unique dynamic system. It changes depending on the variations of physiological conditions and local physical and chemical factors. Large numbers of microflora exist in vagina and lactobacilli are predominant bacteria. Patients feel uncomfortable if the predominant bacteria change. The main causes of the changes can be endogenous and exogenous. Studies showed that menstruation, vaginal douche, contraceptive agents, vaginal medication, number of sexual partners, frequency of intercourse, application of sanitary articles during menstrual period (sanitary towel or tampons) will influence the constitution of vagina flora[7]. The laboratory examinations of vaginal microecosystem in this clinical study are to find out and evaluate whether the application of Hyaluronic acid vaginal gel causes changes of vagina flora and maintains stability of endovaginal environment. 5.5.2.1 Overall evaluation of vaginal microecosystem Clinical evaluation system of vaginal microecosystem is to give an overall evaluation of vaginal microenvironment by describing the intensity, variety of vaginal flora and predominant bacteria, inflammatory response and causative bacteria morphology, and considering the functional parameters (vaginal PH value, H2O2 and leucocyte esterase. In view of limit of the clinical study design, this study evaluated the vaginal microenvironment mainly by the intensity, variety of vaginal flora and predominant bacteria. In the overall evaluation of vaginal microecosystem on the final visit, 29.85% (20/67) of subjects in test group were normal without changes before and after administration, 50.75% (34/67) abnormal without changes. 8.96% (6/67) of subjects became abnormal and 10.45% (7/67) became normal. While in control group, 33.33% (22/66) of subjects were normal without changes before and after administration, 43.94% (29/66) abnormal without changes. 1.52% (1/66) of subjects became abnormal and 21.21% (14/66) became normal. From the above data, 80.6% (54/67) of subjects in Hyaluronic acid vaginal gel group and 77.27% (51/66) in Estriol cream group had no changes on vaginal microenvironment before and after application. The subjects who became abnormal in test group were a little more than that in control group (6 vs 1) while subjects who became normal in control group were a little more than that in test group (14 vs 7). It suggested that Estriol cream had therapeutical effect, promoting the synthesis of epithelium glycogen of vaginal mucosa and the growth of lactobacilli, helping vaginal microenvironment get better. While Hyaluronic acid vaginal gel did not have therapeutical effect, not changing the stability of microenvironment though it was not able to make the microenvironment better. 5.5.2.2 Analysis of intensity, variety of bacterial flora and predominant bacteria On the final visit, 71.6% (48/67), 68.7% (46/67) and 80.6% (54/67) of subjects in test group had no changes in intensity, variety of bacterial flora and predominant bacteria, respectively (before-after administration). While 71.6% (48/67), 73.1% (49/67) and 76.1% (51/67) of subjects in control group had no changes in intensity, variety of bacterial flora and predominant bacteria, respectively (before-after administration). The difference of laboratory examination of vaginal microenvironment between the two groups had no statistical significance. It showed that the products of test group and control group had no influence on the intensity, variety of bacterial flora and predominant bacteria. From the above data, we could also find more subjects became normal in control group. It suggested Estriol cream, as a therapeutic product, can help vaginal microenvironment get better. See Table 5.2 Table 5.2 Changes of vaginal microenvironment laboratory examination indexes on Final visit ----------------------------------------------------------------------------------------------------------------------------------------Indexes Prior to administration → Test group Control group After administration ---------------------------------------------------------------------------------------------------------------------------------------Overall evaluation n (missing) 67 (5) 66 (6) 20 (29.85%) 22 (33.33%) normal→abnormal (%) 6 (8.96%) 1 (1.52%) abnormal→normal (%) 7 (10.45%) 14 (21.21%) abnormal→abnormal (%) 34 (50.75%) 29 (43.94%) 67 (5) 67 (5) normal→normal (%) 34 (50.75%) 44 (65.67%) normal→abnormal (%) 9 (13.43%) 3 (4.48%) abnormal→normal (%) 10 (14.93%) 16 (23.88%) abnormal→abnormal (%) 14 (20.90%) 4 (5.97%) Of microecosystem normal→normal (%) Intensity of n (missing) bacterial flora Variety of n (missing) 67 (5) 67 (5) normal→normal (%) 39 (58.21%) 49 (73.13%) normal→abnormal (%) 9 (13.43%) 1 (1.49%) abnormal→normal (%) 12 (17.91%) 17 (25.37%) abnormal→abnormal (%) 7 (10.45%) 0 (0.00%) bacterial flora Predominant bacteria n (missing) G+ bacillus→G+ 67 (5) bacillus (%) 21 (31.34%) 67 (5) 23 (34.33%) G+ bacillus → others (%) 5 (7.46%) 2 (2.99%) Others→G+ bacillus (%) 8 (11.94%) 14 (20.90%) Others→others (%) 33 (49.25%) 28 (41.79%) ----------------------------------------------------------------------------------------------------------------------------------------Note: baseline- final visit 5.5.2.3 Typical pictures in vaginal microecosystem examinations Test group-- Hyaluronic acid vaginal gel group 012 Prior treatment Normal flora 017 Prior treatment Abnormal flora 017 Post-treatment Normal flora 095 Prior treatment Normal flora 095 Post-treatment Abnormal flora 022 Post-treatment Abnormal flora 022 Prior treatment Abnormal flora 012 Post-treatment Normal flora Control group-- Estriol cream group 001 Prior treatment Normal flora 015 Prior treatment Abnormal flora 027 Prior treatment Normal flora 009 Prior treatment Abnormal flora 001 Post-treatment Normal flora 015 Post-treatment Normal flora 027 009 Post-treatment Abnormal flora Post-treatment Abnormal flora 5.5.3 Vaginal PH value Prior to administration, vaginal PH values were 5.63 in test group and 5.61 in control group. And the difference between the two groups had no statistical significance (P>0.05). Vaginal PH values were 5.30 in test group and 4.87 in control group after administration. The difference between the two groups had statistical significance (P<0.05). It proved that Estriol cream could efficiently reduce vaginal PH value and improve the vaginal health score. Though application of Hyaluronic acid vaginal gel was not able to reduce vaginal PH value, it did not affect the PH value, maintaining the stability of vaginal PH value. See Table 5.3. Table 5.3 Comparison of vaginal PH value ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------Vaginal PH value n (missing) 72 (0) 71 (1) 0.1223 0.9028 Prior to Mean± SD 5.63 ±1.04 5.61 ±0.98 Administration Median 5.45 5.50 Q1; Q3 5.00; 6.50 5.00; 6.50 Min; Max 3.80; 8.00 3.80; 7.50 Vaginal PH value n (missing) 67 (5) 68 (4) 3.1691 0.0019 Final visit Mean± SD 5.30 ±0.84 4.87 ±0.71 Median 5.40 5.00 Q1; Q3 5.00; 6.00 4.50; 5.00 Min; Max 3.50; 7.00 3.00; 7.00 -------------------------------------------------------------------------------------------------------------------Note: 1. Q1: 25% quantile, 2. Q3: 75% quantile, 3. Group comparison used t-test and t was statistic. 5.4 Endometrial thickness Prior to administration, endometrial thickness in test group and control group were 3.19mm and 3.21mm, respectively. After administration, were 3.43mm in test group and 3.52mm in control group. The difference between the two groups had no statistical significance (P>0.05). The endometrial thickness of one subject (No. 003) in Peking University First Hospital, who received Estriol cream, increased to 9mm from 3mm. On follow-up after drug withdrawal, her endometrial thickness reduced gradually and became normal finally. It indicated that a part of Estriol cream could go into blood to cause some influence on endometrium though the application was topical. Thus doctors should be cautious when using it, especially for people hypersensitive to estrogen. See Table 5.4 Table 5.4 Comparison of endometrial thickness ---------------------------------------------------------------------------------------------------------------------Indexes Statistics Group A Group B Statistics p-value ---------------------------------------------------------------------------------------------------------------------mm n (missing) 62 (10) 62 (10) -0.0704 0.9440 Prior to Mean± SD 3.19 ±1.20 3.21 ±1.80 Administration mm n (missing) 57 (15) 59 (13) -0.2661 0.7907 Final visit Mean± SD 3.43 ±1.49 3.52 ±1.93 ---------------------------------------------------------------------------------------------------------------------Note: Group comparison used t-test and statistic was t. 6 Adverse events and side effects and the treatment during the clinical trial Throughout the clinical study, seven adverse events (9.7%) occurred in test group. Peking University: No.050 subject, whose laboratory examination result was VVC, of mild grade with no clinical symptoms, received no relevant treatment and AE have relieved. No.056 subject, whose laboratory examination result was BV, of mild grade with no clinical symptoms, received no relevant treatment and AE have relieved. No. 062 subject had upper respiratory tract infection and relieved after receiving relevant medication. No. 070 subject, whose laboratory examination result was VVC, of mild grade with no clinical symptoms, received no relevant treatment and AE have relieved. Beijing Chaoyang Hospital: No. 073 subject, who had upper respiratory tract infection of mild grade, did not receive relevant treatment and AE relieved. Beijing Shijingtan Hospital: No. 142 subject, who had VVC, relieved after receiving corresponding medication. No. 144 subject, who had severe mixed prolapse of intestinal mucosa, dropped out of the trial and the AE existed till the end. Six adverse events (8.3%) occurred in control group. Peking University First Hospital: No. 003 subject’s B-ultrasonic examination showed her endometrial thickness increased to 9mm from 3mm on final visit. She did not receive relevant treatment because it was mild grade and she finished study. Her B-ultrasonic examination on follow-up visit showed the endometrial thickness reduced, so the AE relieved. Peking University Third Hospital: No. 047 subject’s B-ultrasonic examination on final visit showed cysts of left ovary and right adnexa and she did not receive relevant treatment because the condition was mild grade and she finished study. The follow-up B-ultrasonic examination on follow-up showed that the left ovary cyst still existed while the cyst of right adnexa was relieved. No. 055 subject had cystitis of moderate degree and received new concomitant medication. But she asked to quit for frequent fluctuation of the cystitis. The AE existed at the end of the study. No. 064 subject, whose examination result was BV of mild grade with no clinical symptoms, received no relevant treatment and AE have relieved. No. 067 subject, who had vaginal itching, received concomitant medication. She asked to quit the study and her symptoms relieved at the end. Beijing Chaoyang Hospital: No. 104 subject, who had vulva itching, received no relevant treatment and AE still existed at the end of the study. See Table 6. No. 2 study centers reported more adverse events (8 subjects, 9 cases of AE) than other centers. That was because the investigator’s criteria of AE in this center was stricter than others and he was more careful when asking about AE. Table 6 List of adverse events Center No. Name Group Description Start Date Degree Influence on dosage Relationship With the drug Outcome Date of relieving 1 3 DILI B Endometrial thickness increased 2009-12-30 Mild End of study Probably related Relieved 2 47 WEYU B Left ovary cyst 2010-01-15 Mild End of study Impossibly related Still existed Cyst of right adnexa 2010-01-15 Mild End of study Impossibly related Relieved 2010-04-23 2010-02-18 2010-01-07 2 50 LXFE A VVC 2010-02-08 Mild End of study Suspiciously Relieved 2 55 ZXLA B Cystitis 2010-01-08 Moderate Permanent withdrawal Impossibly related Still existed 2 56 MPYU A BV 2010-01-27 Mild End of study Suspiciously Relieved 2 62 JSLA A Upper respiratory tract infection 2010-02-18 Mild Permanent withdrawal Impossibly related Relieved 2 64 GLLA B BV 2010-03-09 Mild End of study Suspiciously Relieved 2010-NA 2 67 ZHYA B Vaginal itching 2010-02-NA Moderate Permanent withdrawal Suspiciously Relieved 2010-02-NA 2 70 WSHU A VVC 2010-03-29 Mild End of study Suspiciously Relieved NA 3 73 LIHO A Fever 2009-11-25 Moderate Unchanged dosage Impossibly related Relieved 2009-11-30 3 104 ZRHU B vulva itching 2010-04-09 Mild Drug discontinued temporarily Probably related Still existed 4 142 DJHE A VVC 2010-03-30 Mild Unchanged dosage Suspiciously Relieved 4 144 ZPKU A mixed prolapse of intestinal mucosa 2010-04-10 Severe Permanent withdrawal Impossibly related Still existed 2010-NA 2010-02-24 2010-04-02 7 Analysis of clinical trial results The study is a multicenter, randomized, open, positive and parallel controlled study, evaluating the efficacy and safety of Hyaluronic acid vaginal gel (Hyalofemme) for improvement of vaginal dryness. The clinical study results showed that applying Hyaluronic acid vaginal gel and Estriol cream in test group and control group according to the protocol, could remarkably improve the clinical symptoms of vaginal dryness. The final (after administration for 10 times) improvement rate were 84.44% and 89.42%, respectively, without significant statistical differences between them (P>0.05). Mean of rate difference was -3.33% and 95%CI was [-9.40, 2.74], the lower limit of which did not exceed the boundary value of non-inferiority evaluation (15%). Thus we could think that the efficacy of the Hyaluronic acid vaginal gel is not inferior to that of Estriol cream, with no significant difference between them. At the first visit, the improvement rate of vaginal dryness in Hyaluronic acid vaginal gel group and Estriol cream group were 49.17% and 53.53%, and had no statistical significant differences. That meant the two products had a short onset time and they could release the clinical symptoms of vaginal dryness after three times of administration of the products. That the improvement rate on first visit was lower than that on final visit showed the continuous release effect on the symptoms as time of medication increased. The final improvement rate of the secondary efficacy endpoints (itching, dyspareunia and burning sensation) in Hyaluronic acid vaginal gel group and Estriol cream group were 86.23% and 81.97%, 56.96% and 62.33%, 85.83% and 87.87%, respectively. The differences between the two groups had no statistical significance (P>0.05), and FAS results were in line with PPS. Totally 13 adverse events occurred throughout the clinical study, seven in the test group (incidence: 9.7%, 7/72) and six in the control group (incidence: 8.3%, 6/72). The differences between the two groups had no statistical significance (P>0.05). No serious adverse event occurred, and the AEs that were related to the test product or suspicious were in mild degree, which could relieve. The vaginal microecosystem is composed of intravaginal microflora, incretion regulating function and anatomic structure. Normal microflora in vaginal is core content of vaginal microecosystem study. Vaginal microecosystem is a complex and vulnerable system, which may be easily affected by various endogenous and exogenous factors. And vaginal medication is one of the important factors. Once the balance of vaginal microecosystem is broken, a series of clinical symptoms may occur, such as itching, unpleasant odor and abnormal leucorrhea, etc. The clinical study is to evaluate whether applying Hyaluronic acid vaginal gel influences the stability of vaginal environment by investigating the variations of vaginal microecosystem before and after treatment. In the vaginal microecosystem evaluation, no changes of vaginal micro-ecological environment appeared after treatment in 80.6% (54/67) of subjects in Hyaluronic acid vaginal gel group, while 77.27% (51/66) in Estriol cream group. It indicated that applying Hyaluronic acid vaginal gel would not alter the internal environment of vagina and it was able to maintain the homeostasis, with a high safety. Vaginal microecosystem evaluation is a lightspot of this clinical study. It objectively evaluated that no changes occurred in intensity, variety and predominant bacteria of vaginal flora after applying Hyaluronic acid vaginal gel. Mean of vaginal PH value in Hyaluronic acid vaginal gel group was 5.63 prior to treatment and 5.30 after treatment, with no statistical difference. Vaginal PH value after treatment (5.30) in Hyaluronic acid vaginal gel was higher than that in Estriol cream group and the difference between them had statistical significance (P<0.05). It suggested that the hyaluronic acid vaginal gel was not as good as the estriol cream on reducing vaginal PH value. It was mainly because the estrin in the estriol cream could help the growth of lactobacilli in vagina, promoting lactose broken down into lactic acid, to reduce PH value. While the hyaluronic acid vaginal gel had no pharmacological effect. Its biological effect was acted through Hydeal-D®, the derivative of hyaluronic acid, without mechanism to reduce PH value. The endometrial thickness prior to treatment in test group and control group were 3.19mm and 3.21mm, respectively. While 3.43mm and 3.52mm after treatment. The difference between the two group had no statistical difference (P > 0.05). However, the endometrial thickness of the No. 003 subject (applying estriol cream) in Peking University First Hospital increased from 3mm to 9mm. On follow-up after drug withdrawal, the endometrial thickness of the subject reduced gradually and the symptoms were relieved. It indicated that estriol cream had influence on the endometrial thickness. The doctors should apply it strictly in accordance with the indications. Because of its narrow applicability, the doctors should be cautious when using it. The hyaluronic acid vaginal gel did not have hormonelike effect, thus it had no influence on endometrium or hormones-endocrine system and had a high safety. Hyaluronic acid vaginal gel (Hyalofemme), a colorless gel with high hydrating properties, is manufactured by Fidia Advanced Biopolymers, srl, Italy and has been marketed in numerous foreign countries for years. Hyaluronic acid is a natural molecule that can maintain a right level of hydration and plays an important role in tissue repair. The product has no pharmacological effect for it is not a drug but a type Ⅲ medical device. The gel’s hydrating properties are attributed to the characteristics of the hyaluronic acid based biopolymer which releases water molecules to the tissue, thus improving the dry state of the vagina without any irritation of vaginal mucosa. Therefore, the gel is applicable to supplementary treatment of vaginal dryness symptoms induced by any reason. Compared with estriol cream, hyaluronic acid vaginal gel has the characteristics of wider applicable range, no hormonelike effect, higher safety and better acceptability of patients. The clinical trial showed that the hyaluronic acid vaginal gel played a part in repair of vaginal epithelial cells. It could improve the epithelium condition, making the subjects feel better after application. The mechanism need further study. Though the results would be inevitably influenced by the investigator because of the open-label trial, they still had reference value. The certain evaluation of the efficacy and safety of hyaluronic acid vaginal gel depended on further blind trial. 8 Conclusion of the clinical trial To sum up, the hydration and adhesion of hyaluronic acid vaginal gel can improve vaginal dryness throughout the spontaneous recovery of small lesions that caused by friction of vagina. Therefore, the gel is applicable to supplementary treatment of vaginal dryness symptoms induced by any reason. 9 Indications, applicable range, contraindication and attentions Indications: Supplementary treatment of vaginal dryness induced by various reasons and promoting the spontaneous recovery of small lesions caused by frictions of vaginal mucosa Instructions 1. Revolve the vaginal applicator into the tube. 2. Squeeze the tube to fill the applicator to half of it (about 5g). 3. Release the applicator from the tube and plug it in patient’s vagina. The patient should keep crouching or lying position. 4. Impel the applicator until all of the gel is discharged. 5. Clean the applicator after administration. Wash out the remaining gel with water and disinfectants, then rinsing it thoroughly. Finally remove the water from the applicator. 6. Put the applicator into the box for use next time. 7. In case of severe dryness, it is suggested that eject a little gel so as to make enough lubricating before plugging the applicator into vagina. Application frequency Application frequency depends on the dry state of the vaginal mucosa. Patients in the study applied the gel every 3 days, totally 30 days continuously except other prescription. Contraindication Patients who are allergic to the product. Attentions: In case of vaginal infection, patient should consult the doctors before application of the product. The product can be used during menstruation and it will not affect the gynecologic test results. Do not use if packaging is damaged. Do not litter the packaging. Store in a cool and dry place (temperature<40℃)to prevent property changes. Do not use if exceeding valid date. 10 References ⑴ HYALGEL VAGINAL(Hyalofemme)registration materials ⑵ Regulations of medical device clinical trials, SFDA No.5, 2004.1.7 ⑶ Bei Zhengping, Lai Peili, Zhang Bin, Diagnositic criteria of gynecological and obstetrical disease, 2nd version, Science Press, Beijing, 318-319,2006 ⑷ Liu Qin, Jin Pihuan, chief editor. Statistical analysis of classification data and SAS programming. Shanghai: Fudan University Press, 2002. ⑸ Gao Jing, compiled, Hormone replacement therapy and senile vaginitis——evaluation of vagina health score, Foreign Medicine Obstetrics and Gynecology volume, 25(2):101-102, 1998 ⑹ Hong Xiuqin, Zhou Fengjie, Analysis of conjugated estrogen in treatment of 68 patients with postmenopausal urogenital atrophy, the Journal of Practical Medicine, 21(12):1353-1354, 2005. ⑺ Liao Qinping, Female vaginal ecosystem and vaginal microecological evaluation, Journal of Practical Obstetrics and Gynecology, 2010, 26(2): 81-83. 11 Participants of clinical trial Peking University First Hospital Name Position Professional Title Department Telephone no. Liao Qinping Head of the Chief physician Obstetrics and department Gynecology Lin Huaixian Associate chief physician Obstetrics and Gynecology Chen Junya Attending physician Obstetrics and Gynecology Zhang Miao Associate chief physician Obstetrics and Gynecology Peking University Third Hospital Geng Li Chief physician Obstetrics and Gynecology Chen Chief physician Obstetrics and Bingfeng Gynecology Guo Yanli Chief physician Obstetrics and Gynecology Yao Yanjun Senior nurse Obstetrics and Gynecology Beijing Chao-Yang Hospital Attached to Capital Medical University Song Chief physician Obstetrics and Xuehong Gynecology Wang Qiuxi Associate chief physician Obstetrics and Gynecology Liu Jun Attending physician Obstetrics and Gynecology Wei Shuang Nurse-in-charge Obstetrics and Gynecology Beijing Shijitan Hospital Li Hongxia Head of the Chief physician Obstetrics and department Gynecology Yan Associate chief physician Obstetrics and Shuxiang Gynecology Huang Bin Attending physician Obstetrics and Gynecology Xu Zimei Technologist-in-charge Obstetrics and Gynecology Institute of Basic Medical Sciences Chinese Academy of Medical Sciences Han Shaomei Director of Professor Statistics Office 010-65296408 Statistics Office Xu Tao Statistics Office 010-65296408 12 Declaration and signatures of investigators I have read this report. According to my understanding, I confirm that the report has described the process and results of the clinical trial truly and accurately. Study responsible center: Peking University First Hospital Liao Qinping signature: ________ Date: YY-MM-DD Study centers: Peking University Third Hospital Geng Li signature: ________ Date: YY-MM-DD Beijing Chao-Yang Hospital Attached to Capital Medical University Song Xuehong signature: ________ Date: YY-MM-DD Beijing Shijitan Hospital Li Hongxia signature: ________ Date: YY-MM-DD Statistic department: Institute of Basic Medical Sciences Chinese Academy of Medical Sciences Han Shaomei signature: ________ Date: YY-MM-DD 13 Opinions of clinical trial management department of medical institutions Management department of Peking University First Hospital (Stamp) Date: YY-MM-DD Management department of Peking University Third Hospital (Stamp) Date: YY-MM-DD Management department of Beijing Chao-Yang Hospital Attached to Capital Medical University (Stamp) Date: YY-MM-DD Management department of Beijing Shijitan Hospital (Stamp) Date: YY-MM-DD