Novel hydrogel-based ocular drug delivery system for the treatment of conjunctivitis.

PURPOSE: Conventional dosage form like eye drops showed poor therapeutic response and also require frequent dosing. Therefore, developing the dosage form to deliver the drug to the target site without much loss of drug or without causing any systemic side effects is the challenging job for the researchers in pharmaceutical industries. OBJECTIVE: The main aim of the present work was to formulate and evaluate hydrogel-based drug delivery containing combination of neomycin sulphate and betamethasone sodium phosphate in order to provide prolonged release and also better bioavailability of drugs for the treatment of eye infections. METHODS: In this study, poloxamer 407 and chitosan at different concentrations were used as the gelling agents. The prepared formulations were evaluated for clarity, pH, drug content, gelling capacity, gelling temperature and in vitro drug release study. RESULTS: From the preliminary studies, F5 formulation was selected as an optimized formulation. The optimized formulation was further evaluated for ex vivo permeation study, sterility test, HET-CAM and ocular irritation testing using rabbits. Ocular irritation by HET-CAM assay showed that the formulated gel does not cause any irritation to the blood vessels. Draize irritation test was performed using rabbits and results showed that formulation was non-irritant to the eye. CONCLUSION: The formulated hydrogel formulation can be used as an alternative to conventional ophthalmic eye drop formulation of drugs neomycin and betamethasone for the purpose of providing prolonged therapy for the treatment of conjunctivitis.

Resistance to neomycin ototoxicity in the extreme basal (hook) region of the mouse cochlea.

Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO2) in normal culture medium for 19.5 h prior to and after exposure to neomycin (1 mM, 3 h). To study neomycin uptake in the hair cells, cochlear explants were incubated with Neomycin Texas-red (NTR) conjugate. As expected, exposure to neomycin significantly reduced the survival of inner (IHC) and outer hair cells (OHC). IHC survival rate was high in the apical segment and low in the basal segment. OHC were well preserved in the apical and hook regions, with substantial OHC loss in the basal segment. The NTR uptake study demonstrated that the high survival rate in the extreme basal turn OHC was associated with low NTR uptake. Treatment with a calcium chelator (BAPTA), which disrupts the opening of mechanoelectrical (MET) transduction channels, abolished or reduced NTR uptake in the hair cells throughout the cochlea. This confirmed the essential role of MET channels in neomycin uptake and implied that the transduction channels could be impaired in the hook region of the developing mouse cochlea, possibly as a result of the cadherin 23 mutation responsible for the progressive deafness in C57BL/6 mice.

Improvement in adiposity with oligofructose is modified by antibiotics in obese rats.

Given the intimate link between gut microbiota and host physiology, there is growing interest in understanding the mechanisms by which diet influences gut microbiota and affects human metabolic health. Using antibiotics and the prebiotic oligofructose, which has been shown to counteract excess fat mass, we explored the gut microbiota-dependent effects of oligofructose on body composition and host metabolism. Diet-induced obese male Sprague Dawley rats, fed a background high-fat/sucrose diet, were randomized to one of the following diets for 6 wk: 1) high-energy control; 2) 10% oligofructose; 3) ampicillin; 4) ampicillin + 10% oligofructose; 5) ampicillin/neomycin; or 6) ampicillin/neomycin + 10% oligofructose. Combining oligofructose with ampicillin treatment blunted the decrease in adiposity seen with oligofructose. Although ampicillin did not affect total bacteria, ampicillin impeded oligofructose-induced increases in Bifidobacterium and Lactobacillus In contrast, the combination of ampicillin and neomycin reduced total bacteria but did not abrogate the oligofructose-induced decrease in adiposity. Oligofructose-mediated effects on host adiposity and metabolic health appear to be in part dependent on the presence of specific microbial species within the gut.-Bomhof, M. R., Paul, H. A., Geuking, M. B., Eller, L. K., Reimer, R. A. Improvement in adiposity with oligofructose is modified by antibiotics in obese rats.

Conjugation of a Ru(II) arene complex to neomycin or to guanidinoneomycin leads to compounds with differential cytotoxicities and accumulation between cancer and normal cells.

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 µM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 µM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 µM in DU-145 and IC50 = 11.33 µM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 ≫ 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

In vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines.

BACKGROUND: Targeted drug-carrying phage nanomedicines are a new class of nanomedicines that combines biological and chemical components into a modular nanometric drug delivery system. The core of the system is a filamentous phage particle that is produced in the bacterial host Escherichia coli. Target specificity is provided by a targeting moiety, usually an antibody that is displayed on the tip of the phage particle. A large drug payload is chemically conjugated to the protein coat of the phage via a chemically or genetically engineered linker that provides for controlled release of the drug after the particle homed to the target cell. Recently we have shown that targeted drug-carrying phage nanomedicines can be used to eradicate pathogenic bacteria and cultured tumor cells with great potentiation over the activity of the free untargeted drug. We have also shown that poorly water soluble drugs can be efficiently conjugated to the phage coat by applying hydrophilic aminoglycosides as branched solubility-enhancing linkers. RESULTS: With an intention to move to animal experimentation of efficacy, we tested anti-bacterial drug-carrying phage nanomedicines for toxicity and immunogenicity and blood pharmacokinetics upon injection into mice. Here we show that anti-bacterial drug-carrying phage nanomedicines that carry the antibiotic chloramphenicol conjugated via an aminoglycoside linker are non-toxic to mice and are greatly reduced in immunogenicity in comparison to native phage particles or particles to which the drug is conjugated directly and are cleared from the blood more slowly in comparison to native phage particles. CONCLUSION: Our results suggest that aminoglycosides may serve as branched solubility enhancing linkers for drug conjugation that also provide for a better safety profile of the targeted nanomedicine.

Middle ear application of a sodium hyaluronate gel loaded with neomycin in a Guinea pig model.

OBJECTIVE: Establishing methods for topical administration of drugs to the inner ear have great clinical relevance and potential even in a relatively short perspective. To evaluate the efficacy of sodium hyaluronate (HYA) as a vehicle for drugs that could be used for treatment of inner ear disorders. METHODS: The cochlear hair cell loss and round window membrane (RWM) morphology were investigated after topical application of neomycin and HYA into the middle ear. Sixty-five albino guinea pigs were used and divided into groups depending on the type of the treatment. Neomycin was chosen as tracer for drug release and pharmacodynamic effect. HYA loaded with 3 different concentrations of neomycin was injected to the middle ear cavity of guinea pigs. Phalloidin stained surface preparations of the organ of Corti were used to estimate hair cell loss induced by neomycin. The thickness of the midportion of the RWM was measured and compared with that of controls using light and electron microscopy. All animal procedures were pe rformed in accordance with the ethical standards of Karolinska Institutet. RESULT: Neomycin induced a considerable hair cell loss in guinea pigs receiving a middle ear injection of HYA loaded with the drug, demonstrating that neomycin was released from the gel and delivered to the inner ear. The resulting hair cell loss showed a clear dose-dependence. Only small differences in hair cell loss were noted between animals receiving neomycin solution and animals exposed to neomycin in HYA suggesting that the vehicle neither facilitated nor hindered drug transport between the middle ear cavity and the inner ear. One week after topical application, the thickness of the RWM had increased and was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal. CONCLUSION: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss.

Bioactive polymers. LX. Kinetics of delayed release neomycin-xanthan complex.

The kinetics of delayed release neomycin-xanthan complex, and its antimicrobial activity, were studied. Delayed release neomycin (53.13% free neomycin) had zero-order release kinetics, when artificial tear solution was used as eluent. Microbiological tests revealed an activity of 380 units/mg, 10% higher than neomycin in its free form.

A multichannel scala tympani electrode array incorporating a drug delivery system for chronic intracochlear infusion.

We have developed a novel scala tympani electrode array suitable for use in experimental animals. A unique feature of this array is its ability to chronically deliver pharmacological agents to the scala tympani. The design of the electrode array is described in detail. Experimental studies performed in guinea pigs confirm that this array can successfully deliver various drugs to the cochlea while chronically stimulating the auditory nerve.

Tympanostomy tubes and otic suspensions: do they reach the middle ear space?

The treatment of patients with tympanostomy tubes (TTs) and otorrhea with medicated otic suspensions is well known, but confirmation of penetration into the middle ear is difficult. To address this question, we created an in vitro model of the human head and ear and then tested it with 5 different types of liquid exposure: tap water, soapy water, polymyxin B sulfate (Cortisporin), tobramycin and dexamethasone (TobraDex), and ciprofloxacin (Cipro) suspensions. A positive test result corresponded to liquids entering the middle ear through the TT. No positive test result was elicited with tap water (0/20), but soapy water did enter the middle ear (10/40) and was statistically significant (P = 0.0112). Without the use of slight tragal pressure, Cortisporin, TobraDex, and Cipro drops did not consistently pass through the TT (0/20, 1/25, 1/25). By placing the drops with the addition of tragal pressure, a statistically significant difference was obtained for each solution (20/20, 20/20, and 20/20, respectively [P < 0.0001]). We conclude that with a clean external auditory canal, patent TT, and no middle ear fluid, medicated otic suspensions enter the middle ear only when combined with slight tragal pressure.

Bioactive polymers: in vitro and in vivo study of controlled release neomycin.

Neomycin is coupled on xanthan-a polysaccharide of microbial biosynthesis produced by Xanthomonas campestris-through ionic complexation. The kinetics of neomycin release, in vitro, at pH = 8.2 is studied. A controlled release of neomycin, following a zero order kinetics is observed, regardless of the eluent flow. Neomycin complexed on xanthan, administered in a unique daily dose to patients suffering from dysentery in the 100 cases taken in study, has shown a high clinical efficiency as compared with the treatments with ampicillin or furazolidone, administered for 5-10 days or longer.

The nephrotoxic potential of neomycin in the horse.

Neomycin was administered intramuscularly to four normal adult horses at a dose rate of 10 mg/kg bodyweight every 12 h for 10 days (21 doses). The pharmacokinetic behaviour of neomycin with multiple dosing was characterised and a range of blood chemical and urinary parameters examined for evidence of nephrotoxicity. There was evidence of physical renal tubular injury (enzymuria and cylindriuria) within four days of neomycin administration but this subsided following cessation of treatment. No significant functional nephrotoxicity was detected. More severe nephrotoxicity might be expected in ill horses and it is recommended that several clinicopathological results be monitored serially in those horses receiving parenteral neomycin.

Neomycin metabolism in calves.

Disposition of oral neomycin in calves was determined using 14C-labeled neomycin. The influences of age, diet, and method of administration were observed. All calves were killed 96 h after a single oral dose of [14C]neomycin (approximately 30 mg/kg) and the distribution of 14C in excreta and tissues was determined. As indicated by urinary excretion, absorption of neomycin was greater in 3-d-old calves (11.1 +/- 1.8% of the dose) than in 54- to 64-d-old nonruminating calves (1.5 +/- .58% of the dose) dosed similarly. Absorption of neomycin was similar in nonruminating (1.5 +/- .58%) and ruminating (2.13 +/- .62%) calves when the doses were administered in solution via a nippled bottle. In ruminating calves, absorption was somewhat less when the dose was administered on feed via a gelatin capsule (.5 +/- .06% of the dose) than when given in solution via a nippled bottle (2.13 +/- .62% of the dose). In calves dosed at 3 d of age, 14C concentration in the kidneys represented 55 +/- 4.9 ppm of neomycin equivalents. The next highest concentration occurred in the livers, which contained less than 5% of the level in kidneys. Tissue concentrations of 14C were related to absorption (as indicated by urinary excretion). Isolation and characterization (positive-ion fast atom bombardment mass spectroscopy and nuclear magnetic resonance spectroscopy) of 14C compounds in kidneys of calves dosed at 3 d of age indicated that at least 90% of the 14C was present as neomycin. Neomycin was also the major 14C compound in feces of all calves (70 to 80% of the 14C present).(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid mediators and MgATPase modulation causes changes in the cardiovascular effects of vasopressin in lithium carbonate-induced polyuric rats.

The effect of phospholipid and MgATPase modulation was evaluated on the cardiovascular actions of vasopressin in normal and lithium carbonate- (Li2CO3) induced polyuric rats. We examined the effects of the phospholipase inhibitor neomycin, the diacylglycerol kinase II inhibitor R59949 and the MgATPase activator sphingosine on heart rate (HR) and blood pressure (BP) responses to vasopressin analogues lysine vasopressin (LVP) and arginine vasopressin (AVP). R59949 (20 microg/kg) produced an increase while sphingosine (30 microg/kg) caused a decrease in HR responses in both control and polyuric rats. Pretreatment with sphingosine caused significant enhancement of LVP- (10 microg/kg) induced bradycardia in polyuria rats compared with control animals (p < 0.01). R59949 induced a potentiation of vasopressin-induced bradycardia in control animals compared with polyuria rats. Pretreatment with sphingosine and R59949 produced a significant increase in BP per se and potentiated the actions of LVP in control animals, while the response in the lithium-treated animals was attenuated. Neomycin caused a reduction in HR and BP in control and lithium-treated animals. To evaluate the central role of the MgATPase enzyme we used sphingosine, which significantly increased the locomotor activity of lithium-treated animals, suggesting a possible central interaction of lithium and MgATPase (p < 0.05). These results strongly suggest that phospholipid mediators and MgATPase modulation contribute to the alteration of the cardiovascular effects of vasopressin in lithium carbonate-induced polyuric rats.

Bioactive polymers. 70. The kinetics of controlled release of neomycin in an alkaline medium.

Neomycin is coupled on xanthan--a polysaccharide of microbial biosynthesis produced by Xanthomona campestris--through ionic complexation. The kinetics of neomycin release, in vitro, at pH = 8.2 is studied. A release of neomycin, following a zero order kinetics, is observed, regardless of the eluent flow-rate. The neomycin-xanthan complex, protected by a cellulosic membrane, behaves like a monolithic-type device. Diffusion coefficients--increasing with increasing the eluent flow-rate--are also calculated.

Absorption of neomycin from the equine uterus: effect of bacterial and chemical endometritis.

Plasma concentrations of neomycin were measured after intrauterine infusion of 3.3 mg/kg neomycin sulphate. Mares infected two hours previously with an intra-uterine infusion of beta-haemolytic streptococci absorbed approximately 12 per cent of the neomycin in both the oestrous and the luteal phases of the cycle. Normal mares in oestrus absorbed 6 per cent of the neomycin infused and luteal mares absorbed 56 per cent. In infected mares the peak plasma concentrations occurred two hours after neomycin infusion, earlier than in healthy mares. Cervical flushings after neomycin infusion in infected luteal mares revealed an increased reflux of neomycin when compared with healthy mares. Prior infusion of 30 ml of 10 per cent Lugol's iodine into the uterus resulted in 31 per cent of neomycin being absorbed by oestrous mares and 64 per cent by mares in the luteal phase. Peak plasma concentrations occurred 30 minutes after infusion in both phases. In the luteal phase the mares' absorption of neomycin may have been maximal.

Biopharmaceutical assessment of eye drops containing aloe (Aloe arborescens Mill.) and neomycin sulphate.

The subject of the studies was eye drops made of aloe, containing the group of aloe chemical substances of anti-inflammatory use and neomycin sulphate. The aim of the studies was to evaluate the permeability of biologically active aloe substances, determined as aloenin, through synthetic lipophilic and hydrophilic membranes in a standard perfusion apparatus and in vitro verification of the transport possibilities of these substances through the isolated cornea of pig's eye. The permeability process of biologically active aloe substances determined as aloenin, through synthetic lipophilic and hydrophilic membranes, was analyzed using the first-order kinetics. Estimated quotas of permeability rate constant show that the investigated chemical compounds of aloe, included in the eye drops, diffused through the applied membranes. The studies of permeability through isolated pig's cornea proved that biologically active aloe substances could not overcome this biological barrier. On the basis of biopharmaceutical studies it can be concluded that the eye drops containing aloe and neomycin sulphate, due to the lack of permeating abilities through the eye cornea, should be particularly useful in the treatment of inflammations and infections of external parts of the eye, such as conjuctiva, eyelid edges, lacrimal sac and cornea.

Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.