Mixed Granules Research Articles

A New Attempt To Solve the Scale-Up Problem for Granulation Using Response Surface Methodology

Scale-up from lab to production is always problematic for the development of pharmaceuticals. In granulation, an optimal formulation of binder solution determined in a lab scale is often different than that in a production scale. A new mathematical procedure to solve this scale-up problem is assessed. Granules were prepared in the two manufacturing scales (2- and 5-kg scale) by using a highspeed mixer granulator. In the manufacturing process, the binder solution plays an essential role in the formation of granules with desired physical properties, in close conformity with the manufacturing scale. A computerized optimizing technique based on a response surface methodology was developed to study the scale-up problem in the manufacturlng’of granules. For this purpose, a new mathematical function was introduced for the first time, which is namely an integrated optimization function. A universal optimal formulation unaffected by manufacturing scale could be obtained by minimizing the integrated optimization function. Predicted values such as yield, mean granule size, and uniformity of granule size agreed well with experimental ones on both scales. Furthermore, the optimized characteristics measured at the production scale coincided well with those obtained at laboratory scale, suggesting that this approach could be very useful in minimizing scale-up problems.

Granulation Technology for Bioproducts

Introduction. Theory of Granulation. Fluidized Bed Granulation. Spray Drying of Bioproducts. Extrusion and Spheronization. High-Intensity Mixer Granulators. Microencapsulated Enzymes and Live Mammalian Cells. Liquid Phase Granulation. Large Scale Freeze-Drying of Bacterial Concentrates. Fluidized Bed Drying of Granular Bioproducts. Coating of Granular Bioproducts. Granulation Processes for Industrial Enzymes. Process and Equipment Selection.

高速撹はん造粒における造粒特性に及ぼす粉体の濡れ性の影響

The following results were obtained by using a high-speed mixer granulator and investigating the relationship between the granularity and wettability of aluminum hydroxide and calcium carbonate powders that were surface-treated with stearic acid:(1) The granulation limit had a strong relationship to dispersibility in water and to the contact angle of the sample powder. Granulation was possible by using water with powders consisting partly of hydrophilic powders.(2) An induction period of granulation was necessary for seed growth in powders which were highly hydrophobic. This period was influenced by the added water content, mixing speed and the wettability of the sample powder.(3) The fine powder generation ratio of granules was less with powders which were made partly of hydrophobic than non-treated powders.

国内で生産されたテガフール製剤のカプセルおよび包装外部への主薬付着について その２

Contamination of tegafur, an cytotoxic antimalignant agent, in packaging materials of 28 different tegafur capsules available on the Japanese market had been reproted by one of the authors. The study revealed that the tegafur contamination was not been limited to the outer shell of the capsules but extended, in some of the brands, to the exterior of the press-through package (PTP). Suggestions were made one year previously to the manufacturers of the brands to take measures to prevent the contamination from occurring.The purpose of the present study was to follow up and investigate whether any improvement was subsequently made on these brands. The existence of tegafur at the outer shell of tegafur capsules, PTP inner pockets, PTP, and aluminum pillow packs was qualitatively tested by HPLC analysis of most of the brands reported in the previous study. At the surface of most packaging materials, the existence of the active ingredient, tegafur, was demonstrated, and the contamination in some of the brands exceeded that found one year ago. In addition to the previously investigated brands, mixed granules of tegafur and uracil were studied, revealing contaimination of tegafur at the surface of stripped package films. It was suggested that some definitive means should be taken to minimize the contamination and to prevent any potential safety problem from arising by those who continuously handle cytotoxic products.

MR Imaging of Middle Ear Cholesteatomas

We prospectively evaluated MR images of 14 patients who had chronic otitis media and who were suspected of having cholesteatomas on otologic examination and/or on high resolution CT. Cholesteatomas were verified in the middle ear and/or mastoid at surgery in nine patients. Cholesteatomas appeared isointense relative to gray matter on T1-weighted spin echo MR images (T1WI) and hyperintense on T2-weighted spin echo MR images (T2WI) in eight patients. Two cholesterol granulomas appeared hyperintense on both T1WI and T2WI. In three patients with chronic otitis media the associated mixed granulation tissue and fluid collection was revealed as nonspecific, heterogeneous signal intensity in two cases. In the third case the signal intensity was similar to that found in cholesteatomas.

Prolongation of Antipyretic Action and Reduction of Gastric Ulcerogenicity in the Rat by Controlled-Release Granules of Bermoprofen, A New Nonsteroidal Anti-inflammatory Drug

While bermoprofen [(±)-10,11-dihydro-α,8-dimethyl-11-oxodibenz[b,f]oxepin-2-acetic acid], a new nonsteroidal anti-inflammatory drug (NSAID), has potent antipyretic and analgesic activities with a short biological half-life, it shows ulcerogenic activity as a side-effect like other nonsteroidal anti-inflammatory drugs. A bermo-profen preparation was specially designed to prolong its duration of action and to reduce its side effect. Immediate-release granules (IRGs) were prepared by coating particles of Nonpareil 103 with bermoprofen. The IRGs were also coated by spraying a film solution composed of ethylcellulose and hydroxypropylmethylcellulose (3:2), and, thereby, three kinds of retard-release granules (RRGs) were obtained with the coating amounts of 1.5, 2.5, and 4%. Peak plasma bermoprofen levels were seen 0.5, 1, 2, and 3 h after single oral administrations of IRG, RRG(1.5%), RRG (2.5%), and RRG(4%), respectively, in rats. The potency order in ulcerogenic activity was RRG(1.5%) > IRG > RRG(2.5%) > RRG(4%) after a single oral dosage in rats. Then, IRG and RRG(4%) were mixed (in the ratio 3:7, which was calculated from their simulated plasma bermoprofen profiles) in order to get fast onset and long duration of antipyretic action. These mixed granules [i.e., the sustained-release granules (SRGs)] exhibited a flat plasma bermoprofen profile, and longer lasting antipyretic and lower ulcerogenic activities in rats in comparison with IRG or bermoprofen. From these results, it is suggested that a bermoprofen SRG preparation has more durable therapeutic activity and lower side-effects as a NSAID.

Granule Changes of Human Skin Mast Cells Characteristic of Piecemeal Degranulation and Associated With Recovery During Wound Healing In Situ

Human skin mast cells (HSMC) in situ were examined by electron microscopy of surgical biopsy specimens obtained from a wide variety of circumstances. From these studies, it is apparent that the general ultrastructural morphology of normal HSMC is similar to that of human mast cells from other sites, except that the most prevalent granule pattern is that of crystal granules. Scroll granules, particle granules, and mixed granules can also occur in HSMC. Cytoplasmic lipid bodies occur in HSMC extremely rarely, unlike mast cells from lung and gut. Circumstances in which piecemeal degranulation (PMD) of HSMC occurs, such as bullous pemphigoid, examined at high magnifications, revealed typical, focal geographic losses from cytoplasmic granules, often leaving completely empty granule containers in the cytoplasm. Crystal portions of mixed granules were uniquely susceptible to granule losses typified by PMD. Cytoplasmic smooth vesicles were prominent in PMD. These structures were either empty or contained granule-like dense materials and were free in the cytoplasm or attached to granules. HSMC present in wound healing revealed recovery from PMD losses. Typically these granules contained numerous irregular foci of markedly dense new granule products within empty or partially empty granule containers. The morphology of PMD and recovery of HSMC in vivo is contrasted with the morphology of anaphylactic degranulation (AND) and recovery of human mast cells ex vivo.

Particle Size Design Using Computer Optimization Technique

AbstractThe effect of binder solution (amount and composition) on the mean particle size and its distribution of granule was investigated by using a computer optimization technique. The granules were manufactured by two continuous processes, granulating and sizing using a high-speed mixer granulator and a hammer mill, respectively. The particle size distribution pattern of granules was markedly varied with the change in the amount and composition of the binder solution. The distribution pattern could be well expressed by the log-normal distribution model. For designing the optimal particle, a computer optimization technique was applied to the experimental results obtained in this study. The technique was found to be useful for searching the optimal formula in the practical scale.

Heterogeneous Ultrastructure of Human Bronchial Mast Cells: Morphometry Subdivision of Cell Types and Evidence for a Degranulation Gradient

Structural heterogeneity of mast cells in human bronchial mucosa was investigated by examining 100 cells by electron microscopy and morphometry. Differential counts of secretory granules allowed subdivision of mast cells into three groups: (1) 49 cells with greater than 65% mixed granules; (2) 30 cells with greater than 30% scrolled granules; (3) 12 cells with greater than 30% particulate granules. Nine cells showed borderline characteristics. Records of depths of cells in the mucosa showed that most mixed-granule cells occupied middle levels, whereas most scrolled granule cells lay higher up, near the basement membrane. This raised the possibility that scrolled-granule cells may represent partly degranulated mixed-granule cells. Nineteen mast cells were filled with very dense mixed granules, and appeared to correspond to those staining with safranin in paraffin sections. However, morphometry did not produce any criteria for distinguishing these cells sharply from paler mixed-granule cells, which were therefore regarded as degranulating forms of the same type of cell. Evidence was found of a gradient of mast cell degranulation which appeared to increase in magnitude upward from deep submucosa to superficial mucosa. This evidence included finding a significant upward reduction in total granule area per cell, total granule numbers, numbers of mixed granules, and numbers of dense-cored granules. It was concluded that although bronchial mucosal mast cells could be subdivided ultrastructurally into three apparently heterogeneous groups, degranulation was found to produce a wide range of different cell appearances, and could, conceivably, even be responsible for the above grouping, rather than intrinsic mast cell heterogeneity.

Identification of Dense Granule Specific Membrane Proteins in Bovine Platelets that Are Absent in the Chediak-Higashi Syndrome

Platelets from cattle with the Chediak-Higashi syndrome (CHS) have a platelet dense granule deficiency. One hypothesis for the platelet dense granule deficiency is that the granule is simply not formed in CHS megakaryocytes (MK). Alternative hypotheses include that the granule is assembled in CHS MK but a functional amino-nucleotide-cation storage complex cannot be formed or that the dense granule or its precursor fuses with other granules. This study was undertaken to determine if membrane proteins specific for platelet dense granules can be identified in membranes of other granules in CHS platelets. Platelets were disrupted; a mixed-granule fraction and alpha-granule enriched, mitochondrial-enriched, and dense granule-enriched subfractions were obtained. Membrane proteins in these intact granules were radiolabeled and the granule underwent hypotonic lysis. Membrane proteins were extracted from granule "ghosts", separated, and then visualized by autoradiography. Three major proteins were identified in platelet dense granule membrane subfractions. Two of these proteins could be identified in membrane extracts from the mixed-granule fraction from normal platelets. They could neither be identified in extracts from the mixed granule fraction of CHS platelets nor in membranes from alpha granule-enriched and mitochondrial-enriched subfractions. The absence of dense granule membrane proteins in membranes of other organelles within CHS platelets suggests that fusion of dense granules or its precursor with other granules cannot account for the platelet dense granule deficiency in CHS platelets.

Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas.

A morphological study has been carried out on 20 GH-secreting adenomas removed from acromegalic normoprolactinemic patients, on 29 PRL-secreting adenomas removed from hyperprolactinemic patients without signs of acromegaly and on one normal human anterior pituitary gland collected at autopsy. The protein A-gold immunoelectron microscopic technique has been utilized in order to verify the presence of mixed cells producing both GH and PRL (somatomammotrophs) in these pituitary tissues. In the normal pituitary a considerable number of somatomammotrophs (15-20%) was found, thus supporting the idea that these cells are normal components of the human anterior pituitary gland. In 10 GH-secreting adenomas and in 10 PRL-secreting adenomas somatomammotrophs were present in a variable number (from 4 to 20% of the whole cell population in GH adenomas and from 1 to 47% in PRL tumors). It can be concluded therefore that these cells, largely present in all GH/PRL-secreting adenomas, can also be found in GH-secreting and PRL-secreting tumors without clinical evidence of a mixed secretion. Adenomatous somatomammotrophs displayed ultrastructural features of adenomatous somatotrophs and mammotrophs (prominent Golgi complexes, abundant rough endoplasmic reticulum, irregular nuclei). The size and the number of granules were variable. In some cells GH and PRL were stored in distinct secretory granules, in others in mixed granules or both in mixed and distinct granules, thus suggesting that in adenomatous somatomammotrophs the efficiency of the mechanisms of sorting of the two hormones varies from one cell to another.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomass retention in a glucose acidifying anaerobic gas-lift reactor: isolation of the organism responsible for granule formation

A study was undertaken of the microbial composition of aggregates from an acidifying anaerobic gas-lift reactor. For this purpose a simple 100 ml anaerobic gas lift reactor was developed. It was found that the predominant organism in the aggregates was Selenomonas ruminantium. Both, microscopical observations and a newly developed enumeration technique led to the conclusion that the mixed granules consisted mainly of this organism. Grown in pure culture, S. ruminantium was capable of forming aggregates. These aggregates resembled the mixed aggregates both macro- as well as microscopically. Furthermore the fermentation pattern of this pure aggregated culture was similar to that of a mixed aggregated culture.

Subcellular localization of prolactin in the anterior pituitary cells of the female Japanese house bat, Pipistrellus abramus.

We investigated the subcellular localization of PRL and GH in the pituitary gland of the female Japanese house bat by the double immunolabeling procedure using the protein A-gold method combined with electron microscopy and demonstrated a seasonal alteration in the distribution of PRL within the cells. The seasonal changes were related to the different phases of the bats' reproductive cycles. Mammosomatotrophs (MS cells) containing both PRL and GH were constantly present throughout the reproductive cycles of the female bats and they were remarkably hypertrophied during pregnancy. The distribution pattern of PRL and GH within the MS cells was extremely variable owing to the different phases of reproductive cycles of the bats. We divided MS cells into the following four types: the cell containing 1) only mixed granules containing both PRL and GH, 2) both mixed and PRL granules, 3) mixed, PRL and GH granules, 4) both mixed and GH granules. While pure PRL-containing cells were observed in pregnant and lactating bats, they were not observed in bats obtained during prehibernation, midhibernation, and arousal periods. These results suggest that MS cells in the female bats hypertrophy during pregnancy and that some of them may be transformed into PRL-producing cells.

Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis.

Growth hormone [GH] and prolactin [PRL] can be demonstrated simultaneously in electron micrographs by means of the double immunocytochemical labeling technique using colloidal gold particles of two different sizes. This method was used to study biopsy specimens obtained from 15 patients suffering from acromegaly, 11 patients suffering from prolactinomas, and eight biopsy specimens obtained during adenomectomy from the normal, paraadenomatous pituitary tissue. Four granule populations with different immunoreactions were found: (1) granules containing GH only, (2) granules containing PRL only, (3) mixed granules containing GH and PRL, and (4) granules displaying no immunoreactivity. The existence of mixed granules indicated that the two hormones are synthesized by the same cell and in communicating compartments of the cells; i.e., the rough-surfaced endoplasmic reticulum. The number of GH-containing granules (pure GH granules and mixed GH-PRL granules) was greater than that of PRL-containing granules (pure PRL granules and mixed PRL-GH granules) in adenomas causing acromegaly and in the normal pituitary tissue, whereas the opposite was true for prolactinomas. The number of PRL-containing granules was larger in biopsy specimens from patients who had acromegaly and hyperprolactinemia than in patients with acromegaly and normal serum PRL levels.

Impaired morphological development of the dorsal cochlear nucleus in hamsters treated postnatally with α -difluoromethylornithine

The dorsal cochlear nucleus is a highly organized nucleus in the auditory system in which the ramifications of depletion of specific cell types during development can be studied. Granule cells, small interneurons that are located in all layers of the DCN in the adult hamster, proliferate postnatally and are, therefore, potentially vulnerable to anti-mitotic agents that are administered after birth. The present experiments describe the effects of α-difluoromethylornithine, a drug that inhibits proliferation of cerebellar granule cells, on the granule cells in the dorsal cochlear nucleus. As in the cerebellum, the density of granule cells in the dorsal cochlear nucleus is reduced after α-difluoromethylornithine treatment. In hamsters treated with α-difluoromethylornithine (200 or 500 mg/kg subcutaneously (s.c.), twice daily on postnatal days 4–14), the numerical density of granule cells was reduced in the superficial dorsal cochlear nucleus at 15 days; by 40 days this effect was also apparent in the deep layer, suggesting that cells located superficially that would have migrated into the deep dorsal cochlear nucleus had either failed to develop or did not arrive at their final location. This evidence suggests that the cells normally migrate down from the superficial proliferative zone into the deeper layers. In the drug-treated animals, a layer of mixed granule cells and fusiform cells was thinner than in controls probably due to the reduction in interspersed granule cells since the number of fusiform cells was unaffected. There was also a dose-dependent effect on cell growth; fusiform cells were affected at both doses, while giant cells were only affected at the highest dose. Granule cells form a major input to the fusiform cells and their depletion may account for some of the effects on fusiform cell growth. There could also be additional direct actions of α-difluoromethylornithine on this population.

高速攪拌型造粒機の造粒過程及び造粒終点

Granulation processes in high speed mixer granulator were divided into 4 zones to study the granulation mechanism in each zone. This study showed the importance of evaluating the granulation processes from the view of power consumption change and not from the view of granulation time. It was found that the granulation end-point at which a high yield of well-compacted, spherical small grains was obtained, corresponded to the early phase of zone IV during which the power consumption and granule temperature gained stability. Measurement of the granule temperature is, therefore, helpful for determining the granulation end-point. It was further found that the binder content where a high yield of small grains was obtained, resulted in production of well-compacted, spherical small grains. In granulation with a water-ethanol solution as a binder, the grain sphericity tended to become poorer and the small grains coarser as compared with granulation with purified water as a binder.

Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa

Purification of a bactericidal protein (BP) from the cytoplasmic granules of normal human polymorphonuclear leukocytes (PMN) with activity against Pseudomonas aeruginosa is described. Bactericidal activity from acid extracts of a mixed granule population was purified 175-fold by a two-step chromatographic procedure. The first step, dye-ligand affinity chromatography with Matrex-Gel Orange A, was followed by cation-exchange chromatography with Bio-Rex 70 resin, and this combination routinely gave a yield near 80%. Only one peak of bactericidal activity against P. aeruginosa type I was found after each chromatographic step. Characterization of BP showed a single band with an apparent molecular weight of 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified BP was most active against the six strains of P. aeruginosa tested and against Escherichia coli B (a deep rough mutant). Purified BP killed 5 X 10(6) CFU of P. aeruginosa type I per ml at a concentration of 60 to 80 ng/ml. Proteus mirabilis and Staphylococcus aureus were both resistant to the bactericidal activity of BP. BP was shown to be glycosylated by periodic acid staining after gel electrophoresis and to have an isoelectric point near 7.5 by chromatofocusing. The amino acid composition of BP is presented.

Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule.

A morphological study was carried out on five mixed GH- and PRL-secreting pituitary adenomas, surgically removed from acromegalic patients with hyperprolactinemia, in order to verify whether the two hormones were contained in the same cell or in different cells. Double labeling with the protein A-gold immunotechnique was used to visualize the ultrastructural localization of the two hormones on ultrathin sections of the tumors. By means of this high resolution technique we found in all adenomas the presence of numerous (from 50-80% of the whole cell population) mammosomatotrophs, i.e. cells containing simultaneously PRL and GH. The occurrence of cells producing only GH (in four tumors) or only PRL (in one tumor) was also observed. In mixed cells GH and PRL were segregated in the same mixed granule. In one tumor granules positive only for GH together with mixed granules were found in the same cell. Immunofluorescence studies, at the light microscopic level, allowed us to clearly identify mammosomatotrophs only in two tumors. Double labeling using the gold immunotechnique appears therefore to be the most suitable experimental approach to detect the existence of mixed cells in plurihormonal adenomas. Our results support the idea that the frequency of mixed adenomas with mixed cells may be higher than that believed previously. The simultaneous presence of two hormones in the same secretory granule could explain why, in patients having mixed tumors, factors able to stimulate or inhibit the release of one hormone can also stimulate or inhibit the secretion of the other.

Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells.

The isthmus of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice using light and electron microscopy as well as 3H-thymidine radioautography. On the average, the isthmus measured 25 microns in length and was composed of 36 isthmal cells and two enteroendocrine cells. Isthmal cells generally displayed features found in embryonic cells, such as many free ribosomes, scant organelles, and a large reticulated nucleolus, and were, therefore, at an immature stage of development. Isthmal cells could be devoid of secretory granules ("granule-free cells," 2%) or contain a few small, spherical, PA-Schiff-positive, mucous granules in their apex. The granules in some of the cells had a variegated appearance and a diameter averaging 235 nm ("mottled granule cells," 39%); in other cells, the granules had a large diameter, 278 nm, with a pale background and a dense core ("core granule cells," 28%); while in still others they were homogeneously dark and measured 264 nm ("dense granule cells," 12%). Finally, some cells included a mixture of core and dense granules ("mixed granule cells," 14%). One hour after a single injection of 3H-thymidine, 37% of the isthmal cells were labeled. Each of the five isthmal cell types could acquire label and, therefore, divide. After one or more days of continuous 3H-thymidine infusion, all isthmal cells were labeled. Their turnover time was estimated to be 16.1 hr (t1/2 = 11.2 hr). The isthmus is thus composed of several cell types which are turning over rapidly. While all are relatively immature, the various types are thought to represent different developmental stages in the life history of an isthmal cell. A model devised on this basis proposes that the granule-free cells are stem cells, from which mottled granule cells are derived. These in turn evolve into either the dense granule cells of the upper isthmus or the core granule cells of the lower isthmus, or into the mixed granule cells (which are believed to develop eventually into dense granule cells or core granule cells). Maintenance of a steady state requires that the rapid production of isthmal cells be associated with rapid emigration; the dense granule cells presumably going to the pit and the core granule cells to the gland. The turnover of isthmal cells is accordingly described as following a "bidirectional pattern" of renewal.

Tghe Presence of Common Precursor for Basophils and Eosinophils

AbstractWhen human bone marrow cells were incubated in the presence of medium conditioned by PHA‐stimulated leukocytes (PHALCM) in methylcellulose culture, small compact colonies grew. The compact colonies were identified to consist of eosinophils, using Wright and Giemsa staining. However, among som of the eosinophilic colonies, several mixed granule leukocytes containing both basophilic and eosinophilic granules (but not basophils) were identified using astra blue‐biebrich scarlet staining. When some of the eosinophilic colonies were transferred into liquid suspension culture containing PHALCM, the growth of mixed granule leukocytes and basophils was observed in the culture. These results show that basophils and eosinophils may have a common precursor.