Absorption Enhancement of Intranasally Administered Insulin by Sodium Taurodihydrofusidate (STDHF) in Rabbits and Rats

The enhancement of nasal insulin absorption by sodium taurodihydrofusidate (STDHF) was studied in rabbits and rats. Using identical nasal formulations remarkable interspecies differences were observed. The fusidate derivative at 1% (w/v) enhanced nasal insulin bioavailability from 0.9 to 5.2% and from 0.3 to 18.0% in rabbits and rats, respectively. In both species the insulin formulations with STDHF resulted in strong hypoglycemic responses. Coadministration with the trypsin inhibitor aprotinin tended further to increase insulin bioavailability in rats and decrease insulin bioavailability in rabbits; however, these aprotinin effects were not statistically significant. Addition of the aminopeptidase inhibitor bacitracin to the STDHF containing formulation did not have any effect on insulin bioavailability in rats. Hence, STDHF is a potent enhancer of nasal insulin absorption, probably both by facilitating insulin transport through the nasal mucosa and possibly also by inhibiting enzymatic degradation. Further, interspecies differences and, experimental animal conditions can greatly affect nasal drug absorption.     

Nasal Absorption in the Rat. III. Effect of Lysophospholipids on Insulin Absorption and Nasal Histology

The intranasal absorption enhancing and histological effects of a range of lysophospholipids has been investigated in the rat. Blood glucose levels fell rapidly following the administration of insulin (8 IU/kg) in combination with lysophosphatidylcholines (LPC; 0.625% w/v) which had ten or more carbon groups in their fatty acid chain. The effect of the LPC-caproyl (C6) was comparable to that of an unenhanced insulin formulation; the enhancing effect of LPC-decanoyl (C10) was similar to that of an LPC-palmitoyl/stearoyl (C16/C18) for similar concentrations. The effect of LPC-decanoyl was reduced with concentration but was still significant at 0.2% w/v (5mM). Lysophosphatidylglycerol (LPG) had a marked insulin absorption enhancing effect even at 0.0625% w/v. The histological effects of LPC-caproyl were similar to those of an unenhanced insulin formulation, while co-administration of LPC-decanoyl resulted in evidence of epithelial interaction. LPG (0.5% w/v) resulted in similar histological changes as LPC (0.625% w/v) (1), but at 0.0625% w/v no significant changes in epithelial integrity were observed. The length of the fatty acid residue of lysophospholipids was identified as an important factor for intranasal absorption enhancing activity. The nature of the polar head group may also have an influence. Increased insulin absorption was not necessarily accompanied by severe disruption of the nasal epithelium. Careful selection of lysophospholipid type and concentration may enable therapeutic drug levels to be achieved via the nasal route without prohibitive toxic effects.     

Minimization of Shaking-induced Formation of Insoluble Aggregates of Insulin by Cyclodextrins

Abstract

Aggregation is known to complicate insulin delivery and the processing and formulation of biotechnology-derived peptide/protein drugs. Shaking-induced formation of insoluble aggregates in bovine insulin and the potential role of cyclodextrins in preventing such aggregation were investigated. Insulin, dissolved in phosphate buffer, pH 7.2, and preserved with 2 mg/ml of phenol was aggregated, in triplicate, by shaking at 450 rpm for 2.5 days on a gyrotory shaker. Visible aggregation was quantitated by measuring optical density in the visible range on a spectrophotometer. Solutions were then filtered through a 0.22 μ filter and the amount of insulin remaining in filtrate was determined by HPLC. Aggregation increased at lower concentrations, with solutions turning milky at 0.5 mg/ml; HPLC assay of filtrate indicated a complete loss of insulin. Under the same conditions, except for shaking, control solutions exhibited no insulin loss, excluding absorption as a cause of the insulin loss. The use of cyclodextrins (0.5 mg/ml) to stabilize insulin was investigated, α-, β-, γ- and hydroxypropyl-β-cyclodextrin, each at 1.5% level, were used to prevent aggregation. The efficacy of cyclodextrins in preventing aggregation (% insulin aggregated in parentheses), was: hydroxypropyl-β- (15) ～ β- (18) > α- (54). No protection was observed with γ-cyclodextrin.     

Cyclodextrins as Nasal Absorption Promoters of Insulin: Mechanistic Evaluations

The safety and effectiveness of cyclodextrins (CD) as nasal absorption promoters of peptide-like macromolecules have been investigated. The relative effectiveness of the cyclodextrins in enhancing insulin nasal absorption was found to be in the descending order of dimethyl--cyclodextrin (DMCD) > -cyclodextrin (-CD) > -cyclodextrin (-CD), hydroxypropyl--cyclodextrin (HPCD) > -cyclodextrin (-CD). A direct relationship linking absorption promotion to nasal membrane protein release is evident, which in turn correlates well with nasal membrane phospholipid release. The magnitude of the membrane damaging effects determined by the membrane protein or phospholipid release may provide an accurate, simple, and useful marker for predicting safety of the absorption enhancers. In order to estimate further the magnitude of damage and specificity of cyclodextrin derivatives in solubilizing nasal membrane components, the enzymatic activities of membrane-bound 5-nucleotidase (5-ND) and intracellular lactate dehydrogenase (LDH) in the perfusates were also measured. HPCD at a 5% concentration was found to result in only minimal removal of epithelial membrane proteins as evidenced by a slight increase in 5-ND and total absence of LDH activity. On the other hand, 5% DMCD caused extensive removal of the membrane-bound 5-ND. Moreover, intracellular LDH activity in the perfusate increased almost linearly with time. The cyclodextrins are also capable of dissociating insulin hexamers into smaller aggregates, and this dissociation depends on cyclodextrin structure and concentration. Enhancement of insulin diffusivity across nasal membrane through dissociation may provide an additional mechanism for cyclodextrin promotion of nasal insulin absorption.     

Insulin Containing Nanocomplexes Formed by Self-Assembly from Biodegradable Amine-Modified Poly(Vinyl Alcohol)-Graft-Poly(<Emphasis Type="SmallCaps">l</Emphasis>-Lactide): Bioavailability and Nasal Tolerability in Rats

Purpose The bioavailability and local tolerability of insulin containing nanocomplexes from amine-modified poly(vinyl alcohol)-graft-poly(l-lactide) were studied in rats. Histology of the nasal epithelium was studied to document integrity of the mucosa. Methods Nanocomplexes (NC) were prepared by spontaneous self-assembly of insulin and the water-soluble amphiphilic polymer. Changes in blood glucose and insulin blood concentration were monitored in anesthetized rats using a glucose meter and enzyme-linked immunosorbent assay, respectively. Histological sections of the nasal cavity were examined after H&E staining by light microscopy. Results NC reduced blood glucose level in fasted healthy rats by 20% after 50–80 min and in streptozotocin induced diabetic rats by 30% within 75–95 min compared to basal levels. In both animal models significant concentrations of human insulin were detected, with relative bioavailabilities F_rel of 2.8 up to 8.3%. The more hydrophobic, lactic acid grafted polyester were more effective at a threefold higher polymer concentration, increasing the relative bioavailability F_rel of a 5 IU/kg dose from 2.8 to 5.7%. Histological examination of the nasal mucosa after 4 h showed no signs of toxicity at the site of nasal administration. Conclusions These results demonstrate that the NC significantly enhanced insulin absorption, suggesting that amphiphilic biodegradable comb-polymers offer a promising approach for nasal peptide delivery.     

Tolerability and Absorption Enhancement of Intranasally Administered Octreotide by Sodium Taurodihydrofusidate in Healthy Subjects

Nasal sprays containing different concentrations of the somatostatin analogue octreotide and sodium tauro-24,25-dihydrofusidate (STDHF) as an absorption promoter were evaluated in two consecutive pharmacokinetic studies in healthy volunteers to characterize their bioavailability and local tolerability. The concentrations of STDHF were selected on the basis of a phase diagram generated by a dynamic laser light-scattering technique to ensure that the mixture was above the critical micellar concentrations. Compared to a 50-µg subcutaneous injection, the nasal spray formulation without STDHF had a mean relative bioavailability of 17.9%. For nasal formulations containing 3 and 1.65% (w/v) of STDHF, the bioavailability increased to 29.0 and 25.7%, respectively. The enhancement of nasal absorption was dependent on the STDHF concentrations as shown by decreasing the amounts to 1.2 and 0.8% (w/v) for tolerability reasons; the bioavailability was reduced to 15.3 and 20.5% in these cases, respectively. The local tolerability of all STDHF-containing sprays was poor, leading to stinging sensations and lacrimation. The poor local tolerability of the octreotide nasal spray containing different concentrations of STDHF required for effective nasal absorption enhancement appears to be impractical for further clinical development. These findings clearly stress the necessity to investigate tolerability and safety issues of new drug delivery systems in early developmental phases.     

Absorption Enhancing Effect of Labrasol on the Intestinal Absorption of Insulin in Rats

The oral absorption enhancing effect of Labrasol? has been studied in rats using insulin as a model peptide/protein drug. Insulin solution was prepared by dissolving insulin in pH 7.4 buffer followed by the addition of Labrasol. The insulin concentration was 50.0 IU/ml. The test insulin/Labrasol solution was administered to the jejunum, ileum and ascending colon of rats at 10.0 IU/kg. After administration, blood samples were collected for 5 h and serum glucose levels and insulin levels were measured. In another group of rats, insulin solution was injected intravenously at 1.0 IU/kg, and both serum glucose and insulin levels were measured. The pharmacological availability of insulin from Labrasol solution was found to be 3.9, 8.9 and 9.1% following jejunal, ileal and colonic administrations, respectively, by comparing the serum glucose level vs. time profiles obtained after intestinal and i.v. administrations. By comparing the serum insulin levels vs. time profiles, the bioavailability of insulin was found to be 0.25 and 0.20% for intra-ileum and colonic administrations, respectively. The hypoglycemic effect of insulin after intra-ileum administration showed a dose-dependency in the insulin dose range from 10.0 to 1.0 IU/kg. These results suggest the absorption enhancing effect of Labrasol on the intestinal absorption of insulin in rats.     

Effect of Receptor Up-Regulation on Insulin Pharmacokinetics in Streptozotocin-Treated Diabetic Rats

The present study investigated the mechanism by which the disposition of insulin is altered in streptozotocin (STZ)-treated diabetic rats as compared with 48-hr-fasted normal (control) rats. It was shown by an indocyanine green infusion method that the hepatic plasma flow rate (Q _H) in diabetic rats (1.64 ml/min/g liver) is significantly higher than that in control rats (0.982 ml/min/g liver). The portal injection technique revealed that the unidirectional clearance (CL_on), which represents the binding of A₁₄-¹²⁵ I-insulin to surface receptors in the liver, is significantly elevated in diabetic rats, suggesting an increase in the surface receptor number R _T), i.e., up-regulation in the liver. In both control and diabetic rats, the total-body clearance (CL_tot) and steady-state volume of distribution (Vd _ss) of labeled insulin decreased significantly with a simultaneous injection of unlabeled insulin (8 U/kg), confirming that the disposition of insulin is affected largely by specific, saturable receptor-mediated processes. The CL_tot and Vd _ss increased significantly in diabetic rats, while nonspecific portions of these parameters were not changed. From the increases in CL_tot (80%) and Q _H (67%) in diabetic rats, a pharmacokinetic analysis has revealed a 40% increase in the hepatic intrinsic clearance (CL_{int sp}) of A₁₄-¹²⁵ I-insulin via a specific mechanism in diabetic rats. In conclusion, we have provided in vivo evidence for a small increase in CL_{int sp} of insulin in STZ-diabetic rats compared with control rats, which may be caused by an increase in the surface receptor number in the livers of diabetic rats.     

氨氯地平对高血压伴胰岛素抵抗大鼠血清脂联素的影响

目的：观察氨氯地平对高血压伴胰岛素抵抗动物模型血清脂联素浓度的影响。方法：6周龄的雄性Wistar大鼠随机分为4组（每组10只,均以普通标准饲料）。对照组以普通自来水喂养;果糖组（F组）及氨氯地平治疗组以10/果糖水喂养;氨氯地平组分低剂量组（L组）和高剂量组（H组）,果糖水喂养8周后分别以不同浓度的氨氯地平溶液灌胃（1mg·kg^-1·d^-1和10mg·kg^-1·d^-1）。胰岛素抵抗指标采用胰岛素敏感性指数（ISI）=-ln（FBG×FINS）;尾部测量法测血压;放免法测定血清脂联素的浓度。结果：8周后果糖喂养的F、H、L组大鼠血压、空腹胰岛素水平明显升高,ISI、脂联素明显下降,高血压伴胰岛素抵抗大鼠模型成功建立;氨氯地平干预6周后,L、H组较F组空腹胰岛素水平明显下降,ISI、脂联素明显提高;L、H组间对比无明显差异;多元回归分析表明,体质量和胰岛素敏感性是影响脂联素水平的独立因素。结论：氨氯地平可改善高血压伴胰岛素抵抗大鼠的胰岛素敏感性,其机制可能与提高血清脂联素水平有关。     

Vaginal Absorption of Insulin in the Rat: Effect of Penetration Enhancers on Insulin Uptake and Mucosal Histology

The absorption of insulin across the vaginal mucosa into the systemic circulation was studied in ovariectomized rats given subsequent estrogen treatment. Blood glucose levels were determined as an indirect measure of insulin absorption, and the effect of various enhancers on the hypoglycemic response was investigated. In the absence of any enhancer, no decrease in blood glucose levels was observed after vaginal administration of insulin. However, the coadministration of sodium taurodihydrofusidate, polyoxyethylene-9-lauryl ether, lysophosphatidylcholine, palmitoylcarnitine chloride, and lysophosphatidylglycerol significantly increased hypoglycemia, whereas citric acid had little effect. The histological changes in the vaginal epithelium after treatment with the enhancer systems were variable and often severe. While the efficacy of these compounds in promoting the vaginal absorption of insulin is encouraging, their mechanisms of action and long-term histological effects are yet to be defined.     

Absorption Enhancement of Rectally Infused Insulin by Sodium Tauro-24,25-Dihydrofusidate (STDHF) in Rats

The bile salt derivative sodium tauro-24,25-dihydrofusidate (STDHF) has been reported to promote nasal absorption of insulin. In the present study the effect of STDHF on rectal insulin absorption was investigated in rats. At concentrations of 1 and 4% (w/v) it enhanced insulin bioavailability from 0.2 ± 0.2 (control) to 4.2 ± 3.2 and 6.7 ± 2.1%, respectively, as assessed by radioimmunoassay. Insulin preparations with STDHF reduced blood glucose concentrations considerably in a concentration-dependent way. Coadministration of STDHF with Na₂EDTA (0.25%, w/v) tended to increase further insulin bioavailability and hypoglycemic response. Varying the site of rectal administration did not influence these parameters.     

胰岛素肺部给药：各种吸收促进剂对胰岛素经肺吸收的促进作用

通过肺部导入胰岛素溶液的方法研究各种吸收剂对胰岛素肺部吸收的影响,以血糖的下降程度为吸收评价指标（ＰＡ％）。研究发现,２０ ｍｍｏｌ·Ｌ~(－１)胆酸钠（ＰＡ％＝６９．１％）, １％辛酸钠ＰＡ％＝４０．４％）, １％苄泽３５ （ＰＡ％＝４６．６％）,１％苄泽７８（ＰＡ％＝５３．６％）以及稀土化合物氯化钆［ＰＡ％＝３２．２％,（０．０４ｍｇ·ｋｇ~(－１)）和ＰＡ％＝３６.３％, ０．２ ｍｇ·ｋｇ~(－１)）］等能显著增强胰岛素对大鼠血糖的下降作用,而 １％ ＥＤＴＡ（ＰＡ％＝１９．１％）, ５％油酸（ＰＡ％＝２０．８％）和稀土化合物氯化镥（ＰＡ％＝２５.１％, ０.２ ｍｇ·ｋｇ~(－１)）无明显的吸收促进作用。结果表明,选用合适的吸收促进剂有利于胰岛素的肺部吸收。     

Absorption Enhancement of Intrapulmonary Administered Insulin by Various Absorption Enhancers and Protease Inhibitors in Rats

Abstract— The effects of absorption enhancers and protease inhibitors on the pulmonary absorption of insulin were examined by means of an in-situ pulmonary absorption experiment. Absorption enhancers used in this study were sodium glycocholate, linoleic acid-surfactant mixed micelles and N-lauryl-β-d-maltopyranoside whereas aprotinin, bacitracin and soybean trypsin inhibitor were used as protease inhibitors. The absorption of insulin from the lung was evaluated by its hypoglycaemic effect. In the absence of these additives, a slight hypoglycaemic effect was obtained following intrapulmonary administration of insulin. However, we found significant and continuous hypoglycaemic effects after the insulin administration with these additives. N-Lauryl-β-d-maltopyranoside and bacitracin appeared to be more effective for enhancing the pulmonary absorption of insulin than the other adjuvants. These findings suggest that the use of these two adjuvants would be a useful approach for improving the pulmonary absorption of insulin.     

Enhancement of Nasal Absorption of Insulin Using Chitosan Nanoparticles

Purpose. To investigate the potential of chitosan nanoparticles as a system for improving the systemic absorption of insulin following nasal instillation. Methods. Insulin-loaded chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate anions. They were characterized for their size and zeta potential by photon correlation spectroscopy and laser Doppler anemometry, respectively. Insulin loading and release was determined by the microBCA protein assay. The ability of chitosan nanoparticles to enhance the nasal absorption of insulin was investigated in a conscious rabbit model by monitoring the plasma glucose levels. Results. Chitosan nanoparticles had a size in the range of 300–400 nm, a positive surface charge and their insulin loading can be modulated reaching values up to 55% [insulin/nanoparticles (w/w): 55/100]. Insulin association was found to be highly mediated by an ionic interaction mechanism and its release in vitro occurred rapidly in sink conditions. Chitosan nanoparticles enhanced the nasal absorption of insulin to a greater extent than an aqueous solution of chitosan. The amount and molecular weight of chitosan did not have a significant effect on insulin response. Conclusions. Chitosan nanoparticles are efficient vehicles for the transport of insulin through the nasal mucosa.     

Enteral Absorption of Insulin in Rats from Mucoadhesive Chitosan-Coated Liposomes

Purpose. The mucoadhesiveness of polymer-coated liposomes was evaluated to develop a novel drug carrier system for oral administration of poorly absorbed drugs such as peptide drugs. Methods. Multilamellar liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and dicetyl phosphate (DCP) (DPPC:DCP = 8:2 in molar ratio) were coated with chitosan (CS), polyvinyl alcohol having a long alkyl chain (PVA-R) and poly (acrylic acid) bearing a cholesteryl group. The adhesiveness of the resultant polymer-coated liposomes to the rat intestine was measured in vitro by a particle counting method with a Coulter counter. The CS-coated liposomes containing insulin were administered to normal rats and the blood glucose level was monitored. Results. The existence of polymer layers on the surface of liposomes was confirmed by measuring the zeta potential of liposomes. The CS-coated liposomes showed the highest mucoadhesiveness and the degree of adhesion was dependent on the amount of CS on the surface of the liposomes. The blood glucose level of rats was found to be significantly decreased after administration of the CS-coated liposomes containing insulin. The lowered glucose level was maintained for more than 12h after administration of the liposomal insulin, which suggested mucoadhesion of the CS-coated liposomes in the intestinal tract of the rats.     

胰岛素抵抗高血压大鼠红细胞内钙、镁浓度与胰岛素抵抗的关系

目的：探讨胰岛素抵抗高血压大鼠红细胞内钙、镁浓度与胰岛素抵抗的关系。方法：以高果糖饲料喂养雄性SD大鼠建立胰岛素抵抗高血压大鼠模型，测定收缩压(SBP)、空腹血糖(FBG)、空腹血清胰岛素(FINS)和红细胞内钙、镁([Cd^2 ]i、[Mg^2 ]i)浓度，用稳态模型(HOMA)评估胰岛素抵抗(IR)，分析红细胞内钙、镁浓度与胰岛素抵抗的相关性。结果：与对照组相比，高果糖饲料组SBP、FINS、HOMA—IR和[Ca^2 ]i明显升高；[Mg^2 ]i降低；FBG无明显变化(P＞0．05)。高果糖饲料组大鼠的HOMA—IR与SBP和[Ca2 ]i呈显著正相关，与[Mg2 ]i呈显著负相关。结论：高果糖饲料喂养的SD大鼠能形成胰岛素抵抗及高血压。胰岛素抵抗与细胞内钙、镁浓度异常关系密切。     

Confocal Laser Scanning Microscopic Visualization of the Transport of Dextrans After Nasal Administration to Rats: Effects of Absorption Enhancers

Purpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated -cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated -cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated -cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.     

隐形义齿临床应用效果分析

持续性肺动脉高压(persistent pulmonary hypertension of the newborn,PPHN)，又称持续胎儿循环，指多种病因所引起的新生儿出生后肺生障碍，以至于在动脉导管和卵圆孔水平出现右向左分流，从而导致严重的低氧血症和青紫，甚至死亡。它是一种威胁患儿生命的疾病，1000个活产婴儿有1.9例发病［1，2］，死亡率高达20％[3] ，存活者后遗症发生率较高，包括认识障碍、听力损伤和频繁再入院等[4]。1969年，Gersony首先报道该病为“持续性胎儿循环”[5]。1976年Levin以“持续性肺动脉高压”命名该病[6]。     

米力农诱导的胰岛素抵抗对大鼠糖脂代谢的影响

目的　探讨选择性磷酸二酯酶Ⅲ (PhosphdiesteraseⅢ ,PDE3)抑制剂米力农 (milrinone)对大鼠胰岛素分泌、血糖、血浆游离脂肪酸 (freefattyacid ,FFA)的影响和剂量依赖关系 ,及其在胰岛素钳夹状态下对大鼠糖代谢和胰岛素敏感性的影响。方法　由植入导管给予大鼠不同剂量的米力农 ( 1,5 ,2 5 μmoL·kg- 1 )在不同时相测定血糖、血浆FFA和胰岛素水平并与对照组比较。在意识清醒状态下建立大鼠高胰岛素 正常血糖钳夹技术 ,并在钳夹 12 0min时分别经导管给予米力农 ( 2 5 μmoL·kg- 1 )和 2 5 %二甲基亚砜 (DMSO ,对照组 )。采用气相色谱 -质谱仪 (GC MS)测定糖代谢率。结果　 3个不同剂量米力农组血浆FFA浓度明显高于对照组和给药前 ,在注射后 2min ,各组FFA升高的百分数为 :5 0 %、5 2 %、5 5 % ( 1,5 ,2 5 μmoL·kg- 1 )。在 ( 5 ,2 5 )μmoL·kg- 1 组血浆胰岛素水平也明显高于对照组和给药前 ,仅 2 5 μmoL·kg- 1 组血糖浓度高于对照组和给药前。在胰岛素钳夹研究中 ,米力农处理组大鼠血浆FFA明显高于给药前 ( 173± 15vs 6 34± 87μmoL·kg- 1 ) ,肝糖输出 (HGP)也明显高于给药前( - 5 1± 3 1vs 7 5± 2 0mg·kg- 1 ·min)。葡萄糖输注率 (Glucoseinfusionrate,GIR)明显低于对照组和给药前 (