A Platform for the Generation of Site-Specific Antibody-Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines.

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an in silico screening procedure to select for optimal sites in an antibody to which a hydrophobic linker-drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker-drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker-drug while resulting ADCs maintained their cytotoxic potency in vitro. Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Oestrogens promote tumorigenesis in a mouse model for colitis-associated cancer.

BACKGROUND: Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. AIM: To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. DESIGN: We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or ß (Erα or Erß) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. RESULTS: Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erß. CONCLUSIONS: Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.

Preclinical Profile of BYON3521 Predicts an Effective and Safe MET Antibody-Drug Conjugate.

MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody-drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET-expressing cancer cell lines; in moderate and low MET-expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET-amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling.

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17ß-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17ß-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-α is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-α (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controls. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation.

High Prevalence of 5T4/Trophoblast Glycoprotein in Soft Tissue Sarcomas.

The expression of 5T4/trophoblast glycoprotein was evaluated in several histological subtypes of soft tissue sarcoma (STS) to determine whether the prevalence and level of expression of this membrane-associated glycoprotein is sufficient for use in targeted therapies. Tumor tissue microarrays containing cores from different histological subtypes of STS were stained using a standardized immunohistochemical staining method to detect 5T4; the level of staining was assessed using a semi-quantitative scoring method. No 5T4 staining was seen in the angiosarcomas and liposarcomas investigated in this study. 5T4 staining in the other STS subtypes was seen in more than 50% of cases, warranting further investigation into whether this antigen could evoke an anti-tumor immune response or can be used as target for the delivery of more potent toxins through antibody drug conjugates.

An objective and automated method for evaluating abdominal hyperalgesia in a rat model for endometriosis.

Chronic pain and subfertility are the main symptoms of concern in women with endometriosis. In order to find new therapeutic options to suppress the pain, translational animal models are indispensable. We have developed a new automated, experimental setup, with full consideration for animal wellbeing, to determine whether operant behaviour can reveal abdominal hyperalgesia in rats with surgically-induced endometriosis, in order to assess whether abdominal hyperalgesia affect behavioural parameters. Endometriosis was induced by transplantation of uterine fragments in the abdominal cavity. Control groups consisted of sham-operated rats and non-operated rats. We have developed an operant chamber (Skinnerbox) which includes a barrier. The rat can climb the barrier in order to reach the food pellet, increasing in this way the pressure to the abdomen. We show that endometriosis rats collect significantly less sugar pellets when compared with the control rats after the introduction of the barrier. In the Skinnerbox experiment, we showed that in a positive operant setting, the introduction of a barrier results in a contrast of operant behaviour of endometriosis rats and control groups, perchance as a result of abdominal discomfort/hyperalgesia due to surgically-induced endometriosis. This is a promising start for the further development of a refined animal model to monitor abdominal discomfort/hyperalgesia in rats with surgically-induced endometriosis.

Antiangiogenic and vascular-disrupting agents in endometriosis: pitfalls and promises.

It is widely known that angiogenesis plays a key role in endometriotic lesion formation and development. Antiangiogenic treatments aimed at inhibiting new vessel formation have proven efficient in experimental models. However, as antiangiogenic strategies do not target pre-existing pericyte-protected vessels, they require chronic administration and are likely to be beneficial for early-stage disease only or to prevent recurrence after surgery. Moreover, they may have detrimental effects on reproductive function. Vascular-disrupting agents (VDAs) have emerged as a promising new tool for the treatment of tumors. VDAs target established blood vessels, resulting in tumor ischemia and necrosis. These agents may therefore be more efficient against advanced disease. Two major types of VDAs are being developed for cancer: ligand-directed VDAs using antibodies, peptides and growth factors to deliver toxic effectors to tumor endothelium; and small-molecule VDAs exploiting physiological differences between tumor and normal endothelium to induce acute vascular shutdown. The ongoing evolution in genomics and proteomics is revolutionizing the discovery of novel endothelial markers. Several studies suggest that the vasculature of endometriotic lesions may have particular pathophysiological properties, which could be exploited for the development of selective VDAs. The aim of this review is to explore the merits and limitations of vascular therapy for the treatment of endometriosis.

Oral contraceptives prevent the development of endometriosis in the chicken chorioallantoic membrane model.

BACKGROUND: Fundamental and genetic differences between women in the endometrium may cause some to develop endometriosis, whereas others do not. Oral contraceptives (OC) may have an effect on the endometrium, rendering the development of endometriosis less likely. STUDY DESIGN: Endometrium from women using OC (OCE) and menstrual endometrium (ME) from normal cycling women were transplanted onto the chicken chorioallantoic membrane (CAM), and endometriosis-like lesion formation was evaluated. Microarray gene expression profiling was performed to identify differentially expressed genes in the endometrium from these groups. Microarray data were validated by real-time PCR. RESULTS: Less endometriosis-like lesions were formed after transplantation of OCE than after transplantation of ME (p<.05). Most of the differentially expressed genes belong to the TGFbeta superfamily. Real-time PCR validation revealed that inhibin betaA (INHBA) expression was significantly decreased in OCE as compared to ME. CONCLUSION: OC use affects the characteristics of endometrium, rendering it less potent to develop into endometriosis.

Is it time for a paradigm shift in drug research and development in endometriosis/adenomyosis?

BACKGROUND: The drug research and development (R&D) for endometriosis/adenomyosis has been painfully slow. Most completed clinical trials on endometriosis did not publish their results, and presumably failed. While few published trials did report how they foundered, the reasons why they failed are often completely unclear. Surprisingly, there has been no open discussion on why these trials failed. If the causes for these failed trials remain unelucidated, mistakes made in these failed trials may be repeated in the future. Since failure can be infinitely more instructive and educational than success, elucidating the causes for failed clinical trials may yield a treasure trove for future drug R&D. Given our growing understanding of the natural history of ectopic endometrium, it is also important to make an inventory of biologicals/compounds that are currently under development to see where we stand and whether they would stand a better chance of gaining regulatory approval than their predecessors. OBJECTIVE AND RATIONALE: We provide an overview of all compounds under clinical investigation and in development in order to assess the evolution of R&D since the last inventory, reported in 2013. We also have attempted to analyse selected failed clinical trials in the context of published translational/preclinical research and our growing understanding of the natural history of endometriotic/adenomyotic lesions, in the hope that the lessons learned will steer investigators toward the right track in future drug R&D. SEARCH METHODS: We searched ClinicalTrials.gov and a database containing information on drugs gathered daily by Thomson Reuters from a wide range of sources (e.g. patent offices, biomedical literature, congresses, symposia, meetings, company information, regulatory information) for all therapeutic compounds that have undergone or are under clinical trials, or in the developmental stage, and then searched PubMed and Google to determine their publication status using trial identifiers. For trials that were completed at least 2 years ago and have, or have not, published their results, a PubMed search was performed using the name of the therapeutic that has been tested and 'endometriosis' or 'adenomyosis' to identify published preclinical studies prior to the launch of the trial. For those published trials, the cited preclinical studies were also retrieved and scrutinized. OUTCOMES: Despite repeated calls for more transparency, only a small fraction of completed trials on endometriosis has been published. A large number of 'novel' compounds under development are simply repurposed drugs, which seem to be ill-prepared to combat the fibroproliferative nature of endometriosis/adenomyosis. This sobering picture indicates an alarming innovation 'drought' in the drug R&D front, resulting in trickling drug pipelines. Some trials foundered owing to unanticipated serious side-effects, or because attempts were made to suppress a target that can be compensated for by redundant pathways, but many failed in efficacy, indicating that the translational value of the current models is seriously questionable. All existing animal models of endometriosis do not recapitulate the key features of human conditions. WIDER IMPLICATIONS: The glaring innovation drought in drug R&D for endometriosis/adenomyosis should sound alarms to all stake-holders. The failed clinical trials in endometriosis also indicate that some past research had serious deficiencies. In light of the recent understanding of the natural history of ectopic endometrium, it is perhaps time to shift the research paradigm and revamp our research focus and priorities.

Drug Development in Endometriosis and Adenomyosis: It Takes More Than Just Good Science.

The pipelines of pharmaceutical companies are filled with thousands of promising new compounds for a plethora of indications. Yet, a close review of the drugs that have recently been in clinical trials quickly reveals that only a handful of drugs under evaluation in women with endometriosis can be genuinely qualified as truly innovative and breakthrough drugs. Why is there such an industry-wide lukewarm interest in drug research and development for endometriosis/adenomyosis? Why are pharmaceutical companies so reluctant to initiate programs or invest in academic research in endometriosis/adenomyosis? It is evident that a substantial part of the novel druggable targets originate from research in academia. However, only the pharmaceutical industry has the resources and expertise to bring drugs to patients. In other words, we are fully dependent on the pharmaceutical industry to bring new therapeutics to the market. The aim of this editorial is to make scientists from academia aware of the enormous complexity of the drug development process, the drivers that propel pharmaceutical companies to initiate new programs and to prioritize their portfolios, the value of intellectual property rights, and also about the importance of scientific rigor, predictive translational models, and biomarkers. At the same time, the pharmaceutical industry must be made aware of the enormous opportunity at hand, as the current patient population with endometriosis/adenomyosis is just the tip of the iceberg. We hope that the insights presented here will enable the endometriosis/adenomyosis research community to find ways to valorize their knowledge and attract the interest of the industry.

Unraveling the Interaction between Carboxylesterase 1c and the Antibody-Drug Conjugate SYD985: Improved Translational PK/PD by Using Ces1c Knockout Mice.

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody-drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c-/- SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c-/- SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389-98. ©2018 AACR.

Role and regulation of the serum- and glucocorticoid-regulated kinase 1 in fertile and infertile human endometrium.

Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.

The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone.

The human progesterone receptor (PR) is a ligand-dependent transcription factor and two isoforms, (PRA and PRB), can be distinguished. PROGINS, a PR polymorphic variant, affects PRA and PRB and acts as a risk-modulating factor in several gynaecological disorders. Little is known about the functional consequences of this variant. Here, we characterise the properties of PROGINS with respect to transcription, mRNA maturation, protein activity and proliferation. PROGINS is characterised by a 320 bp PV/HS-1 Alu insertion in intron G and two point mutations, V660L in exon 4 and H770H (silent substitution) in exon 5. The Alu element contains a half oestrogen-response element/Sp1-binding site (Alu-ERE/Sp1), which acts as an in-cis intronic enhancer leading to increased transcription of the PROGINS allele in response to 17beta-oestradiol. Moreover, Alu insertions in the human genome are frequently methylated. Our data indicate that the PROGINS-Alu does not affect gene transcription due to DNA methylation. However, the Alu element reduced the stability of the PROGINS transcript compared with the CP allele and does not generate splice variants. The amino acid substitution (V600L) in exon 4 leads to differences in PR phosphorylation and degradation in the two PR variants upon ligand binding, most likely as a result of differences in the three-dimensional structures of the two PR variants. As a consequence, the PR-L660 (PROGINS) variant (1) displays decreased transactivation activity in a luciferase reporter system and (2) is less efficient in opposing cell proliferation in hamster ovarian cells expressing human PRA, when compared with the PR-V660 (most common variant). Taken together, our results indicate that the PROGINS variant of PR is less responsive to progestin compared with the most common PR because of (i) reduced amounts of gene transcript and (ii) decreased protein activity.

Endometriosis is associated with aberrant metabolite profiles in plasma.

OBJECTIVE: To identify metabolites that are associated with and predict the presence of endometriosis. DESIGN: Metabolomics study using state-of-the-art mass spectrometry approaches. SETTING: University hospital and universities. PATIENT(S): Twenty-five women with laparoscopically confirmed endometriosis (cases) and 19 women with laparoscopically documented absence of endometriosis (controls). None of the women included in this study had received oral contraception or GnRH agonists for a minimum of 1 month before blood collection. INTERVENTION(S): Plasma collection. MAIN OUTCOME MEASURE(S): Metabolite profiles were generated and interrogated using multiple mass spectrometry methods, that is, high performance liquid chromatography coupled with negative mode electrospray ionization tandem mass spectrometry, UPLC-MS/MS, and ultra performance liquid chromatography-electroSpray ionization-quadrupole time-of-flight (UPLC-ESI-Q-TOF). Metabolite groups investigated included phospholipids, glycerophospholipids, ether-phospholipids, cholesterol-esters, triacylglycerol, sphingolipids, free fatty acids, steroids, eicosanoids, and acylcarnitines. RESULT(S): A panel of acylcarnitines predicted the presence of endometriosis with 88.9% specificity and 81.5% sensitivity in human plasma, with a positive predictive value of 75%. However, due to data limitations the outcome of the receiver operating characteristic curve analysis was not significant. CONCLUSION(S): A diagnostic model based on acylcarnitines has the potential to predict the presence and stage of endometriosis.

Angiostatic agents prevent the development of endometriosis-like lesions in the chicken chorioallantoic membrane.

A prospective study was performed to determine the effects of the angiostatic compounds anti-hVEGF antibody, TNP-470, endostatin, and anginex on the vascularization and on endometriosis-like lesion formation in the chicken chorioallantoic membrane model. Endometriosis-like lesion formation was significantly impaired after treatment with angiostatic agents, which was associated with decreased vessel densities in the surrounding chorioallantoic membrane and more necrosis in the endometriosis-like lesions.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers.

SYD985 is a HER2-targeting antibody-drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need.

Antiangiogenesis therapy for endometriosis.

It is known that angiogenesis is of pivotal importance for the development of endometriosis. However, in the treatment of endometriosis patients, prevention of endometriosis lesion development only will not be sufficient as a therapy. Treatment options, aimed at interfering with established lesions, have to be developed. In this study we evaluated whether inhibition of angiogenesis by angiostatic therapy is also effective in antagonizing the sustentation of endometriosis. We evaluated the effect of the angiostatic compounds antihuman vascular endothelial growth factor, TNP-470, endostatin, and anginex on the growth of established endometriosis lesions in the nude mouse model. We show that human endometrium in the proliferative endometrium is highly angiogenic and that vascular endothelial growth factor-A is the most important angiogenesis promotory factor. The angiostatic compounds significantly decreased microvessel densities and the number of established endometriosis lesions. In the remaining lesions, the number of pericyte-protected vessels is not different in control and treated mice; however, the number of unprotected vessels was significantly reduced in the groups treated with the angiostatic agents. Our data demonstrate that inhibitors of angiogenesis effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis. This suggests that the use of angiostatic agents may be promising as a therapy for endometriosis.

Pathogenesis of endometriosis.

Many women harbour spots of peritoneal endometriosis without having any symptoms; this is referred to as the phenomenon endometriosis. Some of these women go on to develop symptomatic endometriosis. Although we know the factors potentially involved in the aetiology and pathogenesis of endometriosis, the exact mechanism by which the phenomenon endometriosis develops into the disease endometriosis, with its associated signs and symptoms, remain obscure. The widely accepted theory is Sampson's transplantation theory. Recent findings indicate that certain properties of the endometrium, and the influence of the local environment, are crucial in the development of endometriosis. Early endometriosis lesion formation is described in detail, as this seems to be a key process in the development of peritoneal endometriosis.