Recombinant Human Tissue-type Plasminogen Activator Research Articles

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta).

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Effect of acupuncture on neurological function, cerebral infarction volume, thrombolysis time window and cerebral cell apoptosis signaling pathway in cerebral infarction rats

To observe the effect of acupuncture (Acupunct) on cerebral infarction volume and expression of poly ADP ribose polymerase 1 (PARP1), apoptosis-inducing factor (AIF) and endonuclease G (Endo-G) in the cerebral cortex tissue at different time-points after cerebral ischemia (CI) in acute cerebral infarction rats, so as to explore its underlying mechanisms in prolonging time window of thrombolysis. Forty-eight SD rats were randomly divided into sham operation, model, intravenous thrombolysis (IVT)-4.5 h, IVT-6 h, IVT-9 h, Acupunct＋IVT-4.5 h, Acupunct ＋IVT-6 h, Acupunct＋IVT-9 h groups (n＝6 in each group). The CI model was established by using modified autologous thromboembolism via the right common carotid artery. Two hours after modeling, rats of the Acupunct groups received Acupunct stimulation of "Shuigou" (GV26) and bilateral "Neiguan" (PC6) for 30 min. Thrombolysis was conducted by injection of recombinant human tissue-type plasminogen activator (rt-PA, 10 mg/kg) via caudal vein. The neurological deficit was assessed with reference to Bederson's methods. 2，3，5-triphenyltetrazolium chloride (TTC) staining was used to assess the cerebral infarction volume, and the expression of cerebral PARP1, AIF and Endo-G proteins detected by Western blot. Compared with the sham operation group, the neurological score and percentage of cerebral infarction volume, expression levels of PARP1, AIF and Endo-G proteins were significantly increased in the model group (P<0.01，P<0.05). After the intervention, modeling-induced increase of the aforementioned indexes was reversed in the IVT-4.5 h, Acupunct＋IVT-4.5 h and Acupunct＋IVT-6 h groups (P<0.05), except PARP1 expression of the Acupunct＋IVT 6 h group (P>0.05). The levels of neurological score, percentage of cerebral infarction volume, and AIF expression were significantly lower in both the Acupunct＋IVT 4.5 h and Acupunct＋IVT-6 h groups than in the simple IVT-4.5 h and simple IVT-6 h groups, respectively (P<0.05), and the expression levels of PARP1 and Endo-G proteins were obviously lower in the Acupunct＋IVT-4.5 h group than in the IVT-4.5 h group (P<0.05). Endo-G proteins were obviously lower in the Acupunct＋IVT-9 h group than in the IVT-9 h group (P<0.05). Acupuncture may improve neurological function, reduce cerebral infarction volume and prolong the time window of thrombolysis in CI rats, which may be associated with its effect in suppressing AIF/PARP1/ Endo-G signaling.

Generation and characterization of tissue-type plasminogen activator transgenic rats

To address a species difference in the responsiveness to human recombinant tissue-type plasminogen activator (rt-PA) between rats and humans, tPA transgenic (Tg) rats were generated and characterized. In the rats, transcriptional regulation of tPA was designed under the control of the endogenous tPA promoter. There were no significant differences in hematological parameters between the tPA Tg and non Tg rats. Plasma tPA concentration was significantly increased and serum free PAI-1 was significantly decreased in the tPA Tg rats. Significant overexpression of tPA mRNA in five major organs was also confirmed in the tPA Tg rats. In contrast, the extent of tPA mRNA induction by pathophysiological stimuli (focal cerebral ischemia) was comparable in the two strains. Earlier increase in the plasma D-Dimer level was observed in the tPA Tg rats in a model of thromboembolism compared with the non Tg rats. On the other hand, there was no statistically significant prolongation of bleeding time in a rat model of bleeding between the two strains. rt-PA showed dose-related blood flow restoration in a rat model of thromboembolic stroke in the tPA Tg rats from a dose (1 mg/kg, i.v.) similar to clinical doses for human stroke patients. In conclusion, tPA Tg rats, in which tPA is overexpressed and endogenous fibrinolytic activity is enhanced without hemostatic abnormality, were generated. tPA Tg rats would be beneficial for the pharmacological and the toxicological evaluation of rt-PA and other various fibrinolytic enhancers.

A polymeric micelle magnetic resonance imaging (MRI) contrast agent reveals blood–brain barrier (BBB) permeability for macromolecules in cerebral ischemia-reperfusion injury

Blood–brain barrier (BBB) opening is a key phenomenon for understanding ischemia-reperfusion injuries that are directly linked to hemorrhagic transformation. The recombinant human tissue-type plasminogen activator (rtPA) increases the risk of symptomatic intracranial hemorrhages. Recent imaging technologies have advanced our understanding of pathological BBB disorders; however, an ongoing challenge in the pre-“rtPA treatment” stage is the task of developing a rigorous method for hemorrhage-risk assessments. Therefore, we examined a novel method for assessment of rtPA-extravasation through a hyper-permeable BBB. To examine the image diagnosis of rtPA-extravasation for a rat transient occlusion-reperfusion model, in this study we used a polymeric micelle MRI contrast-agent (Gd-micelles). Specifically, we used two MRI contrast agents at 1h after reperfusion. Gd-micelles provided very clear contrast images in 15.5±10.3% of the ischemic hemisphere at 30min after i.v. injection, whereas a classic gadolinium chelate MRI contrast agent provided no satisfactorily clear images. The obtained images indicate both the hyper-permeable BBB area for macromolecules and the distribution area of macromolecules in the ischemic hemisphere. Owing to their large molecular weight, Gd-micelles remained in the ischemic hemisphere through the hyper-permeable BBB. Our results indicate the feasibility of a novel clinical diagnosis for evaluating rtPA-related hemorrhage risks.

Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain

Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood–brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury.

Analysis of blood pressure, heart rate, NIHSS score, GCS score of cerebral infarction patients with cerebral hemorrhage after recombinant human tissue-type plasminogen activator (rt-PA) therapy

Objective To analyze the blood pressure,heart rate,the US national institutes of health stroke scale(NIHSS) score,Glasgow coma scale(GCS) score for cerebral infarction patients with cerebral hemorrhage after recombinant human tissue-type plasminogen activator (rt-PA) therapy.Methods From April 2011 to April 2013,48 cases with cerebral infarction after intravenous rt-PA thrombolysis were collected.These cases were divided into two groups:the first group (32 cases):without cerebral hemorrhage after thrombolysis; the second group (16 cases):with cerebral hemorrhage after thrombolysis.The systolic blood pressure,diastolic blood pressure,heart rote,NIHSS score and GCS score were compared between the two groups before and after thrombolysis.Results After thrombolysis,the systolic and diastolic blood pressure were significantly increased in cerebral hemorrhage cases compared with before thrombolysis and patients without cerebral hemorrhage.After thrombolysis,the NIHSS score was significantly reduced in the cases without cerebral hemorrhage compared with that before thrombolysis,while the NIHSS score was significantly higher in the cases with cerebral hemorrhage compared with that before thrombolysis.After thrombolysis,the heart rate and GCS score showed no significant difference between the patients with and without cerebral hemorrhage.Conclusions The rising of systolic blood pressure,diastolic blood pressure and NIHSS score helps us determine the early cerebral hemorrhage after thrombolysis in cerebral infarction patients. Key words: rt-PA; Cerebral infarction; Cerebral hemorrhage; Blood pressure; NIHSS score

Pulmonary artery intimal sarcoma: poor 18F-fluorodeoxyglucose uptake in positron emission computed tomography

Intimal sarcoma of the pulmonary artery is a rare malignant tumor that may be misdiagnosed as chronic pulmonary thromboembolism, even if various imaging techniques are used. We report a case of a 58-year-old man with pulmonary artery intimal sarcoma.18F-fleuorodeoxyglucose (FDG) uptake was poor in the mass of the pulmonary artery, and no other hypermetabolic lesions were noted elsewhere. Our presumptive diagnosis was a massive mural thrombus and a concomitant chronic thromboembolism. Intravenous heparin and recombinant human tissue-type plasminogen activator was subsequently administered. However, the patient needed an emergency operation for sudden aggravation of the vital signs, and the tissue diagnosis was intimal sarcoma with poor clinical outcomes.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta).

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Tissue-type plasminogen activator as a therapeutic target in stroke

Background: Ischemic stroke is a leading cause of morbidity and mortality worldwide and recombinant human tissue-type plasminogen activator (tPA) is the prominent therapeutic among very few therapeutics used in its treatment. Due to complications attributed to the drug, most notably transformation of ischemia to hemorrhage, tPA is only used in a small number of ischemic stroke cases, albeit significantly more often in specialized stroke centers. Objective: What are the mechanisms of tPA action and side effects in ischemic stroke, and can the knowledge about these mechanisms aid in making tPA a more efficacious and safe therapeutic or in developing alternative therapeutics? Methods: tPA use and alternative/combination therapies in acute ischemic stroke treatment are summarized. The review focuses on literature concerning tPA neurotoxicity and its implications for further development of tPA as a stroke therapeutic. Results/conclusion: Exogenously administered recombinant tPA and endogenous tPA have both turned into promising therapeutic targets for the stroke patient.

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.

Multitargeted Effects of Statin-Enhanced Thrombolytic Therapy for Stroke With Recombinant Human Tissue-Type Plasminogen Activator in the Rat

Microvascular dysfunction posttreatment of stroke with recombinant human tissue-type plasminogen activator (rht-PA) constrains the therapeutic window to 3 hours. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) promote vascular thrombolysis and reduce the inflammation response. We therefore investigated the neuroprotective effects of a combination of atorvastatin and delayed rht-PA treatment in a rat model of embolic stroke. Rats subjected to embolic middle cerebral artery occlusion were treated with atorvastatin in combination with rht-PA 4 hours after stroke. Magnetic resonance imaging measurements revealed that combination treatment with atorvastatin and rht-PA blocked the expansion of the ischemic lesion, which improved neurological function compared with saline-treated rats. Real-time reverse transcription-polymerase chain reaction analysis of single endothelial cells isolated by laser-capture microdissection from brain tissue and immunostaining showed that combination treatment downregulated expression of tissue factor, von Willebrand factor, protease-activated receptor-1, intercellular adhesion molecule-1, and matrix metalloproteinase-9, which concomitantly reduced cerebral microvascular thrombosis and enhanced microvascular integrity. Combination treatment did not increase cerebrovascular endothelial nitric oxide synthase (eNOS) levels or eNOS activity, and inhibition of NOS activity with N-nitro-L-arginine methyl ester did not block the beneficial effects of combination treatment on stroke. Furthermore, combination treatment compared with thrombolytic monotherapy increased cerebral blood flow and reduced infarct volume in eNOS-null mice. These data demonstrate that combination treatment with atorvastatin and rht-PA exerts a neuroprotective effect when administered 4 hours after stroke and that the therapeutic benefits are likely attributed to its multitargeted effects on cerebrovascular patency and integrity.

Single-chain Tissue-type Plasminogen Activator Is a Substrate of Mouse Glandular Kallikrein 24

Leydig cells of the adult mouse testis express at a detectable level three distinct glandular (tissue) kallikrein genes: mKlk21, mKlk24, and mKlk27. Recently, the proteins encoded by these genes were characterized using active recombinant proteases, but their roles in the mouse testis remained to be determined. The present study showed that among the proteases, mK24 markedly enhanced the activity of human recombinant single-chain tissue-type plasminogen activator when the two were incubated together. This activation was found to be due to proteolytic conversion of the single-chain enzyme to a two-chain form. The expression of tissue-type plasminogen activator in interstitial Leydig cells was demonstrated by RT-PCR and immunohistochemical analyses. The primary culture medium of adult male testicular Leydig cells contained immunoreactive substances recognized by anti-mK24 antibodies. In addition, the same medium was capable of converting the single-chain plasminogen activator to the two-chain protein. These results suggest that mK24 may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis, due not only to its own activity, but also to that of plasmin produced by the single-chain tissue-type plasminogen activator-converting activity of mK24.

Efficacy of thrombolysis in younger and older adult patients suffering their first acute q-wave myocardial infarction.

Advancing age is an independent predictor of increased mortality after acute myocardial infarction (AMI). Several hypotheses have been developed to try to explain this phenomenon, but data available about the efficacy of thrombolytic therapy in older patients are still not conclusive. The goal of this study was to investigate the efficacy of thrombolysis in adult and older patients who suffered their first AMI. Retrospective cohort study. A coronary care unit. The sample included 244 younger (aged <65, n = 166) and older (age 65, n = 78) adult patients suffering their first Q-wave AMI, all receiving thrombolysis with human-recombinant tissue-type plasmin-ogen activator (100 mg total dose within 2.5 hours of the onset of AMI. Infarct size was estimated by isoenzyme creatine kinase-myoglobin (CK-MB) release, measuring the area under the curve as a function of time. ST elevation, the sum of ST elevation above the baseline, and the sum of R wave height in precordial leads V1-V6 were evaluated using 12-lead electrocardiograms. Myocardial reperfusion was calculated when ST-segment elevation decreased more than 60 with respect to the most abnormal peak detected. CK-MB peak level was significantly smaller in younger patients than in older ones (P< .01) and was significantly correlated with increasing age (P< .0001). Area under the 36-hour CK-MB curve was lower in younger patients than in older ones (P< .0001) and was well correlated with increasing age (P< .01). Reperfusion time was significantly shorter in younger patients (P< .05), and age was significantly correlated with reperfusion time (P< .001). Infarct size was greater and reperfusion time was longer in older patients than in younger ones with first Q-wave AMI treated with thrombolysis. Infarct size and reperfusion time were linearly correlated with increasing age. These findings may help explain the increase in mortality due to AMI observed with advancing age.

Treatment of deep cerebral venous thrombosis by local infusion of tissue plasminogen activator

BACKGROUNDTreatment of extensive intracranial venous sinus thrombosis with thrombolytic drugs is described, although the indications for and most efficacious technique for achieving thrombolysis remain uncertain. We report the successful lysis of superficial and deep venous system thrombosis by infusion of recombinant human tissue-type plasminogen activator (rt-PA) into the anterior superior sagittal sinus.CASE DESCRIPTIONA 34-year-old man presented with headaches followed by decreased level of consciousness and left hemiplegia. Angiography showed thrombosis of the superior sagittal and both transverse and straight sinuses with extension into the internal cerebral veins. The superior sagittal sinus was catheterized via a transfemoral route and rt-PA, 25 mg, was infused. There was no significant change in the thrombosis. The catheter was left in place and rt-PA was infused at 1 mg/minute for 19 hours. Repeat angiography showed resolution of the thrombosis. The patient was placed on heparin and then coumadin. He recovered completely.CONCLUSIONSThis report suggests that superselective infusion of thrombolytics into thrombosed intracranial venous sinuses can lyse intracranial venous sinus thrombosis. The thrombolytic agent must be infused for hours. The apparent successful lysis of clot in the deep venous system when infusion was into the superior sagittal sinus might be related to diffusion of rt-PA throughout the intracranial venous system or to improved venous outflow caused by lysis of clot in superficial dural sinuses.

Microsurgical repair after crush-avulsion injury of the femoral vein in rats: prevention of microvascular thrombosis with recombinant human tissue-type plasminogen activator (rt-PA).

Vein thrombosis is often encountered in microsurgery, especially in the case of crush-avulsion injuries. The aim of this study was to investigate the effect of systemic administration of recombinant tissue-type plasminogen activator (rt-PA) on the patency of the femoral vein of the rat, which had previously sustained a crush-avulsion injury. The study consisted of 3 groups of male Wistar rats, 20 animals each. A standardized crush-avulsion injury model was used. After microvascular repair of the femoral vein, the animals received either normal saline (group A), heparin 100 U/kg body weight (group B), or rt-PA 3.5 mg/kg body weight (group C) systemically. Patency tests were performed at 20 minutes, 48 hours, and 1 week after blood flow reestablishment. According to our results, the patency rate of the rt-PA group was significantly higher than in both the control and heparin groups.

Microvascular repair following a modified crush-avulsion injury in a rat model: effect of recombinant human tissue-type plasminogen activator on the patency rate.

The failure rate of replantations following a crush-avulsion type injury is high. This study has been designed to reproduce an effective standardized crush-avulsion injury model to the femoral artery of the rat and evaluate the antithrombotic efficacy of systemic intravenous administration of recombinant human tissue-type plasminogen activator (rt-PA). The crush-avulsion injury was reproduced by using a bulldog clamp and two hemostats and followed by microvascular repair. The animals were divided into three groups of 20 rats each and received either normal saline, heparin 100 U/kg body weight, or rt-PA 3.5 mg/kg body weight intravenously. Patency tests were performed 20 min and 48 h after blood flow reestablishment. Results showed that this experimental crush-avulsion injury model ensures low patency in the control group, whereas systemic rt-PA administration improves the patency rate statistically significantly compared to control and heparin groups at both 20 min and 48 h postrevascularization.

Treatment of a rheumatoid joint with an intra-articular fibrinolytic agent to aid aspiration

A 71-year-old woman presented to the Rheumatology Department with a history of an enlarging, painful swellinp over the right shoulder. She had a long history of rheumatoid arthritis with previous effusions of the right shoulder and left knee requiring aspiration. Two attempts were made at blind aspiration on the ward yielding only 8 and 2 ml of thick fluid respectively. Microbiological culture was negative and she was referred for consideration of arthroscopic lavage. Ultrasound was subsequently requested and revealed a large. high reflectivity subacromial-subdeltoid bursal collection. Attempts to aspirate via a l9G needle and subsequently a IOF drain yielded Ruid containing multiple solid ‘rice body’ particles which caused frequent drain blockage and prevented aspiration of the ma.jority of the Huid (Fig. I). Ten ml of I mg/ml Alteplase (recombinant human tissue-type plasminogen activator. Actilyse. Boehringer Ingelheim) was injected into the collection via the drain which was then clamped and the patient returned to the ward. She experienced a little discomfort due to the presence of the drain but no other il l effects due to the procedure. The following day. 300 ml of yellow fluid was aspirated with ease and no visible solid material identilied. Arthrogaphy demonstrated preferential tilling of the dependent parts of the easily distensible bursa and no residual tilling defects (Fig. 2). Communication with the glenohumeral joint space was demonstrated by contrast medium entering the sheath surrounding the long head of biceps tendon. an extension of the synovial lining of the joint. Decompression of the hursa resulted in superior subluxation of the humeral head and severe nnrrowin_p of the subacromial space suggesting a full thickness rotator

Intelligent peptide-mapping method for recombinant human tissue-type plasminogen activator by lysyl-endopeptidase digestion

A novel peptide-mapping method for recombinant human tissue-type plasminogen activator is described. It involves reduction and S-carboxyamidemethylation of the tissue-type plasminogen activator and digestion of the alkylated protein with lysyl endopeptidase, followed by liquid chromatographic analysis of the digest using a Vydac C4 column with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Twenty-four peptide fragments were clearly resolved, and each peptide was identified by means of a combination of amino acid analysis, automated Edman degradation, and enzymatic deglycosylation. The results indicate that the recombinant tissue-type plasminogen activator consists of 527- and 530-residue polypeptides, due to heterogeneity of N-terminal processing, and exists in one- and two-chain forms, due to limited proteolysis between Arg275 and Ile276. It contains three N-glycosylated sites at Asn117, 184, and 448, and a single O-fucosylated site at Thr61. Two variants of the recombinant tissue-type plasminogen activator were separated by hydrophobic liquid chromatography and peptide mapping of the variants demonstrated that they differed only in N-glycosylation at Asn184. Our method can thus provide information on N- and C-termini, proteolytic cleavage and glycosylation pattern of the target molecule.

Preparation of Excipient-Free Recombinant Human Tissue-Type Plasminogen Activator by Lyophilization from Ammonium Bicarbonate Solution: An Investigation of the Two-Stage Sublimation Phenomenon

Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were ∼4mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1±3.8 kcal/mol, which was ∼10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control.

Single and repeated intravenous toxicity studies of pamiteplase (genetical recombination) in rats and monkeys.

Single and repeated intravenous toxicity studies of Pamiteplase (genetical recombination) YM866, a novel recombinant human tissue-type plasminogen activator, were conducted. No animal died from toxic effects of YM866 after single administration to F344 rats, squirrel monkeys and cynomolgus monkeys. Male and female F344 rats were given YM866 intravenously for 4 weeks at doses of 0 (vehicle), 0.1, 0.3 and 1 mg/kg/day. An increase in platelet count, slight decreases in hemoglobin and hematocrit, increases in plasma phospholipids, total cholesterol and total protein, and liver weight were observed at 1.0 mg/kg. Histopathology revealed no changes in any organ except for hemorrhage at the injection sites. These changes recovered after 4 weeks of withdrawal. Male and female squirrel monkeys were given YM866 intravenously for 4 weeks at doses of 0 (saline), 0 (vehicle), 0.1, 0.3 and 1 mg/kg/day. Prolongation of coagulation time at the injection site was observed at 0.3 mg/kg or more. Subcutaneous hemorrhage and a transient decrease in locomotor activity were observed at 1 mg/kg. Prolongation of coagulation time at the injection site was considered to be related to the pharmacological action of YM866. The results show that the approximate single lethal dose of YM866 is more than 60 mg/kg in rats, and more than 10 mg/kg in squirrel monkeys and cynomolgus monkeys. The no-toxic-effect level of YM866 after repeated administration for 4 weeks in rats and squirrel monkeys is considered to be 0.3 mg/kg.