Polyvinylamine-based capsules: a mechanistic study of the formation using alginate and cellulose sulphate

Capsules based on sodium alginate (SA) and sodium cellulose sulphate (SCS), have been prepared using polyvinylamines (PVAm) of varying intrinsic viscosities. The resulting capsules are relatively dense in nature, revealing a bursting force which is four times that observed for the classical SA/SCS/polymethylene-co-guanidine chemistry. Molar mass cutoffs were typically in the 10-70 kDa range. A mechanistic study was carried out where the reaction time, ionic strength and pH of the reaction mixture, as well as the stoichiometry of the polyanion blend and the PVAm molar mass were varied. It is postulated that both the SA-PVAm and the SCS-PVAm binary interactions contribute to the mechanical properties and the permeability of the resulting capsules. The polyvinylamine-based chemistry offers interesting alternatives to the PMCG system in that it provides a means to produce capsules at low, or zero, ionic strengths. Subtle changes in the pH, or the SA:SCS ratio, can also be used to tune the bursting force quite sensitively. The most appropriate capsules, for transplantation, would likely be formed at polyanion levels of 1.2 wt% with a PVAm molar mass below 17 kDa.     

Alginate-cellulose sulphate-oligocation microcapsules: optimization of mass transport and mechanical properties

Microcapsules based on polyelectrolyte complexation, where the inner phase involves a blend of alginate and sodium cellulose sulphate (SCS), have mechanical and transport properties which are relatively insensitive to the chemical composition of the rigid polyanion. Specifically, the bursting force of 400- and 1000 microm microcapsules increase slightly with the degree of substitution of the SCS, though the molar mass of the SCS appears to influence the transport properties more strongly than its composition. The concentration of the sodium chloride in the gelling batch can be varied rather extensively, with optimum properties at approximately half (i.e. 0.5 M) the level typically employed for the formation of cell-containing microcapsules. This indicates that the microcapsule properties can be tuned for biocompatability, without concern that changes to the polymer microstructure or reaction process conditions would adversely influence the bursting force or molar mass cut-off of the capsules. The alginate-SCS blend, which is typical equimass, can be slightly increased in favour of the SCS (to 55 wt%) if one seeks to mechanically optimize the system. The substitution of the oligocation polymethylene-co-guanidine with pDADMAC seems strongly undesirable. Similarly, the replacement of SCS with sulphoethylcellulose, while possible, offers no important advantages. The overall optimum conditions appear to be for a SCS with a DS of 2, prepared at 1.2 wt% of total cation with alginate. The ideal ratio, for mechanical and transport properties, of SCS to alginate is 55:45 (wt:wt), which represents a subtle modification from the classical formulation with very good biocompatability.     

Polymethylene-co-guanidine based capsules: a mechanistic study of the formation using alginate and cellulose sulphate

Capsules have been prepared based on a polyanion blend of sodium alginate and sodium cellulose sulphate, gelled in the presence of calcium chloride and sodium chloride. In a second step a membrane was formed via the addition of polymethylene-co-guanidine (PMCG), an oligocation. A mechanistic study examined the influences of pH, ionic strength, gelation and reaction times as well as the molar mass of the polyanions on the transport and mechanical properties. The ratio of alginate-to-cellulose sulphate in the polyanion blend was also varied and it was found that both mechanical resistance to compression as well as the pore size of the membrane decreased as the percentage of cellulose sulphate was reduced. The maximum mechanical strength was observed to correspond to the minimum in viscosity of the polyanion blend with, for low NaCl levels, a 3:1 alginate:cellulose sulphate level providing the largest resistance to deformation. The ability to decouple the molar mass cut-off and mechanical resistance is viewed as an important advantage of alginate/cellulose sulphate/PMCG capsules. Capsules were transplanted into mice to a maximum of 102 days, after which animals were sacrificed and capsules retrieved. Over 90% of the capsules were recovered from the peritoneal cavity with the mechanical properties of the explanted capsules observed to decrease as a function of implantation time, likely as a result of ion exchange. The capsules were, however, relatively free from any rejection which is a quite unusual result for cation containing systems. It is believed that the reason PMCG-based microcapsules function well in vivo is that they provide a net negative charge on the surface. Higher molar mass polycations such as poly-L-lysine provide a net positive charge, inducing inflammation.     

Polymethylene-co-guanidine based capsules: A mechanistic study of the formation using alginate and cellulose sulphate

Capsules have been prepared based on a polyanion blend of sodium alginate and sodium cellulose sulphate, gelled in the presence of calcium chloride and sodium chloride. In a second step a membrane was formed via the addition of polymethylene-co-guanidine (PMCG), an oligocation. A mechanistic study examined the influences of pH, ionic strenght, gelation and reaction times as well as the molar mass of the polyanions on the transport and mechanical properties. The ratio of alginate-to-cellulose sulphate in the polyanion blend was also varied and it was found that both mechanical resistance to compression as well as the pore size of the membrane decreased as the percentage of cellulose sulphate was reduced. The maximum mechanical strength was observed to correspond to the minimum in viscosity of the polyanion blend with, for low NaCl levels, a 3:1 alginate:cellulose sulphate level providing the largest resistance to deformation. The ability to decouple the molar mass cut-off and mechanical resistance is viewed as an important advantage of alginate/cellulose sulphate/PMCG capsules. Capsules were transplanted into mice to a maximum of 102 days, after which animals were sacrificed and capsules retrieved. Over 90% of the capsules were recovered from the peritoneal cavity with the mechanical properties of the explanted capsules observed to decrease as a function of implantation time, likely as a result of ion exchange. The capsules were, however, relatively free from any rejection which is a quite unusual result for cation containing systems. It is believed that the reason PMCG-based microcapsules function well in vivo is that they provide a net negative charge on the surface. Higher molar mass polycations such as poly-L-lysine provide a net positive charge, inducing inflammation.     

Modulating drug release and matrix erosion of alginate matrix capsules by microenvironmental interaction with calcium ion.

Effect of calcium gluconate (CG) content on release of dextromethorphan hydrobromide (DMP), model drug, from capsules containing low and medium viscosity grades of sodium alginate (SA) was investigated in different dissolution media. Matrix erosion of the SA matrix capsules in distilled water and pH 7.4 phosphate buffer was compared. Molecular interaction of SA with calcium ion in surface gel layer of the SA matrix capsules was examined using Fourier transform infrared spectroscopy and differential scanning calorimetry. In distilled water and pH 7.4 phosphate buffer, DMP release rate depended on the viscosity grade of SA, whereas a comparable DMP release rate was found in 0.1N HCl. Incorporation of CG into the SA matrix capsules caused a faster drug release in acidic medium because CG acted as a channeling agent in the hydrated insoluble gel matrix of alginic acid. Interaction of calcium ions with carboxyl groups of SA could be formed in surface gel layer of hydrated matrix capsules in distilled water. This led to a more rigid matrix gel structure that caused a slower drug release and matrix erosion. In contrast, the extent of this interaction in pH 7.4 phosphate buffer was less than that in distilled water because the common ion effect and high concentration of sodium ion retarded the hydration of SA and the binding of calcium ions with carboxyl groups of SA. Thus, a small change in drug release and matrix erosion was observed. This finding suggests that microenvironmental interaction between hydrated SA and calcium ion in distilled water could be created in the formulations prepared using low compression force. Moreover, incorporation of CG could moderate drug release and matrix erosion of the SA matrix capsules.     

Influence of alginate characteristics on the properties of multi-component microcapsules

A variety of sodium alginates, differing in molar mass and structural composition, have been evaluated in the preparation of multi-component microbeads and microcapsules. Bead formation occurred by gelation with calcium chloride. Capsules were produced by reacting the pre-formed beads with the oligocation poly(methylene-co-guanidine). Despite the equiponderous (1:1) mixing with a second polyanion, sodium cellulose sulphate, the influence of the alginate properties remains evident. Specifically, the effect of the chemical composition was found to be more significant than that of the molar mass for both the mechanical and transport properties. Furthermore, for alginates of 73% α-l-guluronic acid content less shrinking was observed compared to the 38% guluronic materials. This results in the case of the same encapsulator settings in larger microsphere diameters and thicker membranes accompanied by enhanced mechanical resistance though, also, in a higher permeability for the high-G capsules. However, subsequent coating with lower molar mass alginate allows one to adjust the permeability over a broad range, suitable for cell encapsulation and immunoprotection, without compromising the durability.     

Influence of alginate characteristics on the properties of multi-component microcapsules

A variety of sodium alginates, differing in molar mass and structural composition, have been evaluated in the preparation of multi-component microbeads and microcapsules. Bead formation occurred by gelation with calcium chloride. Capsules were produced by reacting the pre-formed beads with the oligocation poly(methylene-co-guanidine). Despite the equiponderous (1:1) mixing with a second polyanion, sodium cellulose sulphate, the influence of the alginate properties remains evident. Specifically, the effect of the chemical composition was found to be more significant than that of the molar mass for both the mechanical and transport properties. Furthermore, for alginates of 73% alpha-l-guluronic acid content less shrinking was observed compared to the 38% guluronic materials. This results in the case of the same encapsulator settings in larger microsphere diameters and thicker membranes accompanied by enhanced mechanical resistance though, also, in a higher permeability for the high-G capsules. However, subsequent coating with lower molar mass alginate allows one to adjust the permeability over a broad range, suitable for cell encapsulation and immunoprotection, without compromising the durability.     

Intra- and intermolecular reactions of 4,4'-diisocyanatodiphenylmethane with human serum albumin

Diisocyanates are highly reactive industrial chemicals that have been shown to possess toxic activity, including potential for allergic sensitization. To assist in diagnosis of sensitization, immunoassays for diisocyanate-specific antibodies are performed; such assays require preparation of diisocyanate-containing hapten-protein conjugates. Conditions were investigated for formation of conjugates yielding varying degrees of hapten binding. Relative concentrations of haptens and proteins were varied as were pH, temperature, and time of reaction. Quantitation of 4,4'-diisocyanatodiphenylmethane (MDI) binding with human serum albumin (HSA) was assessed by absorbance of the isolated conjugates at 250 nm after determination of the molar extinction coefficient for MDI. At pH 7.4 and 37 degrees C, the binding reaction was found to be biphasic with binding of 5-6 mol of MDI groups/mol of HSA within the first minute, followed by incorporation at a rate of 0.16 mol/min during the next 2 h. Evaluation of reaction products using SDS-PAGE revealed extensive inter- and intramolecular cross-linking of HSA by MDI. Intramolecular cross-linking was accompanied by an increased migration of conjugates from an initial molecular mass of 66 kDa, typical of HSA, to a molecular mass of 44 kDa. The change in migration was also produced by using disuccinimidyl tartarate (DST) as hapten and was eliminated when DST was cleaved with sodium periodate. It was attributed to altered protein shape. Conditions that favored binding of MDI with HSA were a high relative concentration of MDI:HSA, a pH of 9.4, and a temperature of 37 degrees C. Under such conditions it was calculated that 53 mol of MDI were bound per mole of HSA after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitive and selective spectrophotometric assay of gabapentin in capsules using sodium 1, 2-naphthoquinone-4-sulfonate

A simple, sensitive, and selective spectrophotometric method has been developed for the determination of gabapentin (GBP) in capsules. The method is based on the reaction of GBP and sodium 1,2-naphthoquinone-4-sulfonate (NQS) in the presence of Clark and Lubs buffer of pH 11 to form an orange-coloured product which was measured at 495 nm. The parameters that affect the reaction were carefully optimized and under the optimized conditions, linear relationship was obtained in the concentration range of 7.5-75 μg ml(-1) GBP. The molar absorptivity, limits of detection (LOD) and quantification (LOQ) and Sandell sensitivity are also reported. The proposed method was successfully applied to the determination of GBP in capsules with good accuracy and precision and without detectable interference from common excipients. The reliability of the proposed method was further established by parallel determination by the reference method and also by recovery studies. The reaction mechanism is proposed and discussed.     

Chemistry set chemicals: an evaluation of their toxic potential

KM, a 14-year-old female packed 3 empty Sudafed SA capsules with 3 parts sodium chloride and 1 part cobalt chloride obtained from her brother's chemistry set. Following this, she ingested all 3 capsules. While the child had an uneventful hospital course, cobalt serum levels drawn 12 and 22 hr post-ingestion were measured at 7.8 micrograms/dL and 0.7 micrograms/dL, respectively. Due to the limited amount of data on the toxicity of cobalt chloride and the nature of the exposure, the case prompted a review of the potential toxicity of chemistry set chemicals available to consumers. The contents of 3 chemistry sets were evaluated. Each contained either 28, 14, or 9 chemicals. A total of 38 different chemicals were weighted and evaluated to determine whether the amount contained represented a potentially toxic or lethal dose to a 2-year-old, 12 kg child. Fifty-three percent of the chemicals evaluated contained quantities sufficient to result in toxicity. Thirteen percent of chemicals contained potentially lethal quantities, while 18% were considered non-toxic. Sixteen percent of the chemicals could not be evaluated due to lack of data in the literature. Only one chemistry set utilized child resistant closures. However, 65% of all potentially toxic chemicals and 100% of potentially lethal chemicals had appropriate first aid instructions.     

Polyvinylamine hydrochloride-based microcapsules: polymer synthesis, permeability and mechanical properties

Mechanically stable microcapsules, with sizes of 0.4-1.5 mm, have been produced with permeabilities appropriate for applications involving living cells and controlled delivery. Polyvinylamine hydrochloride was employed alone, in miXtures with poly(methylene-co-guanidine) hydrochloride, or as a coating material for pre-formed capsules. The influence of polymer molar mass, the ratio between the two polycations, the coating time, and the capsule size on the properties of the capsules were analysed. The competitive displacement of one polycation with another in the polysaccharide matrix was also documented. The properties of the capsules vary remarkably, depending of the polyelectrolyte combinations used for their preparation. Specifically, capsules could withstand compressive loads of between 0.09-1.67 N, while the permeability varied from 10-120 kDa. Both are within the ranges required for clinical immunosuppressive therapies.     

分子排阻色谱法测定头孢克洛胶囊中高分子杂质的含量

目的 建立分子排阻色谱法测定头孢克洛胶囊中的高分子杂质。方法 采用球状亲水硅胶柱(TSK-GEL?G2500PWXL色谱柱,7.8mm×300mm, 7μm),流动相为磷酸盐缓冲液(pH7.0)[0.02mol/L磷酸氢二钠溶液和0.02mol/L磷酸二氢钠溶液(61:39)]-乙腈(95:5)为流动相,流速为0.8mL/min,检测波长为265nm,以头孢克洛对照品外标法计算高分子杂质的含量。结果 头孢克洛在0.532~21.280μg/mL的浓度范围内,面积与浓度呈良好的线性关系(r=0.9999);最小检出浓度为0.161μg/mL;高分子杂质与头孢克洛峰能有效分离,并先于主峰流出,方法专属性良好。结论 该方法适于测定头孢克洛胶囊中高分子杂质,灵敏度高,重复性好,操作简便。     

Tryptophan-mPEGs: Novel excipients that stabilize salmon calcitonin against aggregation by non-covalent PEGylation

Protein aggregation, which is triggered by various factors, is still one of the most prevalent problems encountered during all stages of protein formulation development. In this publication, we present novel excipients, tryptophan-mPEGs (Trp-mPEGs) of 2 and 5 kDa molecular weight and suggest their use in protein formulation. The synthesis and physico-chemical characterization of the excipients are described. Possible cytotoxic and hemolytic activities of the Trp-mPEGs were examined. Turbidity, 90° static light scatter, intrinsic fluorescence, fluorescence after staining the samples with Nile Red and fluorescence microscopy were used to study the inhibitory effect of the Trp-mPEGs on the aggregation of salmon calcitonin (sCT) in different buffer systems and at various molar ratios. Aggregation of sCT was reduced significantly with increasing concentrations of Trp-mPEG 2 kDa. A 10-fold molar excess of Trp-mPEG 2 kDa suppressed almost completely the aggregation of sCT in 10 mM sodium citrate buffer (pH 6) for up to 70 h. Trp-mPEG 5 kDa also reduced the aggregation of sCT, though less pronounced than Trp-mPEG 2 kDa. Low aggregation of sCT was measured after approximately 10 days in 10 mM sodium citrate buffer, pH 5, with a 10-fold molar excess of Trp-mPEG 2 kDa. This paper shows that Trp-mPEGs are potent excipients in reducing the aggregation of sCT. Trp-mPEGs are superior to dansyl-PEGs concerning the stabilization of sCT in a harsh environment, wherein sCT is prone to aggregation. Trp-mPEGs might therefore also be used for stabilization of other biopharmaceuticals prone to aggregation.     

Characteristics of activation of cathepsin B by sodium salicylate and comparison of catalytic site properties of cathepsins B and H

Attempts to gain greater understanding of characteristics of catalytic site properties of two homologous lysosomal cysteine proteinases, cathepsins B and H, were made by using sodium salicylate (SA) and several specific inhibitors, which extends the studies on the structure-activity relationship and kinetics for the activation of cathepsin B by SA in the previous paper (Yamamoto, K., Takeda, M. and Kato, Y.: Japan. J. Pharmacol. 38, 215-218, 1985). The half-maximal activation of cathepsin B by SA was observed at around a molar ratio of 10(4):1 (SA/cathepsin B). No preincubation time was needed for the SA-stimulated reaction, but the rate of activation was more rapid as pH values in the preincubation mixture decreased. The extent of inactivation of cathepsin B by leupeptin and E-64 significantly decreased in the presence of SA. Catalytic site properties of cathepsins B and H were also distinguished by differences in the extent of inhibition by cysteine proteinase inhibitors. Cathepsin B was more sensitive than cathepsin H to inhibition by antipain, chymostatin, iodoacetic acid and mercuric chloride as well as leupeptin and E-64. Despite the similarity in inhibitory effects of iodoacetic acid and E-64, the rat spleen cathepsin H was characterized by insensitivity to mercuric chloride that had a considerable inhibitory effect on the corresponding enzyme from rat liver and the rat spleen cathepsin B.     

羧甲基壳聚糖金属配合物的研制

应用水溶性的羟甲基壳聚糖与Ca^2 、Fe^2 、Zn^2 进行络合制备金属配合物,探讨反应时间、温度、羧甲基取代度、溶液离子强度、pH等反应条件对络合的影响,考察金属配合物的性质。结果表明：络合反应的最佳pH值为5－7;室温下反应10min络合量趋于饱和;羧甲基壳聚糖对各金属离子络合能力随羧甲基取代度的增大而增强,随着体系的离子强度增大而减弱。     

Permeability and stability of chitosan-based capsules: effect of preparation

Capsules were obtained by interpolymer complexation between chitosan (polycation) and sodium hexametaphosphate (oligoanion). The effect of preparation variables such as the pH, ionic strength as well as the reagent and porogen concentration on the capsule characteristics was evaluated. By decreasing the chitosan/SMP ratio, adding mannitol up to 1% or maintaining the salt concentrations below 0.15 w/v%, the diffusion characteristics can be modulated without disturbing the capsule mechanical stability. Higher concentrations of the cross-linking agent (2.25 w/v%) produced stable capsules only in the absence of electrolyte and low polyol amounts. Furthermore, by increasing the ionic strength, or the pH of the initial chitosan solution, the membrane exclusion limit shifted to higher values concomitant with a significant loss in the membrane compression resistance. The results obtained showed that the capsule characteristics could be independently controlled by manipulating the coacervation conditions.     

Characterization of microcapsules: recommended methods based on round-robin testing

Alginate beads, as well as microcapsules based on alginate, cellulose sulphate and polymethylene-co-guanidine, were produced at diameters of 0.4, 1.0 and 1.5 mm. These standard materials were tested, by independent laboratories, in regards to water activity, bead or capsule size, mechanical resistance and transport behaviour. The water activity and mechanical resistance were observed to increase with bead and capsule size. Transport properties (ingress) were assessed using a variety of low molar mass and macromolecular probes. It was observed that the penetration of Vitamin B12 increased with bead diameter, as did dextran penetration. However, for the membrane-containing microcapsules, larger membrane thickness, observed for the larger capsules, retarded ingress. The authors, who are part of a European working group, recommend that permeability be assessed either using a large range of probes or a broad molar mass standard, with measurements at one or two molar masses insufficient to simulate the behaviour in application. Mechanical compression is seen as a good means to estimate elasticity and rupture of beads and capsules, with the sensitivity of the force transducer, which can vary from #181;N to tens of N, required to be tuned to the anticipated bead or capsule strength. Overall, with the exception of the mechanical properties, the precision in the inter-laboratory testing was good. Furthermore, the various methods of assessing transport properties agreed, in ranking, for the beads and capsules characterized, with gels having smaller radii being less permeable. For microcapsules, the permeation across the membrane dominates the ingress, and thicker membranes have lower permeability.     

Effect of fill weight, capsule shell, and sinker design on the dissolution behavior of capsule formulations of a weak acid drug candidate BMS-309403

Two strengths of BMS-309403 capsules were developed from a common stock granulation. Dissolution testing of the capsules was conducted utilizing the USP apparatus 2 (paddle) with a neutral pH dissolution medium. Unexpectedly, the lower-strength capsules exhibited slower dissolution than the higher-strength capsules filled with the same stock granulation. Higher variability was also observed for the lower-strength capsules. This was found to be mainly caused by a low fill weight in a relatively large size hard gelatin capsule shell. Instead of bursting open, some gelatin capsule shells softened and collapsed onto the granulation, which delayed the release of the active drug. The problem was aggravated by the use of coil sinkers which hindered the medium flow around the capsules. Switching from the gelatin capsule shells to the HPMC (hydroxypropyl methylcellulose) shells reversed the dissolution rate ranking between the two capsule strengths. However, both dissolved at a slower rate initially than the gelatin capsules due to the inherent dissolution rate of the HPMC shells at pH 6.8. Notably, the HPMC shells did not occlude the granulation as observed with the gelatin shells. The study demonstrated that the dissolution of capsule formulations in neutral pH media was significantly affected by the fill weight, sinker design, and capsule shell type. Careful selection of these parameters is essential to objectively evaluate the in vitro drug release.     

Characterization of microcapsules: recommended methods based on round-robin testing

Alginate beads, as well as microcapsules based on alginate, cellulose sulphate and polymethylene-co-guanidine, were produced at diameters of 0.4, 1.0 and 1.5 mm. These standard materials were tested, by independent laboratories, in regards to water activity, bead or capsule size, mechanical resistance and transport behaviour. The water activity and mechanical resistance were observed to increase with bead and capsule size. Transport properties (ingress) were assessed using a variety of low molar mass and macromolecular probes. It was observed that the penetration of Vitamin B12 increased with bead diameter, as did dextran penetration. However, for the membrane-containing microcapsules, larger membrane thickness, observed for the larger capsules, retarded ingress. The authors, who are part of a European working group, recommend that permeability be assessed either using a large range of probes or a broad molar mass standard, with measurements at one or two molar masses insufficient to simulate the behaviour in application. Mechanical compression is seen as a good means to estimate elasticity and rupture of beads and capsules, with the sensitivity of the force transducer, which can vary from microN to tens of N, required to be tuned to the anticipated bead or capsule strength. Overall, with the exception of the mechanical properties, the precision in the inter-laboratory testing was good. Furthermore, the various methods of assessing transport properties agreed, in ranking, for the beads and capsules characterized, with gels having smaller radii being less permeable. For microcapsules, the permeation across the membrane dominates the ingress, and thicker membranes have lower permeability.     

Effect of Fill Weight,Capsule Shell,and Sinker Design on the Dissolution Behavior of Capsule Formulations of a Weak Acid Drug Candidate BMS‐309403

Two strengths of BMS‐309403 capsules were developed from a common stock granulation. Dissolution testing of the capsules was conducted utilizing the USP apparatus 2 (paddle) with a neutral pH dissolution medium. Unexpectedly, the lower‐strength capsules exhibited slower dissolution than the higher‐strength capsules filled with the same stock granulation. Higher variability was also observed for the lower‐strength capsules. This was found to be mainly caused by a low fill weight in a relatively large size hard gelatin capsule shell. Instead of bursting open, some gelatin capsule shells softened and collapsed onto the granulation, which delayed the release of the active drug. The problem was aggravated by the use of coil sinkers which hindered the medium flow around the capsules. Switching from the gelatin capsule shells to the HPMC (hydroxypropyl methylcellulose) shells reversed the dissolution rate ranking between the two capsule strengths. However, both dissolved at a slower rate initially than the gelatin capsules due to the inherent dissolution rate of the HPMC shells at pH 6.8. Notably, the HPMC shells did not occlude the granulation as observed with the gelatin shells. The study demonstrated that the dissolution of capsule formulations in neutral pH media was significantly affected by the fill weight, sinker design, and capsule shell type. Careful selection of these parameters is essential to objectively evaluate the in vitro drug release.