International Journal of Pharmaceutics (v.362, #1-2)

Joseph Howard Rytting (1942–2008) by Alexander T. Florence (1).

The global aim of this research project was to develop a self-nanoemulsifying drug delivery system (SNEDDS) for non-invasive delivery of protein drugs. The specific aim of this study was to develop SNEDDS formulations. An experimental design was adopted to develop SNEDDS. Fluorescent labeled β-lactamase (FITC-BLM), a model protein, was loaded into SNEDDS through solid dispersion technique. The experimental design provided 720 compositions of different oil, surfactant, and co-surfactant at various ratios, of which 33 SNEDDS prototypes were obtained. Solid dispersion of FITC-BLM in SoyPC prepared was able to dissolve in 16 SNEDDS prototypes (approximately 2200 mU BLM in 1 g SNEDDS). SNEDDS NE-12-7 (composition: Lauroglycol FCC, Cremophor EL and Transcutol; ratio: 5:4:3) formed O/W nanoemulsion with mean droplet size in the range of 22–50 nm when diluted with various pH media and different dilution factor with PBS (pH 7.4). The phase diagram of NE-12-7 indicated a broad region of nanoemulsion. BLM-loaded SNEDDS (NE-12-7) stored at 4 °C for 12 weeks indicated 10% loss of BLM activity. A SNEDDS was developed to load FITC-BLM into the oil phase which can spontaneously form O/W nanoemulsion upon the addition of water.
Keywords: Self-nanoemulsifying drug delivery systems (SNEDDS); Solid dispersion; protein delivery; β-Lactamase;

Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs by Sripriya Venkata Ramana Rao; Payal Agarwal; Jun Shao (10-15).
To develop a self-nanoemulsifying drug delivery system (SNEDDS) for protein drugs, and particularly, to test the in vitro transport of β-lactamase (BLM) by SNEDDS across the cell monolayer. Fluorescently labeled BLM (FITC-BLM), a model protein, formulated into 16 SNEDDS preparations through a solid dispersion technique were studied for transport across MDCK monolayer. All the SNEDDS nanoemulsions resulted in higher transport rate than the free solution. The transport rate by SNEDDS depends on the SNEDDS composition. SNEDDS NE-12-7 (oil: Lauroglycol FCC, surfactant: Cremophor EL and a cosurfactant: Transcutol HP) at the ratio of 5:4:3, rendered the highest transportation rate, 33% as compared to negligible transport by the free solution. FITC-BLM solution mixed with the surfactant and the cosurfactant of SNEDDS NE-12-7 or with blank SNEDDS NE-12-7 increased the transport only by 3.3 and 1.5 folds, respectively, compared to free solution alone. It was found that the monolayer integrity was not compromised in the presence of SNEDDS NE-12-7 or its surfactant/cosurfactant. The SNEDDS significantly increased the transport of FITC-BLM across MDCK monolayer in vitro. SNEDDS may be a potential effective delivery system for non-invasive protein drug delivery.
Keywords: MDCK; Protein delivery; β-Lactamase; Self-nanoemulsifying drug delivery system;

Self-nanoemulsifying drug delivery system (SNEDDS) for oral delivery of protein drugs by Sripriya Venkata Ramana Rao; Kavya Yajurvedi; Jun Shao (16-19).
To use self-nanoemulsifying drug delivery system (SNEDDS) to deliver hydrophilic proteins orally. β-Lactamase (BLM), a 29 kDa protein was used as a model protein, and formulated into the oil phase of a SNEDDS through solid dispersion technique. The oral absorption of BLM in rats when delivered by such a SNEDDS was investigated. Oral delivery of 4500 mU/kg of BLM in SNEDDS nanoemulsion resulted in the relative bioavailability of 6.34%, C max of 1.9 mU/ml and mean residence time of 12.12 h which was 1.5-, 2.7- and 1.3-fold higher than that by free solution, respectively. Delivery of BLM in the aqueous phase of the nanoemulsion resulted in a PK profile similar to that by the free solution. BLM when loaded in oil phase of SNEDDS, can significantly enhance the oral bioavailability of BLM. SNEDDS has a great potential for oral protein delivery.
Keywords: β-Lactamase; SNEDDS; Protein oral administration; Pharmacokinetics; Bioavailability;

Biochemical enhancement of transdermal delivery with magainin peptide: Modification of electrostatic interactions by changing pH by Yeu-Chun Kim; Sameer Late; Ajay K. Banga; Peter J. Ludovice; Mark R. Prausnitz (20-28).
Magainin is a naturally occurring, pore-forming peptide that has recently been shown to increase skin permeability. This study tested the hypothesis that electrostatic forces between magainin peptides and drugs mediate drug transport across the skin. Electrostatic interaction between positively charged magainin and a negatively charged model drug, fluorescein, was attractive at pH 7.4 and resulted in a 35-fold increase in delivery across human epidermis in vitro when formulated with 2% N-lauroylsarcosine in 50% ethanol. Increasing to pH 10 or 11 largely neutralized magainin's charge, which eliminated enhancement due to magainin. Shielding electrostatic interactions with 1–2 M NaCl solution similarly eliminated enhancement. Showing the opposite dependence on pH, electrostatic interaction between magainin and a positively charged anti-nausea drug, granisetron, was largely neutralized at pH 10 and resulted in a 92-fold increase in transdermal delivery. Decreasing to pH 5 increased magainin's positive charge, which repelled granisetron and progressively decreased transdermal flux. Circular dichroism analysis, multi-photon microscopy, and FTIR spectroscopy showed no significant pH effect on magainin secondary structure, magainin deposition in stratum corneum, or stratum corneum lipid order, respectively. We conclude that magainin increases transdermal delivery by a mechanism involving electrostatic interaction between magainin peptides and drugs.
Keywords: Electrostatic interaction; Granisetron; Magainin pore-forming peptide; pH effect; Skin permeability enhancement; Transdermal drug delivery;

Quantitative determination of solid-state forms of a pharmaceutical development compound in drug substance and tablets by Yong Xie; Wenle Tao; Henry Morrison; Rick Chiu; Janan Jona; Jan Fang; Nina Cauchon (29-36).
Common analytical techniques including Raman, NIR, and XRD were evaluated for quantitative determination of three solid-state forms (amorphous, Form B and Form C) of a development compound. Raman spectroscopy was selected as the primary analytical technique with sufficient sensitivity to monitor and quantify the neat drug substance alone and in the drug product. A reliable multivariate curve resolution (MCR) method based on the second derivative Raman measurements of the three pure physical forms was developed and validated with 3.5% root mean square error of prediction (RMSEP) for Form B, which was selected as the preferred form for further development. A partial least squares (PLS) algorithm was also used for the multivariate calibration of both the NIR and Raman measurements. The long-term stability of Form B as a neat active pharmaceutical ingredient (API) and in a tablet formulation was quantitatively monitored under various stress conditions of temperature and moisture. Moisture, temperature, excipients and compression were found to have significant effects on the phase transition behavior of Form B.
Keywords: Solid-state forms; Quantitation; Raman spectroscopy; Form stability; Tablet;

Sustained delivery of captopril from floating matrix tablets by Inéz Jiménez-Martínez; Tomás Quirino-Barreda; Leopoldo Villafuerte-Robles (37-43).
The development of a controlled release formulation of captopril has been a challenge for some time. In this work, the in vitro sustained release of captopril from Metolose SH 4000 SR/sodium bicarbonate floating tablets has been studied, varying the proportions of Metolose and bicarbonate. This was studied at two different compaction pressures. Other studied variables include the kinetics of the hydration volume, the matrices floating time and the matrix density. The results show that matrices compacted at 55 MPa float in the dissolution medium for more than 8 h while those compacted at 165 MPa float only when sodium bicarbonate is included in the formulation. The increase of the matrix polymer proportion increases the maximal hydration volume as well as the time to attain this maximum. The matrices hydration volume increases with the inclusion of sodium bicarbonate in the formulation. The matrix density is lower when compacted at 55 MPa. The drug release constant (k) decreases and the exponent indicative of the release mechanism (n) increases with increasing polymer contents. The drug released with time is lesser when sodium bicarbonate is included in the formulation. Carbon dioxide bubbles obstruct the diffusion path and decrease the matrix coherence. The effect of compaction pressure to reduce the drug release rate has to be made clear in further studies.
Keywords: Hydroxypropyl methylcellulose; Captopril; Floating tablets; Release modulation; Hydration kinetics; Compaction pressure;

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): Evasion of MRP-2 mediated efflux by Ritesh Jain; Sheetal Agarwal; Nanda Kishore Mandava; Ye Sheng; Ashim K. Mitra (44-51).
Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.
Keywords: Saquinavir; Multidrug resistance protein-2; Single pass intestinal perfusion; Intestinal absorption; P eff; Effective intestinal permeability; Rat jejunum; Net water flux;

The effect of lubrication on density distributions of roller compacted ribbons by A.M. Miguélez-Morán; C.-Y. Wu; J.P.K. Seville (52-59).
Roller compaction is a continuous dry granulation process for producing free flowing granules in order to increase the bulk density and uniformity of pharmaceutical formulations. It is a complicated process due to the diversity of powder blends and processing parameters involved. The properties of the produced ribbon are dominated by a number of factors, such as the powder properties, friction, roll speed, roll gap, feeding mechanisms and feeding speed, which consequently determine the properties of the granules (size distribution, density and flow behaviour). It is hence important to understand the influence of these factors on the ribbon properties. In this study, an instrumented roller press developed at the University of Birmingham is used to investigate the effect of lubrication on the density distribution of the ribbons. Three different cases are considered: (1) no lubrication, (2) lubricated press, in which the side cheek plates of the roller press are lubricated, and (3) lubricated powder, for which a lubricant is mixed into the powder. In addition, how the powders are fed into the entry region of the roller press and its influence on ribbon properties are also investigated. It is found that the method of feeding the powder into the roller press plays a crucial role in determining the homogeneity of the ribbon density. For the roller press used in this study, a drag angle (i.e., the angle formed when the powder is dragged into the roller press) is introduced to characterise the powder flow pattern in the feeding hopper. It is shown that a sharper drag angle results in a more heterogeneous ribbon. In addition, the average ribbon density depends upon the peak pressure and nip angle. The higher the peak pressure and nip angle are, the higher the average ribbon density is. Furthermore, the densification behaviour of the powder during roller compaction is compared to that during die compaction. It has been shown that the densification behaviour during these two processes is similar if the ribbons and the tablets have the same thickness.
Keywords: Roller compaction; Granulation; Agglomeration; Lubrication; Ribbon;

Lipid composition and grafted PEG affect in vivo activity of liposomal mitoxantrone by ChunLei Li; JingXia Cui; CaiXia Wang; JinXu Wang; YanHui Li; Lan Zhang; Li Zhang; WenMin Guo; YongLi Wang (60-66).
Mitoxantrone was encapsulated into pegylated SUVs using ammonium sulfate gradient method. Four formulations (LM-s, LM-p, LM-m and LM-m-L) were prepared, which were made from different PCs and exhibited different PEG grafting density. In vitro release studies revealed that drug release rate increased with decreased T m of PCs, and reduced PEG polymer coverage. In circulation, the trend towards increased circulation time as T m of PCs and PEG lipid content are elevated is observed. However, it was found that the order of toxicity in balb/c mice was Lm-s < LM-p < LM-m-L < LM-m. Biodistribution studies revealed that the accumulation of LM-s into tumor was ∼12 times as large as that of free MIT. In s-180 tumor model, LM-s exhibited significant antineoplastic effects. Following the injection of LM-s (4 mg/kg), tumor growth was considerably inhibited, resulting in a tumor inhibition ratio of ∼92%. In contrast, the treatment with free MIT exhibited almost no antitumor efficacy. In conclusion, PC composition and PEG grafting density could exert influences on the biological activity of liposomal MIT; and encapsulation of MIT into HSPC/chol SUVs with high PEG grafting density could considerably improve the therapeutic index of MIT.
Keywords: Mitoxantrone; Liposomes; Lipid composition; Drug release; Antitumor efficacy; Toxicity; Polyethylene glycol grafting density;

Stability of the nitrogen mustard mechlorethamine in novel formulations for dermatological use by Wolfgang A. Ritschel; Wei Ye; Lucinda Buhse; John C. Reepmeyer (67-73).
Long term stability measurements were made for the nitrogen mustard mechlorethamine HCl at a concentration of 0.02% in six topical formulations: Aquaphor® ointment, Transcutol®, Labrasol®, 10% Transcutol® in Aquaphor®, 10% Transcutol® in Labrasol®, and Aquaphilic® ointment. The drug decomposed gradually in Aquaphor® ointment at room temperature, dropping to 95% in 4 weeks, 85% in 12 weeks, and 78% in 39 weeks. On the other hand, the drug decomposed rapidly in Aquaphilic® ointment, giving an assay of less than 20% of its initial concentration after 24 h at room temperature. Generally, mechlorethamine HCl was more stable in Aquaphor® ointment than in formulations containing Transcutol® or Labrasol®. However, the addition of the free radical inhibitor, BHT, significantly enhanced the stability of mechlorethamine in Transcutol® and Labrasol® formulations. Four BHT-stabilized Transcutol® and Labrasol® formulations gave assays in ranges of 92–99% at the end of 4 weeks, 77–98% at the end of 12 weeks, and 38–93% at the end of 41 weeks.
Keywords: Mechlorethamine; Nitrogen mustard; Stability; Dermal delivery; Aquaphor®; Transcutol®;

It was of interest to develop a method for solid-state acidity measurements using pH indicators and to correlate this method to the degradation rate of sucrose. Amorphous samples containing lactose 100 mg/ml, sucrose 10 mg/ml, citrate buffer (1–50 mM) and sodium chloride (to adjust the ionic strength) were prepared by freeze-drying. The lyophiles were characterized using powder X-ray diffraction, differential scanning calorimetry and Karl Fischer titremetry. The solid-state acidity of all lyophiles was measured using diffuse reflectance spectroscopy and suitable indicators (thymol blue or bromophenol blue). The prepared lyophiles were subjected to a temperature of 60 °C and were analyzed for degradation using the Trinder kit. The results obtained from this study have shown that the solid-state acidity depends mainly on the molar ratio of the salt and the acid used in buffer preparation and not on the initial pH of the solution. The degradation of sucrose in the lyophiles is extremely sensitive to the solid-state acidity and the ionic strength. Reasonable correlation was obtained between the Hammett acidity function and sucrose degradation rate. The use of cosolvents (in the calibration plots) can provide good correlations with the rate of an acid-catalyzed reaction, sucrose inversion, in amorphous lyophiles.
Keywords: Solid state; Acidity; Amorphous; Sucrose; Decomposition;

The effect of complexation of Cu(II) with P6A peptide and its analogs on their thrombolytic activities by Mei Yang; Guohui Cui; Ming Zhao; Chao Wang; Lili Wang; Hu Liu; Shiqi Peng (81-87).
The complexation of Cu(II) with previously synthesized thrombolytic peptides, Pro-Ala-Lys (6), Arg-Pro-Ala-Lys (7), Ala-Arg-Pro-Ala-Lys (8), Gly-Arg-Pro-Ala-Lys (9) and Gln-Arg-Pro-Ala-Lys (10), resulted in the formation of complexes, Cu(II)-(Pro-Ala-Lys) (6-Cu), Cu(II)-(Arg-Pro-Ala-Lys) (7-Cu), Cu(II)-(Ala-Arg-Pro-Ala-Lys) (8-Cu), Cu(II)-(Gly-Arg-Pro-Ala-Lys) (9-Cu) and Cu(II)-(Gln-Arg-Pro-Ala-Lys) (10-Cu), which was confirmed by UV-Vis, circular dichroism and ESI-MS analyses. Complexes (6–10)-Cu in normal saline (NS) were found to be able to assemble into aggregates, and the size of the aggregates were measured for the next eight consecutive days. It was found that on the 8th day, the diameters of (6–10)-Cu aggregates ranged from 179.21 ± 38.33 nm to 293.46 ± 51.07 nm. The Zeta potentials of (6–10)-Cu aggregates in NS were also examined and it was found that aggregates of (6, 8, 10)-Cu were negatively charged, and 7-Cu and 9-Cu were positively charged. The powders of (6–10)-Cu were analyzed by transmission electron microscopy and were found to have mean particle sizes of 8–15 nm. The in vitro euglobulin lysis, vasodilation and thrombolytic assays indicated that complexation with Cu(II) resulted in a significant increase in the activities of 6–10. The in vivo thrombolytic assays revealed that complexation with Cu(II) resulted in a significant enhancement in the in vivo thrombolytic activities for 6, 7, 8 and 10 at 10 μmol/kg, and 9 at 1 μmol/kg and 0.1 μmol/kg, respectively. These findings suggested that the self-assembly of the Cu(II)–peptide complexes into nano-scale aggregates was beneficial in improving the thrombolytic activity of the peptides.
Keywords: Cu(II)–peptide complex; Self-assembly; Thrombolytic activity;

Improved adhesion to mucosal cells of water-soluble chitosan tetraalkylammonium salts by V. Cardile; G. Frasca; L. Rizza; F. Bonina; C. Puglia; A. Barge; N. Chiambretti; G. Cravotto (88-92).
Chitosan is a natural polymer whose bioadhesive properties make it a useful material for filming over and protecting damaged or sensitive mucosae. Much effort has been expended to develop this employ, and new applications are in the offing. The aim of the present study was to optimize the synthesis under sonochemical conditions of water-soluble chitosan tetraalkylammonium salts and to assess the mucoadhesive properties of the resulting water-soluble cationic polyelectrolytes. Aqueous solutions of several tetralkylammonium chitosan derivatives, viz. N-trimethyl- (1), N-diethylmethyl- (2), N-carboxymethyl- (3) and N-[N,N-diethylaminomethyl(diethyldimethylene ammonium) n ]methylchitosan (4) were tested along with the parent biopolymer and its citric acid salt (5), both at neutral and acidic pH. We used a published technique for evaluating in vitro bioadhesion to isolated buccal cells, a mucosal model that can predict bioadhesive behavior in vivo. Derivatives 1 and 4 gave the best results.
Keywords: Chitosan tetraalkylammonium salts; Ultrasound; Sonochemical reaction; Bioadhesivity; Buccal cells; In vitro test;

Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: Interaction with the transporters on Caco-2 cells by Suresh Katragadda; Ritesh Jain; Deep Kwatra; Sudharshan Hariharan; Ashim K. Mitra (93-101).
In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, l-alanine-ACV (AACV), l-serine-ACV (SACV), l-isoleucine-ACV (IACV), γ-glutamate-ACV (EACV) and l-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (λ z). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p<  0.05). C max(T) (maximum concentration) of SACV was observed to be 39 ± 22 μM in plasma which is 2 times better than VACV and 15 times better than ACV. C last(T) (concentration at the last time point) of SACV was observed to be 0.18 ± 0.06 μM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C max) and eliminated at varying rates (λ z) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections.
Keywords: Acyclovir; Amino acid prodrugs; Oral bioavailability; Caco-2; Transporters;

It is suggested here that liquisolid technique has the potential to be optimized for the reduction of drug dissolution rate and thereby production of sustained release systems. In the present study, propranolol hydrochloride was dispersed in polysorbate 80 as the liquid vehicle. Then a binary mixture of carrier–coating materials (Eudragit RL or RS as the carrier and silica as the coating material) was added to the liquid medication under continuous mixing in a mortar. The final mixture was compressed using the manual tableting machine. The effect of drug concentration, loading factor, thermal treating and aging on release profile of propranolol hydrochloride from liquisolid compacts were investigated at two pH values (1.2 and 6.8). The release rate of propranolol HCl from liquisolid compacts was compared to the release of propranolol HCl from conventional tablets. X-ray crystallography and DSC were used to investigate the formation of any complex between drug and excipients or any crystallinity changes during the manufacturing process. Propranolol HCl tablets prepared by liquisolid technique showed greater retardation properties in comparison with conventional matrix tablets. This investigation provided evidence that polysorbate 80 (Tween 80) has important role in sustaining the release of drug from liquisolid matrices, and a reduction of T g of the polymer can be the reason for the release prolongation of liquisolid tablets. The results also showed that wet granulation had remarkable impact on release rate of propranolol HCl from liquisolid compacts, reducing the release rate of drug from liquisolid compacts. The results showed that aging (liquisolid tablets were kept at 25 °C/75% relative humidity for 6 months) had no effect on hardness and dissolution profile of drug. The kinetics studies revealed that most of the liquisolid formulations followed the zero-order release pattern. X-ray crystallography and DSC ruled out any changes in crystallinity or complex formation during the manufacturing process of liquisolid formulations.
Keywords: Propranolol HCl; Liquisolid compacts; Eudragit; Sustained release;

Effect of particle properties on the flowability of ibuprofen powders by L.X. Liu; I. Marziano; A.C. Bentham; J.D. Litster; E.T.White; T. Howes (109-117).
Powder flowability is one of the key parameters in the pharmaceutical tabletting process. The flowability is affected by both the particles’ properties and the tabletting equipment characteristics. Although it is generally accepted that powder flowability increases with an increase in particle size, quantitative studies and comprehensive theoretical insights into the particle property effects are still lacking. In this paper, ibuprofen, a non-steroidal drug widely used as an anti-inflammatory analgesic was chosen as a model material to assess the effect of particle properties on its flowability. Ibuprofen typically has a needle shaped morphology. The flowability of ibuprofen size fractions was studied in detail using two flow measurement methods. The separated fractions were also compared to magnesium stearate lubricated ibuprofen and its size fractions. The experimental results showed that powder flowability is significantly affected by both the particle size and size distribution. The finest size fraction that is separated from the bulk ibuprofen powder flows better than the bulk powder. For powders with narrow size distributions, the flowability increases significantly with the increase in particle size. In addition, admixing magnesium stearate to ibuprofen not only increases the flow function of the powder, but also reduces the internal friction angle. A theoretical analysis based on the limiting tensile strength of the powder bed was carried out and the flow conditions for particles of different size and shape were developed.
Keywords: Ibuprofen; Flowability; Particle size; Hausner ratio; Shear cell tester;

The aim of the present investigation was to develop controlled release (C.R.) matrix tablet formulations of rifampicin and isoniazid combination, to study the design parameters and to evaluate in vitro release characteristics. In the present study, a series of formulations were developed with different release rates and duration using hydrophilic polymers hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC). The duration of rifampicin and isoniazid release could be tailored by varying the polymer type, polymer ratio and processing techniques. Further, Eudragit L100-55 was incorporated in the matrix tablets to compensate for the pH-dependent release of rifampicin. Rifampicin was found to follow linear release profile with time from HPMC formulations. In case of formulations with HPC, there was an initial higher release in simulated gastric fluid (SGF) followed by zero order release profiles in simulated intestinal fluid (SIFsp) for rifampicin. The release of isoniazid was found to be predominantly by diffusion mechanism in case of HPMC formulations, and with HPC formulations release was due to combination of diffusion and erosion. The initial release was sufficiently higher for rifampicin from HPC thus ruling out the need to incorporate a separate loading dose. The initial release was sufficiently higher for isoniazid in all formulations. Thus, with the use of suitable polymer or polymer combinations and with the proper optimization of the processing techniques it was possible to design the C.R. formulations of rifampicin and isoniazid combination that could provide the sufficient initial release and release extension up to 24 h for both the drugs despite of the wide variations in their physicochemical properties.
Keywords: Rifampicin; Isoniazid; Controlled release; HPMC; HPC; Matrix tablets;

The objective of the present research was to stabilize a novel hemiglutarate ester prodrug of Δ9-tetrahydrocannabinol (THC), in polyethylene oxide (PEO) polymeric matrices produced by hot-melt fabrication, for systemic delivery of THC through the oral transmucosal route. For this purpose, the influence of pH modifiers and antioxidants employed as stabilizing agents in these matrices was investigated. Based on the stability studies, two final formulations were made, and the stability of the active was assessed in these systems. In addition, the bioadhesive properties of PEO matrices were studied as a function of bioadhesive polymer type and concentration, contact time, drug loading and wetting time. Of all of the polymers investigated, bioadhesion was highest with Carbopol® 971p. Bioadhesion increased with bioadhesive polymer concentration and wetting time to a certain level beyond which there was no further contribution. Both the contact time and drug loading influenced the bioadhesion. Severe degradation of the prodrug was observed during storage, even at room temperature (75% at the end of 3 months). Incorporation of the stabilizing agents in the PEO matrices reduced the degradation of the prodrug considerably. Citric acid was the most effective of all of the pH modifiers studied. Among the various antioxidants utilized, degradation was observed least in presence of BHT and ascorbic acid. Only 7.6% and 8.2% of prodrug degraded in these matrices, respectively, as compared to the PEO-only matrices (59.4%) at the end of 3 months at 25 °C/60% RH. The prodrug was very stable in both of the final formulations at the end of the 3 months at 40 °C/75% RH.
Keywords: Bioadhesion; THC; Poly(ethylene oxide); Stability; Prodrug; Antioxidants;

Development and evaluation of novel itraconazole-loaded intravenous nanoparticles by Wei Chen; Bing Gu; Hao Wang; Jun Pan; Weiyue Lu; Huimin Hou (133-140).
The purpose of this study was to present novel intravenous itraconazole-loaded nanoparticles (ITZ-NPs) using human serum albumin (HSA) as drug carrier materials. The ITZ-NPs were prepared by nanoparticle albumin bound technology involving a series of homogenization and lyophilization procedures. The ITZ-NPs powder could be easily reconstituted and provide stable solutions at a wide range of concentrations at 25 °C for 24 h. In safety test, the ITZ-NPs caused mild hemolysis below the concentration of 10 mg/mL and were well tolerated at the dose of 160 mg/kg in mice, indicating better biocompatibility than cyclodextrin formulation of itraconazole (ITZ-CD). The pharmacokinetic parameters of itraconazole and its major metabolite, hydroxyl-itraconazole, of ITZ-NPs had no differences from those of ITZ-CD in mice. For the ITZ-NPs group, the distributions of itraconazole in the lung, liver and spleen were higher than those for ITZ-CD group. It was of significance that ITZ-NPs increased the drug distribution in lung which was always the portal to fungal infection. These results indicate that the ITZ-NPs can be a potential intravenous formulation of itraconazole.
Keywords: Itraconazole; Nanoparticle albumin bound technology; Pharmacokinetics; Tissue distribution;

Low molecular hydrophilic actives such as peptides are typically poorly encapsulated within poly(alkyl cyanoacrylate) nanoparticles when prepared from micellar or microemulsion templates. The aim of the present study was to investigate whether the entrapment of peptides within poly(alkyl cyanoacrylate) nanoparticles could be increased by functionalizing the peptide so that it could copolymerize with the alkyl cyanoacrylate monomer. Peptide and acryloyl functionalized peptide representing the antigenic epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33–41) were synthesized using solid-phase peptide synthesis. Poly(alkyl cyanoacrylate) nanoparticles were prepared to encapsulate either peptide or functionalized peptide using both an aqueous micellar and a water-in-oil microemulsion polymerization template. Using the micellar template, nanoparticles could not be produced in the presence of acryloyl peptide. Rather an agglomerated mass formed on the stirrer. In contrast, nanoparticles could be prepared using both acryloyl and parent peptide using the water-in-oil microemulsion template. Encapsulation efficiency was more than twofold greater for functionalized peptide, being greater than 90%. Encapsulation efficiency of functionalized peptide was also observed to increase with increasing the amount of alkyl cyanoacrylate monomer used for polymerization. A biphasic release profile was observed for the nanoparticles entrapping the non-functionalized peptide with greater than 50% of peptide being released during the first 10 min and with around 90% being released at 6 h. In contrast, less than 10% of the total amount of acryloyl LCMV33–41 entrapped within the nanoparticles was detected in the release media following the initial 10 min, and no further release of peptide was observed up to the termination of the release study at 360 min. The difference in entrapment and release kinetics between the parent and functionalized peptide strongly supports the presumption that most of the acryloyl peptide actually intervened in the copolymerization with alkyl cyanoacrylate monomer and was covalently bound within the nanoparticles instead of being physically entrapped or adsorbed which appeared to be the case for the parent peptide. Thus, functionalizing a peptide so that it can copolymerize with the alkyl cyanoacrylate monomer is a strategy which can be used to increase the entrapment efficiency of peptides within poly(alkyl cyanoacrylate) nanoparticles and also maintain the peptide associated with nanoparticles so that the benefits of nanoparticulate delivery can be exploited.
Keywords: Nanoparticles; Poly(alkyl cyanoacrylate); Peptide; Interfacial polymerization; Microemulsion; Entrapment;

Elaborating the phase behaviour of ethylene oxide oligomers and analogues in 2H, 3H-perfluoropentane by Peter C. Griffiths; Marie Côte; Philippe G.A. Rogueda (147-152).
Pressure metered dose inhalers (pMDIs) are seen as an attractive option for the delivery of active molecules to the bloodstream via the lungs. Formulation issues are important as it is necessary to disperse the active substance into a volatile propellant. The phase behaviour of a range of common formulation agents – excipients – in 2H, 3H-perfluoropentane is reported here, focussing on the effects of simple elaboration of a central ethylene-oxide (EO) moiety. The base series of hydroxyl terminated EO fragments exhibited lower consolute solution temperature (LCST) behaviour that was strongly molecular weight-dependent. Substitution of methyl groups in place of the hydroxyl groups at the terminus of the EO fragment had a pronounced effect on the solubility of the oligomeric or polymeric material, the material becoming fully miscible with the HPFP. Substitution of methyl groups along the backbone of the hydroxyl terminated EO greatly enhanced the solubility of the materials, but the exhibited phase behaviour was still that of LCST. Longer alkyl groups at the terminus of the EO were found to promote the solubility compared to the hydroxyl terminated material, but to a relatively smaller degree compared with the methyl group. It is hoped that these simple “design rules” should facilitate the development of designer excipients tailored for a particular application.
Keywords: Phase behaviour; Fluorinated media; Excipients; Polymers; Surfactants;

Block copolymers of the type poly(caprolactone)-b-poly(ethylene oxide) for the preparation and stabilization of nanoemulsions by Mickael Chausson; Ann-Sophie Fluchère; Emmanuel Landreau; Youssef Aguni; Yves Chevalier; Thierry Hamaide; Nabil Abdul-Malak; Isabelle Bonnet (153-162).
Block copolymers poly(caprolactone)-block-poly(ethylene oxide) are promising non-ionic macromolecular surfactants for the stabilization of emulsions because they display a stronger adsorption and provide an increased long-term stability. But such amphiphilic copolymers should also allow the fabrication of the suspensions according to the emulsification process used. An evaluation of such block copolymers was done regarding the nanoprecipitation and the miniemulsion polymerization processes that both afford aqueous suspensions of nanoparticles. Both the fabrication and the long-term stability were investigated. It was found that the emulsification by means of the nanoprecipitation process was successful when the amphiphilic block copolymer was added into the organic phase. The studies on the structure–activity relationships have shown that a minimum length of the poly(ethylene oxide) block was necessary in order to ensure both the long-term colloidal stability of the suspensions and the instantaneous stability during the preparation process. The length of the hydrophobic block was a parameter of less relevance, but a minimum length was required for the copolymers to be soluble in the organic phase. The miniemulsion polymerization process using block copolymer emulsifiers could be adapted to the incorporation of large loads of vitamin E acetate used as a hydrophobe stabilizer.
Keywords: Block copolymers; Emulsifier; Nanoparticles; Nanoprecipitation; Miniemulsion polymerization;

Understanding mechanical properties of pharmaceutical solids at the submicron scale can be very important to pharmaceutical research & development. In this paper, the hardness of individual particles of various pharmaceutical solids including sucrose, lactose, ascorbic acid, and ibuprofen was quantified using the atomic force microscopy (AFM) nanoindentation. Effects of data variation and indentation size or peak load on hardness are evaluated. The results show acceptable reproducibility and indicate that data variation may be primarily from the inhomogeneous nature of the samples. Different extents of indentation size or peak load effect on hardness were observed for the samples. With consideration of both data variation and indentation size effects, the hardness values of different samples were compared at similar contact depths or peak loads. The hardness ranked as: ascorbic acid > sucrose > lactose ≈ ibuprofen, at contact depths from ∼40 to 400 nm or peak loads ranging from ∼16 to 70 μN. Additionally, the potential implication of particle hardness to compact hardness and tableting performance was discussed.
Keywords: Atomic force microscopy; Nanoindentation; Particle; Hardness; Pharmaceutical solids;

Solid microemulsion preconcentrate (NanOsorb) of artemether for effective treatment of malaria by Medha Joshi; Sulabha Pathak; Shobhona Sharma; Vandana Patravale (172-178).
A microemulsion preconcentrate was formulated on the basis of solubility of artemether (ARM) in the various oily phases and surfactants and phase diagrams. Various solid adsorbents were evaluated for their ability yield solid microemulsion preconcentrates (NanOsorb-ARM). NanOsorb-ARM on dilution yielded microemulsion with average globule size of 183 nm and polydispersity index of 0.498 when determined using photon correlation spectroscopy. The antimalarial activity of NanOsorb-ARM, ARM solution and marketed ARM formulation (Larither®) was evaluated in Plasmodium berghei infected mice as per Peter's four day protocol. The acute lethal dose and the subacute toxicity of NanOsorb-ARM were determined as per the method suggested in Organization for Economic Cooperation and Development (OECD) guidelines. The NanOsorb-ARM exhibited significantly higher antimalarial activity (P  < 0.05) as compared to the marketed formulation of artemether (Larither®). Surprisingly, placebo NanOsorb also showed significantly higher antimalarial activity as compared to Larither® indicating that excipients used for the formulation of NanOsorb may have antimalarial activity. Subacute toxicity studies demonstrated that NanOsorb-ARM is comparatively safer than artemether oily solution with respect to survival, gross pathology, hematology and serum biochemistry in mice of both the genders.
Keywords: Malaria; Artemether; Solid microemulsion preconcentrate; Antimalarial activity; Oral dosage form;

Development of SMEDDS using natural lipophile: Application to β-Artemether delivery by Sagar D. Mandawgade; Shobhona Sharma; Sulabha Pathak; Vandana B. Patravale (179-183).
The objective of the present investigation was to formulate self-microemulsifying drug delivery systems (SMEDDS) using a novel, indigenous natural lipophile (N-LCT) as an oily phase. SMEDDS based on N-LCT and commercially available modified oil (Capryol 90) were formulated and their application in improving the delivery of a lipophilic anti-malarial drug, β-Artemether (BAM) was also evaluated. BAM-loaded SMEDDS were characterized with respect to mean globule size and in vitro drug release profile in comparison to the marketed formulation (Larither®). Comparative in vivo anti-malarial performance of the developed SMEDDS was evaluated against the (Larither®) in Swiss male mice infected with lethal ANKA strain of Plasmodium berghei. The parameters studied were percent parasitemia, activity against time and animal survival period. Both the BAM–SMEDDS showed excellent self-microemulsification efficiency and released >98% of the drug in just 15 min whereas (Larither®) showed only 46% drug release at the end of 1 h. The mean globule size for optimized BAM–SMEDDS was <100 nm. The anti-malarial studies revealed that BAM–SMEDDS resulted in significant improvement in the anti-malarial activity (P  < 0.05) as compared to that of (Larither®) and BAM solubilized in the oily phases and surfactant. The developed SMEDDS highlight safety for use and potential applications of indigenous natural lipophile in the development of novel colloidal drug carriers.
Keywords: Self-microemulsifying drug delivery systems (SMEDDS); Indigenous natural lipophile; β-Artemether; In vivo anti-malarial efficacy;

The goals of this study were to determine the solubility values of solid organic solutes in pure HFA-134a and in HFA-134a/ethanol cosolvent systems (0–20%, w/w), and to investigate the relationship between these solubilities and a solute's physico-chemical properties. A direct inject on-line HPLC method was used to determine the solubility of 21 solutes in HFA-134a/ethanol. The samples were allowed to equilibrate for at least 48 h. The filtered sample was injected directly on an analytical HPLC column through a manual injector interface, and analyzed at an appropriate solute wavelength via HPLC. The solutes display diverse physico-chemical properties and yielded solubility values that ranged over four orders of magnitude. In general, a linear–linear solubility relationship was observed as the fraction of ethanol increased. The effects on solubilization ranged from 1.3 to 99.4 times when 20% (w/w) ethanol was introduced, relative to pure HFA-134a. A regression equation utilizing a solute's hydrogen bonding potential resulted in a significant correlation to the slope obtained from a linear model for solubility in HFA-134a with 0–20% (w/w) ethanol, and may be useful for pre-formulation studies.
Keywords: HFA-134a; Solubility; Physico-chemical properties; MDI; Ethanol;

Liposomes are lipid vesicles largely investigated in the past 30 years as pharmaceutical carriers. In the development of new liposome-based formulations, the study of liposome surface properties remains a crucial step. For this purpose, microscopy techniques can provide useful information, although each such technique suffers from some limitations. Here, we have used cold field emission gun-scanning electron microscopy (cFEG-SEM) to acquire detailed images of liposome surface. In particular, we observed PEGylated and non-PEGylated liposomes in different size ranges. In the case of nanosized liposomes (mean diameter about 200 nm), a morphological evaluation of the whole preparation was obtained. On the other hand, in the case of giant liposomes (mean diameter about 2 μm), it was possible to observe the different surface ultrastructures of the two formulations. In particular, a regular and only slightly wrinkled surface was observed in the case of non-PEGylated liposomes, while a very irregular surface ultrastructure was visible in the case of PEGylated liposomes. This study shows, for the first time, the potential of cFEG-SEM as a new and powerful tool to obtain information on liposome morphology and, at least in the case of giant liposomes, on ultrastructure of the liposome surface.
Keywords: Liposomes; Microscopy; Surface morphology; PEGylated liposomes;

Diblock copolymers of ethylene oxide and 1,2-butylene oxide in aqueous solution by Maria Elenir N.P. Ribeiro; Samira A. de Oliveira; Nágila M.P.S. Ricardo; Shao-Min Mai; David Attwood; Stephen G. Yeates; Colin Booth (193-196).
The dependence of log(cmc) on hydrophobic block length n was examined for E m B n copolymers (E = oxyethylene, B = oxybutylene, subscripts denote number-average block lengths in repeat units) with n in the range 30–76. Combination with published data for E m B n diblock copolymers with shorter E-blocks shows two changes of slope in the log(cmc)–n plot corresponding to the onset of unimolecular micelle formation at n  ≈ 12 and completion of this process at n  ≈ 30. The results are discussed with reference to published data for E m L n and E m CL n (L from d,l-lactide; CL from ɛ-caprolactone) copolymers, which show similar behaviour.
Keywords: Block copolymers; Critical micelle concentration; Unimolecular micelles;

Particle size-dependent triggering of accelerated blood clearance phenomenon by Hiroyuki Koide; Tomohiro Asai; Kentaro Hatanaka; Takeo Urakami; Takayuki Ishii; Eriya Kenjo; Masamichi Nishihara; Masayuki Yokoyama; Tatsuhiro Ishida; Hiroshi Kiwada; Naoto Oku (197-200).
A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2 nm in diameter) or PEGylated liposomes (119, 261 or 795 nm) were preadministered into BALB/c mice. Three days after the preadministration [3H]-labeled PEGylated liposomes (127 nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24 h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2–795 nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7–31.5 nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.
Keywords: Polyethylene glycol; Liposomes; Accelerated blood clearance; Polymeric micelles; Nanocarriers;

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