Significance of unfolding thermodynamics for predicting aggregation kinetics: a case study on high concentration solutions of a multi-domain protein.

PURPOSE: To enable aggregation rate prediction over a broad temperature range for complex multi-domain proteins at high concentrations. METHODS: Thermal unfolding, non-isothermal kinetics and storage stability studies were conducted on a model multi-domain protein (MDP) at moderate to high concentrations (25-125 mg/mL) over a broad temperature range (4-40°C). RESULTS: Storage stability studies indicated the aggregation of MDP in solution to be a second order process. Application of Arrhenius kinetics to accelerated stability data resulted in underestimation of the aggregation rate under refrigerated conditions. Additional studies undertaken to understand the mechanism of the aggregation process highlighted the association of the monomer (or the aggregation competent species) to be the rate-limiting step for aggregation over the temperature range studied. Thermal unfolding studies in the presence of urea were used to calculate the heat capacity change upon unfolding (Δcp,un). The resulting value of Δcp,un when used in the extended Lumry-Eyring model resulted in a more accurate and a conservative estimate of the aggregation rate under refrigerated condition. Some complicating factors for the aggregation rate prediction for multi-domain proteins at high concentration are discussed. CONCLUSIONS: The work highlights (i) the significance of incorporating unfolding thermodynamics in protein aggregation rate prediction, (ii) the advantages and challenges associated with the use of the extended Lumry-Eyring (ELE) model for rate prediction and (iii) the utility of using the Arrhenius and the ELE models in tandem during product development.

Formation of reactive impurities in aqueous and neat polyethylene glycol 400 and effects of antioxidants and oxidation inducers.

A 2,4-dinitrophenylhydrazine (DNPH) precolumn derivatization high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed to quantify levels of formaldehyde and acetaldehyde in polyethylene glycol (PEG) solutions. Formic acid and acetic acid were quantified by HPLC-UV. Samples of neat and aqueous PEG 400 solutions were monitored at 40°C and 50°C to determine effects of excipient source, water content, pH, and trace levels of hydrogen peroxide or iron metal on the formation of reactive impurities. The effects of antioxidants were also evaluated. Formic acid was the major degradation product in nearly all cases. The presence of water increased the rate of formation of all impurities, especially formic acid as did the presence of hydrogen peroxide and trace metals. Acidic pH increased the formation of acetaldehyde and acetic acid. A distribution of unidentified degradation products formed in neat PEG 400 disappeared upon addition of HCl with corresponding increase of formic acid, indicating they were likely to be PEG-formyl esters. Other unidentified degradation products reacted with DNPH to form a distribution of derivatized products likely to be PEG aldehydes. Antioxidants butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate d-alpha tocopheryl polyethylene glycol-1000 succinate, and sodium metabisulfite were effective in limiting reactive impurity formation, whereas ascorbic acid and acetic acid were not.

An active film-coating approach to enhance chemical stability of a potent drug molecule.

Peliglitazar, a PPAR α/γ agonist, was found to undergo acid as well as base catalyzed degradation. The acid catalyzed degradation led to the formation of benzylic alcohol and glycine carbamate and the base catalyzed degradation led to formation of p-hydroxyanisole and an amine degradant. In capsule formulations, the capsules with the lowest drug-loading exhibited maximum instability even at 25 °C/60% RH storage condition. Incorporation of pH-modifiers to maintain 'micro-environmental pH' acidic did not prevent the formation of the base-catalyzed degradants. Traditional dry granulated tablet formulation which is qualitatively similar to the capsule formulations showed the presence of acid-catalyzed degradants even without the presence of an acidifying agent. On the other hand, traditional wet granulated tablet formulation showed mainly base-catalyzed degradants. Stability problems of the tablet formulation were aggravated because the potent molecule required low tablet strengths which resulted in low drug to excipient ratio. To stabilize the molecule, an active film-coating approach was explored. In this approach, the drug was sprayed with the coating material onto non-active containing tablet cores. This approach of trapping the drug particles into the coating material provided tablets with satisfactory chemical stability. The stability enhancement observed in the active coating approach is attributed to the higher drug to excipient ratio in the film coat of non-reactive coating material compared to that in the traditional dry or wet granulated formulations.

Effect of antioxidants and silicates on peroxides in povidone.

Reactive peroxides in povidone often lead to degradation of oxidation-labile drugs. To reduce peroxide concentration in povidone, the roles of storage conditions, antioxidants, and silicates were investigated. Povidone alone and its physical mixtures with ascorbic acid, propyl gallate, sodium sulfite, butylated hydroxyanisole (BHA), or butylated hydroxytoluene (BHT) were stored at 25 °C and 40 °C, at 11%, 32%, and 50% relative humidity. In addition, povidone solution in methanol was equilibrated with silicates (silica gel and molecular sieves), followed by solvent evaporation to recover povidone powder. Peroxide concentrations in povidone were measured. The concentration of peroxides in povidone increased under very-low-humidity storage conditions. Among the antioxidants, ascorbic acid, propyl gallate, and sodium sulfite reduced the peroxide concentration in povidone, whereas BHA and BHT did not. Water solubility appeared to determine the effectiveness of antioxidants. Also, some silicates significantly reduced peroxide concentration in povidone without affecting its functionality as a tablet binder. Porosity of silicates was critical to their ability to reduce the peroxide concentration in povidone. A combination of these approaches can reduce the initial peroxide concentration in povidone and minimize peroxide growth under routine storage conditions.

Reactive impurities in excipients: profiling, identification and mitigation of drug-excipient incompatibility.

Reactive impurities in pharmaceutical excipients could cause drug product instability, leading to decreased product performance, loss in potency, and/or formation of potentially toxic degradants. The levels of reactive impurities in excipients may vary between lots and vendors. Screening of excipients for these impurities and a thorough understanding of their potential interaction with drug candidates during early formulation development ensure robust drug product development. In this review paper, excipient impurities are categorized into six major classes, including reducing sugars, aldehydes, peroxides, metals, nitrate/nitrite, and organic acids. The sources of generation, the analytical method for detection, the stability of impurities upon storage and processing, and the potential reactions with drug candidates of these impurities are reviewed. Specific examples of drug-excipient impurity interaction from internal research and literature are provided. Mitigation strategies and corrective measures are also discussed.

Design of tablets for the delayed and complete release of poorly water-soluble weak base drugs using SBE7M-ß-CD as a solubilizing agent.

The challenge of designing a delayed-release oral dosage form is significantly increased when the drug substance is poorly water soluble. This manuscript describes the design and characterization of a novel controlled-release film-coated tablet for the pH-triggered delayed and complete release of poorly water-soluble weak base drugs. Delivery of weak bases is specifically highlighted with the use of dipyridamole and prazosin as model compounds. Tailored delayed release is achieved with a combination of an insoluble but semipermeable polymer and an enteric polymer, such as cellulose acetate and hydroxypropyl cellulose phthalate, respectively, as coatings. The extent of the time lag prior to complete release depends on the film-coating composition and thickness. Complete release is achieved by the addition of a cyclodextrin, namely SBE7M-ß-CD with or without a pH modifier added to the tablet core to ensure complete solubilization and release of the drug substance. The film-coating properties allow the complex formation/solubilization to occur in situ. Additionally, the drug release rate can be modulated on the basis of the cyclodextrin to drug molar ratio. This approach offers a platform technology for delayed release of potent but poorly soluble drugs and the release can be modulated by adjusting the film-coating composition and thickness and/or the cyclodextrin and pH modifier, if necessary.

Effect of force feeder on tablet strength during compression.

Mechanical strength of tablets is an important quality attribute, which depends on both formulation and process. In this study, the effect of process variables during compression on tablet tensile strength and tabletability (the ratio of tensile strength to compression pressure) was investigated using a model formulation. Increase in turret and force feeder speeds reduced tablet tensile strength and tabletability. Turret speed affected tabletability through changes in dwell time under the compression cam and the kinetics of consolidation of granules in the die cavity. The effect of force feeder was attributed to the shearing of the granulation, leading to its over-lubrication. A dimensionless equation was derived to estimate total shear imparted by the force feeder on the granulation in terms of a shear number. Scale-independence of the relationship of tabletability with the shear number was explored on a 6-station Korsch press, a 16-station Betapress, and a 35-station Korsch XL-400 press. The use of this relationship, the exact nature of which may be formulation dependent, during tablet development is expected to provide guidance to the scale-up and interchangeability of tablet presses.

Modeling of pan coating processes: Prediction of tablet content uniformity and determination of critical process parameters.

We developed an engineering model for predicting the active pharmaceutical ingredient (API) content uniformity (CU) for a drug product in which the active is coated onto a core. The model is based on a two-zone mechanistic description of the spray coating process in a perforated coating pan. The relative standard deviation (RSD) of the API CU of the coated tablets was found to be inversely proportional to the square root of the total number of cycles between the spray zone and drying zone that the tablets undergo. The total number of cycles is a function of the number of tablets in the drying zone, the spray zone width, the tablet velocity, the tablet number density, and the total coating time. The sensitivity of the RSD to various critical coating process parameters, such as pan speed, pan load, spray zone width, as well as tablet size and shape was evaluated. Consequently, the critical coating process parameters needed to achieve the desired API CU were determined. Several active film coating experiments at 50, 200, and 400 kg using various pan coaters demonstrated that good correlation between the model predictions and the experimental results for the API CU was achieved.

Colloidal phase behavior of pH-responsive, amphiphilic PEGylated poly(carboxylic acid)s and effect on kinetic solubility under acidic conditions.

PEGylated poly(carboxylic acid)s, PEG-b-PCAs, were evaluated as additives for solubilized oral formulations of weakly acidic compounds. Micelles of poly(ethylene glycol)-block-poly(acrylic acid), PEG-b-PAA, and poly(ethylene glycol)-block-poly(methacrylic acid), PEG-b-PMAA, were prepared. Fluorescence spectroscopy and dynamic light scattering revealed that both polymers assemble into nanoscopic structures (< 200 nm) in acidic media and exhibit pH-sensitive colloidal phase behavior. Using a solvent evaporation technique, the block copolymers and corresponding PCA homopolymers were incorporated into PEG3350-based solid dispersions. The kinetic solubility profile of a BMS compound, BMS-A (Seq ~ 12.5 µg/mL at pH 1.1) in 0.1 N HCl was monitored as a function of polymer composition. While BMS-A precipitated rapidly in 0.1 N HCl in the absence of PEG-b-PCAs, a supersaturated level of ca. 400 µg/mL was maintained for variable lengths of time in the presence of PEG-b-PCAs. Although the kinetic solubility of BMS-A was also enhanced in the presence of the PCA homopolymers, the relative magnitude and duration of supersaturation as a function of polymer composition suggests that micellar solubilization, rather than specific interaction, contributes to enhanced solubility of BMS-A in 0.1 N HCl. Under acidic conditions, pH-responsive PEG-b-PCAs may offer the kinetic supersaturation necessary to minimize precipitation of compounds which have limited solubility in acidic milieu.

Release mechanisms of a sparingly water-soluble drug from controlled porosity-osmotic pump pellets using sulfobutylether-beta-cyclodextrin as both a solubilizing and osmotic agent.

The purpose of this work is to delineate the release mechanisms of a sparingly water-soluble drug, prednisolone (PDL), from a microporous or controlled porosity-osmotic pump pellet (CP-OPP) using sulfobutylether-beta-cyclodextrin (CD) as both a solubilizing and osmotic agent. All factors, osmotic and diffusional, influencing drug release as described by the Theeuwes and Zentner equation were partially demonstrated in an earlier paper1 and are further quantitatively evaluated here to determine whether the equation may be applied to CP-OPPs. The PDL release rate from the CP-OPPs containing precomplexed PDL follows the zero-order kinetics for up to 30-40% of drug release during the first 1-2 h and subsequently nonzero order kinetics. The zero-order drug release phase reveals the main contribution is from osmotic pumping with a negligible diffusion component, resulting from the nearly constant driving forces in the system. The nonzero order drug release phase is associated with the dynamic changes in the system (e.g., declining osmotic driving force and greater diffusion component with time). In addition, the parameters related to membrane characteristics were determined, and the effect of viscosity was evaluated for the pellet system. The membranes coated on the CP-OPPs are less permeable to water or solutes than the membranes coated on the previously reported tablets. The viscosity due to the CD decreases as a function of CD concentration, which partly affects the observed drug release profiles. The viscosity effect of CD is significant and captured in a hydraulic permeability term.

Co-solubilization of poorly soluble drugs by micellization and complexation.

The use of combined approach of surfactants and cyclodextrins in solubilization of poorly soluble drugs has been described in literature. In this report, a mathematical model has been developed to provide the quantitative basis for this approach. First, by way of hypothetical examples and simulations, the influence of various interaction parameters on the phase solubility profile is presented. Additionally, the model results are compared with (a) results reported by Yang et al., with NSC-639829, sodium lauryl sulfate (SLS) and sulfobutyl-ether-beta-cyclodextrin ((SBE)(7M)-beta-CD) and (b) solubility of methylprednisolone, a model poorly soluble drug, in the presence of its water-soluble 'surfactant-like' prodrug, methylprednisolone 21-hemisuccinate, and (SBE)(7M)-beta-CD. The model shows good agreement with experimental data. Furthermore, theoretical simulations show that the combined solubility is less than the sum of the individual solubility values in cyclodextrins and surfactants. Based on the hypothetical case and the two examples, the factors affecting the phase solubility profile in mixed solutions of surfactants and cyclodextrins are presented. Finally, the limitations of the model to explain co-solubilization by surfactants and cyclodextrins are discussed.

Effect of sodium lauryl sulfate in dissolution media on dissolution of hard gelatin capsule shells.

PURPOSE: Sodium lauryl sulfate (SLS) is a commonly used surfactant in dissolution media for poorly water soluble drugs. However, it has occasionally been observed that SLS negatively impacts the dissolution of drug products formulated in gelatin capsules. This study investigated the effect of SLS on the dissolution of hard gelatin capsule shells. METHODS: The USP paddle method was used with online UV monitoring at 214 nm (peptide bond). Empty size #0 capsule shells were held to the bottom of the dissolution vessel by magnetic three-prong sinkers. RESULTS: SLS significantly slowed down the dissolution of gelatin shells at pH < 5. Visually, the gelatin shells transformed into some less-soluble precipitate under these conditions. This precipitate was found to contain a higher sulfur content than the gelatin control sample by elemental analysis, indicating that SLS is part of the precipitate. Additionally, the slowdown of capsule shell dissolution was shown to be dependent on the SLS concentration and the ionic strength of the media. CONCLUSIONS: SLS interacts with gelatin to form a less-soluble precipitate at pH < 5. The use of SLS in dissolution media at acidic pH should be carefully evaluated for gelatin capsule products.

When can cyclodextrins be considered for solubilization purposes?

This communication is intended to address the question: How can one determine, with minimum experimentation, if cyclodextrins (CDs) might be the right choice as solubilization enhancers for a given poorly water-soluble drug? The cyclodextrin utility number, U(CD), a dimensionless number, is introduced to assess the feasibility of the use of CDs in dosage forms. U(CD) is a lumped-parameter consisting of the dose of the drug, the workable amount of CD, the binding constant, and the drug solubility in the absence of CDs. U(CD) was been extended to ionizable drugs that show synergistic increase in solubility due to ionization and complexation. U(CD) is a guiding and not a predictive tool that the formulator can use.

Design of pH-independent controlled release matrix tablets for acidic drugs.

The rate and extent of drug release from most controlled release systems are influenced by the pH of the dissolution medium for drugs with pH-dependent solubility. This dependency of drug release on pH may lead to additional inter- and intra-subject variability in drug absorption. In the present study, a pH-independent controlled release matrix system for acidic drugs was designed by incorporating release-modifiers in the formulation. Controlled release matrix tablets were prepared by compression of divalproex sodium, Methocel K4M and Eudragit E 100 or Fujicalin as the release-modifier. For formulations without any release-modifier, the extent and rate of drug release at pH 6.8 was much higher than that at pH 1.0. Formulations containing Eudragit E 100 provided drug release that was essentially independent of pH. This was achieved because Eudragit E 100 significantly increased the drug release in acidic medium and slightly decreased the release rate at higher pH. The increased release in the acidic medium can be attributed to the elevation of the micro-environmental pH in the swollen polymer gel layer. Formulations containing Fujicalin were less effective than those containing Eudragit E 100. This was attributed to the relative inability to elevate the pH and shorter residence time of Fujicalin in the matrix relative to Eudragit E 100.