Dual Functionality of Poloxamer 188 in Freeze-Dried Protein Formulations: A Stabilizer in Frozen Solutions and a Bulking Agent in Lyophiles.

Poloxamer 188 (P188) was hypothesized to be a dual functional excipient, (i) a stabilizer in frozen solution to prevent ice-surface-induced protein destabilization and (ii) a bulking agent to provide elegant lyophiles. Based on X-ray diffractometry and differential scanning calorimetry, sucrose, in a concentration-dependent manner, inhibited P188 crystallization during freeze-drying, while trehalose had no such effect. The recovery of lactate dehydrogenase (LDH), the model protein, was evaluated after reconstitution. While low LDH recovery (∼60%) was observed in the lyophiles prepared with P188, the addition of sugar improved the activity recovery to >85%. The secondary structure of LDH in the freeze-dried samples was assessed using infrared spectroscopy, and only moderate structural changes were observed in the lyophiles formulated with P188 and sugar. Thus, P188 can be a promising dual functional excipient in freeze-dried protein formulations. However, P188 alone does not function as a lyoprotectant and needs to be used in combination with a sugar.

Drug Phase Transformation and Water Redistribution during Continuous Tablet Manufacturing: A Case Study of Carbamazepine Dihydrate.

In recent years, continuous tablet manufacturing technology has been used to obtain regulatory approval of several new drug products. While a significant fraction of active pharmaceutical ingredients exists as hydrates (wherein water is incorporated stoichiometrically in the crystal lattice), the impact of processing conditions and formulation composition on the dehydration behavior of hydrates during continuous manufacturing has not been investigated. Using powder X-ray diffractometry, we monitored the dehydration kinetics of carbamazepine dihydrate in formulations containing dibasic calcium phosphate, anhydrous (DCPA), mannitol, or microcrystalline cellulose. The combined effect of nitrogen flow and vigorous mixing during the continuous mixing stage of tablet manufacture facilitated API dehydration. Dehydration was rapid and most pronounced in the presence of DCPA. The dehydration product, amorphous anhydrous carbamazepine, sorbed a significant fraction of the water released by dehydration. Thus, the dehydration process resulted in a redistribution of water in the powder blend. The unintended formation of an amorphous dehydrated phase, which tends to be much more reactive than its crystalline counterparts, is of concern and warrants further investigation.

Mannitol as an Excipient for Lyophilized Injectable Formulations.

The review summarizes the current state of knowledge of mannitol as an excipient in lyophilized injectable small and large molecule formulations. When compared with glycine, the physicochemical properties of mannitol make it a desirable and preferred bulking agent. Though mannitol is a popular bulking agent in freeze-dried formulations, its use may pose certain challenges such as vial breakage or its existence as a metastable crystalline hemihydrate in the final cake, necessitating appropriate mitigation strategies. The understanding of the phase behavior of mannitol in aqueous systems, during the various stages of freeze-drying, can be critical for the optimization of freeze-drying cycle parameters in multi-component formulations. Finally, using a decision tree as a guiding tool, we demonstrate the use of orthogonal techniques for attaining a stable and cost-effective lyophilized drug product containing mannitol.

Role of arginine salts in preventing freezing-induced increase in subvisible particles in protein formulations.

While arginine hydrochloride (ArgHCl) has emerged as a potential stabilizer of protein drugs in liquid formulations, the purpose of this manuscript was to evaluate its stabilization potential in frozen solutions. The phase behavior of frozen ArgHCl solutions was investigated by differential scanning calorimetry and low temperature powder X-ray diffractometry. The aggregation of ß-galactosidase was evaluated following freeze-thaw cycling in ArgHCl solutions with and without mannitol. ArgHCl (5% w/v) was retained amorphous in frozen aqueous solutions and effectively inhibited protein aggregation even after 5 freeze-thaw cycles. Annealing frozen arginine solution (5% w/v) containing mannitol (10% w/v) induced mannitol crystallization which in turn facilitated crystallization of ArgHCl. The stabilizing effect of ArgHCl was completely lost in the presence of mannitol. Use of alternate arginine salts (aspartate, glutamate, and acetate) allowed selective crystallization of mannitol while arginine was retained amorphous and stabilized the protein.

Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations.

This review aims to provide an overview of the current knowledge on protein stabilization during freezing and freeze-drying in relation to stress conditions commonly encountered during these processes. The traditional as well as refined mechanisms by which excipients may stabilize proteins are presented. These stabilizers encompass a wide variety of compounds including sugars, sugar alcohols, amino acids, surfactants, buffers and polymers. The rational selection of excipients for use in frozen and freeze-dried protein formulations is presented. Lyophilized protein formulations are generally multicomponent systems, providing numerous possibilities of excipient-excipient and protein-excipient interactions. The interplay of different formulation components on the protein stability and excipient functionality in the frozen and freeze-dried systems are reviewed, with discussion of representative examples of such interactions.

Freezing-induced protein aggregation - Role of pH shift and potential mitigation strategies.

The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) ß-galactosidase (ß-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to -25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and ß-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.

Crystallization of Cyclophosphamide Monohydrate During Lyophilization.

The purpose of this study was to investigate the phase behavior of cyclophosphamide (CPA) during various stages of lyophilization, with special emphasis on obtaining crystalline CPA monohydrate (CPA-MH) in the lyophilized product. Subambient differential scanning calorimetry and low-temperature X-ray diffractometry (LTXRD) were used to study the phase behavior of CPA solution (3.7% w/v). In situ lyophilization in LTXRD chamber was used to monitor the phase transitions occurring during the drying stages. Finally, the implications of these findings were confirmed by freeze-drying the aqueous solution in a laboratory-scale freeze-dryer. The results suggested that CPA remains amorphous during freeze concentration, with a Tg' of -50°C. However, its crystallization as CPA-MH can be induced by annealing the frozen solution between -5°C and -10°C. In situ lyophilization in LTXRD showed that the CPA-MH crystallized during annealing, rapidly dehydrated during primary drying, thereby causing structural collapse. The dehydration of CPA-MH can be prevented by lowering the escaping tendency of water molecules from the crystal lattice of CPA-MH by maintaining the chamber pressure to 300, 400, or 500 mTorr. This study highlights the relationship of process parameters used during lyophilization with the solid form of lyophilized CPA.

Impact of Drug-Polymer Miscibility on Enthalpy Relaxation of Irbesartan Amorphous Solid Dispersions.

PURPOSE: Drug-polymer miscibility has been proposed to play a critical role in physical stability of amorphous solid dispersions (ASDs). The purpose of the current work was to investigate the role of drug-polymer miscibility on molecular mobility, measured as enthalpy relaxation (ER) of amorphous irbesartan (IBS) in ASDs. METHODS: Two polymers, i.e. polyvinylpyrrolidone K30 (PVP K30) and hydroxypropyl methylcellulose acetate succinate (HPMCAS), were used to generate ASDs with 10% w/w of the polymer. Drug-polymer miscibility was determined using melting point depression (MPD) method. Molecular mobility was assessed from ER studies at a common degree of undercooling (DOU) (Tg - 13.0°C ± 0.5°C). RESULTS: IBS exhibited higher miscibility in PVP K30 as compared to HPMCAS at temperature > 140°C. However, extrapolation of miscibility data to storage temperature (62°C) using Flory-Huggins (F-H) theory revealed a reversal of the trend. Miscibility of IBS was found to be higher in HPMCAS (2.6%) than PVP K30 (1.3%) at 62°C. Stretched relaxation time (τß) of 17.4365 h and 7.0886 h was obtained for IBS-HPMCAS and IBS-PVP K30 ASDs, respectively. CONCLUSION: Miscibility of drug-polymer at storage temperature explained the behavior of the molecular mobility, while miscibility near the melting point provided a reverse trend. Results suggest that drug-polymer miscibility determined at temperatures higher than the storage temperature should be viewed cautiously.

Effect of cyclophosphamide on the solid form of mannitol during lyophilization.

Mannitol is a commonly used bulking agent in lyophilized formulations. It can crystallize into multiple solid forms during lyophilization thereby exhibiting phase heterogeneity and variability in product performance. In this manuscript, we studied the effect of cyclophosphamide (CPA), an anticancer drug, on the solid form of mannitol during lyophilization from aqueous solutions. Freeze-concentration studies were performed in the DSC while lyophilization was performed in a lab scale freeze dryer. DSC experiments revealed two-stage crystallization of mannitol (1.5% w/v) during freeze-concentration, evident as two distinct exothermic events (at -18.2°C and -30°C) in the cooling curve. This was complemented by two eutectic melting endotherms in the subsequent heating curve. Addition of CPA (4.0% w/v) completely inhibited the exotherm at -18.2°C, but enhanced the enthalpy of exotherm at -30°C by five folds. Likewise, only one eutectic melting endotherm was observed in the subsequent heating curve. Lyophilization of the solution containing only mannitol, yielded a mixture of ß- (major) and δ- (minor) polymorphs of mannitol. However, in the presence of CPA, only δ-polymorph was observed in the lyophilized sample. This selective favoring of the metastable δ-polymorph over the stable ß-polymorph, was explained by altered freezing kinetics of the solution in presence of CPA. The study provides mechanistic insights into solute crystallization behaviour during lyophilization of multi-component systems.

Counter-intuitive effect of non-crystallizing sugars on the crystallization of gemcitabine HCl in frozen solutions.

In this study, the effect of four non-crystallizing sugars, namely fructose, trehalose, sucrose and raffinose, was assessed on the crystallization of gemcitabine hydrochloride (GHCl) in frozen solutions. Aqueous solutions containing GHCl (50 mg/mL) and a sugar at varying concentrations (10-60 mg/mL) were frozen in situ in DSC and analyzed in the subsequent heating run. Crystallization propensity of GHCl was quantified in terms of reduced crystallization temperature (RCT) as a function of sugar type and concentration. Multivariate analysis option in JMP(®) software was employed for calculating correlation between the variables. All sugars inhibited GHCl crystallization in a concentration dependent manner. At equal concentration, fructose (with the lowest Tg') exerted the strongest inhibitory effect, whereas raffinose (with the highest Tg') exerted the weakest inhibitory effect. Additionally, RCT showed a poor correlation with Tg' (r=0.2327). Thus, the inhibitory effect of sugars could not be described by their anti-plasticization effect. This counter-intuitive behavior was explained by the inhibitory effect of sugars on ice crystallization, which increased the unfrozen water content (UWC) in the freeze concentrate, thereby lowering the supersaturation of GHCl. This was established by observing a good correlation (r=0.9666) between RCT and ln(1/UWC). Additionally, reduced diffusion kinetics of GHCl in presence of sugar molecules was also postulated. This study highlights the importance of unfrozen water towards governing the crystallization behavior of solutes in multi-component frozen systems.

Impact of tert-butyl alcohol on crystallization kinetics of gemcitabine hydrochloride in frozen aqueous solutions.

The effect of tert-butyl alcohol (TBA) on isothermal crystallization kinetics of gemcitabine hydrochloride (GHCl) in frozen aqueous solutions was assessed by cold-stage microscopy. Addition of TBA (0%-5%, w/w) increased the value of Johnson-Mehl-Avrami rate constant (1.3-33.3 h⁻¹) and reduced the Avrami exponent (2.5-1.0). Thermodynamic parameters [enthalpy (ΔH(‡)), entropy (ΔS(‡)), and free energy (ΔG(‡)) of activation], calculated using Arrhenius and Eyring-Polanyi equations, established that TBA (2%, w/w) accelerated GHCl crystallization by reducing its ΔH(‡) (53.9 cf. 96.5 kJ/mol⁻¹) and ΔG(‡) (68.5 cf. 74.9 kJ/mol⁻¹). Further, to explore insights into the effect of TBA on nucleation and crystal growth of GHCl, crystallization kinetics data were deconvolved using Finke-Watzky model. This revealed that addition of TBA decreased ΔH(‡) of nucleation and increased ΔS(‡) of crystal growth, thereby reducing ΔG(‡) of nucleation and crystal growth by 11.7% and 4.2%, respectively. Finkey-Watzky model also predicted a reduction in the crystal size upon TBA addition, which was confirmed by comparing particle size of GHCl lyophilized in the presence and absence of TBA. In conclusion, TBA reduces ΔG(‡) of nucleation and crystal growth in a differential manner, thereby enhancing the crystallization kinetics of GHCl and affecting its morphological features.

Differential effect of buffering agents on the crystallization of gemcitabine hydrochloride in frozen solutions.

The purpose of this study was to evaluate the differential effect of buffering agents on the crystallization of gemcitabine hydrochloride (GHCl) in frozen solutions. Four buffering agents, viz. citric acid (CA), malic acid (MA), succinic acid (SA) and tartaric acid (TA) were selected and their effect on GHCl crystallization was monitored using standard DSC and low temperature XRD. Onset of GHCl crystallization during heating run in DSC was measured to compare the differential effect of buffering agents. Glass transition temperature (Tg'), unfrozen water content in the freeze concentrate and crystallization propensity of the buffering agents was also determined for mechanistic understanding of the underlying effects. CA and MA inhibited while SA facilitated crystallization of GHCl even at 25 mM concentration. Increasing the concentration enhanced their effect. However, TA inhibited GHCl crystallization at concentrations <100mM and facilitated it at concentrations ≥100 mM. Lyophilization of GHCl with either SA or TA yielded elegant cakes, while CA and MA caused collapse. Tg' failed to explain the inhibitory effects of CA, MA and TA as all buffering agents lowered the Tg' of the system. Differential effect of buffering agents on GHCl crystallization could be explained by consideration of two opposing factors: (i) their own crystallization tendency and (ii) unfrozen water content in the freeze concentrate. In conclusion, it was established that API crystallization in frozen solution is affected by the type and concentration of the buffering agents.

SNEDDS curcumin formulation leads to enhanced protection from pain and functional deficits associated with diabetic neuropathy: an insight into its mechanism for neuroprotection.

Curcumin has shown to be effective against various diabetes related complications. However major limitation with curcumin is its low bioavailability. In this study we formulated and characterized self nano emulsifying drug delivery system (SNEDDS) curcumin formulation to enhance its bioavailability and then evaluated its efficacy in experimental diabetic neuropathy. Bioavailability studies were performed in male Sprague Dawley rats. Further to evaluate the efficacy of formulation in diabetic neuropathy various parameters like nerve function and sensorimotor perception were assessed along with study of inflammatory proteins (NF-κB, IKK-ß, COX-2, iNOS, TNF-α and IL-6). Nanotechnology based formulation resulted in prolonged plasma exposure and bioavailability. SNEDDS curcumin provided better results against functional, behavioural and biochemical deficits in experimental diabetic neuropathy, when compared with naive curcumin. Further western blot analysis confirmed the greater neuroprotective action of SNEDDS curcumin. SNEDDS curcumin formulation due to higher bioavailability was found to afford enhanced protection in diabetic neuropathy. FROM THE CLINICAL EDITOR: In this study the authors formulated and characterized a self-emulsifying drug delivery system for formulation to enhance curcumin bioavailability in experimental diabetic neuropathy. Enhanced efficacy was demonstrated in a rat model.

Novel lipid based oral formulation of curcumin: development and optimization by design of experiments approach.

The clinical utility of curcumin (CRM) is limited due to its poor oral bioavailability. Lipid based oral formulations (LBOFs) are emerging as useful oral drug delivery systems for 'difficult to deliver' molecules like CRM. In present study, we report novel Type IV LBOF for CRM using Gelucire 44/14, Labrasol, Vit. E TPGS and PEG 400 with superior CRM loading and enhanced oral bioavailability. The optimization of LBOF for CRM loading and post dilution droplet size was carried out by design of experiments (DoE) approach with Box-Behnken design. Oral bioavailability of optimized LBOF (O-LBOF) was evaluated in male Sprague-Dawley (SD) rats at a dose of 250 mg/kg. Raw CRM (control) showed C(max) and AUC(0-∞) of 32.29 ng/ml and 38.07 ng h/ml, respectively. O-LBOF improved C(max) and AUC(0-∞) by 11.6 and 35.8 folds respectively over control.

Bioavailability of a lipidic formulation of curcumin in healthy human volunteers.

Numerous publications have reported the significant pharmacodynamic activity of Curcumin (CRM) despite low or undetectable levels in plasma. The objective of the present study was to perform a detailed pharmacokinetic evaluation of CRM after the oral administration of a highly bioavailable lipidic formulation of CRM (CRM-LF) in human subjects. Cmax, Tmax and AUC0-¥ were found to be 183.35 ± 37.54 ng/mL, 0.60 ± 0.05 h and 321.12 ± 25.55 ng/mL respectively, at a dose of 750 mg. The plasma profile clearly showed three distinct phases, viz., absorption, distribution and elimination. A close evaluation of the primary pharmacokinetic parameters provided valuable insight into the behavior of the CRM after absorption by CRM-LF. CRM-LF showed a lag time (Tlag) of 0.18 h (around 12 min). Pharmacokinetic modeling revealed that CRM-LF followed a two-compartment model with first order absorption, lag time and first order elimination. A high absorption rate constant (K01, 4.51/h) signifies that CRM-LF ensured rapid absorption of the CRM into the central compartment. This was followed by the distribution of CRM from the central to peripheral compartment (K12, 2.69/h). The rate of CRM transfer from the peripheral to central compartment (K21, 0.15/h) was slow. This encourages higher tissue levels of CRM as compared with plasma levels. The study provides an explanation of the therapeutic efficacy of CRM, despite very low/undetectable levels in the plasma.

Comparative oral bioavailability advantage from curcumin formulations.

The aim of the present study was to study the oral bioavailability of seven different formulations of curcumin (CRM). CRM formulations viz. aqueous suspension, micronized suspension, nanosuspension, amorphous solid dispersion, hydroxypropyl-ß-cyclodextrin (HP-ß-CD) inclusion complex, combination with piperine, and spray-dried CRM-milk composite were compared for oral bioavailability in male Sprague-Dawley rats at a CRM dose of 250 mg/kg body weight using a validated high-performance liquid chromatography method. Aqueous suspension provided a C max and AUC(0 - t) of 28.9 ng/ml and 26.9 ng h/ml, respectively. In comparison, statistically significant increase in the oral bioavailability was obtained with the nanosuspension, HP-ß-CD inclusion complex, and amorphous solid dispersion with 251%, 567%, and 446% increase in terms of AUC(0 - t) and 405%, 415%, and 270% in terms of C max. However, no significant increase in AUC(0 - t) and C max was observed with piperine and micronized suspension. The milk composite reduced the oral bioavailability of CRM (10% and 37% in terms of AUC(0 - t) and C max). A statistically significant increase in the T max was observed with piperine and in HP-ß-CD complex, while the T max was reduced for nanosuspension. The results provide interesting insights into the role of solubility enhancement and metabolism inhibition, for improving the oral bioavailability of CRM.