Lotta Salminen

Ocular inserts for topical delivery

Abstract In spite of extensive pharmacological and clinical data pointing to the usefulness and advantages of solid devices for topical drug delivery to the eye, liquid, and to a smaller extent, gel-type preparations appear to enjoy the continued interest of manufacturers and ophthalmologists. In the present update, the authors discuss the advantages, disadvantages and requirement for sucess of ocular inserts, and examine the few inserts which are available on the market or are being developed by pharmaceutical companies for drug delivery. The article discusses S.O.D.I., Ocusert ® , Collagen Shields, Ocufit ® , Minidisc and Nods ® with special attention to biological/ clinical performances, and potential for future applications and developments.

Comparison of an Immortalized Human Corneal Epithelial Cell Line and Rabbit Corneal Epithelial Cell Culture in Cytotoxicity Testing

The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40. Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity. Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium. Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results. However, the LDH test seemed less reliable due to great variation. The use of serum was found to result in lower toxicity of the compounds in both tests. The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA. The human cell line is a promising in vitro alternative in oculotoxicity testing.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Teleconsultations between general practitioners and ophthalmologists in Finland

We carried out a study of the value of videoconferencing in consultations between general practitioners GPs and ophthalmologists in Finland. We used ISDN lines 128 kbit s between the Primary Health Care Centre in Ikaalinen and the ophthalmology clinic at the Tampere University Hospital TAUH. Questionnaires covering both clinical and technical matters were given to patients and doctors after the consultation. During the 10-month study, consultations were carried out successfully for 23 of 24 patients 96 . Most consultations 84 took less than 15 min. If we had not had this system, the GP would have made 21 referrals to an ophthalmologist. After teleconsultation, six patients were sent to the TAUH, so the system saved 15 referrals. Twenty-two patients 92 thought that videoconferencing was a reliable tool for GPs. Our system proved to be a valuable and reliable tool for the GPs in ophthalmology consultations and continuing education.

A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 test

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.

β-Blocking effects of timolol at low plasma concentrations

The concentration‐effect relationship of 0.25 mg intravenous timolol with and without pretreatment with 100 mg quinidine was studied in six healthy young volunteers with a randomized, double‐blind, crossover study design. Blockade of cardiac β‐adrenoceptors was assessed by determining the dose ratios (DR) of isoproterenol infusions required to increase heart rate by 25 beats/min before and after timolol infusion. The logarithm of timolol concentration in plasma was linearly related to the logarithm (DR–1) of isoproterenol infusion, with a mean Pearson correlation coefficient of 0.89 ± 0.11 (± SD; n = 24) at timolol concentrations well below 1 ng/ml. The increases in cyclic adenosine monophosphate (cAMP) and norepinephrine plasma levels caused by isoproterenol infusions were attenuated after timolol. Quinidine administration increased timolol plasma levels and cardiac β‐blocking effects by 10% to 40%. It was concluded that timolol at concentrations below 1 ng/ml in plasma competitively antagonizes cardiac and noncardiac effects of isoproterenol infusions. Timolol effects are augmented after quinidine administration. The β‐blockade occurring at low plasma levels can explain side effects and actions of ocularly applied timolol.

EVALUATION OF ADVERSE OCULAR EFFECTS OF 5-FLUOROURACIL BY USING HUMAN CORNEAL EPITHELIAL CELL CULTURES

5-Fluorouracil (5-FU) is commonly used in ophthalmology for suppressing fibroblast activity after glaucoma surgery. Adverse effects on corneal epithelial cells have been reported to relate to 5-FU therapy. The effects of 5-FU were evaluated in vitro on SV40-immortalized human corneal epithelial cell (HCE) cultures with two cytotoxicity tests: WST-1 assay as an index of cell proliferation, and lactate dehydrogenase (LDH) assay as an index of plasma membrane integrity. The cells were exposed to 5-FU with various concentrations in serum-free medium and in medium containing 15 % (v/v) fetal bovine serum (FBS) for 1, 24, 48 and 72 hours. One-hour exposure had no effects on HCE cells. Longer exposures caused dose-dependent inhibition of cell proliferation. Exposure to 5 mg/ml 5-FU lowered cell number to 50 % of controls after 24-hour treatment and resulted to complete cell death after 72 hours. Serum protected the cells for 24 hours, but after longer exposure times the protective nature of serum disappeared. 5-FU had only minor effects on LDH release. The LDH leakage was at its peak after 48-hour treatment.

Effects of Tamoxifen and Toremifene on ALDH1 and ALDH3 in Human Retinal Pigment Epithelial Cells and Rat Liver

Aldehydehydrogenase is a NAD(P)-dependent enzyme with wide distribution virtually in all animal tissues (Vasiliou and Marselos, 1989; Lindahl, 1992). Different consti- tutiveexpressed ALDHs are found in liver, stomach, brain, kidney, skin and eye (Vasil and Marselos, 1989; Pappas et al., 1997). In general, ALDHs are located especially in org with a high content of epithelial cells. An increased interest has been shown r the last years for aldehyde dehydrogenase-3 (ALDH3) activity in the cornea where h higher constitutive specific activity is detected compared to the liver cells (Boesct al., 1996). High levels of ALDH3 activity occurs in the cornea from baboon, cow, hn, opossum, pig and sheep (King and Holmes, 1997). The same study reports also tpresence of ALDH1 as the 1-2% of human lens soluble protein. Furthermore, retina and real pigment epithelial (RPE) cells have been shown to play an important protective role fthe photosensitive cells of the eye against photo- and chemical toxicity.

Wireless picture transfer as a tool of primary health care.

Sir, We are investigating the benefits of amobile telemedicine systemfor patients whoare treated intheir ownhomes bynurses andgeneral practitioners. The systemis basedona digital still camera anda personal communicator, allowingstill images tobetransmittedtoa server locatedinahospital. It alsoallows adoctor tobook medical examinations, hospital admissions andmanage patient data generally. The applicationuses objectorientedprogramming techniques, for boththe mobile client andthe server. The connectionto the server is achievedthroughthe GSMmobile phone networkusing standardInternet protocols (TCP/IP). Personal communicators provide amethodfor realtime data transfer for staff who visit patients intheir homes, even whenthere are no telecommunications other thanGSM. Twonurses visitingpatients intheir homes inthetown of Ikaalinen, Finland, were equippedwitha digital still camera (QV-7000SX, Casio)1 andapersonal communicator (9110, Nokia)2. Whentransfer of visual informationwas needed, nursestookthepictures, whichwerethensent via aninfraredconnectionfromthedigital still cameratothe communicator. The pictures were forwardedas anemail attachment toa secure server inthe townof Ikaalinen. Consultingdoctorswerethenabletoreadtheemailmessages andviewthe pictures using commerciallyavailable software (e.g. Adobe PhotoShopand Netscape Navigator) andholdatelephoneconversationwiththesender (Fig1). If the general practitioner felt unable to advise, he or she couldeither transfer the picture to the appropriatespecialist or ask thepatient tovisit the health centre. Nurses couldalsoconsult byusing the voice connectionor use other communicationapplications, suchas fax, whichwere includedinthe communicator. Other functionssuchasacalendar, notes andcontacts list were usedtoassist inroutine tasks. The screenof the personal communicator couldalso be usedfor receiving visual informationsuchas manuals, working lists, drawings or medical record documents, as well as Webbased information.

Laser treatment of cultured retinal pigment epithelial cells - Evaluation of the cellular damage in vitro

PURPOSE Evaluation of the effects of laser photocoagulation on cultured primary retinal pigment epithelial cells. METHODS Cells were treated by a diode laser (678 nm) with 800 and 1600 mW for 0.186 second. Cell toxicity was tested by the WST-1 assay, and the uptakes of glutamate and gamma-aminobutyric acid (GABA) were measured. RESULTS Laser photocoagulation (1600 mW) caused cell damage and the mitochondrial enzyme activity evaluated by a WST-1 test significantly decreased by 20%-30%. Laser treatment caused a dose-dependent decrease in glutamate uptake but increased GABA uptake. CONCLUSIONS Laser treatment and the laser-induced increase in temperature influence transport processes in retinal pigment epithelial cells and may cause cell damage in the posterior part of the retina.