Alternative delivery of insulin via eye drops.

BACKGROUND: Various insulin delivery systems have been considered for systemic absorption other than injections. Although the ocular route has been suggested, its use is limited by the amount of insulin absorbed systemically via eyes. In order to improve the absorption rate of insulin into systemic circulations, the effects of pH and absorption enhancers were studied with rabbit eyes. METHODS: Insulin eye drops were instilled into eyes of rabbits, and their blood glucose levels were measured with a Glucoscan 200 Meter (Lifescan, Mountain View, CA). RESULTS: Systemic absorption of insulin via the ocular route was much more prominent at higher pH (8.0) than at lower pH (3.5). When absorption enhancers such as glycocholate and fusidic acid were added, the insulin absorption increased further markedly. CONCLUSION: It is feasible to administer insulin through the eyes at pH 8.0 with low concentrations of glycocholate or fusidic acid to achieve the therapeutic efficacy of insulin to lower blood glucose to normal levels.

Ocular hypotensive actions of haloperidol, a dopaminergic antagonist.

Haloperidol, a dopaminergic antagonist, was found to be very effective in suppressing the intraocular pressure recovery curve of rabbits infused with 20% saline intravenously. Haloperidol was at least equipotent, if not more potent, in lowering IOP and had much longer duration of action compared with timolol maleate. In the cat model, haloperidol increased aqueous humor (AH) outflow initially, followed by a long-lasting suppression of AH formation. Since haloperidol does not block beta-adrenergic receptors and the ophthalmic dose required to lower IOP is only 6.5% of the antipsychotic dose, haloperidol might be a good drug for glaucoma treatment with the possibility of minimal side effects.

Ocular hypotensive effects of N-demethylated carbachol on open angle glaucoma.

Ocular hypotensive actions of N-demethylated carbachol hydrochloride (DMC) were studied in patients with open angle glaucoma; 1% pilocarpine hydrochloride solution was used as a reference. Although 1% pilocarpine seemed to be slightly more potent that 6% and 9% DMC solutions, DMC solutions were devoid of the noticeable side effects produced by pilocarpine. In all cases, maximum ocular hypotensive actions were observed six hours after the single-drop medication and were determined as 4.2, 4.6, and 4.8 mm Hg with 6% DMC, 9% DMC, and 1% pilocarpine, respectively.

Ocular hypotensive effects of timolol in cat eyes.

The effects of timolol on the aqueous humor (AH) formation and AH outflow in cat eyes were simultaneously studied with a continuous infusion method under constant intraocular pressure. The rate of AH formation was reduced 28%, 56%, and 71%, respectively, by 0.005%, 0.025%, and 0.15% of timolol solution infused intracamerally. There was no significant change of AH outflow after the administration of timolol. It was noted that the pupil was dilated by 0.025% and 0.15% timolol solutions. To determine the action mechanism of timolol to inhibit AH formation, experiments of carbonate dehydratase inhibition were performed with various concentrations of timolol. No appreciable enzyme inhibition was noted with timolol at concentrations up to 0.5%. It is thus concluded that the action mechanism of timolol to inhibit AH formation differs from that of carbonate dehydratase inhibitors.

Effects of 5-(N,N-diethylamino)-n-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) on cardiovascular systems.

The effects of 5-(N,N-diethylamino)-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) were studied pharmacologically on smooth muscle, skeletal muscle, blood vessel and cardiac preparations. In all cases, TMB-5 inhibited muscle contractions induced by muscle stimulants such as acetylcholine, norepinephrine, KCl and BaCl2, indicating that the muscle inhibition induced by TMB-5 is unrelated to specific receptors. TMB-5 was found to be most potent in inhibiting skeletal muscles (at 10(-6)-10(-5) M level) and least effective in inhibiting smooth muscles (at 10(-4)-10(-3) M level). The potency of vascular inhibition was in between these two levels (at 10(-4) M level). The ability of TMB-5 to raise the threshold of cardiac arrhythmias was quite good at 7X10(-7)-7X10(-6) M. It is concluded that TMB-5 could be a good antiarrhythmic agent with some skeletal muscle relaxation action.

Effects of alpha 1 and alpha 2 activation of adrenergic receptors on aqueous humor dynamics.

Effects of phenylephrine (alpha 1-adrenergic agonist), prazosin (alpha 1-adrenergic antagonist), clonidine (alpha 2-adrenergic agonist), and yohimbine (alpha 2-adrenergic antagonist) on aqueous humor (AH) dynamics were studied with a cat eye model. Phenylephrine (130 microgram/ml) inhibited AH outflow (67% at 90 min. period) more than AH formation (26% at the same period) indicating the intraocular pressure (IOP) might be raised by the administration of phenylephrine. Prazosin (0.1 microgram/ml) produced effects opposite to those of phenylephrine (55% reduction of AH formation and 25% reduction of AH outflow at 3 hr. period) suggesting the alpha 1-adrenergic receptor is responsible for increases rather than decreases of IOP. Both clonidine (10 microgram/ml) and yohimbine (0.1-1.0 microgram/ml) inhibited AH formation (60% inhibition) more than AH outflow (no inhibition for clonidine and 40% inhibition for yohimbine) to lower IOP. The conventional theory of receptor antagonism does not seem to function at alpha 2-receptor sites.

Systemic administration of calcitonin through ocular route.

Calcitonin has been used clinically to treat hypercalcemia, Vitamin D intoxication, osteolytic bone metastases and increased skeletal remodeling in Paget's disease. In general calcitonin is given every 6 to 12 hrs intramuscularly or subcutaneously. It has been found in this study that the same results can be achieved by giving calcitonin through eyes as ophthalmic solutions. When 25 microliters of 0.05% calcitonin was given as eyedrops to New Zealand white rabbits, it did not reach the concentration achieved by i.v. administration at the same dose level. The systemic absorption of calcitonin did not reach the level achieved by i.v. administration even though the eyedrop concentrations were increased 2-fold (0.1%) to 10-fold (0.5%). When absorption enhancers such as BL-9 and Brij-78 were added to calcitonin eyedrops, however, the systemic absorption of calcitonin was enhanced markedly. BL-9 (0.5%) increased calcitonin (0.5%) absorption 16-20 fold and raised blood concentration of calcitonin above levels achieved by i.v. injection (25 microliters, 0.05%) with 0.5% calcitonin eyedrops instillation. Effects of Brij-78 (0.5%) were even more impressive. It increases calcitonin absorption 22-24 fold and raised the blood concentration of calcitonin above the levels achieved by i.v. injection (25 microliters 0.05%) with 0.15% and 0.5% calcitonin eyedrops instillation. These results indicate that the therapeutic level of calcitonin can be reached through the ocular route.

Prevention of organophosphate intoxication with presynaptic cholinergic blockers.

In Vivo cardiovascular neuromuscular preparation of rats was used to study the prophylaxis and mechanism of diisopropyl fluorophosphate (DFP) intoxication by presynaptic cholinolytic agents (ganglionic blocking agents), including hexamethonium, trimethaphan and mecamylamine. The lethal action of DFP was partially or completely prevented by pretreatment of rats with hexamethonium (10 mg/kg), trimethaphan (80 mg/kg) and mecamylamine (30 mg/kg). Combined use of these drugs with 2-PAM (100 mg/kg) improved further the prophylaxis of DFP lethality. The mechanism of prophylaxis could be due to the reduction of acetylcholine turnover and accumulation at the synaptic cleft as hemicholinium prevented the DFP intoxication efficiently. In all cases, DFP caused cardiovascular suppression before neuromuscular blockade indicating that the cardiovascular system is much more sensitive than the neuromuscular junction to DFP intoxication.

Contribution of circulating acetylcholine to sensory nerve conduction augmentation.

We previously discovered that sensory nerve action potential amplitudes increased during isometric muscle contraction and that this response could be blocked with tourniquet isolation of the contraction source. The hypothesis for this study was that a circulating factor was responsible for this effect. In this prospective study, baseline and post intravenous injection of serial sural nerve action potential recordings were made in the leg of 8 rabbits. The sequence of the injections was randomized: 1) normal saline placebo, 2) 0.01 mg/kg acetylcholine (ACh) 3) 200 mg/kg Na acetate, 4) 260 mg/kg Na lactate, and 5) 20 mg/kg choline. Results showed there was a 3.8 microV increase in the sural nerve response 6 min after ACh injection compared to baseline at rest (p = .01, power = .9, analysis of variance (ANOVA), repeated measures). There were no significant changes in the amplitudes of the sural nerve after injection of the remaining agents or placebo (p = .33 to .81, ANOVA, repeated measures). In conclusion, circulating ACh is the only agent tested thus far that appears to be responsible for this effect. In addition, the amplitude and temporal curve of this response is similar to that seen after exercise in human subjects. The clinical importance of this study is that ACh plays a role in this newly discovered sensory regulatory mechanism controlled by the motor system.

Systemic delivery of enkephalin peptide through eyes.

Leu-enkephalin is one of the important peptides which could become useful drugs in the clinics because of its analgesic action and its availability in mass quantity through biotechnology production. It is found in this study that enkephalin can be effectively absorbed systemically through eyes without using a surfactant as absorption enhancer. Enkephalin at 0.125% (31.25 micrograms/25 microliter) reached a plateau of blood concentration at 11.5 ng/ml in 3-4 hrs and stayed high for 8-9 hrs. In contrast, the blood concentration of enkephalin declined rapidly after i.v. administration with a T1/2 of less than 30 min and reached the lowest point at 22 ng/ml in 5 hrs. With higher concentrations at 1% (.25 mg/25 microliter) and 5% (1.25 mg/25 microliter) similar absorption kinetics was observed except that they reached higher plateau of blood concentration at 72 ng/ml and 233 ng/ml, respectively.

Effects of N-demethylated carbachol on iris and ciliary muscles.

The tertiary nitrogen derivative of carbachol, N-demethylated carbachol, was synthesized to produce a drug that could be used as an alternative to pilocarpine in the treatment of open-angle glaucoma. Unlike pilocarpine, N-demethylated carbachol appeared to be stable in alkaline solutions. Since pilocarpine has to be dissolved in an acidic medium (pH 5.5) to prevent its destruction, less than 10% of its molecules are in an unionized form. On the other hand, N-demethylated carbachol is very stable at pH values higher than 7.4 and thus has more molecules (greater than 10%) in an unionized, penetrable form than does pilocarpine in high pH media. N-Demethylated carbachol acted as a full agonist on isolated dog iris and ciliary muscle preparations, whereas pilocarpine produced only 49 and 38% of the maximum tissue responses, respectively. These findings suggest that N-demethylated carbachol may be able to affect the outflow facility to a greater extent than is achievable with pilocarpine at high doses.

Chemotherapy of neuroblastoma in mice with anticancer agents.

Neuroblastoma-inoculated A/J mice were treated with various anticancer chemotherapeutic agents, including cyclophosphamide, daunorubicin, vincristine, alpha-bungarotoxin, dihydroxytryptamine, and diaminopropane. Cyclophosphamide and diaminopropane inhibited neuroblastoma as effectively as bromoacetylcholine and bromacetate. The effectiveness of these drugs could be related to the inhibition of ornithine decarboxylase, a rate-limiting enzyme for the synthesis of polyamines.

Improvement of systemic absorption of insulin through eyes with absorption enhancers.

A painless, simple, and practical method has been developed for systemic delivery of peptide drugs through eyes. In order to increase the amount of peptides absorbed into systemic circulation, effects of enhancers on insulin absorption into systemic circulation and reduction of blood glucose concentration have been studied. Saponin showed the highest potency to enhance insulin absorption, while Tween 20 was the least effective. The efficacy order of seven compounds studied can be arranged as follows: saponin greater than fusidic acid greater than BL-9 = EDTA greater than glycocholate = decamethonium greater than Tween 20. Since 1% of saponin reached the maximum effect to enhance insulin absorption and decrease blood glucose concentration, not more than 1% of saponin should be used for enhancing peptide absorption through eyes.

Effects of glucose on neuroblastoma in vitro and in vivo.

Because neuroblastoma cells in vitro are sensitive to changes in glucose levels in growth media, the effects of glucose levels and dietary carbohydrate on the sensitivity of neuroblastoma cells to chemotherapy were studied. In vitro, 20 microM bromoacetylcholine, 3mM 1,3-diaminopropane, and 5 mM 5-fluorouracil were added to the growth medium of strain N2a neuroblastoma cells cultured in Dulbecco's modified Eagle medium with high glucose (4.5 g/L, HG), normal glucose (1.0 g/L, NG), or low glucose (0.1 g/L, LG). Diaminopropane and 5-fluorouracil had some cytotoxic effect on cells in all media. Bromoacetylcholine killed cells in all media, but at a concentration of 20 microM was most effective in LG medium. Mice (A/Jax) were inoculated with neuroblastoma cells for in vivo studies. Mice could not tolerate a carbohydrate-free diet, while a high-carbohydrate diet caused no change in survival time. When mice on a high carbohydrate diet were treated with cyclophosphamide (100 mg/kg, ip) or 5-fluorouracil (125 mg/kg, ip) they died faster than drug-treated mice on a normal diet. Oral chlorpropamide or cimetidine did not prolong survival time. Analysis of blood glucose levels showed neuroblastoma significantly lowered blood glucose levels (p less than 0.05), while chlorpropamide had no significant effect. It is proposed that manipulation of plasma glucose to lower levels will not be effective in enhancing the chemotherapy of neuroblastoma.

Use of C1300 neuroblastoma cells to evaluate the protective value of hexamethonium, trimethaphan, hemicholinium, and triethylcholine against diisopropyl phosphorofluoridate toxicity.

Our intent was to evaluate the C1300 neuroblastoma cell as an in vitro system for studying the mode of action and efficacy of drugs used to treat or prevent organophosphate intoxication. The anticholinergic drugs hexamethonium, trimethaphan, and hemocholinium and the triethylcholine and cholinesterase/reactivator 2-pyridine aldoxime methochloride (2-PAM) have been shown to be effective in preventing intoxication by diisopropyl phosphorofluoridate (also known as diisopropyl fluorophosphate, DFP) in vivo. We determined their efficacy in preventing cell death (as measured by trypan blue exclusion) of neuroblastoma cells alone or in combination. We also determined their efficacy in reversing the cytotoxic effects of DFP on cell DNA synthesis (as measured by [3H]-thymidine incorporation), cell RNA synthesis (as measured by [3H]uridine incorporation), and on cell protein synthesis (as measured by [3H]leucine incorporation). The maximal nontoxic doses of the drugs in vitro were determined. All anticholinergic agents studied reduced the cytotoxicity of DFP using one or more parameters. 2-PAM, the cholinesterase reactivator, enhanced the cytotoxicity of DFP on cultured cells at a high concentration (1 mg/mL) and reduced it at a lower concentration (0.3 mg/mL). All four anticholinergic agents were capable of enhancing the uptake of [3H]thymidine. Only hexamethonium and hemicholinium reversed DFP inhibition of DNA synthesis. RNA synthesis was not affected by any anticholinergic agent and no agent reversed DFP inhibition of RNA synthesis. Protein synthesis was enhanced by every anticholinergic agent except hemicholinium; the inhibition of protein synthesis by DFP was reversed by trimethaphan and triethylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of D-timolol on intraocular pressure, heart rate, cardiac contractility, and tracheal muscle function.

Introduction of L-timolol maleate into the clinics for glaucoma treatment has been a great success as it produces relatively few side effects, such as miosis, ciliary spasm and headache, which are associated with the use of pilocarpine. Recent reports indicated, however, that L-timolol produces asthmatic attacks, cardiovascular suppression and central depression. D-Timolol was found to be as effective as L-timolol in lowering the intraocular pressure and aqueous humor production. However it was much less potent than L-timolol in inhibiting cardiac contractility and heart rate stimulated by isoproterenol and was less active in blocking beta-adrenergic receptors in tracheal muscle. It is suggested that the D-isomer of timolol could be used to treat glaucoma instead of L-isomer without producing untoward side effects in cardiovascular and bronchial tissues.

Characterization of unique ADTN-catecholamine binding sites in the iris root-ciliary body of rabbits.

A binding site for tritiated 2-amino-6, 7-dihydroxy-1, 2,3,4-tetrahydronaphthalene (ADTN) has been partially characterized in the rabbit iris root-ciliary body. Binding of ADTN is proportional to protein content and requires at least 60 minutes to reach equilibrium. Binding is saturable, with a Kd of 27 +/- 1 nM and a Bmax of 2.1 +/- .3 pmol/mg protein (mean +/- SEM). Dopamine competes for this site with a Ki of 100 nM and apomorphine with a Ki of 180 nM. This site is not blocked by L-timolol, phenoxybenzamine, or by several DA1 and DA2 antagonists. It appears to be a new type of catecholamine binding site, of a type not observed outside the anterior eye. It is possible that some of the effects of dopamine on intraocular pressure are mediated through this binding site.

Review: effects of nitric oxide on eye diseases and their treatment.

Both underproduction and overproduction of nitric oxide (NO) could lead to various eye diseases. It is known that endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are activated in normal tissues to produce NO for physiological functions. Thus, underproduction of NO results in various eye diseases which could be corrected by providing NOS substrates or NO donors to lower the intraocular pressure, increase ocular blood flow, relax ciliary muscle, etc. On the other hand, immunological NOS (iNOS) is inducible only in pathological conditions by endotoxins, inflammation, and certain cytokines, such as interleukin-1 (IL-1), IL-6, TNF (tumor necrosis factor) and the like. Once induced, iNOS will produce large amounts of NO for long periods of time, so that NO is converted into NO2, nitrite, peroxynitrite and free radicals to induce pathophysiological actions, such as optic nerve degeneration and posterior retinal degeneration lesion, which lead to glaucoma, retinopathy, age-related macular degeneration (AMD), myopia, cataracts and uveitis. To treat/prevent these eye diseases, inhibitors of iNOS activity and/or iNOS induction could be tried.

Efficacy of oxymetazoline eye drops in non-infectious conjunctivitis, the most common cause of acute red eyes.

Oxymetazoline (OMZ) is an alpha-adrenergic agonist which causes potent vasoconstriction in various tissues. Thus, its effects on red eye conjunctivitis induced by histamine and arachidonic acid were tested in rabbit eyes. It was found that a brief conjunctivitis induced by 0.01% of histamine can be eliminated effectively by 0.001, 0.004, and 0.016% of OMZ. The long lasting conjunctivitis induced by 0.3% arachidonic acid can also be markedly reduced by 0.004, 0.008, and 0.016% of OMZ. These results indicate that OMZ is a potent, long acting decongestant for conjunctival red eye syndrome.

Feasibility of glucagon eyedrops for clinical use in hypoglycemia.

Although sufficient amount of glucagon can be taken up into systemic circulation through ocular route, the concentration of eyedrops needed at 7.5% would be too high and the price of the doses would be too expensive to be desirable. Attempts have been made to reduce the doses of glucagon needed for systemic delivery through ocular route by adding absorption enhancers such as 0.5% BL-9 or 0.5% Brij-78. With these absorption enhancers, the effective concentrations of glucagon eyedrops can be lowered to 0.05% in rabbit eyes and could be lowered to 1.25% in human eyes which would be reasonable to be used in the clinics.