Association of oral lichen planus with hepatic disorders and hepatocellular carcinoma: systematic review and meta-analysis.

BACKGROUND: Oral lichen planus (OLP) is a prevalent autoimmune chronic inflammatory disease of unknown etiology. The importance of the association between hepatic disease and OLP lies in the fact that many of these disorders (HC, HB, cirrhosis, hepatic steatosis) behave as risk factors for hepatocellular carcinoma. MATERIAL AND METHODS: We searched PubMed, Embase, Web of Science, and Scopus for studies published before January 2022. We evaluated the quality of studies (Joanna Briggs Institute tool). We performed meta-analyses, investigated the heterogeneity between studies, and we also carried out subgroups, meta-regression, and small-study effects analyses. 146 studies (21,187 patients) were included in this study. Our study aims to evaluate current evidence on the prevalence and magnitude of association between hepatic diseases (especially those with risk of malignancy), hepatocellular carcinoma and OLP. RESULTS: Our results suggest that patients with OLP present a significant tendency to the development of hepatitis B (OR=1.62, 95%CI=1.01-2.40, p=0.02), hepatitis C (OR=4.09, 95%CI=2.77-6.03, p<0.001), cirrhosis (OR=5.58, 95%CI=1.83-16.96, p=0.002), hepatic steatosis (OR=5.71, 95%CI=0.97-33.60, p=0.05) and hepatocellular carcinoma (OR=3.10,95%CI=1.14-8.43, p=0.03). CONCLUSIONS: Patients with OLP should be investigated to rule out the presence of hepatic disease, which can lead to hepatocellular carcinoma, allowing an early diagnosis that would help to a better approach to liver disease and a notable improvement in prognosis in terms of both progression and severity.

Dysplasia in oral lichen planus: relevance, controversies and challenges. A position paper.

BACKGROUND: Patients with oral lichen planus (OLP) have an increased risk of oral cancer. For this reason, OLP is classified as an oral potentially malignant disorder. However, the precise personal (or individual) risk is unknown. Recent meta-analytical studies have reported that dysplastic OLP may transform to cancer in around 6% of cases, while the rate of transformation is lower (<1.5%) in non-dysplastic cases. The presence of epithelial dysplasia has emerged as the most powerful indicator for assessing cancer risk in oral potentially malignant disorders in routine practice. However, the general acceptance of epithelial dysplasia as an accompanying histologic feature in OLP is subject to great controversy. Many pathologists consider the presence of dysplasia as a criterion to exclude OLP when routinely reporting on this disease. This practice, widespread among oral pathology professionals, has resulted in the underestimation of the potential for malignancy of OLP. MATERIAL AND METHODS: A review of the literature was carried out in order to critically analyze the relevance, controversies and challenges encountered across the diagnosis of epithelial dysplasia in OLP. RESULTS: 12 studies have been published examining dysplastic changes in OLP, reporting figures ranging from 0.54% to 25% of cases with dysplasia in the first diagnostic biopsy. The diagnosis of dysplasia in the OLP poses an additional difficulty due to the fact that the affected oral epithelium per se develops changes related to autoimmune aggression. Among the most frequent histological features of OLP that develops dysplasia are basal cell hyperplasia with basaloid appearance, loss of basal cells polarity, cellular and nuclear pleomorphism and irregular stratification. CONCLUSIONS: Epithelial dysplasia should not be considered an exclusion criterion for OLP; its evaluation requires experienced pathologists in this field.

Significance of cytoplasmic cyclin D1 expression in oral oncogenesis.

OBJECTIVES: To examine cytoplasmic cyclin D1 expression levels in oral carcinogenesis and evaluate their possible oncogenic significance and their clinicopathological and prognostic implications. MATERIALS AND METHODS: Immunohistochemical analysis of 69 oral squamous cell carcinomas (OSCCs) was performed, revealing 23 with cytoplasmic cyclin D1 expression. We analyzed the association of the percentage of cyclin D1-positive cells and the intensity of expression with TNM classification, tumor stage, differentiation degree, cell morphology, and Ki-67 expression. RESULTS: Cytoplasmic cyclin D1 expression was associated with advanced tumor stage, poor differentiation, elevated Ki-67 expression, and the presence of invasive cell morphology, indicators of a poor prognosis. An association was observed between nuclear and cytoplasmic expressions of cyclin D1. CONCLUSIONS: Cytoplasmic expression of cyclin D1 appears to possess functions related to increased cell migration and invasion in OSCC.

Outcomes of oral lichen planus and oral lichenoid lesions treated with topical corticosteroid.

OBJECTIVE: To determine corticosteroid treatment effectiveness in patients with oral lichen planus/oral lichenoid lesions (OLP/OLL). MATERIAL AND METHODS: Twenty-one patients with OLP and eighty-one patients with OLL received 0.05% clobetasol propionate (CP) or 0.05% triamcinolone acetonide (TA) in aqueous solution (AS) or orabase (OB), evaluating responses to treatment and follow-up compliance. RESULTS: Lesions were atrophic (72 of 102; 70.6%), extensive (58 of 100; 58%), producing eating difficulties (62 of 102; 60.8%), and spontaneous pain (30 of 102; 29.4%); 50 patients (49%) received CP-AS. The mean ± SD percentage of follow-ups attended was 43 ± 32%. Symptom remission was achieved in 46% of patients receiving CP-AS, 36.36% of those receiving TA-AS, 20% of those receiving CP-OB, and 25% of those receiving TA-OB. Follow-up compliance was poor in 66.7% of patients. Among 51 patients with continuous symptoms, 64.7% evidenced total remission at treatment completion; among 33 with intermittent symptoms, 73.1% had outbreaks 2-3 times/year and 51.5% controlled outbreaks with <6 corticosteroid applications. Adverse effects were observed in seven patients (6.8%) (moon face, hirsutism, capillary fragility) in induction stage, subsiding with dose; among 15 patients under maintenance treatment for >6 months, one showed hypothalamic-pituitary-adrenal (HPA) axis inhibition but not adrenal insufficiency. CONCLUSIONS: Our treatment proved highly effective and safe. Recall programs are desirable to enhance follow-up compliance.

Is oral cancer incidence among patients with oral lichen planus/oral lichenoid lesions underestimated?

BACKGROUND: Oral lichen planus (OLP) and oral lichenoid lesions (OLL) are considered potentially malignant disorders with a cancer incidence of around 1% of cases, although this estimation is controversial. The aim of this study was to analyze the cancer incidence in a case series of patients with OLP and OLL and to explore clinicopathological aspects that may cause underestimation of the cancer incidence in these diseases. METHODS: A retrospective study was conducted of 102 patients diagnosed with OLP (n = 21, 20.58%) or OLL (n = 81) between January 2006 and January 2016. Patients were informed of the risk of malignization and followed up annually. The number of sessions programmed for each patient was compared with the number actually attended. Follow-up was classified as complete (100% attendance), good (75-99%), moderate (25-74%), or poor (<25% attendance) compliance. RESULTS: Cancer was developed by four patients (3.9%), three males and one male. One of these developed three carcinomas, which were diagnosed at the follow-up visit (two in lower gingiva, one in floor of mouth); one had OLL and the other three had OLP. The carcinoma developed in mucosal areas with no OLP or OLL involvement in three of these patients, while OLP and cancer were diagnosed simultaneously in the fourth. Of the six carcinomas diagnosed, five (83.3%) were T1 and one (16.7%) T2. None were N+, and all patients remain alive and disease-free. CONCLUSIONS: The cancer incidence in OLP and OLL appears to be underestimated due to the strict exclusion criteria usually imposed.

An update on the implications of cyclin D1 in oral carcinogenesis.

Cyclin D1 promotes cell cycle progression during G1 phase, a key event in G1-S transition. The protein is encoded by gene CCND1, located in chromosomal band 11q13. Cyclin D1 plays key roles in cell biology, including cell proliferation and growth regulation, mitochondrial activity modulation, DNA repair, and cell migration control. CCND1 gene and its protein cyclin D1 are frequently altered by different molecular mechanisms, including amplification, chromosomal translocations, mutations, and activation of the pathways involved in cyclin D1 expression, alterations which appear to be essential in the development of human cancers, including oral carcinoma. This is the first published review of the specific features of cyclin D1 overexpression in oral oncogenesis. Starting with the physiological regulation of cyclin D1, there is an evaluation of its functions, overexpression mechanisms, and the implications of the oncogenic activation of CCND1/cyclin D1 in oral squamous cell carcinoma. The potential diagnostic and prognostic value of cyclin D1 is reviewed. The influence of CCND1/cyclin D1 on tumor size and clinical stage is reported, and an update is provided on the utilization of cyclin D1 as therapeutic target and on the combination of cyclin D1 inhibitors with cytotoxic agents. Future research lines in this field are also proposed.

Asymmetrical proliferative pattern loss linked to cyclin D1 overexpression during malignant transformation of the lip epithelium.

BACKGROUND: There is inadequate knowledge on the involvement of oncogenic mechanisms linked to the cyclin (CCND1) gene in lip squamous cell carcinoma (LSCC). OBJECTIVE: The aim of this study was to analyse the implication of cyclin D1 in the malignant transformation of lip lesions. METHODS: We immunohistochemically studied 45 actinic cheilitis cases (15 mild dysplasia, 15 moderate dysplasia, 15 severe dysplasia/carcinoma in situ), 30 LSCC cases with adjacent non-tumour epithelium and 15 normal oral epithelium samples for detection of cyclin D1, ß-catenin and Ki-67. RESULTS: Cyclin D1 and Ki-67 expressions were significantly increased in the basal layer of premalignant epithelia and peripheral layers of tumour nests vs. CONTROLS: Premalignant epithelia had lost their asymmetrical proliferative pattern. CONCLUSION: Lip carcinogenesis was associated with loss of the asymmetrical proliferative pattern, a preventive mechanism against lip oncogenesis, and with cyclin D1 overexpression.

Is periodontitis a risk factor for cognitive impairment and dementia? A case-control study.

BACKGROUND: Dementia is a multi-etiologic syndrome characterized by multiple cognitive deficits but not always by the presence of cognitive impairment. Cognitive impairment is associated with multiple non-modifiable risk factors but few modifiable factors. Epidemiologic studies have shown an association between periodontitis, a potentially modifiable risk factor, and cognitive impairment. The objective of this study is to determine whether clinical periodontitis is associated with the diagnosis of cognitive impairment/dementia after controlling for known risk factors, including age, sex, and education level. METHODS: A case-control study was conducted in Granada, Spain, in two groups of dentate individuals aged >50 years: 1) cases with a firm diagnosis of mild cognitive impairment or dementia of any type or severity and 2) controls with no subjective memory loss complaints and a score >30 in the Phototest cognitive test (screening test for cognitive impairment). Periodontitis was evaluated by measuring tooth loss, plaque and bleeding indexes, probing depths, and clinical attachment loss (AL). RESULTS: The study included 409 dentate adults, 180 with cognitive impairment and 229 without. A moderate and statistically significant association was observed between AL and cognitive impairment after controlling for age, sex, education level, oral hygiene habits, and hyperlipidemia (P = 0.049). No significant association was found between tooth loss and cognitive impairment. CONCLUSION: Periodontitis appears to be associated with cognitive impairment after controlling for confounders such as age, sex, and education level.

Asymmetrical proliferative pattern loss during malignant transformation of the oral mucosa.

OBJECTIVES: The aim of this study was to study the loss of asymmetrical proliferation in oral tumorigenesis. MATERIALS AND METHODS: Samples: 92 oral squamous cell carcinomas (OSCC) with associated non-tumor epithelia (NTE). NTE and tumor were classified as distant from or close to the invasion point. Immunohistochemistry was performed using Mib-1 antibody. Ki-67 was assessed in basal, parabasal layer, medium and upper third, counting total and positive cells. Proliferative patterns were classified according to the ki-67 expression: 1 = expression in parabasal layers of well-differentiated tumor nest (WDTN); 2 = expression in parabasal and basal layers of WDTN; 3 = ki-67 expression in <20% cells in tumor tissue without WDTN; 4 = ki-67 expression in ≥20% of cells in tumor tissue without WDTN; and 5 = ki-67 expression exclusively found in basal layers of WDTN. RESULTS: Ki-67 expression was highest in parabasal layers of both close and distant NTE (39.7 ± 27.6 and 30.1 ± 20) and was also elevated in the close (43.4 ± 21.3) and distant (48.8 ± 21.9) tumor tissue samples. Close tumors largely corresponded to proliferation patterns 2 and 4, while distant tumors generally followed pattern 4. Of the 92 close NTE samples, 23 showed reduced basal proliferation with increased parabasal proliferation. Tumors derived from these epithelia followed patterns 2 (52%, 12/23 cases) or 4 (30.4%, 7/23 cases). Parabasal proliferation in distant NTE was significantly increased in patients with multiple vs. single tumors (36.7% vs. 25.4%; P = 0.032). CONCLUSION: The change from asymmetrical to symmetrical division appears to be an oncogenic mechanism in oral carcinogenesis.

ß-catenin in oral cancer: an update on current knowledge.

ß-Catenin is a multiple function protein. These functions derive from its interactions with other cell proteins, both on the cell membrane, in the cytoplasm and in the nucleus. ß-Catenin forms a complex with the adhesion molecule E-cadherin, promoting cell-cell adhesion and thereby preventing the cell dissociation that is required for cancer invasion and progression mechanisms. There is also a dynamic pool of cytoplasmic ß-catenin that serves as connection between the extracellular microenvironment and the nucleus. Cytoplasmic ß-catenin acts as a transcription factor for the nucleus in the canonical Wnt pathway, activating the transcription of various genes. Structural or functional alterations of ß-catenin can promote cancer progression. This review addresses the current knowledge on the implications of ß-catenin in the development of oral cancer.

Atraumatic restorative treatment and Carisolv use for root caries in the elderly: 2-year follow-up randomized clinical trial.

OBJECTIVES: New preventive and treatment strategies are required to address the high prevalence of caries among the elderly. The main objective of this study was to analyze the effectiveness of Carisolv® gel to improve the clinical behavior of restorations obtained by atraumatic restorative treatment (ART) in root caries of elderly patients. A secondary objective was to determine the factors associated with the failure of ART restorations after a 2-year follow up. MATERIAL AND METHODS: A randomized controlled trial with 2-year follow-up was designed for this purpose. Candidate caries lesions were randomly assigned to an ART group for root caries treatment with the conventional ART technique, filling with glass ionomer, or an ART + Carisolv® gel for the same ART plus the application of a caries solvent (Carisolv®). Evaluations were conducted at 6, 12, and 24 months. RESULTS: A total of 81 restorations were performed, 37 in the ART group and 44 in the ART + Carisolv® gel group, with 22 and 26 restorations, respectively, surviving at the end of the 24-month follow-up. Survival rates at 24 months did not significantly differ between ART (63 %) and ART + Carisolv® gel (62 %) restorations. The best model for predicting the failure of the restorations included the number of tooth-brushings/day, the presence or not of prosthesis, the anterior or posterior location of the tooth, and the baseline plaque level. CONCLUSION: The application of Carisolv does not modify the survival rate of ART restorations in elderly patients. CLINICAL RELEVANCE: The use of Carisolv gel does not improve the outcomes of atraumatic restorative treatment.

The cancer stem cell hypothesis applied to oral carcinoma.

It has been proposed that the development of tumors is based exclusively on the activity of cancer stem cells (CSCs) leading to a new model of carcinogenesis, the CSC hypothesis, in opposition to the conventional model of clonal evolution. The new model may help to explain the high mortality of oral cancer, unchanged over the past decades, the low response to treatment and the tendency of oral squamous cell carcinoma (OSCC) patients to develop multiple tumors. However, a more profound understanding of the molecular pathways involved in maintaining the stem cell (SC) state and of their alterations is required to elucidate the mechanisms underlying the development of tumors and metastatic spread, but research into SC biopathology is hampered by the lack of specific markers for identifying SCs and CSCs in tissues and for establishing topographic relationships with their lineage. We review current knowledge on stem cells in relation to oral cancer, including their possible origins, focusing on the CSC hypothesis of oral tumorigenesis and attempts being made to identify oral stem cells.

Expression of proliferative markers in ameloblastomas and malignant odontogenic tumors.

OBJECTIVE: To compare the proliferative activity in ameloblastoma and malignant odontogenic tumors, as assessed by Ki-67 immunostaining and determine whether expression of substance P (SP) and NK-1 receptor (NK-1R) is related to cell proliferation in these tumors. MATERIALS AND METHODS: Immunohistochemistry was used to evaluate protein expression in 44 benign and malignant odontogenic tumors from 39 patients. Immunohistochemistry was performed with anti-SP, anti-NK-1R, and anti-Ki-67 monoclonal antibodies, and the clinical and pathological data of the patients with odontogenic tumor were evaluated. RESULTS: Expression of Ki-67 in malignant odontogenic tumors was significantly higher than in ameloblastomas (P < 0.001), and the expression level was associated with higher expression of NK-1R. Among the ameloblastomas, there was significantly higher expression of Ki-67 in peripheral ameloblastic-like cells (3.3 ± 4.1) than in stellate reticulum-like cells (2.6 ± 3.7) (P = 0.04). In the majority of tissue locations of the malignant tumors, expression of SP and NK-1R was positively correlated with higher expression of Ki-67. CONCLUSION: These findings show that the expression level of Ki-67 in ameloblastomas was positively correlated with the rate of growth of odontogenic tumors. Overexpression of NK-1R complex in malignant odontogenic tumors could be part of the trigger stimulus that results in higher proliferative activity of the tumor.

E-cadherin in non-tumor epithelium adjacent to oral cancer as risk marker for the development of multiple tumors.

Our aim was to find out whether the loss of E-cadherin is a risk factor for the development of multiple tumours in the oral cavity and whether it could serve as a diagnostic marker for oral premalignant fields. We studied 77 oral squamous cell carcinomas (SCC) with associated non-tumour epithelia from 61 patients. Immunohistochemical studies (antibody NHC-38) were used to investigate E-cadherin expression, which was completely lost in basal (48% of cases) and parabasal (43%) layers of non-tumour epithelia close to the tumour and in basal (47%) and parabasal (38%) layers of non-tumour epithelia distant from the tumour. In multiple tumours E-cadherin expression was significantly lower than in single tumours in the basal, parabasal layers, and the middle third of close (p=0.002, <0.001, <0.001) and distant (p=0.041, p<0.001, p=0.005) non-tumour epithelia, respectively. Downregulation of E-cadherin may be valuable as a risk marker for the development of multiple tumours in the oral cavity and for the diagnosis of premalignant fields.

Molecular findings in oral premalignant fields: update on their diagnostic and clinical implications.

The development of multiple oral tumours, seen in up to 30% of patients with a primary oral squamous cell carcinoma, is sometimes attributable to the presence of genetically altered premalignant fields and has important prognostic implications. Molecular techniques available for the definitive diagnosis of such a field (loss of heterozygosity analysis of 3p, 9p and 17p and study of TP53 tumour suppressor gene mutation) are expensive, complex and not universally available, hampering their routine application. Nevertheless, molecular diagnosis is essential for modern assessment of the risk of multiple tumours and for decisions on the appropriate preventive and therapeutic approaches. This article reviews current knowledge on molecular findings in premalignant fields in the oral cavity and oropharynx and provides an update on criteria for their identification, discussing the clinical and therapeutic implications.

Cell apoptosis and proliferation in salivary glands of Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome (SS) occurs associated with parotid neoplasm, non-Hodgkin's B-cell lymphoma, which could impair the condition or be life-threatening for patients. The aim of this work was to analyze cell proliferation and apoptosis modifications in acinar, ductal and inflammatory infiltrate in salivary glands (SG) in patients with Sjögren Syndrome, keratoconjunctivitis, or stomatitis sicca or in healthy subjects, to establish parameters that indicate the likelihood of malignancy of the disease in populations at risk. METHODS: A study was performed with n = 58 histological samples of lower lip SG from patients diagnosed with SS, keratoconjunctivitis, or stomatitis sicca (SICCA) and from healthy subjects (C). Ki67 and caspase-3 immunolabeling were performed. RESULTS: The most important result was significant differences between the three study groups in Ki67 and caspase-3 markers (P < 0.0001) in infiltrated lymphocytes. CONCLUSION: The results of this work are indicative of a high degree of proliferation (85%) in infiltrated lymphocytes (IL) associated with SS which, according the literature, could be considered a risk. Furthermore, the markers used in this work are widely known and represent a lower cost than others and can be used to determine risk groups within the population of SS patients, enabling their follow-up.

Methylation in oral cancer and pre-cancerous lesions (Review).

There is considerable interest in the analysis of epigenetic alterations in cancer, including oral cancer and pre-cancerous lesions. These processes affect or inactivate the functions of genes without altering their structure or sequence. One example is the methylation of the promoter region of some genes involved in cell cycle control. Knowledge of methylation patterns is very important for understanding the expression of genes in normal and pathological situations. This review provides an update on research into this issue in oral cancer and pre-cancerous lesions. A greater understanding of this epigenetic alteration could not only assist the diagnosis and prognosis of oral cancer but could also open up novel therapeutic approaches. The presence of methylation in specific tumour suppressor genes could modify their function and alter cell cycle control, so the patients could have an increased risk of developing cancer and also a higher degree of malignancy. The most frequently and extensively studied methylated genes in oral premalignant lesions are p16, MGMT, RARß2, E-cadherin and DAP-kinase.

Analysis of Ki-67 expression in oral squamous cell carcinoma: why Ki-67 is not a prognostic indicator.

OBJECTIVES: To analyze the prognostic value of Ki-67 in oral cancer and its relationship with Ki-67 expression in precancerous epithelium. MATERIAL AND METHODS: We studied 79 tumors from 65 patients. Immunohistochemistry study with Mib-1 monoclonal antibody was used to detect Ki-67 expression in tumor tissue and adjacent non-tumor tissue. The influence of different variables on survival was studied with univariate and multivariate analyses. RESULTS: Ki-67 expression was significantly higher in well-differentiated versus poorly-differentiated carcinomas. The survival time of these patients was affected by the clinical presentation, T, N, stage, and surgical treatment. Ki-67 expression had no impact on survival. An association was found between the parabasal expression of Ki-67 in adjacent non-tumor epithelium and Ki-67 expression in the tumor. CONCLUSIONS: Ki-67 lacks prognostic value, probably because it is a marker of the total fraction of proliferating cells, corresponding not only to cells in constant proliferation but also to proliferating cells destined for terminal differentiation.

Potential role of HDAC inhibitors in cancer therapy: insights into oral squamous cell carcinoma.

Histone deacetylases (HDACs) are enzymes that are involved in the remodeling of chromatin and play a key role in the epigenetic regulation of gene expression. In addition, the activity of non-histone proteins can be regulated through HDAC-mediated hypoacetylation. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and several classes of HDAC inhibitors have been found to have potent and specific anti-cancer activities in preclinical studies. Many researchers have highlighted the need to determine specific HDAC isotypes related to different tumor types in order to develop selective drugs for specific isoforms and avoid side effects. We summarize recent advances in the understanding of the molecular events that underlie the epigenetic changes in different tissue carcinomas, focusing on oral squamous carcinoma. The role of epigenetics in oral squamous cancer remains poorly understood, and further descriptive studies of specific HDAC member alterations are required to form the basis for future clinical trials.

HPA-suppressive effects of aqueous clobetasol propionate in the treatment of patients with oral lichen planus.

BACKGROUND: Oral topical corticosteroids have potential to produce inhibition of the hypothalamus-pituitary-adrenal (HPA) axis. OBJECTIVE: To assess whether clobetasol propionate (CP) in aqueous solution causes HPA inhibition. PATIENTS AND METHODS: Sixty-two patients with oral lichen planus or oral lichenoid lesions presenting with severe lesions were treated with topical oral 0.05% CP plus 100,000 IU/cm(3) nystatin in aqueous solution. Initial treatment of three 5-min mouthwashes (10 mL) daily was reduced, when the response was deemed complete or excellent, to a maintenance treatment of one 5-min mouthwash on alternate days for 6 months; treatment was then withdrawn and patients were followed up for 1 year. HPA function was assessed by plasma cortisol measurement and adrenocorticotropin (ACTH) stimulation at the end of the initial and maintenance treatment regimens. RESULTS: The HPA axis was more frequently inhibited during initial (53/62; 85.5%) vs. maintenance (2/49; 4%) regimens of aqueous CP. LIMITATIONS: In patients with morning plasma cortisol levels between 3 and 18 microg/dL, a normal result for the ACTH stimulation test only moderately reduces the possibility that a patient has secondary adrenal insufficiency. This can be considered a minor limitation in our study, as only three patients required additional assessment with the ACTH stimulation test. CONCLUSIONS: Hypothalamus-pituitary-adrenal inhibition is substantial during initial treatment with aqueous CP three times daily.