Development of cationic nanocrystals for ocular delivery.

A cationic nanocrystal formulation containing dexamethasone acetate nanocrystals (0.05%) and polymyxin B (0.10%) for ophthalmic application was produced using a self-developed small scale method for wet bead milling. The formulation developed offers the advantage of increased saturation solubility of the drug (due to the nano-size of the crystals) and increased residence time in the eye (due to small size and increased mucoadhesion by the cationic charge) resulting ultimately in potential increased bioavailability. Characterization of the nanosuspensions by photon correlation spectroscopy (PCS) and transmission electron microscopy showed that the production method was successful in achieving dexamethasone crystals in the range of about 200-250nm. The physical stabilization of the nanocrystals and generation of the positive charge were realized by using cetylpyridinium chloride (CPC) and benzalkonium chloride (BAC) at the concentration of 0.01%. In contrast to other cationic excipients, they are regulatorily accepted due to their use as preservatives. The drug polymyxin B also contributed to the positive charge. Positive zeta potentials in the range +20 to +30mV were achieved. Isotonicity was adjusted using NaCl and non-ionic excipients (glycerol, sorbitol, dextrose). Physical and chemical stabilities were monitored for a period of 6months at room temperature, 5°C and 40°C. Particle size of the bulk population assessed by PCS remained practically unchanged over 6months of storage for the various formulations without isotonicity agents, and for the CPC-containing formulations with non-ionic isotonicity excipients. The chemical content also proved stable after 6months for all 3 temperatures evaluated. In vitro investigation of mucoadhesion was tested using mucin solutions at different concentrations, and the generated negative zeta potential was used as a measure of the interaction. The zeta potential reversed to about -15mV, indicating distinct interaction. The results show the potential of increased mucoadhesion of such cationic nanocrystals compared to standard eye drop formulations. The positively charged nanocrystal formulation also showed no in vitro cytotoxicity as assessed on fibroblast cell culture. In summary, 3 formulation candidates were identified being a promising alternative for ocular delivery with increased performance compared to what is presently available.

Nanocrystals for dermal penetration enhancement - Effect of concentration and underlying mechanisms using curcumin as model.

Nanocrystals have received considerable attention in dermal application due to their ability to enhance delivery to the skin and overcome bioavailability issues caused by poor water and oil drug solubility. The objective of this study was to investigate the effect of nanocrystals on the mechanism of penetration behavior of curcumin as a model drug. Curcumin nanocrystals were produced by the smartCrystals® process, i.e. bead milling followed by high pressure homogenization. The mean particle size of the curcumin crystals was about 200nm. Stabilization was performed with alkyl polyglycoside surfactants. The distribution of curcumin within the skin was determined in vitro on cross-sections of porcine skin and visualized by fluorescent microscopy. The skin penetration profile was analyzed for the curcumin nanosuspension with decreasing concentrations (2%, 0.2%, 0.02% and 0.002% by weight) and compared to nanocrystals in a viscous hydroxypropylcellulose (HPC) gel. This study demonstrated there was minor difference between low viscous nanosuspension and the gel, but low viscosity seemed to favor skin penetration. Localization of curcumin was observed in the hair follicles, also contributing to skin uptake. Looking at the penetration of curcumin from formulations with decreasing nanocrystal concentration, formulations with 2%, 0.2% and 0.02% showed a similar penetration profile, whereas a significantly weaker fluorescence was observed in the case of a formulation containing 0.002% of curcumin nanocrystals. In this study we have shown that curcumin nanocrystals prepared by the smartCrystal® process are promising carriers in dermal application and furthermore, we identified the ideal concentration of 0.02% nanocrystals in dermal formulations. The comprehensive study of decreasing curcumin concentration in formulations revealed that the saturation solubility (Cs) is not the only determining factor for the penetration. A new mechanism based also on the concentration of the nanocrystals on skin surface was proposed.

Amorphous cyclosporin A nanoparticles for enhanced dermal bioavailability.

Cylosporin A (CyA) was formulated as amorphous nanoparticle suspension to increase dermal penetration, e.g. applicable in psoriasis. The suspension consisted of 5% CyA in water, stabilized with vitamin E polyethylene glycol succinate (TPGS, Kolliphor TPGS) and was produced by bead milling. The diameter of the bulk population was about 350 nm, laser diffraction diameter 99% was 690 nm. The suspension was physically stable over one year of storage at room temperature, and most important the amorphous state also remained stable. Despite the high dispersitivity and related large surface area in contact with water, the drug content reduced only by 5% over 1 year of storage. i.e. the formulation is feasible as commercial product with expiry date. The CyA nanoparticles and µm-sized CyA particles were incorporated into hydroxypropylcellulose (HPC) gels and the penetration studied into fresh pig ear skin applying the tape stripping method. At tape number 30, the penetrated cumulative amount of CyA from nanoparticles was 6.3 fold higher compared to the µm-sized raw drug powder (450.1 µg/cm(2) vs. 71.3 µg/cm(2)). A theoretical mechanism is presented to explain the observed superiority in penetration. Based on amorphous CyA nanoparticles, dermal formulations for improved dermal CyA delivery seem to be feasible.

Simple low-cost miniaturization approach for pharmaceutical nanocrystals production.

Systematic screening for optimal formulation composition and production parameters for nanosuspensions consumes a lot of time and also drug material when performed at lab scale. Therefore, a cost-effective miniaturized scale top down approach for nanocrystals production by wet bead milling was developed. The final set-up consisted of 3 magnetic stirring bars placed vertically one over the other in a 2 mL glass vial and agitated by a common magnetic stirring plate. All of the tested actives (cyclosporin A, resveratrol, hesperitin, ascorbyl palmitate, apigenin and hesperidin) could be converted to nanosuspensions. For 4 of them, the particles sizes achieved were smaller than previously reported on the literature (around 90 nm for cyclosporin A; 50 nm for hesperitin; 160 nm for ascorbyl palmitate and 80 nm for apigenin). The "transferability" of the data collect by the miniaturized method was evaluated comparing the production at larger scale using both wet bead milling and high pressure homogenization. Transferable information obtained from the miniaturized scale is minimum achievable size, improvements in size reduction by reduction of beads size, diminution kinetics and potentially occurring instabilities during processing. The small scale batches also allow identification of optimal stabilizer types and concentrations. The batch size is 0.5 mL, requiring approximately 50 mg or 5 mg of drug (5% and 1% suspension, respectively). Thus, a simple, accessible, low-cost miniaturized scale method for the production of pharmaceutical nanocrystals was established.

Industrial concentrates of dermal hesperidin smartCrystals®--production, characterization & long-term stability.

Industrial concentrates of hesperidin nanocrystals (5.0% nominal concentration) were produced applying the smartCrystal(®) combination technology of wet bead milling and subsequent high pressure homogenization. Stabilization was performed by Kolliphor(®) P 188, preservation by Euxyl PE 9010 and glycerol. Physical and chemical stability were monitored over 1.5 years of storage at 4-6 °C. The size of the bulk population stayed unchanged with about 250 nm (photon correlation spectroscopy). Absence of crystal growth by Ostwald ripening and absence of agglomerates were shown by laser diffraction (LD) and light microscopy. The LD diameter 90% was still 0.7 µm after 1.5 years. Despite the large surface of the nanosuspension in contact with the water phase, the chemical content proved also stable, only a reduction by 0.15% from 5.70% to 5.55% content was observed. The nanocrystals kept their crystalline state unchanged as shown by X-ray diffraction. The saturation solubility of the nanosuspension was more than triple compared to the raw drug powder in water. The data show the availability of a stable hesperidin concentrate as intermediate for industry to produce dermal formulations.

Chromatic modulation of luminance visual evoked potential latencies in healthy subjects and patients with mild vision disorders.

OBJECTIVE: To study whether and how color modulates luminance visual evoked potentials (VEPs). METHODS: We studied pattern-reversal luminance VEPs to red/black and blue/black checkerboards with identical luminance contrast values (mixed luminance and chromatic components) (isocontrast color VEP, in brief, IVEPs) in 25 healthy subjects and two groups of patients with mild vision disorders (23 with glaucoma and 25 with optic neuritis). We then compared these with the standard color VEPs to pure chromatic contrast red/green and blue/yellow gratings (CVEPs). RESULTS: In healthy subjects, VEPs to red/black checkerboards and red/green gratings were slower than those obtained with blue/black checkerboards and blue/yellow gratings. Both procedures (IVEPs and CVEPs) differentiated patients with vision disorders from healthy subjects and distinguished between the two different vision disorders. Red/black checkerboards and red-green gratings elicited slower VEPs in patients with optic neuritis and blue/black checkerboards and blue/yellow gratings elicited slower VEPs in patients with glaucoma. IVEPs appeared more stable and ample than CVEPs. The contrast indices normalized CVEP and IVEP latencies in the same subject and showed a positive correlation between CVEP and IVEP latencies in healthy subjects and in patients with optic neuritis, but not in patients with glaucoma. CONCLUSIONS: Our study confirms the usefulness of CVEPs in detecting and differentiating mild vision disorders. IVEPs to colored pattern-reversal luminance checkerboards are equally effective in distinguishing between various vision disorders possibly because colors can modulate VEP latencies to luminance contrast stimuli. SIGNIFICANCE: IVEPs can be useful in differentiating the various vision disorders and are easier than CVEPs to test in a routine clinical setting.

Fast voluntary neck movements in patients with cervical dystonia: a kinematic study before and after therapy with botulinum toxin type A.

OBJECTIVE: To study fast voluntary neck movements in patients with cervical dystonia (CD) before and after therapy with botulinum toxin type-A (BTX-A). METHODS: A selected sample of 15 patients with CD (with prevalent torticollis) and 13 age-matched control subjects performed both right and left rotational, and flexion and extension neck movements as fast as possible. Movements were recorded with a motion analysis system (SMART, BTS). Movement time, angular amplitude, and peak angular velocity were analyzed. In patients, rotational neck movements were pooled as "pro-dystonic" (toward the dystonic side) and "anti-dystonic" (toward the non-dystonic side). Results obtained in patients before BTX-A treatment were compared with those of control subjects. The effect of BTX-A treatment was evaluated by comparing movement performance before and after treatment. RESULTS: Before receiving BTX-A, patients performed pro- and anti-dystonic movements with lower peak angular velocity than control subjects. Pro-dystonic movements had a reduced angular amplitude. Anti-dystonic movements showed an abnormally long movement time. Flexion and extension movements required longer movement times, but the other kinematic variables were normal. After BTX-A injections, pro-dystonic movement amplitude and anti-dystonic movement peak angular velocity increased, whereas flexion and extension movements remained unchanged. CONCLUSIONS: Before BTX-A injection patients with CD perform fast voluntary neck movements abnormally and BTX-A injections improved their peak velocity and amplitude. SIGNIFICANCE: Kinematic studies can detect specific neck movement disturbance in patients with CD, and can quantify both the severity of clinical picture and the effect of BTX-A injections in these patients.

Nuclear Regulatory Authority low energy germanium detection system: performance for the uranium individual monitoring.

The lung counter facility of the Nuclear Regulatory Authority (ARN) is presented. A calibration was carried out using the Lawrence Livermore National Laboratory (LLNL) phantom. This phantom is provided with a pair of lungs and lymph nodes containing uranium homogeneously distributed and a set of four overlay plates covering a chest wall thickness (CWT) ranging from 1.638 to 3.871 cm. Individual organ calibration factors were acquired for 235U photopeaks energies and for each effective chest thickness. Using these factors, a collection of theoretical fitting curves were found. A counting efficiency formulae and a curve for simultaneously active lymph nodes and lung was obtained and checked through measures. Background measurements of the chamber with and without volunteer persons were performed in order to obtain the detection limits (DL) of the system. As this task involves the knowledge of the volunteers CWTs, these magnitudes were determined through formulae selected from the literature taking into account the detection system characteristics. The deviation in the CWT assigned to an individual, generated by applying different equations, produces variations up to 33% in the estimations of the incorporated activity and DL. An analysis of the changes in efficiencies as consequences of the detectors locations and CWT was also performed. This reveals that the DL of the camera (detectors, shield and blank phantom) is between 2.7 and 6.4 Bq of 235U, which implies 4.9 and 11.5 mg lung burden of natural uranium. An estimation of the minimum detectable intake performed with the DL considering blank persons shows that a system with the characteristics described is only adequate for non-routine individual monitoring.

Argentine intercomparison programme for personal dosimetry.

An Intercomparison Programme is being carried out in Argentina for individual monitoring services. The programme was designed to test, on a voluntary basis, the performance of the laboratories that provide individual monitoring services for X and gamma radiation fields in the range from low-level dose up to 100 mSv. Irradiations were performed in full accordance with ISO 4037-3 recommendations by the Regional Reference Centre for Dosimetry (CRRD), belonging to Atomic Energy Commission (CNEA) and the Physical Dosimetry Laboratory of the Nuclear Regulatory Authority (ARN). At the same time, several items have been asked to each participant pertaining to the action range, the detectors' characteristics, the laboratory procedures, the existence of an algorithm and its use for the dosemeter evaluation. In this study the evolution of the laboratories' performance throughout the programme, based on ISO 14146 acceptance criteria, is shown.

Results of the regional intercomparison exercise for the determination of operational quantity HP(10) in Latin America.

Several intercomparison exercises were organised by the International Atomic Energy Agency (IAEA) on the determination of operational quantities at the regional or interregional basis. In the Latin American region an intercomparison for the determination of the operational quantity Hp(10) was completed mid-2004, as a follow-up to previous exercises carried out during the 1990s. Eighteen individual external monitoring services from nineteen Member States participated in the first phase. The second phase grouped 15 services that had participated in the first phase. Dosemeter irradiations in photon beams were done by four Secondary Standard Dosimetry Laboratories (SSDLs) of the region. The preparation of this exercises involved an audit by the IAEA SSDL, where reference irradiations were provided to all participants for verification of their systems. During the first phase (2002-2003) only 9 out of 18 services met the performance requirements for such monitoring services. Necessary corrective actions and procedure verification were implemented. During the second phase (2004) 11 out of 15 services fulfilled the performance criteria. This intercomparison shows that there has been improvement in the second phase and most participants demonstrated a satisfactory performance of the quantity tested.

Luminance and chromatic visual evoked potentials in type I and type II diabetes: relationships with peripheral neuropathy.

The objective of the study was to investigate the subclinical visual deficit in type I and II diabetes, and its relationship with peripheral neuropathy. Thirty-two healthy volunteers, 20 patients with type I diabetes and 30 patients with type II diabetes were studied in a clinical neurophysiology setting. Luminance (VEPs) and chromatic visual evoked potentials (CVEPs) were recorded, with white-black, grey-black, red-green and blue-yellow sinusoidal gratings. The peak latencies of the VEP positive wave and CVEP negative wave were recorded. Ten patients with type I and 8 with type II diabetes had peripheral neuropathy. VEPs were slower in patients with type II diabetes and CVEPs were slower in patients with type I and type II diabetes than in controls. Blue-yellow CVEPs were slower in type II than in type I diabetes. VEPs and red-green CVEPs were slower in patients with diabetes with neuropathy than in those without. In conclusion, we found that visual system impairment differs in diabetes with and without peripheral neuropathy.

Topiramate and cortical excitability in humans: a study with repetitive transcranial magnetic stimulation.

Repetitive transcranial magnetic stimulation (rTMS) delivered at 5 Hz frequency and suprathreshold intensity progressively increases the size of muscle evoked potentials (MEPs) and the duration of the cortical silent period (CSP) in normal subjects. The aim of this study was to evaluate the effects of topiramate (TPM) at different doses on cortical excitability variables tested with rTMS. We tested the facilitation of the MEP size and CSP duration evoked by focal rTMS in eight patients before and after treatment with TPM at different doses for chronic neuropathic pain. In each patient, rTMS (5 Hz frequency-120% resting motor threshold) was applied at baseline and during the TPM induction phase (drug intake schedule: week I 25 mg/day, week II 50 mg/day, week III 75 mg/day, week IV 100 mg/day) and total TPM plasma concentrations were measured. The effects on the MEP size of 5 Hz-rTMS delivered over repeated sessions were tested in eight control subjects. TPM had no effect on the resting motor threshold. Antiepileptic treatment at increasing doses abolished the normal rTMS-induced MEP facilitation. ANOVA showed that this was a dose-related effect. Accordingly, in patients receiving TPM at higher doses (75 and 100 mg) rTMS failed to elicit the MEP facilitation. TPM left the progressive lengthening of the CSP during the rTMS train unchanged. In control subjects, rTMS applied over repeated sessions elicited a constant increase in MEP size. Our results suggest that TPM modulates the excitatory intracortical interneurons probably by altering rTMS-induced synaptic potentiation. These drug-induced effects are related to TPM doses and plasma concentrations. In conclusion, rTMS may be useful for quantifying the effectiveness of antiepileptic drugs and for assessing individual responses to different drugs but acting through similar mechanisms, thus combining functional neurophysiological information and laboratory data.

Effects of repetitive transcranial magnetic stimulation in a patient with fixation-off sensitivity.

Aim of the present study was to evaluate the acute and long-term effects of low-frequency repetitive transcranial magnetic stimulation (rTMS) on focal epileptiform interictal EEG activity in a patient with fixation-off sensitivity and partial epilepsy. Real and sham rTMS were delivered over the vertex. Two trains of 500 stimuli per day were delivered at 0.33 Hz frequency and threshold intensity for five consecutive days. The number of posterior EEG spikes and spike-and-wave complexes/min before and after the application of rTMS were compared in a blinded manner. In our patient, real-rTMS induced a long-lasting decrease in the number of posterior EEG spikes and spike-and-wave complexes/min. Despite the limitations of a single case report, our study confirms that low-frequency rTMS significantly reduces interictal focal epileptic activity over time.

Surface electromyography and mechanomyography recording: a new differential composite probe.

The objective of the study was to develop a new surface probe for differential mechanomyographic (MMG) and electromyographic (EMG) recording. Differential amplification is commonly used in electromyography to improve the signal-to-noise ratio. A new composite probe was developed with two electrodes (EMG) and two identical piezo-electric membranes (MMG) to be positioned on muscle. The probe had two built-in fixed-gain differential amplifiers: one to amplify the electric signal and the other to amplify the vibration signal. A similar non-differential MMG probe was used for comparisons. Burst muscular activity was recorded using the non-differential and differential probes and was used to test the performance of the two probes in suppressing artifacts of non-muscular origin. Power spectrum analysis of signals from the two probes showed that differential amplification significantly improved the signal-to-noise ratio in MMG recordings and significantly suppressed artifacts (power difference > 90%). The composite probe allowed simultaneous differential recording of MMG and EMG signals from the same muscular site. It recorded muscular activity more efficiently than the non-differential probe and could therefore be useful in studying fatigue and neuromuscular diseases.

Multisphere neutron spectrometric system with thermoluminescence dosemeters: sensitive improvement.

In this work, a neutron spectrometric system based on a set of moderating spheres with thermoluminescence detectors (TLD) is presented. The system at the Nuclear Regulatory Authority (ARN) Dosimetry Laboratory consists of 12 solid spheres made of high-density polyethylene (p = 0.95 g x cm(-3)), with diameters ranging from 2" to 12" and TLD sensitive to thermal and gamma radiation, namely TLD-600 and TLD-700, located at the centre of the spheres. The neutron response matrix for this Bonner Sphere Spectrometer (BSS) was calculated using the MCNP-IVB code and the library ENDF/B-VI in the energy range between thermal neutrons and 100 MeV. The neutron spectrum was obtained using the LOUH182 unfolding code. The improvement in sensitivity of the system is based on the election of a different heating cycle of the TLD that allows an increase in sensitivity by a factor of 2.6 compared with the standard laboratory treatment. The system response for the calibration with an Am-Be source is presented.

A new clinical scale of carpal tunnel syndrome: validation of the measurement and clinical-neurophysiological assessment.

OBJECTIVE: To validate a new clinical scale of carpal tunnel syndrome (CTS). The scale is based on clinical history and physical examination findings and includes two figures. The first is a score determined by clinical history and objective findings. The second evaluates the presence/absence of pain as a dichotomous categorical score. METHODS: One hundred and sixty-eight consecutive idiopathic CTS hands were studied in two centers (Rome, Siena). We compare the results of the historical-objective scale (Hi-Ob scale) with the results of other validated measurements of CTS severity: (1) the Italian version of the Boston Carpal Tunnel Questionnaire, (2) the neurophysiological classification adopted by the Italian CTS study group. Furthermore, for the Hi-Ob scale the intra-observer and inter-observer evaluations were assessed. RESULTS: The main Hi-Ob parameter was positively related to the conventional validated measurements. Conversely, the category 'PAIN' of the Hi-Ob scale appeared unrelated to the other clinical and electrophysiological parameters. Intra- and inter-observer evaluation showed the reproducibility of the Hi-Ob assessment. CONCLUSIONS: Our data show that the Hi-Ob scale is a reliable measurement which may be useful in CTS evaluation either for clinical or for scientific purposes.

Sympathetic skin response and cardiovascular autonomic function tests in Parkinson's disease and multiple system atrophy with autonomic failure.

The relationship between sympathetic skin response (SSR) and cardiovascular autonomic function tests (CVTs) was investigated in 15 patients with idiopathic Parkinson's disease (PD), 15 patients with clinical evidence of multiple system atrophy (MSA) with autonomic failure, and in 15 healthy control subjects. SSR was elicited by electrical stimulation of the right and left median nerves and simultaneously recorded on the palms of both hands. CVTs included the following sympathetic and parasympathetic tests: orthostatism, head-up tilt, cold pressor test, deep breathing, Valsalva maneuver, and hyperventilation. The SSR was normal in all patients with PD and control subjects but was abnormal or absent in all patients with MSA. For patients with MSA, SSR latency was significantly longer and amplitude was significantly smaller than that of patients with PD and control subjects. For patients with PD, SSR did not differ from that of control subjects. In these patients, SSR latency was significantly longer and SSR amplitude was smaller when the side with more marked motor symptoms was stimulated, both ipsilaterally and contralaterally to the side of stimulation. A statistically significant difference in SSR latencies and amplitudes was found between patients with PD and control subjects only when motor asymmetries were considered. CVTs showed severe sympathetic and parasympathetic hypofunction in patients with MSA, but not in patients with PD or control subjects. No correlation was found between SSR and CVTs that assess sympathetic function in patients and control subjects. SSR is indicated as an additional test for the evaluation of sympathetic degeneration in patients with MSA.

A new color vep procedure discloses asymptomatic visual impairments in optic neuritis and glaucoma suspects.

OBJECTIVE: To evaluate the reliability of visual evoked potentials obtained with a set of multiple chromatic and achromatic patterns (C-VEPs) in differentiating asymptomatic perifoveal retinal impairment from central conduction impairment. METHODS: We propose a set of colored pattern stimuli that allows relatively differential activation of the magnocellular and parvocellular pathways. The system runs on a standard Pentium PC with peripherals that present stimuli and collect, analyze and print data. P1 latencies of C-VEPs obtained with achromatic (black/white) and chromatic (blue/black and red/black isocontrast) checkerboards were evaluated in normal subjects and patients with subclinical retinal impairment (glaucoma suspects) or mild neural conduction impairment (optic neuritis), none of whom had subjective visual defects. RESULTS: The procedure evoked robust cortical signals and statistically distinguished the 3 groups of subjects. The achromatic and chromatic stimuli used distinguished controls from glaucoma suspects and patients with optic neuritis. Glaucoma suspects had greater impairment of C-VEPs to blue/black checkerboards whereas patients with optic neuritis had greater impairment of responses to red/black stimuli. CONCLUSIONS: Our data suggest that chromatic patterns (color/ black, red and blue), that may activate the parvocellular and magnocellular systems differentially but not selectively, can distinguish between mild perifoveal or foveal conduction impairment. They have the additional advantage of evoking large, stable responses across all the subjects.