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In the in vitro experiment using a luminal, mucosal, and fecal fluid/extract from jejunum and colon of a rat, Lys18-residue modified mono-PEG(2k)-sCT (Lys18-PEG(2K)-sCT) exhibited a longer half-life than salmon calcitonin (sCT) in a colonic fluid and its extract. A physical adsorption study showed that Lys18-PEG(2K)-sCT had lower adsorption in the...
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... sCT was more rapidly degraded in both the jejunal fluid and its mucosal extract than Lys 18 -PEG 2k -sCT with only $ 9% and $ 12% remaining after 6 and 12 min, respectively, after incubation (data not shown). However, in the colonic fluid, the rate of Lys 18 - PEG 2k -sCT degradation was slower than sCT, whereas the degradation of Lys 18 -PEG 2k -sCT in the mucosal extract was the slowest, as shown in Figure 1. The degradation half-lives (and rate constant, k, as in Table 1) of sCT and Lys 18 -PEG 2k -sCT were 8.7 and 17.7 min, respectively, in the colonic fluid, whereas it was 37.6 and 84.5 min, respectively, in its mucosal extract. ...
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Background
The aim of this study was to investigate the performance and predictive value of hypocalcemia in severe COVID-19 patients.
Methods
We retrospectively investigated the clinical and laboratory characteristics of severe COVID-19 patients. 107 patients were divided into hypocalcemia group and normal serum calcium group. The clinical and lab...
Citations
... delivery, with the effect of PEs (CG and C 10 ) being more impressive in colonic versus jejunum mucosae. The stability of native sCT was also shown to be 5-fold higher in rat colonic fluid than in jejunal (Mansoor et al., 2005), providing an additional tool to increase peptide permeability and bioavailability. Possible reasons for the increased colonic susceptibility to many enhancers include a more delicate membrane composition than the jejunum due to lack of colonic exposure to food components, bile salts, and pancreatic enzymes. ...
Salmon calcitonin (sCT, MW 3432Da) is a benchmark molecule for an oral peptide delivery system because it is degraded and has low intestinal epithelial permeability. Four dry emulsion minisphere prototypes (SmPill®) containing sCT were co-formulated with permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10) or coco-glucoside (CG), or with a pH acidifier, citric acid (CA). Minispheres protected sCT from thermal degradation and the released sCT retained high bioactivity, as determined by cyclic AMP generation in T47D cells. Pre-minisphere emulsions of PEs combined with sCT increased absolute bioavailability (F) compared to native sCT following rat intra-jejunal (i.j.) and intra-colonic (i.c.) loop instillations, an effect that was more pronounced in colon. Minispheres corresponding to ~2000 I.U. (~390μg) sCT /kg were instilled by i.j. or i.c. instillations and hypocalcaemia resulted from all prototypes. The absolute F (i.j.) of sCT was 11.0, 4.8, and 1.4 % for minispheres containing NaTDC (10μmol/kg), CG (12μmol/kg) or CA (32μmol/kg) respectively. For i.c. instillations, the largest absolute F (22% in each case) was achieved for minispheres containing either C10 (284μmol/kg) or CG (12μmol/kg), while the absolute F was 8.2% for minispheres loaded with CA (32μmol/kg). In terms of relative F, the best data were obtained for minispheres containing NaTDC (i.j.), a 4 fold increase over sCT solution, and also for either C10 or CG (i.c.), where there was a 3 fold increase over sCT solution. Histology of instilled intestinal loops indicated that neither the minispheres nor components thereof caused major perturbation. In conclusion, selected SmPill® minisphere formulations may have the potential to be used as oral peptide delivery systems when delivered to jejunum or colon.
... Thus, by manipulating the amphiphilic properties using PEG based oligomers and by controlling the location of attachment, the oral absorption of therapeutic proteins or peptides has been successfully achieved. This strategy had been successfully applied to other proteins, such as insulin (HIM-2) and calcitonin etc [42][43][44][45]. In another study, the intestinal absorption phenomenon of PEGylated lactoferrin was shown to be nonselective; it was believed that the native lactoferrin was transported via lymphatic system from the intestinal lumen [46,47]. ...
Oral delivery of therapeutics is extremely challenging. The digestive system is designed in a way that naturally allows the degradation of proteins or peptides into small molecules prior to absorption. For systemic absorption, the intact drug molecules must traverse the impending harsh gastrointestinal environment. Technologies, such as enteric coating, with oral dosage formulation strategies have successfully provided the protection of non-peptide based therapeutics against the harsh, acidic condition of the stomach. However, these technologies showed limited success on the protection of therapeutic proteins and peptides. Importantly, inherent permeability coefficient of the therapeutics is still a major problem that has remained unresolved for decades. Addressing this issue in the context, we summarize the strategies that are developed in enhancing the intestinal permeability of a drug molecule either by modifying the intestinal epithelium or by modifying the drug itself. These modifications have been pursued by using a group of molecules that can be conjugated to the drug molecule to alter the cell permeability of the drug or mixed with the drug molecule to alter the epithelial barrier function, in order to achieve the effective drug permeation. This article will address the current trends and future perspectives of the oral delivery strategies.
... In particular, as demonstrated with salmon calcitonin, increasing PEG size, i.e. over 5 kDa, increases peptide stability but decreases its bioactivity probably by interfering with receptor binding [218]. The promising results obtained with pegylated VIP [217], GLP-1 [216,219,220] and calcitonin [221] have paved the way for development of PEGmodified PACAP derivatives as potent neuroprotective agents. ...
In neurological insults, such as cerebral ischemia and traumatic brain injury, complex molecular mechanisms involving inflammation and apoptosis are known to cause severe neuronal cell loss, emphasizing the necessity of developing therapeutic strategies targeting simultaneously these two processes. Over the last decade, numerous in vitro and in vivo studies have demonstrated the unique therapeutical potential of pituitary adenylate cyclase-activating polypeptide (PACAP) for the treatment of neuronal disorders involving apoptotic cell death and neuroinflammation. The neuroprotective activity of PACAP is based on its capacity to reduce the production of deleterious cytokines from activated microglia, to stimulate the release of neuroprotective agents from astrocytes and to inhibit pro-apoptotic intracellular pathways. However, the use of PACAP as a clinically applicable drug is hindered by its peptidic nature. As most natural peptides, native PACAP shows poor metabolic stability, low bioavailability, inadequate distribution and rapid blood clearance. Moreover, injection of PACAP to human can induce peripheral adverse side effects. Therefore, targeted chemical modifications and/or conjugation of PACAP to different macromolecules are required to improve the pharmacokinetic and pharmacological properties of PACAP. This review presents the chemical, biochemical and pharmacological strategies that are currently under development to convert PACAP from a hypophysiotropic neurohormone into a clinically relevant neuroprotective drug.
... A nasal spray formulation of salmon calcitonin is currently being marketed by Novartis, and research groups are trying to find methods of prolonging its half-life by PEGylation. PEGylation at Lys (18) has been reported to result in maximal biological activity (defined as in vitro cAMP-releasing activity and in vivo hypocalcemic efficacy), and a number of successful attempts have been made to achieve the selective PEGylation sCT and to identify alternative routes for administrating Lys(18)-PEG-sCT (per nasal or oral) to improve drug delivery [90][91][92][93]. 6.4.1 CDP791 (alacizumab Pegol) CDP791 is a PEGylated di-Fab′ against VEGF receptor-2 (VEGFR-2) and inhibits the angiogenesis pathway. ...
PEGylation is a pharmaceutical technology that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Research groups are striving to improve the consistencies of PEGylated drugs and to PEGylate commercialized proteins and small organic molecules. Furthermore, the PEGylations of novel medications, like oligonucleotides and antibody fragments, are being pursued to improve their bioavailabilities. This active research in the PEGylation field and the continued growth of the biopharmaceutical market predicts that PEGylated drugs have a bright future.
... It is obvious however, that as the molecular weight of the polymer is increased over that of the active molecule and with use of sitespecific conjugation, accurate characterization of conjugate purity and efficiency of the synthetic process are paramount. Unlike previously published polymer systems where linear PEG was used for the synthesis sCT conjugates [21,22,31,32], comb-shaped PolyPEG® 6.5 K has an advantage over other polymers in that the length can be varied in more than one dimension, increasing the length of the methacrylate backbone accompanied by additional PEG teeth of the same or different molecular weights [16,17]. It can also be tailored to incorporate active end-groups for specific amino acid targeting. ...
Salmon calcitonin (sCT) was conjugated via its N-terminal cysteine to a comb-shaped end-functionalized poly(poly(ethylene glycol) methyl ether methacrylate) (PolyPEG, 6.5 kDa), and to linear PEG (5 kDa). Conjugate molecular weight and purity was assessed by SEC-HPLC and MALDI-TOF MS. Bioactivity of conjugates was measured by cyclic AMP assay in T47D cells. Calcium and calcitonin levels were measured in rats following intravenous injections. Stability of conjugates was tested against serine proteases, intestinal and liver homogenates and serum. Cytotoxicity of conjugates was assessed by lactate dehydrogenase (LDH) assay and by haemolytic assay of rat red blood cells. Results showed that the two conjugates were of high purity with molecular weights similar to predictions. Both conjugates retained more than 85% bioactivity in vitro and had nanomolar EC(50) values similar to sCT. While both sCT-PolyPEG(6.5 K) and sCT-PEG(5 K) were resistant to metabolism by serine proteases, homogenates and serum, PolyPEG (6.5 K) was more so. Although both conjugates reduced serum calcium to levels similar to those achieved with sCT, PolyPEG(6.5 K) extended the T(1/2) and AUC of serum sCT over values achieved with sCT-PEG and sCT itself. None of PolyPEG, PEG or methacrylic acid displayed significant cytotoxicity. PolyPEG may therefore have potential to improve pharmacokinetic profiles of injected peptides.
... A number of oral delivery approaches for sCT are described in several reports. One of them is the chemical modification of calcitonin to produce prodrugs and analogues (8,9,10,11). It is plausible that this approach may protect CT against degradation by proteases and other enzymes present at the mucosal barrier (8). ...
... After biotinylation, a heterogeneous mixture of seven possible biotin modified sCTs (one tri-bio-sCT (Cys 1 , Lys 11 , Lys 18 ); three di-bio-sCT (Cys 1 and Lys 11 ; Cys 1 and Lys 18 ; Lys 11 and Lys 18 ); three mono-bio-sCT (Cys 1 or Lys 11 or Lys 18 ) might be obtained. To prepare the Lys-bioconjugated sCTs, Lys-amine specific conjugation reaction conditions conducted in an organic medium, as previously described (5,9,10,28). Figure 3 clearly shows that the primary aminedirected mono-biotinylation of sCT results in the two different isomers of mono-and di-biotin-sCT. ...
... Furthermore, the use of absorption enhancers is limited by the fact that once cell membranes are permeabilized or tight junctions opened, transport is enhanced not only for peptide and protein drugs but also for undesirable molecules present in the gastrointestinal tract (35). Another approach is site-specific bioconjugation with PEG of sCT to help overcome enzymatic barrier in intestine or colon (9,10). This conjugation technology has been used to improve the therapeutic efficacy of sCT. ...
This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.
... In this respect, the development of an orally available exendin-4 presents an interesting challenge, because such a product would improve patient convenience and therapeutic efficacy. However, the oral deliveries of many therapeutic peptides, including that of exendin-4, are profoundly restricted by their physicochemical characteristics, e.g., their proteolytic instabilities and limited abilities to traverse biological barriers111213. We recently proposed a novel, potent, enzyme-resistant, orally active GLP-1 analogue1213, and hypothesized that site-specific covalent coupling with biotin (vitamin H) can facilitate the receptor-mediated intestinal absorption of peptide via the sodium-dependent multivitamin transport (SMVT) system12131415. The chemical modification of lysine residues of GLP-1 with biotin molecules resulted in the enhancement of intestinal stability, Caco-2 cell permeability, and hypoglycemic potentials followed by oral administration [12]. ...
An orally active glucagon-like peptide-1 (GLP-1) formulation would have great advantages over conventional injectable therapies for the treatment of diabetic patients. Because GLP-1 absorption in the intestine is restricted by its natural physiological characteristics, biotinylated exendin-4 analogues might useful as orally active GLP-1 receptor agonists. Three different biotinylated exendin-4 analogues, Lys(27)-Biotin-Exendin-4 (MB1-Ex-4), Lys(12)-Biotin-Exendin-4 (MB2-Ex-4), and Lys(12, 27)-Biotin-Exendin-4 (DB-Ex-4) were prepared, and their biological activities and enzymatic stabilities were studied in vitro. The hypoglycemic effects and pharmacokinetics of these analogues after oral administration were evaluated in db/db mice and Sprague-Dawley rats, respectively. These biotinylated exendin-4 analogues preserved GLP-1 receptor binding affinity and stimulated insulin secretion in RIN-m5F murine insulinoma cells and in isolated rat islets, respectively, and were as potent as exendin-4. In particular, DB-Ex-4 showed 9.0-fold better stability against rat intestinal fluid than exendin-4. When 0.1, 1, and 10 microg/mouse of DB-Ex-4 were orally administered, mean total hypoglycemic degrees (HGD) were increased by 36.8+/-1.2, 46.9+/-1.8, and 54.3+/-4.5%, respectively, whereas 1 microg/mouse of native exendin-4 showed an increase of 8.8+/-7.3%. This study demonstrates that biotinylated exendin-4 analogues are absorbed in the intestine and that they have biological efficacies of exendin-4. Furthermore, it indicates that biotinylated exendin-4 analogues could be used as potential oral antidiabetic agent for the treatment of type 2 diabetes.
... Recently, we demonstrated that PEGylation of salmon calcitonin (sCT) greatly increases its proteolytic stability [14][15][16], extends its biological half-life [17][18][19], and improves its anti-osteoporosis potency [19][20][21]. In the present study, we examined the benefits of PEGylation on the efficacy of the intrapulmonary delivery of sCT. ...
... Plasma samples were obtained by centrifuging at 10,000 rpm for 10 min after being treated with heparin. Total calcium levels were measured using a colorimetric Ca assay, which was based on the o-cresolphthalein complexation method, as previously described [19][20][21]. Total Ca decreases (D%) in plasma were calculated using a modification of the method described by Hirai et al. [30], as follows: D% = [(AUC control, 0-480 min − AUC test, 0-480 min ) / AUC control, 0-480 min ] × 100. ...
The purpose of this study was to demonstrate the biological potentials of PEGylated salmon calcitonin (PEG-sCT) derivatives administered intratracheally and their dependences on PEG Mw (1, 2, 5 kDa). Initially, three different PEG-sCT derivatives were site-specifically synthesized by attaching PEG to the Lys(18)-amine. In an attempt to examine the pulmonary feasibilities of these derivatives, the following evaluations were undertaken to determine their; (i) proteolytic resistances to pulmonary enzymes, (ii) bioactivities, and (iii) pulmonary pharmacokinetic and pharmacologic profiles. The results obtained showed that the pulmonary stabilities and pharmacokinetic properties of these derivatives were greatly improved by increasing PEG Mw. PEG-sCTs had 10.5-, 40.1-, and 1066.0-fold greater stabilities than that of sCT in rat lung homogenates. Moreover, all pharmacokinetic parameters (AUC(inf), C(max), t(1/2), and others) of these derivatives in endotracheally cannulated rats were significantly improved by PEGylation. Specifically, C(max) values increased on increasing PEG Mw, i.e., 78.1+/-21.1, 102.9+/-9.1, and 115.2+/-5.7 for 1, 2, 5 kDa, respectively, vs. 54.8+/-3.9 ng/mL for sCT. Their circulating t(1/2) values also increased to 53.9+/-6.0, 100.7+/-21.7, and 119.4+/-13.7 min, respectively, vs. 34.6+/-7.6 min for sCT. Despite having the best properties, Lys(18)-PEG(5k)-sCT was found to have significantly lower hypocalcemic efficacy than other PEG-sCTs, probably due to its reduced intrinsic bioactivity ( approximately 30% vs. sCT). Rather, Lys(18)-PEG(2k)-sCT showed the most promising pulmonary potential because of its well-preserved bioactivity (>80% of sCT). Taken together, our findings suggest that the site-specific substitution to peptides like sCT with a PEG of an appropriate size offers optimized therapeutic potential by dual advantages, i.e., (i) increased proteolytic stability and (ii) extended circulating half-life in terms of intrapulmonary delivery.
... In particular, Lys 18 -PEG-sCT was found to have the most prominent potential in terms of biological activity and proteolytic stability [20,21] versus the other PEGylated sCT isomers. Furthermore, the sCT conjugate modified with a PEG of 2 kDa molecular weight (MW) has displayed much more effective hypocalcemic efficacies than other conjugates prepared with lower (1 kDa) or higher MW (5, 10, and 12 kDa) PEGs in the various delivery routes [21,23,27,28]. The goal in this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of this Lys 18 -PEG 2K -sCT. ...
The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys(18)) salmon calcitonin (Lys(18)-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC(1,11)-sCT), which was FMOC protected at Cys(1)- and Lys(11)-amines among three PEGylation sites including Lys(18)-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC(1,11)-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC(1,11)-sCT. The optimized reaction resulted in the high production yield of Lys(18)-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys(18)-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys(18)-PEG-sCT conjugate with a high yield.
... Our previous studies demonstrated that the PEGylation of salmon calcitonin (sCT) improves its enzymatic stability [25][26][27], in vivo hypocalcemic efficacy [27,28] and its pharmacokinetics [29] vs. sCT. These findings suggest that, if a PEGylated sCT had comparable intestinal permeability and bioactivity, it would show superior in vivo therapeutic efficacy, due to (i) increased stability in the intestinal system and (ii) attenuated bioclearance. ...
... Our previous studies demonstrated that the PEGylation of salmon calcitonin (sCT) improves its enzymatic stability [25][26][27], in vivo hypocalcemic efficacy [27,28] and its pharmacokinetics [29] vs. sCT. These findings suggest that, if a PEGylated sCT had comparable intestinal permeability and bioactivity, it would show superior in vivo therapeutic efficacy, due to (i) increased stability in the intestinal system and (ii) attenuated bioclearance. ...
... sCT was PEGylated, as described previously [25][26][27][28]30]. Briefly, 2.3 mg of mPEG 2K -SPA was added to 1 ml of sCT (2 mg/ml) in a 50 mM phosphate buffer solution (pH 7.5). ...
Peptides like salmon calcitonin (sCT) are subjected to aggressive proteolytic attack by various intestinal enzymes, and fractions that enter the systemic circulation via the intestinal route are rapidly inactivated by tissue accumulation and glomerular filtration. Here, we describe the beneficial effects of the Lys(18)-amine specific PEGylation of sCT on the intestinal delivery of sCT. Two key properties were enhanced by the PEGylation process: (i) the resistance of sCT to intestinal enzymes and (ii) the systemic clearance of sCT that had entered the circulation. Initially, we evaluated the cAMP-secreting activities of PEG(2K)-sCT isomers substituted at Cys(1)-, Lys(11)- or Lys(18)-amine position in T47D cells, and found that sCT PEGylated at Lys(18)-amine (Lys(18)-PEG(2K)-sCT) had the highest bioactivity. We then investigated the stability of Lys(18)-PEG(2K)-sCT in the presence of intestinal enzymes, its abilities to traverse the intestinal membrane, its pharmacokinetic behavior and in vivo hypocalcemic efficacy. Results show that Lys(18)-PEG(2K)-sCT has significantly increased resistance to pancreatic peptidases and brush-border peptidases. Despite the molecular size increase caused by PEGylation, Lys(18)-PEG(2K)-sCT was found to have an intestinal permeability similar to that of unmodified sCT (p>0.59) over an apical concentration range 12.5-100 microM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that (125)I-labeled Lys(18)-PEG(2K)-sCT markedly resists liver accumulation and glomerular filtration; levels were reduced by 75% and 50% vs. sCT. Finally, the hypocalcemic efficacy of intestinally administered Lys(18)-PEG(2K)-sCT, measured as total serum calcium in a rat model, was 5.8 and 3.0 times that of sCT at 100 and 200 IU/kg (p<0.025). Our findings suggest that this site-specific conjugation of peptides with PEG of proper size enhances pharmacokinetic properties by increasing their abilities to resist both proteolysis and systemic clearance without significantly reducing their membrane permeabilities or bioactivities. We believe that this concept, namely, dual effects by PEGylation, has great potential value because it presents a practical means of enhancing the efficacies of the peroral/intestinal pharmacologic route.
