Circulating biomarkers in glaucoma, age-related macular degeneration, and diabetic retinopathy

Biomarkers to predict the altering physiological conditions over the period leading toward the ocular disorders are of major importance in therapeutics. Isolation and validation of the biomarkers specific to ocular diseases are a challenging task. Glaucoma is a neurodegenerative disease of the eye where the correlation of biomarkers in circulating fluid may be made specific for the eye. However, conditions such as wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR), circulating biomarkers might be having some degree of overlap with other conditions like cancer where a common factor such as angiogenesis is involved. Diabetes, a systemic disorder affecting the target organs such as eye, kidney, heart, and nervous system can be predicted using common circulating biomarkers. However, these markers need to be validated along with various stages of disease progression to enable the possibility of targeted pharmacological interventions apart from good glycemic control alone. This review compiles the attempts made to correlate such circulating biomarkers in the ocular conditions such as glaucoma, AMD, and DR in the search for a surrogate marker for diagnostic and prognostic value. To make biomarkers for the common convenience, genetic markers are excluded from this review

Synthesis, Characterization, and Biological Evaluation of 99mTc(CO)3-Labeled Peptides for Potential Use as Tumor Targeted Radiopharmaceuticals

During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared [99mTc(CO)3(H2O)3]+ intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10�2 M. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30–35% at 30 min of incubation attaining maximum value of about 40–60% after 2–4 h incubation). A good percentage of quick internalization was also observed in avb3-receptor-positive B16F10 mouse melanoma cell line (14–16% after 30 min of incubation and 25–30% after 2–4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. 99mTc(CO)3-tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, 99mTc(CO)3-hexa-Pep exhibited the highest tumor to muscle and tumor to blood ratios among the three. The preliminary results with these amino acid–based peptides are encouraging enough to carry out further experiments for targeting tumor

Ocular kinetics and safety of intravitreally injected angiotensin converting enzyme inhibitor lisinopril

Abstract Background and objectives The study investigated the intravitreal safety and vitreous disposition of lisinopril, an angiotensin converting enzyme inhibitor in rabbits for its projected use in retinopathy. Methods For the safety study, following the baseline ERG recording and fundus photography, 40 µg/50 µl of lisinopril sterile injection was injected unilaterally in the rabbit eyes (n = 4), where other eye served as a control. The electroretinogram and fundus images were obtained at 24, 48, 72 and 168 h following the intravitreal injection. For pharmacokinetics evaluation of the lisinopril, one eye of each rabbit (n = 4) received an intravitreal injection of lisinopril (40 µg/50 µl). The concentration of lisinopril in the ocular tissues, humours, plasma, lung, kidney and liver were measured through ESI-LC-MS/MS. Results Upon the electroretinography studies, no significant difference was observed in the ERG pattern in the lisinopril injected eye when compared to the baseline of the respective animals till the 7th day of the study. In the fundus imaging, no morphological changes were observed in the retina of the animal. The concentration of the lisinopril was found to be above to the IC50 in the retina-choroid till 36 h. The concentration found in the plasma and body tissues were many folds less than the IC50 of the lisinopril. Conclusions Intravitreal injection of 40 µg/50 µl of lisinopril found to be safe in the rabbit eye as evidenced by the electroretinography and fundus imaging studies. The average half-life of lisinopril is 12.6 h and the above-mentioned dose able to sustain its IC50 value till the 36 h

Involvement of Renin-Angiotensin System in Retinopathy of Prematurity - A Possible Target for Therapeutic Intervention.

Examining the Retinal Renin Angiotensin System (RRAS) in the ROP neonates and analyzing the possibility of modulating the RRAS to prevent the progression in Oxygen Induced Retinopathy (OIR) model.Vitreous of ROP patients (n = 44, median age 5.5 months) was quantified for RRAS components, VEGF, HIF-1α and compared with age matched control. The involvement of RRAS in ROP was tested in the rat model of OIR and compared with normoxia. Expressions of RAS components, VEGF and HIF-1α in retina were analyzed using qPCR and retinal structure and function was also analyzed. Effect of Angiotensin Converting Enzyme Inhibitor (ACEI) and Angiotensin Receptor Blocker (ARB) was evaluated and compared with Bevacizumab which served as a positive control. Drug penetration into retina was confirmed by liquid chromatography coupled ESI-tandem mass spectroscopy (LC-MS/MS).Multifold increase in the expression of RAS components in human vitreous and rat retina showed their involvement in ROP. ERG & fundus studies in OIR revealed the altered function of retina and were successfully prevented by ARB (telmisartan), ACEI (lisinopril) and bevacizumab. Retinal analysis revealed the presence of ACEI and ARB in their therapeutic levels.This study for the first time demonstrates the upregulated level of RAS components in human ROP vitreous and further that the pharmacological intervention in RRAS can functionally and structurally preserve retina against the progression of ROP in the OIR model

Protein concentration of RAS components and angiogenic factors.

<p>Human vitreous of disease (n = 44) and control patients (n = 12) were subjected for the quantification of (A) VEGF- vascular endothelial growth factor, (B) HIF1α-Hypoxia inducible factor 1 alpha, (C) angiotensinogen, (D) ACE-Angiotensin Converting Enzyme, (E) ANG II- angiotensin II and (F) Renin. All concentrations were normalized as per mg/protein. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using unpaired student t-test.</p

ERG ‘b’ & ‘a’ wave.

<p>‘b’ wave significantly decreased (*p≤0.01) in disease control group (6A). Fig 6B depicting the improved ‘b’ wave response in all treatment group in comparison with disease control. Fig 6C & 6D showing the ‘a’ wave response of various groups. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using mann-whitney test (n = 9).</p

Protein concentration of RAS components and angiogenic factors.

<p>Human vitreous of disease (n = 44) and control patients (n = 12) were subjected for the quantification of (A) VEGF- vascular endothelial growth factor, (B) HIF1α-Hypoxia inducible factor 1 alpha, (C) angiotensinogen, (D) ACE-Angiotensin Converting Enzyme, (E) ANG II- angiotensin II and (F) Renin. All concentrations were normalized as per mg/protein. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using unpaired student t-test.</p

ERG, OPs & Fundus images.

<p>Representative graphs for (A) electroretinogram, (B) oscillatory potential. Whereas (C1–6) represents (C1) disease control, (C2) normoxia, (C3) bevacizumab, (C4) lisinopril, (C5) telmisartan group fundus images.</p

Tortousity Index.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168809#pone.0168809.g004" target="_blank">Fig 4A</a> is showing the significant increase in tortousity index of disease control as compared to normoxia (p≤0.01), whereas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168809#pone.0168809.g004" target="_blank">fig 4B</a> is depicting the significant fall in the TI of treatment groups in comparison with disease control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168809#pone.0168809.g004" target="_blank">Fig 4C</a> & 4D tortousity index of veins in various groups, where no significant difference was observed. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using unpaired student t-test (n = 9).</p

Representative fundus images.

<p>Fig 1 shows the fundus images of human subjects with stage 4A (A) and 4B (B) ROP. The stages were classified according to ICROP (International classification of Retinopathy of Prematurity).</p