Effects of lignocaine in endometriosis: a clinical and cellular investigation

Sammanfattning: BACKGROUND; Endometriosis is a chronic inflammatory disease of unknown origin that can cause severe dysmenorrhea, chronic pain and impaired quality of life. Lignocaine has antiinflammatory properties and pertubation with lignocaine could be beneficial in the treatment of endometriosis-associated pain. The Endometriosis Health Profile-30 (EHP-30) questionnaire is specific for endometriosis and evaluates quality of life. One scale on the EHP-30 core questionnaire has earlier failed to demonstrate any responsiveness. OBJECTIVE; The objectives of the included studies were to evaluate the effect of pertubation with Ringer-Lignocaine on dysmenorrhea and quality of life in women with endometriosis. The safety and the pharmacokinetics of lignocaine after pertubation were to be investigated. Another objective was to evaluate the responsiveness of the EHP-30 questionnaire in a Swedish sample. In the final study, the objective was to evaluate the effect of lignocaine on cytokine expression and secretion in vitro in peritoneal fluid macrophages and endometriotic stromal cells. DESIGN; Study I and III were double-blinded, randomised and controlled trials. Study II and IV were prospective observational studies and the fifth study was an experimental in vitro study on human cells. SETTING; The studies were performed at three gynaecological outpatient units in Stockholm, Sweden and at the research laboratory at Uppsala Akademiska Hospital, Sweden. POPULATION; Eligible patients had endometriosis as diagnosed by laparoscopy, dysmenorrhoic pain > VAS 50 mm (visual analogue scale) and patent Fallopian tubes. 42 women were included in study I, III and IV and of these, 25 were also included in study II. Peritoneal fluid and samples from endometriotic cysts were collected from 15 women (patients with endometriosis n=9, and healthy controls n=6) during surgery for clinical reasons in study V. METHODS; The participants in study I-IV were randomised sequentially to pre-ovulatory pertubations with placebo (n=18) or lignocaine 1.0 mg/ml (n=24) during three consecutive menstrual cycles. The pertubation procedure comprised passing study solution through the uterine cavity and the Fallopian tubes via an intracervical placed balloon catheter. In study I, the effect on pain was evaluated using a VAS scale before and after the treatments and up to nine menstrual cycles after the last pertubation. Success was defined as a reduction of pain ≥ 50% on the VAS scale. Fisher’s exact test was used to compare the success rates between the treatment and the placebo groups. In study II, serum samples were collected at 0, 5, 15 and 30 minutes after pertubation. The serum samples were analysed for the concentration of lignocaine with a LCMS-SIM method. The effect on quality of life was evaluated with the EHP-30 questionnaire before and after the treatments in study III. The changes in scores from baseline to follow-up at six and twelve months were compared between the lignocaine and placebo groups with Mann Whitney U test. In study IV, the changes on the EHP-30 questionnaire after three pertubations were compared with the patients’ self-estimated change in pain intensity. The responsiveness to change for the EHP-30 questionnaire was evaluated with effect sizes and significance of change (paired t-test). The change in scores between those who improved (improved group) and those who did not improve (stable group) were compared with independent t-test. In study V, macrophages from the peritoneal fluid and cells from the inside of the endometriotic cysts capsules were isolated and cultivated for 24 h-48 h in medium with and without the supplement of lignocaine to a final concentration of 0.1 mg/ml or 1.0 mg/ml. Relative gene expression of MCP-1, IL-6 and IL-8 were evaluated with RT-PCR and compared between treated and untreated cells with Wilcoxon matched pairs. The concentration of MCP-1, IL-6 and IL-8 in cell culture media was measured using ELISA and were compared between treated and untreated cells with Wilcoxon matched pairs. RESULTS; Study I; In the Intention To Treat analysis in study I, the success rate was 41.7 % (10 of 24) in the treatment group compared to 16.7 % (3 of 18) in the placebo group (p=0.10). In the Per Protocol analysis, the proportion of subjects with success in the treatment group was 45 % (9 of 20) compared to 7.1 % (1 of 14) in the placebo group (p=0.024). Of the nine patients in the lignocaine group that fulfilled the criteria for success after three pertubations, four had an effect persisting after nine months. Study II; Low levels of lignocaine were detected in the serum samples following pertubation of 10 mg lignocaine hydrochloride. The highest observed concentration was seen after 30 minutes with a mean of 0.050 μg/ml and an individual maximum of 0.124 μg/ml. Study III; After six months there was a significant difference between the lignocaine (n=19) and the placebo (n=16) groups on the dimension social support (median -18.8 vs. -6.3, p=0.034) whereas there were no differences for the other dimensions after six or twelve months. Study IV; The change in scores were significant for all dimensions (p=0.04-0.0002) except sexual intercourse (p=0.29) for improved patients in contrast to the patients in the stable group where there were no significant changes in any dimension (p=0.16-0.63). The effect sizes were large (>0.8) on all core scales except self image (0.51) for the improved patients and small on all scales in the stable group (-0.17-0.35). There were significant differences between the improved and the stable group considering change in most of the core EHP-30 scores. Study V; The gene expression and protein secretion of IL-8 in endometriotic stromal cells after incubation with lignocaine 0.1 mg/ml was significantly decreased after 24 h compared to control (p=0.03 and p=0.02). Macrophages from healthy controls had a significantly lower gene expression of all tested cytokines (p=0.04) after treatment with lignocaine but there were no significant differences on protein level. Macrophages from patients with endometriosis (n=5) showed diverging results since three samples showed increased gene expression of one or two cytokines after lignocaine treatment. CONCLUSIONS; The first study indicates that pertubation with lignocaine is an efficient, non-hormonal treatment option for some patients with dysmenorrhea and endometriosis. The serum levels of lignocaine following pertubation of 10 mg lignocaine hydrochloride are detectable but low. Pertubation with lignocaine is safe and have no adverse events related to lignocaine. Study III indicates that pertubations with lignocaine might improve the social support dimension of quality of life in patients with endometriosis. EHP-30 is responsive to improvement on all scales on the core questionnaire and is acceptable, understandable and applicable in our Swedish sample. Lignocaine can affect the gene expression and secretion of the pro inflammatory cytokine IL-8 in vitro. Macrophages from patients with endometriosis might be dysregulated. The data presented in this thesis indicate that due to differences at cellular level, nearly half of the endometriosis population may benefit from lignocaine pertubations.