Liquid growth hormone: preservatives and buffers

Growth hormone (GH) treatment is a successful medical therapy for children and adults with GH deficiency as well as for growth retardation due to chronic renal disease, Turner syndrome and in children born small for gestational age. For all of these conditions, treatment is long term and patients receive daily subcutaneous injections of GH for many years. Patient compliance is therefore of critical importance to ensure treatment benefit. One of the major factors influencing compliance is injection pain. Besides the injection device used, pain perception and local tissue reaction following injection are dependent on the preservative used in the formulation and the concentration of GH. Injection pain may also be related to the buffer substance and injection volume. A liquid formulation of GH, Norditropi SimpleXx, has been developed that dispenses with the need for reconstitution before administration. The formulation uses phenol (3 mg/ml) as a preservative (to protect product from microbial degradation or contamination) and histidine as a buffer. Alternative preservatives used in other GH formulations include m-cresol (9 mg/ml) and benzyl alcohol (3-9 mg/ml). Buffering agents include citrate and phosphate. Phenol has been successfully used as a preservative in drug formulations for more than 50 years and is considered a safe and effective agent which complies with strict international requirements for preservatives in drug formulations. In toxicological studies, no or only mild local reactions have been observed following subcutaneous administration of phenol (7.5 mg/ml), m-cresol (3-4 mg/ml) and benzyl alcohol (9 mg/ml). No general toxicity reactions were observed after subcutaneous administration of these agents. Clinical evaluation of the preservatives and buffers used in Norditropin SimpleXx showed that pain perception was similar between formulations containing phenol and benzyl alcohol, whereas m-cresol was associated with more painful injections than benzyl alcohol. Furthermore, patients reported more pain following injection of a citrate-buffered solution than after a histidine-buffered solution. More pain was also reported following large volume injections and following injections with solutions containing high protein concentrations. In summary, optimization of the preservative and buffer content of a liquid GH formulation may reduce injection pain and lead to improved patient compliance.     

Localized treatments using commercial dust and liquid formulations of fipronil against Coptotermes formosanus （Isoptera： Rhinotermitidae） in the laboratory

Use of proper application methods and formulations of termiticides are im- portant to reduce their negative impact to the environment. In this study, we conducted laboratory experiments to determine the effect of localized treatments with commercial dust and liquid formulations of fipronil against Formosan subterranean termites, Coptoter- mes formosanus Shiraki. The test arena consisted of a specially designed 16-chambered structure with a center chamber connected to 5 foraging chambers that themselves were connected to 10 additional foraging chambers. One peripheral chamber received a liquid or dust treatment and termites were released in the center chamber. Results showed that 〉 91% of the termites were dead within the 9-d test period despite the localized treatment of only 1 foraging chamber. Termites that were still alive after 9 d were transferred to an untreated dish and held for 10 more days. The majority of those termites were dead and the rest were moribund on day 19. Regardless of the specific dish treated, both formulations of fipronil were found to be highly efficacious. Termites did not exhibit repellency to either formulation. Our results suggest that localized （or spot） treatment with either commer- cially available dust or liquid formulations of fipronil can be a viable option for control of a termite infestation where complete soil drenching is not desirable.     

Solution Formulation Development of a VEGF Inhibitor for Intravitreal Injection

PF-00337210 is a potent, selective small molecule inhibitor of VEGFRs and has been under consideration for the treatment of age-related macular degeneration. An ophthalmic solution formulation intended for intravitreal injection was developed. This formulation was designed to maximize drug properties such that the formulation would precipitate upon injection into the vitreous for sustained delivery. As a parenteral formulation with additional constraints dictated by this specialized delivery route, multiple features were balanced in order to develop a successful formulation. Some of these considerations included low dosing volumes (≤0.1 mL), a limited repertoire of safe excipients for intravitreal injection, and the unique physical chemical properties of the drug. The aqueous solubility as a function of pH was characterized, buffer stressing studies to select the minimal amount of buffer were conducted, and both chemical and physical stability studies were executed. The selected formulation consisted of an isotonic solution comprised of PF-00337210 free base in a citrate-buffered vehicle containing NaCl for tonicity. The highest strength for regulatory toxicology studies was 60 mg/mL. The selected formulation exhibited sufficient chemical stability upon storage with no precipitation, and acceptable potency and recovery through an intravitreal dosing syringe. Formulation performance was simulated by precipitation experiments using extracted vitreous humor. In simulated injection experiments, PF-00337210 solutions reproducibly precipitated upon introduction to the vitreous so that a depot was formed. To our knowledge, this is the first time that a nonpolymeric in situ-forming depot formulation has been developed for intravitreal delivery, with the active ingredient as the precipitating agent.     

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.     

Reformulation of a new vancomycin analog: An example of the importance of buffer species and strength

The purpose of this research was to use our previously validated dynamic injection apparatus as a rapid method for screening pH-adjusted formulations of a new vancomycin analog, Van-An, for their potential to precipitate upon dilution. In 1 vial, Van-An was reconstituted according to the manufacturer’s instructions. In a separate vial, the Van-An formulation’s existing phosphate buffer species was supplemented with acetate buffer, which has a pKa in the desired range: between the pH values of the formulation (pH 3.9) and blood (pH 7.4). The formulations were injected using the dynamic injection apparatus into a flowing stream of isotonic Sorensen’s phosphate buffer at rates of 0.25, 0.5, 1, and 2 mL/min. The peaks obtained with the spectrophotometer were reproducible for each injection rate/formulation combination. For the phosphate-buffered formulation, the least amount of precipitation was obtained at the 0.25 mL/min injection rate. Acetate buffer was able to substantially reduce such precipitation, even at the highest injection rate. The opacity peaks for the formulation with the acetate addition were significantly smaller (P<.05) than those obtained for the unaltered formulation at all 4 injection rates. The results suggest that acetate is a better buffer species than phosphate for the pH range defined. Furthermore, we present evidence to support a generally applicable approach to screening new formulations of drug products that may be clinically useful for reducing the incidence of phlebitis in humans. Published: January 13, 2006     

Evaluation of Shellac and Sucrose Ester Fruit Coating Formulations that Support Biological Control of Post-harvest Grapefruit Decay

Fruit coating formulations of bleached and unbleached shellac were developed that support survival of the yeast Candida oleophila . These and commercial formulations based upon sucrose esters were evaluated for their ability to promote surface colonization of citrus for the biological control of post-harvest decay. The type of shellac did not affect yeast survival in the liquid formulation; however, a pH 7.6 was essential for satisfactory recovery from the liquid over 24 h. The replacement of morpholine and aqueous ammonia, which aid dissolution of shellac, with equally efficacious sodium or potassium hydroxide improved yeast survival, as did replacing the surfactant oleic acid with polyoxyethylene (20) sorbitan monooleate ( polysorbate 80). When formulations were applied to grapefruit, those that initially facilitated higher numbers of yeasts on the fruit surface later developed epiphytic populations that were significantly greater during the most critical first 2 weeks after harvest. As a group, coating formulations that incorporated sucrose esters favored the development of yeast populations to a greater extent than those based upon shellac and, over 6 months, grapefruit with these coatings were slower to decay. However, shellac formulations incorporating sodium hydroxide significantly extended the storage life of grapefruit compared with similar formulations with morpholine.     

Optimization of formulation variables of benzocaine liposomes using experimental design

This study aimed to optimize, by means of an experimental design multivariate strategy, a liposomal formulation for topical delivery of the local anaesthetic agent benzocaine. The formulation variables for the vesicle lipid phase uses potassium glycyrrhizinate (KG) as an alternative to cholesterol and the addition of a cationic (stearylamine) or anionic (dicethylphosphate) surfactant (qualitative factors); the percents of ethanol and the total volume of the hydration phase (quantitative factors) were the variables for the hydrophilic phase. The combined influence of these factors on the considered responses (encapsulation efficiency (EE%) and percent drug permeated at 180 min (P%)) was evaluated by means of a D-optimal design strategy. Graphic analysis of the effects indicated that maximization of the selected responses requested opposite levels of the considered factors: For example, KG and stearylamine were better for increasing EE%, and cholesterol and dicethylphosphate for increasing P%. In the second step, the Doehlert design, applied for the response-surface study of the quantitative factors, pointed out a negative interaction between percent ethanol and volume of the hydration phase and allowed prediction of the best formulation for maximizing drug permeation rate. Experimental P% data of the optimized formulation were inside the confidence interval (P < 0.05) calculated around the predicted value of the response. This proved the suitability of the proposed approach for optimizing the composition of liposomal formulations and predicting the effects of formulation variables on the considered experimental response. Moreover, the optimized formulation enabled a significant improvement (P < 0.05) of the drug anaesthetic effect with respect to the starting reference liposomal formulation, thus demonstrating its actually better therapeutic effectiveness.     

Preparation and characterization of Eudragit Retard nanosuspensions for the ocular delivery of cloricromene

The purpose of this study was to improve the stability of cloricromene (AD6) in ophthalmic formulations and its drug availability at the ocular level. To this end, AD6-loaded polymeric nanoparticle suspensions were made using inert polymer resins (Eudragit RS100 and RL100). We modified the quasi-emulsion solvent diffusion technique by varying some formulation parameters (the drug-to-polymer ratio, the total drug and polymer amount, and the stirring speed). The chemical stability of AD6 in the nanosuspensions was assessed by preparing some formulations using (unbuffered) isotonic saline or a pH 7 phosphate buffer solution as the dispersing medium. The formulations were stored at 4°C, and the rate of degradation of AD6 was followed by high performance liquid chromatography (HPLC). The obtained nanosuspensions showed mean sizes and a positive surface charge (ζ-potential) that make them suitable for an ophthalmic application; these properties were maintained upon storage at 4°C for several months. In vitro dissolution tests confirmed a modified release of the drug from the polymer matrixes. Nanosuspensions prepared with saline solution and no or lower amounts of surfactant (Tween 80) showed an enhanced stability of the ester drug for several months, with respect to an AD6 aqueous solution. Based on the tecnological results, AD6-loaded Eudragit Retard nanoparticle suspensions appear to, offer promise as a means to improving the shelf life and bioavailability of this drug after ophthalmic application. Published: March 24, 2006     

Effects of Formulation and Host Nematode Density on the Ability of In Vitro-Produced Pasteuria Endospores to Control its Host Belonolaimus longicaudatus

The effect of nematode population density at the time of application and formulations of in vitro-produced Pasteuria spp. endospores on the final population density of Belonolaimus longicaudatus was studied in an 84-d-long pot bioassay. The experiment utilized a factorial design consisting of 30 or 300 B. longicaudatus /100 cm(3) of sandy soil and three formulations of in vitro-produced Pasteuria spp. endospores (nontreated, granular, or liquid). No differences were observed in percent endospore attachment between nematode inoculum levels during either trial. Granular and liquid formulations of in vitro-produced endospores suppressed nematode population densities by 22% and 59% in the first trial and 20% and 63% in the second, respectively compared with the nontreated control. The liquid formulation increased percent endospore attachment by 147% and 158%, respectively, compared with the granular formulation. The greatest root retention by the host plant was observed at the lower B. longicaudatus inoculation level following application of the liquid formulation. While both the granular and liquid formulations reduced B. longicaudatus population densities in the soil, the liquid spore suspension was most effective.     

Investigations into,and development of,a lyophilized and formulated recombinant human factor IX produced from CHO cells

Objectives

To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4–8 °C.

Results

NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance.

Conclusions

Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, l-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

     

Improving the biological efficacy of small droplets of permethrin by the addition of silicon-based surfactants

Oil-soluble and oil-dispersible silicon-based, non-ionic surfactants were incorporated in two oil-based formulations of permethrin. At the optimal concentration of surfactant, 40 μm droplets of the insecticide were twice as effective against whitefly (Trialeurodes vaporariorum) larvae as droplets without surfactant. The addition of a surfactant to a formulation containing 10 g/litre permethrin resulted in a spray mixture which was at least as effective as the same formulation containing 100 g/litre permethrin without surfactant. These improvements in efficacy were not attributable to droplet spread or perimeter on the leaf surface, or to differences in the surface tension of the mixtures. The implications of these results for ultra-low volume spraying are discussed. A statistical model based on the zero-cell of the Poisson distribution is described and used to analyse the bioassay results.     

Preformulation Studies on Solid Self-Emulsifying Systems in Powder Form Containing Magnesium Aluminometasilicate as Porous Carrier

The influence of alkaline and the neutral grade of magnesium aluminometasilicate as a porous solid carrier for the liquid self-emulsifying formulation with ibuprofen is investigated. Ibuprofen is dissolved in Labrasol, then this solution is adsorbed on the silicates. The drug to the silicate ratio is 1:2, 1:4, and 1:6, respectively. The properties of formulations obtained are analyzed, using morphological, porosity, crystallinity, and dissolution studies. Three solid self-emulsifying (S-SE) formulations containing Neusilin SG2 and six consisting of Neusilin US2 are in the form of powder without agglomerates. The nitrogen adsorption method shows that the solid carriers are mesoporous but they differ in a specific surface area, pore area, and the volume of pores. The adsorption of liquid SE formulation on solid silicate particles results in a decrease in their porosity. If the neutral grade of magnesium aluminometasilicate is used, the smallest pores, below 10 nm, are completely filled with liquid formulation, but there is still a certain number of pores of 40–100 nm. Dissolution studies of liquid SEDDS carried out in pH = 1.2 show that Labrasol improves the dissolution of ibuprofen as compared to the pure drug. Ibuprofen dissolution from liquid SE formulations examined in pH of 7.2 is immediate. The adsorption of the liquid onto the particles of the silicate causes a decrease in the amount of the drug released. Finally, more ibuprofen is dissolved from S-SE that consist of the neutral grade of magnesium aluminometasilicate than from the formulations containing the alkaline silicate.KEY WORDS: dissolution, ibuprofen, labrasol, magnesium aluminometasilicate, self-emulsifying powder     

Controlled Microwave Processing Applied to the Pharmaceutical Formulation of Ibuprofen

The first successful development of controlled microwave processing for pharmaceutical formulations is presented and illustrated with a model drug (ibuprofen) and two excipients (stearic acid and polyvinylpyrrolidone). The necessary fine temperature control for formulation with microwave energy has been achieved using a uniquely modified microwave oven with direct temperature measurement and pulse-width modulation power control. In addition to comparing microwave and conventional heating, the effect of the presence of liquid (water) in aiding the mixing of the drug and excipient during formulation was also investigated. Analysis of the prepared formulations using differential scanning calorimetry and dissolution studies suggest that microwave and conventional heating produce similar products when applied to mixtures of ibuprofen and stearic acid. However, the differences were observed for the ibuprofen and polyvinylpyrrolidone formulation in terms of the dissolution kinetics. In all cases, the presence of water did not appear to influence the formulation to any appreciable degree. The application of controllable microwave heating is noteworthy as fine temperature control opens up opportunities for thermally sensitive materials for which microwave methods have not been feasible prior to this work.     

Influence of Formulation Factors on the Aerosol Performance and Stability of Lysozyme Powders: a Systematic Approach

With the growing interest in developing biologics for pulmonary delivery, systematic fast screening methods are needed for rapid development of formulations. Due to the labile nature of macromolecules, the development of stable, biologically active formulations with desired aerosol performance imposes several challenges both from a formulation and processing perspective. In this study, spray-freeze-drying was used to develop respirable protein powders. In order to systematically map the selected design space, lysozyme aqueous pre-formulations were prepared based on a constrained mixture design of experiment. The physicochemical properties of the resulting powders were characterized and the effects of formulation factors on aerosol performance and protein stability were systematically screened using a logic flow chart. Our results elucidated several relevant formulation attributes (density, total solid content, protein:sugars ratio) required to achieve a stable lysozyme powder with desirable characteristics for pulmonary delivery. A similar logical fast screening strategy could be used to delineate the appropriate design space for different types of proteins and guide the development of powders with pre-determined aerodynamic properties.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

Mode of action of a surfactant–polymer formulation to support performance of the entomopathogenic nematode Steinernema carpocapsae for control of diamondback moth larvae (Plutella xylostella)

The use of entomopathogenic nematodes on cabbage leaves against larvae of the diamondback moth (DBM) Plutella xylostella requires the addition of formulation adjuvants to achieve satisfying control. Without adjuvants nematodes settle in the tank mix of backpack sprayers causing uneven distribution. The polymers arabic and guar gum, alginate and xanthan were used in concentrations between 0.05 and 0.3% to retard sedimentation of Steinernema carpocapsae. Arabic gum had no effect, guar gum prevented sedimentation at 0.3% but the effect dropped significantly at lower concentration. At 0.05%, xanthan prevented nematode sedimentation better than alginate. Deposition of nematodes on the leaves was significantly increased by the addition of any of the polymers. Spraying nematodes on leaves with an inclination of 45° without the addition of any formulation resulted in 70% run-off. Adding 0.2% alginate or xanthan reduced the losses to <20%. The use of a surfactant–polymer formulation significantly reduced defoliation by DBM larvae. Visual examinations provided evidence that nematodes are not ingested by DBM larvae. Invasion of S. carpocapsae is an active process via the anus. The function of the formulation is not to prolong nematode survival, but to provide environmental conditions which enable rapid invasion of the nematodes. Nematode performance was improved by selection of the best surfactant in combination with xanthan and by optimisation of the concentrations of the surfactant Rimulgan® and the polymer xanthan. The best control results were achieved with Rimulgan® at 0.3% together with 0.3% xanthan, causing DBM mortality of >90% at 80% relative humidity and >70% at 60%. The formulation lowered the LC₅₀ from 12 to 1 nematode/larva. The viscosity of the surfactant–polymer formulations correlated well with nematode efficacy, prevention of sedimentation and adherence to the leave. This physical parameter can therefore be recommended for improvement of nematode formulations to be used for foliar application against DBM.     

Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants

AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C. METHODS AND RESULTS: The aw of the preservation medium of C. sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG). Moreover, several protectant substances at different concentrations were evaluated. Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells. Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels. C. sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations. Moreover, liquid formulations of C. sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C. When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C. sake formulations. Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples. CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C. sake cells as they maintain the viability >70% for 4 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy. Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production.     

Statistical optimization of gastric floating system for oral controlled delivery of calcium

The development of an optimized gastric floating drug delivery system is described. Statistical experimental design and data analysis using response surface methodology is also illustrated. A central, composite Box-Wilson design for the controlled release of calcium was used with 3 formulation variables: X₁ (hydroxypropyl methylcellulose [HPMC] loading), X₂ (citric acid loading), and X₃ (magnesium stearate loading). Twenty formulations were prepared, and dissolution studies and floating kinetics were performed on these formulations. The dissolution data obtained were then fitted to the Power Law, and floating profiles were analyzed. Diffusion exponents obtained by Power Law were used as targeted response variables, and the constraints were placed on other response variables. All 3 formulation variables were found to be significant for the release properties (P<,05), while only HPMC loading was found to be significant for floating properties. Optimization of the formulations was achieved by applying the constrained optimization. The optimized formulation delivered calcium at the release rate of 40 mg/hr, with predicted n and T_50% values at 0.93 and 3.29 hours, respectively. Experimentally, calcium was observed to release from the optimized formulation with n and T_50% values of 0.89 (±0.10) and 3.20 (±0.21) hours, which showed an excellent agreement. The quadratic mathematical model developed could be used to further predict formulations with desirable release and floating properties.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.