Development of a Lyophilized Parenteral Pharmaceutical Formulation of the Investigational Polypeptide Marine Anticancer Agent Kahalalide F

Kahalalide F is a novel antitumor agent isolated from the marine mollusk Elysia rufescens; it has shown highly selective in vitro activity against androgen-independent prostate tumors. The purpose of this study was to develop a stable parenteral formulation of kahalalide F to be used in early clinical trials. Solubility and stability of kahalalide F were studied as a function of polysorbate 80 (0.1%-0.5% w/v) and citric acid monohydrate (5-15 mM) concentrations using an experimental design approach. Stabilities of kahalalide F lyophilized products containing crystalline (mannitol) or amorphous (sucrose) bulking agents were studied at +5°C and +30°C ±60% relative humidity (RH) in the dark. Lyophilized products were characterized by infrared (IR) spectroscopy and differential scanning calorimetry (DSC). Recovery studies after reconstitution of kahalalide F lyophilized product and further dilution in infusion fluid were carried out to select an optimal reconstitution vehicle. It was found that a combination of polysorbate 80 and citric acid monohydrate is necessary to solubilize kahalalide F. Lyophilized products were considerably less stable with increasing polysorbate 80 and citric acid monohydrate concentrations, with polysorbate 80 being the major effector. A combination of 0.1% w/v polysorbate 80 and 5 mM citric acid monohydrate was selected for further investigation. Lyophilized products containing sucrose as a bulking agent were more stable compared to the products containing mannitol. The glass transition temperature of the sucrose-based product was determined to be + 46°C. The amorphous state of the product was confirmed by IR analysis. A solution composed of Cremophor EL, ethanol, and water for injection (5%/5%/90% v/v/v CEW) kept kahalalide F in solution after reconstitution and further dilution with 0.9% w/v sodium chloride (normal saline) to 1.5 μg/m. A stable lyophilized formulation was presented containing 100 μg of kahalalide F, 100 mg sucrose, 2.1 mg citric acid monohydrate, and 2 mg polysorbate 80 to be reconstituted with a vehicle composed of 5%/5%/90% v/v/v CEW and to be diluted further using normal saline.     

Formulation for a novel inhaled peptide therapeutic for idiopathic pulmonary fibrosis

A caveolin-1 scaffolding domain, CSP7, is a newly developed peptide for the treatment of idiopathic pulmonary fibrosis. To develop a CSP7 formulation for further use we have obtained, characterized and compared a number of lyophilized formulations of CSP7 trifluoroacetate with DPBS and in combination with excipients (mannitol and lactose at molar ratios 1:5, 70 and 140). CSP7 trifluoroacetate was stable (>95%) in solution at 5 and 25?°C for up to 48?h and tolerated at least 5 freeze/thaw cycles. Lyophilized cakes of CSP7 trifluoroacetate with excipients were stable (>96%) for up to 4?weeks at room temperature (RT), and retained more than 98% of the CSP7 trifluoroacetate in the solution at 8?h after reconstitution at RT. The lyophilized CSP7 formulations were stable for up to 10?months at 5?°C protected from moisture. Exposure of the lyophilized cakes of CSP7 to 75% relative humidity (RH) resulted in an increase in the absorbed moisture, promoted crystallization of the excipients and induced reversible formation of CSP7 aggregates. Increased molar ratio of mannitol slightly affected formation of the aggregates. In contrast, lactose significantly decreased (up to 20 times) aggregate formation with apparent saturation at the molar ratio of 1:70. The possible mechanisms of stabilization of CSP7 trifluoroacetate in solid state by lactose include physical state of the bulking agent and the interactions between lactose and CSP7 trifluoroacetate (e.g. formation of a Schiff base with the N-terminal amino group of CSP7). Finally, CSP7 trifluoroacetate exhibited excellent stability during nebulization of formulations containing mannitol or lactose.     

Some Properties of Diazepam-Polyethylene Glycol 6000 Solid Dispersions and their Modification in the Presence of Stearic Acid of Polysorbate 80

Abstract

Physical mixtures and melts of diazepam and polyethylene glycol 6000 (PEG 6000) have been studied by Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis (DTA). Problems were encountered in determining the precise position of the eutectic which contained <30% diazepam. Melts contained amorphous diazepam and, immediately after preparation, an unstable form of PEG 6000 which transformed on storage to a more stable form, probably folded crystals. Dissolution rates were determined by constant surface area methodology and were greatest in melts containing 15% diazepam. The inclusion of 1 or 5% polysorbate 80 or 1% stearic acid into the melts slightly increased the dissolution rates from dispersions containing 5, 10 or 15% diazepam but especially from dispersions containing 20% diazepam. A limited 4-week ageing study indicated that age-induced changes depended on both the storage temperature and diazepam concentration. The inclusion of either stearic acid or polysorbate 80 appeared not to protect dissolution rates against ageing     

Preparation, characterization, and stability of liposome-based formulations of mitoxantrone

The preparation, characterization, and stability of lyophilized liposome-based formulation of mitoxantrone was investigated. Mitoxantrone was entrapped inside small, unilamellar liposomes composed of dioleoylphosphocholine (DOPC), cholesterol, and cardiolipin. The mean vesicle size and drug entrapment efficiency of the liposomes were ~ 150 nm and ~ 99%, respectively. Less than 1% of drug was lost and mean vesicle size remained unchanged after sterile filtration. The pre-lyophilized (pre-lyo) formulations were characterized by a differential scanning calorimetric (DSC) method. Results showed that the glass transition temperatures (Tg') increased as the molar ratios of sucrose:lipid and trehalose:lipid in the formulations were increased. The maximum Tg' of the pre-lyo formulations containing 10:1 sucrose:lipid and trehalose:lipid molar ratios were - 37°C and - 41°C, respectively. After reconstitution of the lyophilized cake of the sucrose-containing formulation, the mean vesicle size was comparable to pre-lyo liposome size. In vitro release studies showed that less than 2% of mitoxantrone was released after an extensive dialysis against phosphate buffered saline (PBS) at 37°C, indicating that the mitoxantrone was highly associated and retained inside the liposomes. Short-term stability studies of the sucrose-containing formulations revealed that the reconstituted and eight-fold diluted formulations were stable for up to 8 hours at room temperature. Long-term stability studies of lyophilized liposomal mitoxantrone showed that the lyophilized formulation was stable for up to 13 months after storage at refrigerated condition.     

Development and evaluation of nanostructured lipid carriers of cytarabine for treatment of meningeal leukemia

Lipid matrix based carriers are able to provide sustained release, increase the drug transport into cancer cells and overcome the drug resistance. Therefore, nanostructured lipid carriers (NLC) were prepared and coated with polysorbate 80 to overcome the blood brain barrier for achieving effective treatment of meningeal leukemia. NLC were prepared by melt emulsification followed by ultrasonication, producing particles of 90.7 +/- 4.28 nm size with appreciable amount of drug entrapment (49.5 +/- 2.24%), considering the hydrophilic nature of the drug. The polysorbate 80 coated cytarabine loaded NLC (Cyt-NLC) thus produced were non-aggregated and had almost spherical, smooth and uniform shape. Results of DSC and XRD studies indicated that Cyt was entrapped inside the lipid as molecular dispersion. In-vitro release pattern showed initial fast release (15.87 +/- 1.524% in 1 h) followed by sustained release upto 72 h (89.90 +/- 1.11%). In-vitro cell line studies demonstrated that blank NLC showed no significant cytotoxic effects on leukemic EL-4 cells whereas Cyt-NLC exhibited concentration dependent cytotoxicity. At 48 and 72 h, cytotoxicity of Cyt-NLC was found to be significantly more than that of Cyt solution and the percentage cell viability decreased with increasing concentration of Cyt-NLC. The lyophilized Cyt-NLC formulation was found to be stable with respect to size and total drug content at refrigerated condition (2-8 degrees C) for 3 months. These results suggest that polysorbate 80 coated Cyt-NLC can be explored for treatment of meningeal leukemia owing to their ability of sustained drug release and improved cytotoxic effect in leukemic EL-4 cell line.     

Real time stability and viability prediction of the anticancer BCG after lyophilization

To test if trehalose could be a better cryoprotectant for BCG than the usually used lactose and predict viability of BCG during shelf-life, BCG was suspended into three stabilizer systems containing 15% w/v trehalose, trehalose–gelatin mixture (in ratio, 30:1 w/w) or lactose. Each formula was lyophilized and several quality parameters were tested before and after lyophilization including sterility, safety, viability, morphology and moisture content. Samples of lyophilized formulae were tested for their reconstitution time and others were charged to stability chambers at 5?°C for the performance of real time study. Shelf-life of each formula was estimated and correlation between moisture content and loss in viability was established at each time interval of the real time stability study. Sterility, safety and morphology were retained after lyophilization. Just after lyophilization, minute diminish in viability was observed in the presence of each stabilizer (0.02–0.05%). There was no significant difference in reconstitution time of the three lyophilized formulae. Lactose BCG had the highest shelf-life among the used cryoprotectants during the real time stability studies. Also, moisture content was highly correlated to viability with correlation coefficient ranged between 0.97 and 0.99 and so, the former could be used for prediction of viability throughout the vaccine shelf-life.     

Physical Properties of Granules Containing Polysorbate 80

The friability and crushing load of granules containing polysorbate 80 were determined. It was found that while polysorbate 80 decreased granule hardness, as indicated by the load required to crush it, friability values increased to a maximum then decreasing at higher polysorbate 80 concentration. Thus the use of granule friability to measure granule strength may be erroneous unless good correlation between granule friability and direct crushing weight was obtained.

Direct measurement of granule strength tends to vary with granule shape and size giving a rather wide scatter of results. For overcoming this difficulty, tablet triturates could be prepared and the crushing strength determined. The crushing strength of the tablet triturates was found to be similar to that of granules but with a smaller scatter and more easily handled.

Studies of other physical properties of the granules containing polysorbate 80 were also made. Small amounts of the nonionic surfactant (0.002 - 0.2%) generally improved granule fluidity as characterised by the orifice flow velocity and the angle of repose of the granules.     

Development of a topical suspension containing three active ingredients

The objective of this study was to develop a topical suspension that contains sarafloxacin hydrochloride (1 mg/mL), triamcinolone acetonide (1 mg/mL), and clotrimazole (10 mg/mL), and is stable at room temperature (15-28°C) for clinical usage. Due to the difference in the physicochemical properties and chemical stability profiles of these three active ingredients, it is a challenge to develop a stable suspension formulation containing these three drugs. In this study, the stability of these drugs in different buffer solutions was determined under different accelerated isothermal conditions. The Arrhenius equation was subsequently utilized to predict the room-temperature stability of these three drugs in these buffer solutions. By knowing the room-temperature solubility of the drugs in the buffer solution, the stability of the drugs in suspension was predicted. As a result, a 0.02 M phosphate buffer (pH 7.0) containing 0.02% (w/v)polysorbate 20, 1% (w/v) NaCl, and 0.1% (w/v) EDTA was determined to be an acceptable medium. In addition, 0.35% (w/v) high-viscosity carboxymethylcellulose (HV-CMC) was first selected as the suspending agent to enhance the redispersibility of the suspension. Stability data further supported that all three drugs were stable in the suspension containing HV-CMC with less than 5% potency loss for at least 6 months at 40°C and 12 months at 25°C. However, the viscosity drop of this HV-CMC formulation at 25°C and 40°C became a product stability concern. To improve the viscosity stability of the suspension, the medium-viscosity carboxymethylcellulose (MV-CMC) was selected to replace the HV-CMC as the suspending agent. The optimal combination of MV-CMC and sodium chloride in achieving the most desirable dispersion properties for the formulation was determined through the use of a 3² factorial design. The optimal formulation containing 1% MV-CMC and 1% sodium chloride has shown improved viscosity stability during storage and has been used for clinical studies.     

Formulation of a charcoal suspension for intratumoral injection. Study of galenical excipients

To tattoo human breast cancer prior to chemotherapy, radiotherapy, or surgery, thus allowing a better localization of the remaining tumor by the surgeon, we developed a formulation containing 10% charcoal suspended in water for parenteral preparations. The present study concerns a new step in the development of the charcoal suspension. We sought to determine whether the addition of various excipients could improve the formulation properties and affect the labeling of tumor by the suspension. We have tested surfactants (egg lecithin, polysorbate 80, Cremophor EL, and Pluronic F68), isotonisants (sugars such as glucose and mannitol), polysaccharides (dextrans 20 and 40), and Cabosil, a pyrogenated silica. Except for glucose and mannitol, which were added at a 5% concentration, the other excipients were added at a 0.1% concentration. they were dissolved in water for parenteral injection and sterilized at 120°C for 20 min. We then measured diffusion in vivo in mammary tumor. In vivo, when injected intratumorally in mice, a greater diffusion of charcoal particles was noted within the tumor (in the case of egg lecithin, polysorbate 80, dextran 20 and 40, and glucose) and sometimes in some organs (e.g., Cremophor EL and mannitol). Pluronic F68 slightly improved the stability of the suspension and did not lead to marked diffusion at the injection site, but it showed slight toxicity and cannot be used in the formulation. We concluded that the best formulation was an aqueous 10% micronized peat charcoal suspension.     

Influence of surfactants in aqueous-based polymeric dispersions on the thermomechanical and adhesive properties of acrylic films

Good adhesion between a polymeric film and the surface of a solid substrate is critical to the performance of coated pharmaceutical products. Previous research has shown that tablet wettability by an organic-based cellulosic solution could predict the extent of film-tablet adhesion. Using an aqueous-based acrylic polymeric dispersion, the current study investigated the relationship between film adhesion and tablet wettability. Up to 10% (w/w based on dry polymer weight) polysorbate 80 or sorbitan monooleate was incorporated into the film-coating formulations. While the contact angle between the polymeric dispersion and the tablet surface was dependent on the type and concentration of surfactants added to the coating formulation, no correlation between tablet wettability and polymer adhesion could be established. The addition of surfactants to formulations containing the hydrophobic plasticizer tributyl citrate (TBC) caused lowering of the glass transition temperature of the polymer. Increased force of adhesion, elongation at adhesive failure, and adhesive toughness, however, were noted only in the TBC-plasticized films containing polysorbate 80. These findings demonstrate that our understanding of the mechanisms involved in film-tablet adhesion is still quite limited.     

Physical Properties of Granules Containing Polysorbate 80

Abstract

The friability and crushing load of granules containing polysorbate 80 were determined. It was found that while polysorbate 80 decreased granule hardness, as indicated by the load required to crush it, friability values increased to a maximum then decreasing at higher polysorbate 80 concentration. Thus the use of granule friability to measure granule strength may be erroneous unless good correlation between granule friability and direct crushing weight was obtained.

Direct measurement of granule strength tends to vary with granule shape and size giving a rather wide scatter of results. For overcoming this difficulty, tablet triturates could be prepared and the crushing strength determined. The crushing strength of the tablet triturates was found to be similar to that of granules but with a smaller scatter and more easily handled.

Studies of other physical properties of the granules containing polysorbate 80 were also made. Small amounts of the nonionic surfactant (0.002 - 0.2%) generally improved granule fluidity as characterised by the orifice flow velocity and the angle of repose of the granules.     

Influence of Surfactants in Aqueous-Based Polymeric Dispersions on the Thermomechanical and Adhesive Properties of Acrylic Films

Good adhesion between a polymeric film and the surface of a solid substrate is critical to the performance of coated pharmaceutical products. Previous research has shown that tablet wettability by an organic-based cellulosic solution could predict the extent of film-tablet adhesion. Using an aqueous-based acrylic polymeric dispersion, the current study investigated the relationship between film adhesion and tablet wettability. Up to 10% (w/w based on dry polymer weight) polysorbate 80 or sorbitan monooleate was incorporated into the film-coating formulations. While the contact angle between the polymeric dispersion and the tablet surface was dependent on the type and concentration of surfactants added to the coating formulation, no correlation between tablet wettability and polymer adhesion could be established. The addition of surfactants to formulations containing the hydrophobic plasticizer tributyl citrate (TBC) caused lowering of the glass transition temperature of the polymer. Increased force of adhesion, elongation at adhesive failure, and adhesive toughness, however, were noted only in the TBC-plasticized films containing polysorbate 80. These findings demonstrate that our understanding of the mechanisms involved in film-tablet adhesion is still quite limited.     

Formulation of cellulose film containing permeation enhancers for prolonged delivery of propranolol hydrocloride

The aim of this study was to evaluate the capacity of cellulose films enriched with oleic acid and polysorbate 80 to enhance the transdermal permeation of propranolol hydrochloride. Polymeric films were prepared by casting and drying aqueous solutions of hydroxypropylmethylcellulose or carboxymethylcellulose and characterized in chemical–physical properties, such as drug content, thickness, morphology and water uptake capacity. In vitro transport experiments were performed in order to evaluate the permeation enhancing ability of oleic acid and polysorbate 80. All carboxymethylcellulose films showed lower cumulative amounts of drug permeated than hydroxypropylmethylcellulose. Moreover, films containing both oleic acid and polysorbate 80 provided a greater permeation in comparison to film without permeation enhancers or only with one of these. The results obtained confirm that propranolol hydrochloride permeation can be easily modulated by varying the cellulose and enhancer type used for film preparation.     

A new reconstitutable oral paediatric hydrocortisone solution containing hydroxypropyl-β-cyclodextrin

Hydrocortisone (HC) despite a low aqueous solubility and a very poor palatability is frequently used unlicensed in paediatric practice. Hence a reconstitutable taste masked hydrocortisone solution with the potential to be easily produced extemporaneously was developed. Excipients for the reconstitutable dry powder mix were selected based on their aqueous solubility, compatibility, safety profile in children and stability at the optimum pH for HC. Formulations were visually inspected and pH was checked. The chemical and microbiological stability was assessed by a validated HPLC method and the European Pharmacopeia tests. A taste assessment study was performed on 20 young healthy adults to determine the optimum sweetener. HC was flavored (orange tangerine), preserved (methyl paraben sodium salt/potassium sorbate), adjusted to pH 4.2 (citric acid buffer) and included in a 1:6 hydroxypropyl-β-cyclodextrin (HP-β-CyD) complex which allowed complete solubilization of the drug following reconstitution within 1?min of handshake. Neotame 0.075% was found to be the sweetener of choice to mask the unpalatable taste and aftertaste of HC. All formulations tested at different storage conditions were found to be chemically stable after reconstitution with a HC recovery of >95% for 1 month. Microbiological assessment showed that the selected preservative combination was efficient and the presence of preservative ensured the recommended acceptance criteria for microbiological quality after reconstitution with repetitive sampling. The successfully developed 5?mg/mL reconstituted oral palatable paediatric HC solution was stable for 1 month after reconstitution and has the potential to facilitate dosing, acceptability, availability and affordability.     

Influence of Certain Additives on the Photostabilizing Effect of Dimethyl Sulfoxide for Sodium Nitroprusside Solutions

The influence of certain pharmaceutical adjuvants on the photostabilizing effect of dimethyl sulfoxide for a buffered solution of sodium nitroprusside was investigated. Dimethyl sulfoxide in a concentration of 10% v/v was found to exercise its effect as a photoprotective agent in the presence of methylparaben, sodium sulfite, sodium chloride, destrose, PEG 300, Tween 80, citric acid and sodium edetate. In the absence of dimethyl sulfoxide, sodium sulfite produced the most deleterious effect on the photostability of sodium nitroprusside solution. The photoprotective action of dimethyl sulfoxide appeared to be slightly enhanced by the presence of sodium edetate, methylparaben, sodium chloride or citric acid.     

Electrochemical determination of dissolution rates of lyophilized pharmaceutical formulations.

We present a method to measure dissolution times for rapidly dissolving lyophilized formulations. K4Fe(CN)6 was lyophilized in formulations containing sucrose, salts, and Tween 20. Dissolution of the lyophilized powders was measured by monitoring the time dependence of the oxidation of Fe(CN)6(4-) ion at the surface of a platinum rotating disk electrode. Reconstitution of lyophilized K4Fe(CN)6 formulations with aqueous solutions of 0.03% Tween 20 altered the time of dissolution for all cases. Salt and sucrose formulations without Tween 20 dissolved more slowly in a Tween 20 solution than in water alone. In contrast, formulations containing Tween 20 dissolved faster in the Tween 20 solution when compared to dissolution in water.     

Influence of excipients on moisturizing effect of urea

Water is the most important molecule contained in the skin and is bound to the intracellular hygroscopic substances called natural moisturizing factors (NMF). The clinical characteristic of xerosis is rough and/or coarse skin. This anaesthetic alteration necessitates cosmetic products application. In this study, we tested the efficiency of urea incorporated into six different emulsions (O/W) and 10 different gels.

Skin of 10 healthy women (20 to 45 years) was treated using 50 mg of emulsion or gel containing 5% of urea. A skin surface of 16 cm² was chosen in the area of the forearm. The gain in moisturizing was performed measuring the skin electrical capacity using a corneometer (Courage & Khazaka, model CM 825), one hour after treatment.

Sodium carboxymethyl cellulose gel has the least moisturizing effect. On the other hand, the mixture of polyacrylamide and C13-14 isoparaffin polysorbate 85 can be a good vehicle in the treatment of skin dehydration. Adding various oils (6%) or collagen in aqueous solution does not improve the efficiency of the tested products.

Moisturizing effect of gels (polyacrylamine with C13-14 isoparaffin polysorbate 85) is higher than the one of emulsions (L/H).     

Development of gel-forming lyophilized formulation with recombinant human thrombin

The objective of this work was development and evaluation of gel-forming lyophilized formulation with recombinant human thrombin for topical administration. The influence of pH, ionic strength and buffer type on protein stability was evaluated as part of the pre-formulation screening studies. Results indicated an optimal pH from 6.0 to 7.0 and increased stability with increasing content of sodium chloride. The tested buffer types had no significant effect on thrombin stability. For further development, thermosensitive Pluronic® F-127 was employed as a bulking and gelling agent. Physical and mechanical characterization and viscosity measurement confirmed the gel-forming properties of the formulation at the application temperature of 32?°C. Several techniques (addition of well-soluble polyols, different freezing protocols and reconstitution under vacuum) were tested to decrease the reconstitution time. The obtained results revealed that a vacuum in the vial headspace is crucial for acceptable reconstitution. The freeze drying process has no negative impact on recombinant thrombin stability, and this was confirmed by reverse-phase-HPLC, activity assay and optical density measurements.     

Design of an i.v. formulation of an unstable prodrug candidate for prostate cancer treatment: solution chemistry of N-(glutaryl-hyp-ala-ser-cyclohexylglycyl-gln-ser-leu)-doxorubicin

The chemical degradation of N-(glutaryl-hyp-ala-ser-cyclohexylglycyl-gln-ser-leu)-doxorubicin (henceforth referred to as doxorubicin peptide conjugate 1) was studied in buffered aqueous solution. The pH-rate profile of degradation shows that the doxorubicin conjugate is most stable between pH 5 and 6. The dependence of log k_obsd on pH in acidic medium is characteristic of specific acid-catalysis of the sugar hemiaminal of 1 (as in the case of doxorubicin). Isolation of degradates and structural determination shows that the degradation at lower pH values yields the water-insoluble aglycone doxorubicinone, supporting the mechanism of acid-catalyzed loss of the amino sugar. At pH higher than 5, a more complicated degradation pattern is observed, including the loss of the amino sugar and the aromatization of the saturated ring to give 7,8-dehydro-9,10-desacetyldoxorubicinone as one of the major products. Around the pH of maximum stability in solution, the rate of degradation of 1 is significantly greater than that for doxorubicin, which rules out the formulation of a room temperature solution product with a sufficiently long shelflife for market use. Design of a stable lyophilized formulation for sterile reconstitution based on the physicochemical properties of 1 is described.     

Influence of Certain Additives on the Photostabilizing Effect of Dimethyl Sulfoxide for Sodium Nitroprusside Solutions

Abstract

The influence of certain pharmaceutical adjuvants on the photostabilizing effect of dimethyl sulfoxide for a buffered solution of sodium nitroprusside was investigated. Dimethyl sulfoxide in a concentration of 10% v/v was found to exercise its effect as a photoprotective agent in the presence of methylparaben, sodium sulfite, sodium chloride, destrose, PEG 300, Tween 80, citric acid and sodium edetate. In the absence of dimethyl sulfoxide, sodium sulfite produced the most deleterious effect on the photostability of sodium nitroprusside solution. The photoprotective action of dimethyl sulfoxide appeared to be slightly enhanced by the presence of sodium edetate, methylparaben, sodium chloride or citric acid.