Development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues.

Protegrin antimicrobial peptides possess activity against gram-positive and gram-negative bacteria and yeasts. An extensive structure-activity relationship (SAR) study was conducted on several hundred protegrin analogues to gain understanding of the relationship between the primary and secondary structure of the protegrins and their antimicrobial activities, and to identify a protegrin analogue for clinical development. Native sequence protegrins are cationic, amphiphilic peptides that are characterized by the presence of a beta-sheet structure that is maintained by two disulfide bridges. The presence of the beta-sheet is key to the stability of the protegrin structure; linearized analogues or analogues that have amino acid substitutions that eliminate hydrogen bonding across the beta-sheet have reduced activity, especially in the presence of physiological concentrations of NaCl. Also, maintaining amphiphilicity of the beta-sheet is key; analogues with substitutions of polar amino acids in the hydrophobic face have reduced activity. Analogues with reduced positive charge tend to be less active, an observation that is more marked for gram-negative than gram-positive bacteria, and may implicate binding to lipopolysaccharide as a key mechanistic step in the killing of gram-negative bacteria. A very large number of amino acid substitutions are tolerated by the protegrin structure, implying that overall structural features such as amphiphilicity, charge, and shape are more important to activity than the presence of specific amino acids. This lack of importance of specific stereochemistry is supported by the fact that completely D-amino acid substituted protegrins are fully potent. Based on the SAR studies, and on the microbiological data from an animal model, one protegrin analogue, IB-367, was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy.

IB-367, a protegrin peptide with in vitro and in vivo activities against the microflora associated with oral mucositis.

Although the microflora associated with oral mucositis initiated by cytotoxic therapy is not well characterized, several studies suggest that reduction of the microbial load in the oral cavity has some clinical benefit. The MICs of IB-367, a synthetic protegrin analog, ranged from 0.13 to 64 microgram/ml for gram-positive bacteria (Streptococcus mitis, Streptococcus sanguis, Streptococcus salivarius, and Staphylococcus aureus) and from 0.06 to 8 microgram/ml for gram-negative species (Klebsiella, Escherichia, and Pseudomonas). IB-367 exhibited rapid, microbicidal activity against both log- and stationary-phase cultures of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. At concentrations near the MICs for these two organisms (4 and 2 microgram/ml, respectively), IB-367 reduced viability by more than 3 logs in less than 16 min. Similarly, IB-367 effected a 4-log reduction of the endogenous microflora in pooled human saliva within 2 min at 250 microgram/ml, a concentration readily attained by local delivery. After nine serial transfers at 0.5x the MIC, the MIC of IB-367 for MRSA and P. aeruginosa increased only two to four times. In a phase I clinical study with healthy volunteers, IB-367 was well tolerated, with no detectable systemic absorption. One hour after treatment with 9 mg of IB-367, the prevalence of gram-negative bacteria and yeast was reduced, and the density of the predominant gram-positive oral flora was decreased 1,000 times. IB-367's properties (speed of killing, breadth of spectrum, and lack of resistance) make the compound a strong candidate for the prophylaxis of oral mucositis. Phase II clinical trials with IB-367 are under way for this indication in immunocompromised subjects.

Reliability of drop size from multi-dose eye drop bottles: is it cause for concern?

PURPOSE: Responses to topically applied ocular drugs vary between patients. The volume of drug instilled is of particular importance as one of many sources of response variation, but the reliability of drop volume from eye drop bottles is unknown. Hence, the repeatability of drop volume and factors affecting this for a variety of drug manufacturers were considered in this study. METHODS: Nineteen bottles, one from each primary manufacturer in the UK, were examined. The mass of all drops expelled from each bottle was measured with respect to the bottle type, handling angle, drop number, drug and concentration. The accuracy (repeatability and trueness) of drops from each bottle was also evaluated. RESULTS: Drop volume varied significantly between drug manufacturers, ranging from 33.8 microliters to 63.4 microliters. The handling angle of the bottle also influenced drop volume, with angles less than 60 degrees giving smaller drops. Drop number exhibited no significant effect upon drop volume. However, the drug type and its concentration did significantly affect the volume of the drop expelled from the bottle, with higher concentrations giving rise to larger drops. Repeatability coefficients across the range of bottles varied between +/- 2.24 microliters and +/- 10.76 microliters (mean +/- 5.07 microliters). CONCLUSIONS: It is well reported that drug volume instilled has a significant effect on the degree of response. However, there are currently no official regulations concerning eye drop volume in either the UK or the USA. Since drop volume has been shown to vary significantly depending upon a variety of factors, it may be appropriate that the regulatory bodies consider the consequences of variable drop size.

Ocular effects of antimuscarinic compounds: is clinical effect determined by binding affinity for muscarinic receptors or melanin pigment?

Although antimuscarinic drugs are being used with increasing frequency in clinical practice for the purposes of mydriasis and cycloplegia, the extent of their actions varies considerably between different compounds. Investigation of the binding characteristics of these agents revealed that as their reported clinical potency increased, so did their specific binding affinity for muscarinic receptors in the iris sphincter and ciliary muscle and their nonspecific binding affinity for melanin pigment. However, the affinity of each drug for melanin pigment was much lower than for the muscarinic receptors. Therefore, although binding to melanin can significantly influence the overall response, differences in the clinical effect of various compounds appear to be primarily due to their differences in specific affinity for muscarinic binding sites.

A novel system for the objective classification of iris colour and its correlation with response to 1% tropicamide.

Iris colour can provide an enormous amount of information about an individual. In addition to changes with pathological conditions, the colour of the iris can be a particularly useful indicator of how well a person will respond to a topically applied ocular drug. Until recently, classification of iris colour has been subjective, ranging from a basic description ('light' and 'dark') to more detailed grading systems, such as a comparison with preset photographic standards. However, variability within observers and differences in the interpretation between observers can influence the results. Objective techniques, in this respect, possess several advantages. They are able to detect differences in colour that subjective techniques are incapable of and they provide continuous data rather than discrete categories, thus improving the accuracy of drug response predictions. This study assessed iris colour by objective means. Slit-lamp photographs of various coloured irides were taken under standardised conditions. The slides were then scanned into a computer and the colour analysed using a calibrated software package. To establish the optimum colour parameter to be used for predictions of drug response, several parameters were calculated and compared with the subject response to 1% tropicamide (maximum change in pupil size, time to maximum change and total duration of effect). Many parameters had strong correlations with drug response, but the parameters 'z', 'b' (the proportion of blue in the image) and 'y' (the proportion of yellow in the image) were found to exhibit the highest correlations. They also showed better correlations with drug response than did a current iris colour grading system.

Validating a "pillar and collar" technique for measuring the edge lift of rigid contact lenses.

PURPOSE: The clinical importance of the edge lift of rigid contact lenses is often neglected, possibly due to previous difficulties in its measurement. A new method of measuring axial edge lift (AEL) and radial edge lift (REL) using standard contact lens verification equipment, such as an optical spherometer, a thickness gauge, and contact lens V gauge, is described. METHODS: The technique was validated for trueness (accuracy) and precision (repeatability) by measuring the edge lift of a number of monocurve lenses, manufactured both with and without a normal edge finish. RESULTS: Edge lift was measured to an accuracy of 0.01 mm. CONCLUSIONS: As long as a mean of eight independent measurements of back optic zone radius (BOZR), sagitta, and one measurement of center thickness are taken, the pillar and collar technique is capable of producing accurate and repeatable measurements of the edge lift of a rigid contact lens.

"Pillar and collar" technique for measuring the axial edge lift of multicurve rigid lenses.

BACKGROUND: The method of using an optical spherometer and a lens thickness gauge, in combination with a pillar and collar, has previously been shown to be repeatable and accurate (to 0.01 mm) in measuring the edge lift of rigid monocurve lenses. PURPOSE: This paper goes on to validate the technique for measurements taken on edge-finished multicurve rigid lenses. METHODS: The axial edge lift (AEL) of a series of multicurve rigid lenses having known values of AEL was measured using an optical spherometer, a thickness gauge, and contact lens V gauge in a similar way as was described in the companion paper. RESULTS: The results show that as long as a correction factor of 0.01 mm is applied, as found in the previous paper, the technique will allow satisfactory verification of the edge lift. CONCLUSIONS: The technique is accurate and reproducible, even in multicurve lens designs, provided that a calibrating correction factor of 0.01 mm is applied to account for methodological error. It will also indirectly allow the accuracy of manufacture of the peripheral curves and diameters of any rigid contact lens to be verified.

Evaluation of the pinhole pupillometer.

Evaluation of pupil diameter is used in many clinical and research fields to aid diagnosis of neurological disorders and to monitor pharmacological effects upon the eye. Methods used to determine pupillary diameter have evolved from simple scale measurements to much more complex systems, supposedly improving accuracy and repeatability. However, many techniques are restricted to the consulting room or research laboratory due to the size of the equipment, its cost, or the expertise required. We have evaluated a portable pupillometer originally developed by Broca, capable of precise measurements on fixed, dilated pupils, that has the potential to be used by unsupervised patients. Luminance levels will still need to be controlled because, although luminance does not influence the mechanics of the pupillometer itself, it significantly affects the natural pupil. The pinhole size should ideally be 1.1 mm in diameter, since smaller pinholes underestimate and larger holes overestimate pupil size. The pupillometer also exhibited reasonable accuracy in fixed pupils dilated with tropicamide. Pupil measurements were overestimated by 0.5% using the 1.1 mm pinholes (insignificant if the pupil can be measured to the nearest 0.25 mm) and both intra- and inter-visit repeatabilities were relatively good. The results from this study therefore indicate that the pinhole pupillometer may be of use in pupil research, since it facilitates more frequent pupillary measurements over much longer time intervals than are currently achievable.

Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity.

Protegrin-1 (PG-1) is a cysteine-rich, 18-residue beta-sheet peptide isolated from porcine leukocytes with antimicrobial activity against a broad range of microorganisms. The MICs of PG-1 against representative gram-positive and gram-negative bacteria ranged from 0.12 to 2 microg/ml. At these levels, PG-1 was rapidly bactericidal in vitro, reducing the number of viable CFU of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa by more than three log units in less than 15 min. Resistance to PG-1 did not develop after 11 subculturings of P. aeruginosa or 18 subcultures of MRSA in Mueller-Hinton broth containing PG-1 at one-half the MIC. Under similar conditions of serial passage, the MICs of norfloxacin and gentamicin against P. aeruginosa increased 10 and 190 times, respectively. Similarly, the MIC of norfloxacin against MRSA increased 85 times. Immunocompetent mice inoculated intraperitoneally (i.p.) with P. aeruginosa or S. aureus exhibited 93 to 100% mortality in the vehicle control group compared with 0 to 27% mortality in animals that received a single i.p. injection of PG-1 (0.5 mg/kg of body weight). Mice inoculated with S. aureus by intravenous (i.v.) injection and dosed 0 to 60 min later with a single i.v. injection of PG-1 (5 mg/kg) had a mortality of 7 to 33%, compared to a mortality of 73 to 93% in the vehicle controls. In leukopenic mice inoculated i.v. with vancomycin-resistant Enterococcus faecium, mortality was 87% in the vehicle control group and 33% in animals that received a single i.v. injection of PG-1 (2.5 mg/kg). Taken together, these data indicate that PG-1 has potential for use as an antimicrobial agent in the treatment of local or systemic infections caused by clinically relevant pathogens.

Eye drop container delivery: a source of response variation?

Variations in response to topically applied ocular drugs (e.g. mydriatics and cycloplegics) and their possible aetiology have been studied previously. It appears that individual patient differences, external influencing factors and the characteristics of the particular drug may all govern the response. One factor worthy of note in this regard is the possibility of variation in the drug volume instilled. Single-use eye drop containers known as Minims are commonly used during ophthalmic diagnostic procedures. However, in this study the drop volume was shown to vary considerably, depending upon the angle at which the Minims container was held, the particular drug and the number of drops previously expelled from the unit.

Validation of methods for the assessment of cataract progression in the Roche European-American Anticataract Trial (REACT)

The Roche European-American Anticataract Trial (REACT) will assess the effect of antioxidants on progression of cataract in humans. This report evaluates the methods used in REACT. Seventy three subjects (139 eyes) with cortical (C), posterior subcapsular (P), nuclear (N) or mixed cataract were seen twice within two weeks for eye examinations, assessments of visual function, lens photographs and CCD images. The degree of cataract and nuclear color (NC) were assessed with subjective (LOCS III) and objective (computerized, CASE 2000 CCD) methods. Repeat visit values were used to calculate intraclass correlation coefficients (r1) and 95% tolerance limits (TL). A clinically significant change (CSC) was defined as one step in LOCS III. The relative power of each method to detect cataract change and sample sizes needed to achieve statistically significant results were calculated. The r1 values for visual function tests ranged from 0.76 to 0.88; if these tests of visual function were used to detect a clinically significant change in cataract severity, sample sizes of 840 to 2707 per group would be needed. The r1 values for LOCS III were 0.88 to 0.97, and sample sizes ranged from 50 to 135 per group. The r1 values for the CCD were 0.93 to 0.98, and sample sizes ranged from 1 to 42 with poorer values relating to measurement of P. We conclude that the methods used in REACT are reproducible. The analytical algorithms in the image analysis programs did not permit differentiation between C and P opacification; therefore, P cataract is best measured with LOCS III. REACT sample sizes are adequate to detect a difference of 0.2 LOCS III units/year between the mean rates of cataract progression in two groups.

Displacement threshold hyperacuity as a predictor of postsurgical visual performance in patients with cataract.

PURPOSE: To evaluate the ability of Displacement Threshold Hyperacuity (DTH) in predicting the postoperative visual outcome in patients awaiting cataract surgery and discriminating between the relative performance of the neural and optical components of the visual system. To establish eventually the cut-off DTH values to be used clinically in the preoperative evaluation of patients with cataract. METHODS: Forty-five subjects admitted for extracapsular cataract extraction with implant were examined preoperatively and postoperatively. In addition to DTH, logMAR visual acuity (VA), contrast sensitivity, and a cataract classification system were used to obtain a more comprehensive assessment of the patient's visual performance. RESULTS: DTH was found to be sensitive to decreased macular function but relatively unaffected by the severity of the cataract; higher thresholds were associated with denser opacities. A preoperative threshold of 50 seconds of arc was found to be the upper limit under which normal macular function is likely. DTH sensitivity and specificity for decreased macular function was 1.00 and 0.8, respectively. CONCLUSIONS: If preoperative DTH is 50 seconds of arc or lower, the subject will achieve a postoperative logMAR VA better than 0.3 (Snellen equivalent 20/40). On the contrary, higher DTH seems to give no reliable information about the patient's visual function.

Assessing vision behind cataract--a review of methods.

The clinician involved in cataract assessment is often faced with a deterioration in visual performance which may or may not be totally attributable to the cataract. Neural changes may occur concurrently and contribute toward the reduced performance. This paper reviews the methods which attempt to discriminate between changes due to cataract development and changes produced by neural degeneration. New techniques have been developed with more emphasis on quantitative rather than qualitative assessment. These techniques are discussed critically with particular reference to their ability to distinguish between the contributions of optical and neural change. This has particular application in predicting the visual outcome after cataract surgery.

Simple clinical techniques to evaluate visual function in patients with early cataract.

Among the 80 subjects who were recruited with normal retinal and neural function, 54 had cataract and a visual acuity (VA) better than 6/24. The 26 age-matched subjects had clear media. Contrast sensitivity (CS) at low and intermediate spatial frequencies was measured using the Pelli-Robson letter chart. Two measures of glare disability (GD) were obtained using the Mentor Brightness Acuity Tester (BAT) in conjunction with a logMAR VA chart and the Pelli-Robson chart. Although CS is predominantly affected at high spatial frequencies in early cataract, we found that some subjects had reduced scores on the Pelli-Robson chart. This CS loss could not be predicted from VA measurements and was particularly found in subjects with posterior subcapsular cataract. High GD scores were found in a number of subjects with relatively good VA and could not be predicted from results of VA or CS. We suggest that CS and GD measurements using the Pelli-Robson chart and the BAT provide valuable information regarding the management of patients with early cataract.

Comparing clinical tests of visual function in cataract with the patient's perceived visual disability.

Conventional techniques for assessing the visual function of cataract patients include visual acuity (VA), contrast sensitivity (CS) and glare disability (GD). The extent to which these measurements provide accurate information about a patient's perceived visual disability is not known. In this study, binocular and monocular VA and CS and monocular GD measurements were made using commercially available techniques on 33 cataract patients. VA was measured using a Ferris-Bailey LogMAR chart and CS by the Pelli-Robson letter CS chart. Glare disability was measured using the Mentor Brightness Acuity Tester in conjunction with both the LogMAR and Pelli Robson charts. Each patient's perceived visual disability was quantified using a 20-point questionnaire about the effect of vision on everyday activities. There was little correlation between subjective visual disability and monocular or binocular VA measurements. Measurements of binocular CS, however, were highly correlated with the patient's perceived visual disability, particularly their subjective assessment of the effect of vision on their mobility-orientation. We suggest that binocular CS measurements using the Pelli-Robson chart provide useful additional information regarding the need for surgery in cataract patients.

Assessing the effect of cataract: a clinical evaluation of the Opacity Lensmeter 701.

The efficacy of a new clinical instrument which measures light backscatter from the crystalline lens--the Opacity Lensmeter 701--was assessed. Repeated measurements were made on 83 normal subjects in the age range 15 to 82 years, and on 38 eyes of subjects with cataract but normal retinal and neural function. The cataract scores were compared against results of Logmar visual acuity (VA) and glare disability measurements from the same subjects. The Lensmeter was found to be simple to use and gave quick, repeatable, objective measurements of light backscatter. Scores correlated well with normal age changes in noncataractous lenses and in cataracts of pure nuclear morphology, but not in cortical or posterior subcapsular cataracts.