Clinical Pharmacology Perspective on Development of Adeno-Associated Virus Vector-Based Retina Gene Therapy.

Adeno-associated virus (AAV) vector-based gene therapy is an innovative modality being increasingly investigated to treat diseases by modifying or replacing defective genes or expressing therapeutic entities. With its unique anatomic and physiological characteristics, the eye constitutes a very attractive target for gene therapy. Specifically, the ocular space is easily accessible and is generally considered "immune-privileged" with a low risk of systemic side effects following local drug administration. As retina cells have limited cellular turnover, a one-time gene delivery has the potential to provide long-term transgene expression. Despite the initial success with voretigene neparvovec (Luxturna), the first approved retina gene therapy, there are still challenges to be overcome for successful clinical development of these products and scientific questions to be answered. The current review paper aims to integrate published experience learned thus far for AAV-based retina gene therapy related to preclinical to clinical translation; first-in-human dose selection; relevant bioanalytical assays and strategies; clinical development considerations including trial design, biodistribution and vector shedding, immunogenicity, transgene expression, and pediatric populations; opportunities for model-informed drug development; and regulatory perspectives. The information presented herein is intended to serve as a guide to inform the clinical development strategy for retina gene therapy with a focus on clinical pharmacology.

Machine learning-guided covariate selection for time-to-event models developed from a small sample of real-world patients receiving bevacizumab treatment.

Therapeutic outcomes in patients with metastatic colorectal cancer (mCRC) receiving bevacizumab treatment are highly variable, and a reliable predictive factor is not available. Progression-free survival (PFS) and overall survival (OS) were recorded from an observational, prospective study after 5 years of follow-up, including 46 patients with mCRC receiving bevacizumab treatment. Three vascular endothelial growth factor (VEGF)-A and two intercellular adhesion molecule-1 genes polymorphisms, age, gender, weight, dosing scheme, and co-treatments were collected. Given the relatively small number of events (37 [80%] for the PFS and 26 [57%] for the OS), to study the effect of these covariates on PFS and OS, a covariate analysis was performed using statistical and supervised machine learning techniques, including Cox regression, penalized Cox regression techniques (least absolute shrinkage and selection operator [LASSO], ridge regression, and elastic net), survival trees, and survival forest. The predictive performance of each method was evaluated in bootstrapped samples, using prediction error curves and the area under the curve of the receiver operating characteristic. The LASSO penalized Cox-regression model showed the best overall performance. Nonlinear mixed effects (NLME) models were developed, and a conventional stepwise covariate search was performed. Then, covariates identified as important by the LASSO model were included in the base NLME models developed for PFS and OS, resulting in improved models as compared to those obtained with the stepwise covariate search. It was shown that having gene polymorphisms in VEGFA (rs699947 and rs1570360) and ICAM1 (rs1799969) are associated with a favorable clinical outcome in patients with mCRC receiving bevacizumab treatment.

Consensus Statement Regarding the Efficacy and Safety of Long-Term Low-Dose Colchicine in Gout and Cardiovascular Disease.

Over the last decade, evidence has demonstrated that long-term, low-dose colchicine (0.5 mg daily) is effective for preventing gout flare and cardiovascular (CV) events in a wide range of patients. Given the potentially expanding use of colchicine in CV disease, we here review and update the biologic effects and safety of colchicine based on recent data gathered from bench and pharmacodynamic studies, clinical reports, controlled clinical trials, and meta-analyses, integrated with important studies over the last 50 years, to offer a consensus perspective by experts from multiple specialties familiar with colchicine's long-term use. We conclude that the clinical benefits of colchicine in gout and CV disease achieved at low dose do not sustain serum levels above the upper limit of safety when used in patients without advanced renal or liver disease or when used concomitantly with most medications. Further, data accrued over the last 50 years strongly suggest that the biologic effects of long-term colchicine do not increase the risk of cancer, sepsis, cytopenia, or myotoxicity.

Pharmacogenetics in Model-Based Optimization of Bevacizumab Therapy for Metastatic Colorectal Cancer.

Vascular endothelial growth factor A (VEGF-A) and intercellular adhesion molecule 1 (ICAM-1) are significant regulators of angiogenesis, an important biological process involved in carcinogenesis. Bevacizumab, an anti-VEGF monoclonal antibody (MAB), is approved for the treatment of metastatic Colorectal cancer (mCRC), however clinical outcomes are highly variable. In the present study, we developed a pharmacokinetic (PK), a simplified quasi-steady state (QSS) and a pharmacokinetic/pharmacodynamic (PK/PD) model to identify potential sources of variability. A total of 46 mCRC patients, who received bevacizumab in combination with chemotherapy were studied. VEGF-A (rs2010963, rs1570360, rs699947) and ICAM-1 (rs5498, rs1799969) genes' polymorphisms, age, gender, weight, and dosing scheme were investigated as possible co-variates of the model's parameters. Polymorphisms, trough, and peak levels of bevacizumab, and free VEGF-A were determined in whole blood and serum. Data were analyzed using nonlinear mixed-effects modeling. The two-compartment PK model showed that clearance (CL) was significantly lower in patients with mutant ICAM-1 rs1799969 (p < 0.0001), inter-compartmental clearance (Q) was significantly higher with mutant VEGF-A rs1570360 (p < 0.0001), and lower in patients with mutant VEGF-A rs699947 (p < 0.0001). The binding QSS model also showed that mutant ICAM-1 rs1799969 was associated with a lower CL (p = 0.0177). Mutant VEGF-A rs699947 was associated with a lower free VEGF-A levels, prior to the next dose (p = 0.000445). The above results were confirmed by the PK/PD model. Findings of the present study indicated that variants of the genes regulating angiogenesis might affect PK and PD characteristics of bevacizumab, possibly influencing the clinical outcomes.

Ex vivo evaluation of degradation rates of metronidazole and olsalazine in distal ileum and in cecum: The impact of prandial state.

PURPOSE: Evaluate ex vivo the bacterial metabolism induced degradation rates of mesalamine (negative control), metronidazole and olsalazine in distal ileum and in cecum. METHODS: The contents of distal ileum and cecum were collected during colonoscopy under anaerobic conditions from twelve healthy adults in the fasted and in the fed state. To eliminate potential effects of enzymes that may exist in the fluid of lower intestine, each sample was ultracentrifuged and the precipitate was diluted with a volume of normal saline equivalent to that of the supernatant, after ultracentrifugation of intestinal contents from which the specific precipitate had been obtained. Degradation of the three model drugs in individual materials was evaluated anaerobically. RESULTS: Mesalamine was stable in all cases. Degradation rates of metronidazole and olsalazine were higher in cecum than in distal ileum, only in the fasted state; no trend could be observed in the fed state. Degradation rates of metronidazole and olsalazine were decreased in the fed state in the cecum; no trend could be observed in distal ileum. CONCLUSIONS: In the fasted state, bacterial activity is higher in cecum than in distal ileum. Food residues decrease bacterial metabolism degradation rates of drugs in cecum.

Characterization of Contents of Distal Ileum and Cecum to Which Drugs/Drug Products are Exposed During Bioavailability/Bioequivalence Studies in Healthy Adults.

PURPOSE: Characterize the contents of distal ileum and cecum in healthy adults under conditions simulating the bioavailability/bioequivelance studies of drug products in fasted and fed state. METHODS: Twelve males participated in a two-phase crossover study. Phase I: subjects remained fasted overnight plus 4.5 h in the morning prior to colonoscopy. Phase II: subjects remained fasted overnight, consumed breakfast in the morning, and abstain from food until colonoscopy, 4.5 h after breakfast. Upon sampling, volume, pH and buffer capacity were measured; after ultracentrifugation, supernatant was physicochemically characterized and non-liquid particles diameter was measured. RESULTS: In distal ileum, pH is ~8.1 and size of non-liquid particles is ~200 µm, regardless of dosing conditions; in fed state, liquid fraction was lower whereas osmolality and carbohydrate content were higher. In cecum, the environment was similar with previously characterized environment in the ascending colon; in fasted state, size of non-liquid particles is smaller than in distal ileum (~70 µm). Fluid composition in distal ileum is different from cecum, especially in fasted state. CONCLUSION: Differences in luminal environment between distal ileum and cecum may impact the performance of orally administered products which deliver drug during residence in lower intestine. Dosing conditions affect cecal environment more than in distal ileum.

Versican but not decorin accumulation is related to malignancy in mammographically detected high density and malignant-appearing microcalcifications in non-palpable breast carcinomas.

BACKGROUND: Mammographic density (MD) and malignant-appearing microcalcifications (MAMCs) represent the earliest mammographic findings of non-palpable breast carcinomas. Matrix proteoglycans versican and decorin are frequently over-expressed in various malignancies and are differently involved in the progression of cancer. In the present study, we have evaluated the expression of versican and decorin in non-palpable breast carcinomas and their association with high risk mammographic findings and tumor characteristics. METHODS: Three hundred and ten patients with non-palpable suspicious breast lesions, detected during screening mammography, were studied. Histological examination was carried out and the expression of decorin, versican, estrogen receptor α (ERα), progesterone receptor (PR) and c-erbB2 (HER-2/neu) was assessed by immunohistochemistry. RESULTS: Histological examination showed 83 out of 310 (26.8%) carcinomas of various subtypes. Immunohistochemistry was carried out in 62/83 carcinomas. Decorin was accumulated in breast tissues with MD and MAMCs independently of the presence of malignancy. In contrast, versican was significantly increased only in carcinomas with MAMCs (median ± SE: 42.0 ± 9.1) and MD (22.5 ± 10.1) as compared to normal breast tissue with MAMCs (14.0 ± 5.8), MD (11.0 ± 4.4) and normal breast tissue without mammographic findings (10.0 ± 2.0). Elevated levels of versican were correlated with higher tumor grade and invasiveness in carcinomas with MD and MAMCs, whereas increased amounts of decorin were associated with in situ carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs. CONCLUSIONS: The specific accumulation of versican in breast tissue with high MD and MAMCs only in the presence of malignant transformation and its association with the aggressiveness of the tumor suggests its possible use as molecular marker in non-palpable breast carcinomas.

Non-palpable breast carcinomas: correlation of mammographically detected malignant-appearing microcalcifications and epidermal growth factor receptor (EGFR) family expression.

Malignant-appearing microcalcifications (MAMCs) represent one of the earliest mammographic findings of non-palpable breast carcinomas (NPBCs). In the present study, we have evaluated the expression of all EGFR family members in NPBCs and its possible association with MAMCs. Three hundred and fifty patients with non-palpable suspicious breast lesions detected during screening mammography were studied. EGFR family proteins' expression was found to be present since the preclinical phase of breast carcinomas and was strongly correlated (except HER-3) with MAMCs. The co-expression pattern of EGFR family members combined with other molecular prognostic factors and the mammographic appearance might predict the natural history of NPBCs.

Acute lymphoblastic leukemia relapse in the breast diagnosed with gray-scale and color Doppler sonography.

Extramedullary relapses of acute lymphoblastic leukemia (ALL) in children and young adults are rare and in most cases are localized in the central nervous system, testes, or both. We describe a rare case of extramedullary relapse of ALL in the breast of a 17-year-old girl. The patient, who had been diagnosed with ALL 1 year before and had been in complete remission for 5 months, was admitted to the hospital for investigation of a mass in her left breast. On clinical examination, she had a large, palpable, nontender mass in her left breast. Mammography revealed a very dense mass. On gray-scale and power Doppler sonography, the appearance of the mass was consistent with malignancy. Histopathologic examination of a surgical biopsy specimen permitted accurate diagnosis of ALL.

Non-palpable breast carcinomas: correlation of mammographically detected malignant-appearing microcalcifications and molecular prognostic factors.

Screening mammography has greatly increased the number of non-palpable breast carcinomas diagnosed in asymptomatic women. Malignant-appearing microcalcifications represent one of the earliest mammographic findings of non-palpable breast carcinomas. Many studies have attempted to correlate radiological and histological features of malignant-appearing microcalcifications. In the present study, we evaluated the association between mammographically detected malignant-appearing microcalcifications and the expression profile of selected biological markers in non-palpable breast carcinomas. Two hundred and eighty patients with non-palpable suspicious breast lesions that were detected during screening mammography were studied. All patients underwent mammographically-guided needle localization-excision breast biopsy. Histological examination showed 74 (26.4%) carcinomas of various subtypes. Immunohistochemistry was carried out in 58/74 carcinomas by using a panel of monoclonal and polyclonal antibodies against estrogen receptor (ER), progesterone receptor (PR), HER-2/neu, Bcl-2, Bax, Fas and DNA fragmentation factor (DFF). Malignant-appearing microcalcifications was the major mammographic finding in 45/58 (77%) patients. Nuclear ER positivity (65.5%) and PR positivity (46.5%) of non-palpable breast carcinomas were statistically correlated with malignant-appearing microcalcifications (p < 0.01 and p < 0.05, respectively). Statistically significant associations were also found between malignant-appearing microcalcifications and HER-2/neu positivity (p < 0.01), Bax positivity (p < 0.01), Fas positivity (p < 0.05) and DFF positivity (p < 0.01), whereas no statistical correlation was found with Bcl-2 positivity (p > 0.05). Malignant-appearing microcalcifications detected during screening mammography represent a diagnostic, prognostic and therapeutic challenge. The mammographic/biological associations and their potential implications in the management of women with non-palpable breast carcinomas are thoroughly discussed.