MULTICOMPARTMENT SYSTEM OF NANOCAPSULE-IN-NANOCAPSULE TYPE, FOR ENCAPSULATION OF A LIPOPHILIC AND HYDROPHILIC COMPOUND, AND THE RELATED PRODUCTION METHOD
20210196630 · 2021-07-01
Inventors
Cpc classification
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
A multicompartment system of nanocapsule-in-nanocapsule type based on hyaluronic acid derivative is designed for encapsulation of peptides and/or hydrophobic active compounds, either simultaneously or separately, where surfactants, emulsifiers and/or stabilizers are not required for the system stability. The system functions as a carrier which enables protection of sensitive hydrophilic substances against aggressive external environment, and the resulting degradation and deactivation, and makes it possible to concurrently administer active substances of varied hydrophilicity. A method is provided of producing a multicompartment nanocapsule-in-nanocapsule system in the form of water-in-oil-in-water double emulsion.
Claims
1-12. (canceled)
13. A multicompartment system of nanocapsule-in-nanocapsule type, in a form of water-in-oil-in-water double emulsion, for concurrent delivery of hydrophilic and lipophilic compounds, the multicompartment system comprising: a) a liquid oil core for transport of a lipophilic compound, containing oil selected from the group including: oleic acid, isopropyl palmitate, fatty acids, natural extracts and oils, such as corn oil, linseed oil, soybean oil, argan oil, or their mixtures; beneficially oleic acid; b) a capsule or many capsules with aqueous core, embedded in an oil core, for transport of a hydrophilic compound; c) a stabilizing shell for both the capsule with oil core and the inner capsule with water core, consisting of a hydrophobically modified polysaccharide selected from a group comprising: derivatives of chitosan, oligochitosan, dextran, carrageenan, amylose, starch, hydroxypropyl cellulose, pullulan and glycosaminoglycans, hyaluronic acid, heparin sulfate, keratan sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate; beneficially derivatives of hyaluronic acid; d) outer capsule with a diameter below 1 μm, stable in aqueous solution; and e) active substance.
14. The multicompartment system of claim 13, wherein a degree of hydrophobic side chains substitution in a hydrophobically modified polysaccharide ranges from 0.1 to 40%.
15. The multicompartment system of claim 13, wherein the stabilizing shells for the capsule with oil core and the capsule with water core (inner capsule) consist of hydrophobically modified sodium hyaluronate, Hy-Cx, with a formula: ##STR00003##
16. The multicompartment system of claim 13, wherein the transported lipophilic compound may be a fluorescent dye, fat-soluble vitamin, or hydrophobic drug.
17. The multicompartment system of claim 13, wherein the transported hydrophilic compound may be a fluorescent dye, water-soluble vitamin, protein or hydrophilic drug; advantageously: insulin.
18. The multicompartment system of claim 17, wherein insulin is in a concentration of 0.005-20.000 of insulin units per 1 ml of the capsule suspension.
19. A method of producing a multicompartment system of nanocapsule-in-nanocapsule type, in the form of water-in-oil-in-water double emulsion, as defined in claim 13, the method comprising: a) during the first step invert emulsion of water-in-oil (W/O) type is produced by mixing an aqueous solution of hyaluronic acid dodecyl derivative Hy-Cx, described by the above formula, with a non-toxic oil constituting about 0.1-99.9% of the mixture volume, by exposition to ultrasounds (sonication) or to mechanical stimuli, advantageously—mixing or shaking, with aqueous phase to oil phase volume ratio ranging from 1:10 to 1:10000; advantageously approx. 1:100; b) during the second step, water droplets suspended in the continuous oil phase receive hyaluronate coating, with W/O phase emulsion to aqueous phase volume ratio ranging from 1:10 to 1:10000; advantageously approx. 1:100; and c) as a result, the water-in-oil-in-water (W/O/W) double emulsion system is produced by exposition to ultrasounds (sonication) or to mechanical stimuli, advantageously—mixing or shaking, wherein, the aqueous phase applied is based on aqueous solution of hydrophobically modified polysaccharide selected from a group comprising: derivatives of chitosan, oligochitosan, dextran, carrageenan, amylose, starch, hydroxypropyl cellulose, pullulan and glycosaminoglycans, and particularly hyaluronic acid, heparin sulfate, keratan sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate; advantageously derivatives of hyaluronic acid with pH in the range of 2-12, concentration of 0.1-30 g/L and ionic strength in the range of 0.001-3 mol/dm.sup.3, and the oil phase contains oil selected from the group including: oleic acid, isopropyl palmitate, fatty acids, natural oils, in particular linseed oil, soybean oil, argan oil, or their mixtures; beneficially oleic acid, notably, the process is carried out without using any small-particle surfactants.
20. The method of claim 19, wherein pulsed sonication is carried out with impulse duration twice as short as the duration of the interval between two consecutive impulses.
21. The method of claim 19, wherein the encapsulated lipophilic compound is contained in the oil core and the encapsulated hydrophilic compound is comprised in the water core of the nanocapsule.
22. The method of claim 19, wherein the content of ionic groups in the polysaccharide is not lower than 20 mol %, and is greater than 60 mol-% (calculated per one mer).
23. The method of claim 19, wherein during the first and second step, sonication is continued for 15-60 minutes, at a temperature of 18° C.-40° C., for at least 60 min to obtain invert emulsion, and at least 30 min to obtain double emulsion, at a temperature of 25-30° C.
24. Application of the multicompartment system of claim 13, for transport of lipophilic compounds and hydrophilic compounds, where the lipophilic compound may be a fluorescent dye, fat-soluble vitamin, or a hydrophobic drug, while the hydrophilic compound may be a fluorescent dye, water-soluble vitamin, protein or a hydrophilic drug.
Description
DESCRIPTION OF THE TABLES AND FIGURES
[0058] The object of the invention is shown in the examples and figures, listed below:
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[0090] The invention is illustrated by the following non-limiting examples
EXAMPLE I
Method of Making Inverted Emulsion of Water-in-Oil Type
[0091] In order to produce inverted emulsion (W-O type), water-ethanol solution of hyaluronic acid dodecyl derivative was applied. The presence of the volatile organic solvent was to enable polymer chains to achieve extended conformation (to produce the inverted emulsion). The solvent subsequently was to be evaporated.
[0092] Solution of hyaluronic acid dodecyl derivative (degree of hydrophobic side chains substitution from 4.5%) was prepared in physiological saline (concentration approx. 7.5 g/L). The neutral solution was then ethanolized and a mixture with 2:3 volume ratio was obtained.
[0093] Concurrently a pre-emulsion was prepared by mixing oleic acid with aqueous solution of sodium chloride (c=0.15 mol/dm.sub.3), at volume ratio of 100:1. The system was subjected to shaking for 10 minutes in a vortex type shaker, and subjected to sonication for 30 minutes in an ultrasonic cleaner (pulsed mode, 1 s ultrasounds, 2 s interval) in room temperature. As a result of sonication, a milk-white emulsion was produced.
[0094] Water-ethanol solution of hyaluronic acid dodecyl derivative was gradually added drop by drop to the pre-emulsion, for 5 minutes. The whole mixture was subjected to sonication for 30 min in pulsed mode, in an open bottle, in order to evaporate the ethanol.
[0095] Size distributions measured using dynamic light scattering (DLS) show that the system contained many molecular fractions. It was impossible to measure zeta potential (ξ) indicating stability of the system (highly unstable measurements). Furthermore, the bottle contained visible spherical bubbles with diameters exceeding 1 mm (
EXAMPLE II
Method of Making Inverted Emulsion of Water-in-Oil Type, after Decreasing the Content of Aqueous Phase in the Water-Ethanol Solution
[0096] Pre-emulsion was prepared as described in Example I. Water-ethanol solution of hyaluronic acid dodecyl derivative was added gradually, however aqueous phase to ethanol phase volume ratio of 1:2 was applied.
[0097] In order to evaporate the ethanol, the system was subjected to sonication at a higher temperature (about 34° C.).
[0098] Initially white suspension could be seen in the oil; after the system was introduced into the cuvette used in DLS measurements, the suspension transformed into bubbles with diameters exceeding 1 mm (
[0099] After the sizes were measured in DLS apparatus, 2 large water drops were observed in the cuvette. Zeta potential could not be measured
[0100] Based on the results presented in Examples I and II, it was concluded that ethanol adversely affected production of the emulsion; at the next step alcohol was eliminated from the system.
EXAMPLE III
Method of Making Inverted Emulsion of Water-in-Oil Type, after Eliminating Alcohol from the System
[0101] Inverted emulsion of water-in-oil type was prepared by mixing a solution of hyaluronic acid dodecyl derivative (c=4.7 g/L) in physiological saline (c.sub.NaCl=0.15 mol/dm.sup.3) with oleic acid, at a volume ratio of 1:100. The system was subjected to shaking and sonication, as described in Example I, however sonication process continued for one hour.
[0102] A milk-white emulsion was obtained, and its stability was measured on the day and five days after the emulsification. The DLS tests showed high stability of the initial system (ξ=−33±21.7 mV). The molecular sizes were characterized by narrow distribution. After five days, the distribution describing molecule sizes shifted towards smaller molecules; additionally, another small maximum could be observed. After five days there was a significant decrease in the turbidity of the sample (
EXAMPLE IV
Inverted Emulsion Imaging with Cryoscopic Transmission Electron Microscopy
[0103] Inverted emulsion was prepared following the procedure described in Example III, however the inner aqueous phase contained sodium tungstate (VI), in order to enhance contrast during the imaging examination. Two days later the emulsion was examined using transmission electron microscopy technique, supplemented with cryoscopy device. Analysis of the acquired images confirms presence of spherical molecules with a diameter of approx. 250 nm (
EXAMPLE V
Method of Making Double Emulsion
[0104] Inverted emulsion was prepared as in Example III, however dodecyl derivative of fluorescein isothiocyanate (FITC) labeled hyaluronic acid was applied at a concentration of 2 g/L, and sonication continued for 30 minutes.
[0105] Double emulsion was obtained by mixing inverted emulsion constituting 0.4% volume of the mixture with dodecyl derivative of rhodamine isothiocyanate (RhBITC) labeled hyaluronic acid at a concentration of 1 g/L in physiological saline. The system was subjected to shaking for 10 minutes in a vortex type shaker, and subjected to sonication in room temperature for 30 minutes, in accordance with the parameters described in Example I. Analysis of molecule-size distributions in DLS tests shows there are molecules with diameters of 500-600 nm, while zeta potential measurement confirms stability of the obtained system (ξ=−44.6±3.33 mV). After seven days of observations no significant changes were shown in molecule sizes or the value of zeta potential (ξ=−44.6±3.08 mV) (
EXAMPLE VI
Double Emulsion Imaging with Confocal Microscopy
[0106] Labeled polysaccharides were applied to visualize the structures obtained in Example V, using confocal microscopy. Because of the spectral characteristics both dyes can be excited with lasers of varied wavelength (488 nm and 561 nm), and emissions can be observed in other microscope channels. It was shown that FITC is not excited by the laser corresponding to RhB (and vice versa); RhB signal was not observed in FITC channel, and FITC signal was not identified in the channel corresponding to rhodamine emission.
[0107] By applying the derivative containing FITC in the first W-O type emulsion, and the derivative containing RhBITC at the second stage to produce double emulsion, it was possible to visualize the obtained structures and confirm their morphology.
[0108] Images from confocal microscope (100× lens, 488 nm and 561 nm lasers) confirm presence of a “layered” sheath—observation of signal from all the channels and the channel characteristic for FITC (
EXAMPLE VII
Double Emulsion Imaging with Cryoscopic Transmission Electron Microscopy
[0109] Double emulsion was prepared following the procedure described in Example V, however the inner aqueous phase contained sodium tungstate (VI), in order to enhance contrast during the imaging examination. After two days a sample was examined using transmission electron microscopy technique, and cryoscopy device. Analysis of the acquired images confirms presence of spherical molecules with a diameter of approx. 600 nm (
EXAMPLE VIII
Encapsulation of Hydrophilic Dye in the Inner Aqueous Phase
[0110] Double emulsion was prepared as described in Example V, however inverted emulsion was prepared from water solution of hyaluronic acid dodecyl derivative with concentration of 4.5 g/L in physiological saline mixed with calcein solution (c.sub.kalc=2 g/L) at 3:1 volume ratio. Analysis of molecule sizes based on results of DLS measurements confirmed the formulation obtained was stable (ξ=−32.5±6.58 mV) and contained molecules with hydrodynamic diameters of approx. 600 nm (
[0111] Confocal microscopy images (observation in all the channels) confirm that a nanocapsule-in-nanocapsule system was obtained, which is shown by a signal visible in both channels, and overlapping within the molecules observed (
PRZYKŁAD IX
Optimization of Double Emulsion Composition
[0112] In order to optimize the sizes and composition of the obtained system, a change was introduced in the volume ratio of aqueous and oil phase in the inverted emulsion, which was made as described in Example VIII, with aqueous phase to oil phase volume ratio of 30:1. Double emulsion was obtained by mixing the inverted emulsion and dodecyl derivative of rhodamine isothiocyanate labeled hyaluronic acid with a concentration of 1 g/L. The content of the inverted emulsion in the mixture amounted to 0.1% volume. Sonication was conducted as described in Example V. The obtained system was characterized by narrow distribution of molecule sizes (
EXAMPLE X
Long-Term Stability of Double Emulsion
[0113] Stability of the water-in-oil-in-water double emulsion produced using hyaluronic acid dodecyl derivative was tested over a period of 11 weeks. The parameters of the system were examined in specified points of time using dynamic light scattering technique and confocal microscopy. The capsules were produced as described in Example IX.
[0114] The obtained system was characterized by monomodal molecule size distribution (
EXAMPLE XI
Preparation and Visualization of Double Emulsion Containing Dissolved Fluorescent Dyes
[0115] Inverted emulsion was made by mixing oleic acid with solution of hyaluronic acid dodecyl derivative, in physiological saline, as described in Example IX, with Nile Red dye dissolved in the oil phase (c=0.85 g/L), and calcein dissolved in the aqueous phase (c=0.17 g/L). Double emulsion was produced as described in Example IX.
[0116] The obtained molecules were characterized by hydrodynamic diameter similar to that in the molecules formed in Example X (
[0117] Visualization performed using confocal microscope showed that a nanocapsule-in-nanocapsule system was formed (overlapping signal from both fluorescence channels) (
EXAMPLE XII
1) Preparation of Insulin Solution:
[0118] 21.66 mg of insulin (Sigma Aldrich) was dissolved in 1 ml 0.15M NaCl (addition of 4 μl 3M HCl, pH ˜1.9), i.e. approx. 600 UI/ml (3.56 mg=100 UI)*.
[0119] The process produced clear insulin solution which retained the lucid form when stored at a temperature of 4° C. (two-week observations).
[0120] Subsequently, insulin solution was prepared with an addition of a dye, i.e. Neutral Red (C=1 g/l in 0.15M NaCl) (180 μl insulin solution+20 μl dye solution).
[0121] No negative effect of the dye added to insulin solution was observed.
2) Preparation of Capsules
[0122] a) Emulsion 1:
[0123] In accordance with the procedures described above in this invention, Emulsion 1 was obtained following the formula: 3.6 ml of oleic acid was emulsified with 100 μl of HyC12 solution (C=4.6 g/l in 0.15M NaCl) and 20 μl of insulin solution with a dye; the process was carried using Vortex-type shaker (10 min) and ultrasounds (pulsed mode, 30 min).
[0124] b) Emulsion 2:
[0125] Emulsion 2 was made from 6 ml of HyC12 solution (C=1 g/l in 0.15M NaCl) and 12 μl of Emulsion 1. The mixture was emulsified using Vortex shaker (10 min) and ultrasounds (30 min, pulsed mode).
[0126] Milk-white emulsion was obtained.
[0127] 1 ml of the capsules contained 0.01 μl of insulin solution, i.e. 0.0061 units of insulin per 1 ml of the capsules.
3) Characteristics:
[0128] The obtained W/O/W emulsion consisted of suspended molecules with hydrodynamic diameter of up to 180 nm. It was highly stable, as shown by the high value of zeta potential. The capsules were stored at a temperature of 4° C. After one week a small outflow of the oil phase to the surface was observed along with dilution of the emulsion. Measurements performed using dynamic light scattering (DLS) technique showed a slightly reduced modular value of zeta potential and a decrease in the molecule sizes. The results are presented in Table 1 and in
TABLE-US-00001 TABLE 1 Summary measures of hydrodynamic diameters (volume means) and zeta potentials in the W/O/W system, on the day as well as one and two weeks after the emulsion was produced. dv [nm] Zeta potential [mV] Time [week] [Diss. 100x] [Diss. 100x] 0 173 ± 6 −45 ± 3 1 165 ± 14 −37 ± 1 2 165 ± 11 −38 ± 4
EXAMPLE XIII
1) Preparation of Insulin Solution.
[0129] The insulin solution from Example 1 was condensed with additional solution of 49.73 mg of insulin, and acidified with an addition of 6 μl of muriatic acid (C=3 mol/dm.sup.3) in order to obtain a clear solution, which was then subjected to shaking in Vortex shaker for 5 min
[0130] The obtained insulin had a concentration of 81.34 mg/ml (2284.75 UI).
[0131] The first component of Emulsion 1 was prepared by mixing 30 μl of HyC12 solution (C=15 g/l in 0.15M NaCl) with 80 μl of insulin solution and 10 μl of the dye (Neutral Red, C=3.5 mg/ml in 0.15M NaCl).
Emulsion 1:
[0132] A mixture of 120 μl of the first component of Emulsion 1 and 3.6 ml of oleic acid was subjected to shaking in Vortex shaker for 10 min, and then to sonication in pulsed mode, for 30 min
Emulsion 2:
[0133] A mixture of 20 μl of Emulsion 1 and 2 ml of HyC12 solution (C=5 mg/ml in 0.15M NaCl) was subjected to shaking in Vortex shaker for 10 min, and then to sonication in pulsed mode, for 30 min. The obtained milky, viscous and very dense emulsion contained 0.49 units of insulin per 1 ml.
Characteristics:
[0134] The obtained capsules were characterized by good stability, reflected by the high values of zeta potentials. The encapsulated dye also influenced these high values. The capsules were stored at a temperature of 4° C. After one and two weeks the emulsion retained its stability. Following one week (and later) measurements of hydrodynamic diameters, high dispersion indicator, and confocal microscopy show that aggregates and larger structures are formed, and there is no evidence of monodispersity in the sample.
[0135] For the purpose of the measurements the capsules were diluted (100×) with 0.15M NaCl solution. The results are shown in Table 2 and
TABLE-US-00002 TABLE 2 Summary measures of hydrodynamic diameters (volume means) and zeta potentials in the W/O/W system, on the day as well as one, two and three weeks after the emulsion was produced. dv [nm] Zeta potential [mV] Time [week] [Diss. 100x] [Diss. 100x] Day 1 313 ± 51 −59 ± 0 1 883 ± 265 −53 ± 2 2 1062 ± 178 −51 ± 3 3 668 ± 40 −48 ± 2
EXAMPLE XIV
Emulsion 1: Produced Following the Procedure Described in Example 2
Emulsion 2:
[0136] 10 μl of Emulsion 1 and 2 ml HyC12 (C=2.5 mg/ml; 0.15M NaCl) were subjected to shaking in Vortex shaker for 10 min and then to sonication in pulsed mode for 30 min.
[0137] The obtained milky and viscous emulsion contained 0.245 units of insulin per 1 ml.
Characteristics:
[0138] The obtained capsules were characterized by good stability, shown by the high values of zeta potentials. The encapsulated dye also influenced these high values. The capsules were stored at a temperature of 4° C.
[0139] After one week the emulsion retained its stability. The low PDI values reflect monodispersity of the samples and a lack of tendency for aggregation.
[0140] For the purpose of the measurements the capsules were diluted (100×) with 0.15M NaCl solution. The results are listed in Table 3 and
TABLE-US-00003 TABLE 3 Summary measures of hydrodynamic diameters (volume means) and zeta potentials in the W/O/W system, on the day and one week after the emulsion was produced. dv [nm] Zeta potential [mV] Time [week] [Diss. 100x] [Diss. 100x] Day 1 339 ± 32 −51 ± 2 1 437 ± 26 −43 ± 2
EXAMPLE XV
[0141] Preparation of insulin solution: following the procedure described in Example 2.
[0142] The first component of Emulsion 1 was prepared by mixing 60 μl of HyC12 solution (C=7.5 mg/ml in 0.15M NaCl) with 50 μl of insulin solution and 10 μl of the dye (Neutral Red C=3.5 mg/ml in 0.15M NaCl).
Emulsion 1:
[0143] A mixture of 120 μl of the first component of Emulsion 1 and 3.6 ml of oleic acid was subjected to shaking in Vortex shaker for 10 min, and then to sonication in pulsed mode, for 30 min
Emulsion 2:
[0144] A mixture of 10 μl of Emulsion 1 and 2 ml of HyC12 solution (C=2.5 mg/ml in 0.15M NaCl) was subjected to shaking in Vortex shaker for 10 min, and then to sonication in pulsed mode, for 30 min. The obtained milky, viscous and very dense emulsion contained 0.154 units of insulin per 1 ml.
Characteristics:
[0145] The obtained capsules were characterized by good stability, reflected by the high values of zeta potentials. The encapsulated dye also influenced these high values. The capsules were stored at a temperature of 4° C.
[0146] After one week the emulsion retained its stability. The obtained distributions of hydrodynamic diameters show that initially there were aggregates which disintegrated after one week. For the purpose of the measurements the capsules were diluted (100×) with 0.15M NaCl solution. The results are shown in Table 4 and
TABLE-US-00004 TABLE 4 Summary measures of hydrodynamic diameters (volume means) and zeta potentials in the W/O/W system, on the day and one week after the emulsion was produced. dv [nm] Zeta potential [mV] Time [week] [Diss. 100x] [Diss. 100x] Day 1 615 ± 66 −50 ± 1 1 476 ± 28 −45 ± 2
EXAMPLE XVI
1) Preparation of Insulin Solution.
[0147] The insulin solution obtained in Example 4 was condensed by adding 94 mg of insulin, and acidified with 4 μl 3M of muriatic acid in order to obtain a clear solution, which was subsequently subjected to shaking in Vortex shaker for 5 min.
[0148] The obtained insulin solution had a concentration of 200 mg/ml (5617.98 UI).
[0149] The first component of Emulsion 1 was prepared by mixing 20 μl of HyC12 solution (C=7.5 mg/ml; 0.15M NaCl) with 100 μl of insulin solution
Emulsion 1:
[0150] A mixture of 120 μl of the first component of Emulsion 1 and 3.6 ml of oleic acid was subjected to shaking in Vortex shaker for 10 min, and then to sonication in pulsed mode, for 30 min.
Emulsion 2:
[0151] A mixture of 10 μl of Emulsion 1 and 1 ml of HyC12 solution (C=1.5 mg/ml in 0.15M NaCl) was subjected to shaking in Vortex shaker for 20 min, and then to sonication in pulsed mode, for 35 min.
[0152] The obtained milky, viscous and very dense emulsion contained 1.5 units of insulin per 1 ml.
Characteristics:
[0153] The obtained capsules were characterized by good stability, which was shown by the high values of zeta potentials. The capsules were stored at a temperature of 4° C. After one week the emulsion retained its stability. The distribution of hydrodynamic diameter sizes is narrow.
[0154] For the purpose of the measurements, the capsules were diluted (100×) with 0.15M NaCl solution. The results are presented in Table 5 and
TABLE-US-00005 TABLE 5 Summary measures of hydrodynamic diameters (volume means) and zeta potentials in the W/O/W system, on the day and one week after the emulsion was produced. dv [nm] Zeta potential [mV] Time [week] [Diss. 100x] [Diss. 100x] Day 1 276 ± 17 −39 ± 3 1 350 ± 13 −46 ± 4
[0155] *3.56 mg=100 UI [© 2011, “Drug Discovery and Evaluation: Methods in Clinical Pharmacology”, Editors: Vogel, H. Gerhard, Maas, Jochen, Gebauer, Alexander]
EXAMPLE XVII
Inducing Type 1 Diabetes
[0156] A group of 30 male Wistar rats, ranging in mass from 180 to 200 g, were anesthetized with thiopental (50 mg/kg of body mass); subsequently streptozotocin (STZ) dissolved in phosphate buffer was injected via tail vein, at the rate of 60 mg/kg of body mass. The final volume of the injected solution amounted to 1 ml/kg of body mass. Blood glucose was measured three days after streptozotocin injection. Each of the animals was found with blood glucose level exceeding 450 mg % which reflected the fact that insulin-producing β cells in the pancreas were damaged. During this time the animals had unlimited access to fodder and water.
Assessment of Encapsulated Insulin Activity
[0157] Twelve hours before the glucose tolerance test, the rats were divided into five groups of six animals (a total of 30 animals), with fodder no longer available. The animals continued to have unlimited access to water. The experiment was conducted in the following groups: [0158] 1. Control group: 2 g of glucose per 1 kg of body mass, administered via a gastric tube. [0159] 2. Insulin group: 7.5 units per 1 kilogram and 2 g of glucose per kg of body mass, administered concurrently via a gastric tube. [0160] 3. Control group: 0.5 g of glucose per 1 kg of body mass, administered via a gastric tube. [0161] 4. Insulin group: 11.25 units per one kilogram delivered 20 minutes prior to the administration of 0.5 g of glucose per 1 kg of body mass via a gastric tube. [0162] 5. Insulin group: 11.25 units per 1 kilogram and 0.5 g of glucose per kg of body mass, administered concurrently via a gastric tube.
[0163] Insulin was administered in an encapsulated form in W/O/W system obtained following the procedure described in Example 5.
[0164] In each group glucose levels were measured in blood samples collected from tail veins, at the following points of time: 0; 15; 30; 45; 60; 75; 90; 105; 120 (and 135 in groups 1 and 2). Glucose measurements were conducted using Bionime Rightest® GM100 glucose meter.
[0165] The results of glucose level measurements are shown in Tables 6-10 and in
TABLE-US-00006 TABLE 6 List of results of glucose level measurements in Group 1, expressed in mg/dl - glucose 2 g/kg only. Time [min] Mass Glucose concentration [mg/dl] Lp. [g] 0 15 30 45 60 75 90 105 120 135 1 160 361 481 600 600 600 544 550 494 481 458 2 163 242 522 600 600 600 600 548 515 431 423 3 152 188 355 493 516 564 558 500 521 481 445 4 174 165 350 520 600 600 600 578 516 426 406 5 178 153 331 436 524 537 512 492 460 416 358 6 178 138 267 424 476 485 457 357 306 258 185 Lp. = No. Czas [min] = Time [min] Waga [g] = Weight [g] Stężenie glukozy [mg/dl] = Glucose concentration [mg/dl]
TABLE-US-00007 TABLE 7 List of results of glucose level measurements in Group 2 - insulin (7.5 u/kg) and glucose (2 g/kg) concurrently. Time [min] Mass Glucose concentration [mg/dl] Lp. [g] 0 15 30 45 60 75 90 105 120 135 1 167 417 600 600 600 562 517 521 464 436 419 2 146 238 426 530 600 564 536 494 495 454 460 3 161 208 470 547 563 530 496 473 495 465 417 4 164 155 337 455 519 513 461 451 441 442 428 5 167 141 419 527 527 497 472 480 427 434 384 6 163 145 259 421 600 465 396 376 353 357 324
TABLE-US-00008 TABLE 8 List of results of glucose level measurements in Group 3 - glucose 0.5 g/kg only. Time [min] Mass Glucose concentration [mg/dl] Lp. [g] 0 15 30 45 60 75 90 105 120 1 175 382 569 495 493 495 457 456 415 434 2 190 155 270 265 289 260 255 222 212 203 3 166 141 311 317 295 283 274 269 283 263 4 178 98 208 215 208 186 187 177 161 152 5 175 98 219 262 255 223 182 170 145 122 6 184 80 148 190 174 167 141 121 109 93
TABLE-US-00009 TABLE 9 List of results of glucose level measurements in Group 4 - insulin (11.25 u/kg) 20 minutes before glucose (0.5 g/kg) Time [min] Mass Glucose concentration [mg/dl] Lp. [g] 0 15 30 45 60 75 90 105 120 1 180 104 148 145 131 129 112 102 100 92 2 185 100 187 197 181 193 191 196 173 157 3 185 120 219 250 254 258 250 234 237 229 4 182 275 333 337 336 351 350 332 335 304 5 179 91 163 209 191 173 150 137 129 117 6 179 90 158 137 122 109 95 86 79 85
TABLE-US-00010 TABLE 10 List of results of glucose level measurements in Group 5 - insulin (11.25 u/kg) and glucose (0.5 g/kg) concurrently Time [min] Mass Glucose concentration [mg/dl] Lp. [g] 0 15 30 45 60 75 90 105 120 1 180 341 472 452 424 402 403 380 369 333 2 180 226 301 357 345 347 367 332 337 330 3 167 110 209 189 189 169 156 144 122 122 4 166 100 209 216 238 215 194 186 176 179 5 175 97 171 190 194 194 175 164 158 147 6 190 83 147 174 166 153 140 119 125 115
[0166] Based on the measurements, the surface area below the glucose curve was calculated. Mean value was computed for each group and compared to the relevant control group, whereby the percent proportion was calculated in relation to the control group, i.e. Group 2 to Control Group 1, and Groups 4 and 5 to Control Group 3 (Table 11).
TABLE-US-00011 TABLE 11 Results of the measurements of surface areas below the glucose curve for Groups 2, 4 and 5 (fields P2, P4, P5) by reference to the relevant control group (P1 and P3). Percent change in the surface below the glucose curve (%) Group 2 Group 4 Group 5 (P2/P1).sup.a (P4/P3).sup.a (P5/P3).sup.a 84.8 61.0 76.2 .sup.arelates to surface areas below glucose curves in Groups 1-5.
Final Conclusions:
[0167] 1. The findings show positive effect produced by encapsulated insulin in the glucose curve in animals with streptozotocin-induced type 1 diabetes. [0168] 2. The observed effect was more visible in the case of lower glucose dose which suggests a necessity to increase the number of units of insulin in the formulation. [0169] 3. More beneficial effect is produced by administration of encapsulated insulin 20 minutes before glucose administration.