Phase Ⅰ Clinical Trial Of BmrhGM-CSF Produced By Silkworm Bioreactor And Its Oral Absorption Mechanism Study

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is an acid glycoprotein containing 127 amino acid residues and has a molecular weight of 14.4 kDa to 32 kDa. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) can be expressed in Escherichia coli, yeast, and mammalian cells. rhGM-CSF acts on precursor cell proliferation in bone marrow. It also stimulates granulocytes, monocytes, and colony formation; and induces hyperplasy of macrophages. This protein is primarily used in bone marrow transplantation, tumor chemotherapy, and the treatment of agranulocytosis related to AIDS. Currently, the rhGM-CSF is administered by injection in clinical settings.There are some limits and side effects induced by injection preparation. We expressed hGM-CSF in silkworm pupae bioreactor named as BmrhGM-CSF. It was purified and determined to have molecular weigh of 29 kDa, and its biological activity was measured both in vitro and in vivo. The results from preclinical studies demonstrated that BmrhGM-CSF could stimulate the colony formation of human bone marrow cells in vitro. The orally administered BmrhGM-CSF was shown to be absorbed into blood of mice, beagles, and macaques. It also could function as an active cytokine. (1) In hematogenesis-inhibited mice, orally administered BmrhGM-CSF stimulated the colony formation in hemopoietic tissues on the spleen surface and DNA synthesis in the bone marrow. (2) Hematogenesis-inhibited beagles treated with 60Co and orally administered BmrhGM-CSF had white blood cell counts reached 50% of their baseline values by 9th day, and recovered to the normal levels by 12th day. (3) The oral administration of BmrhGM-CSF to cyclophosphamide-treated macaques increased leukocyte production in a manner similar to that of Leucomax injections.This research was based on the results mentioned above mainly involving phase I clinical trial of BmrhGM-CSF and the absorption mechanism of BmrhGM-CSF by oral administration.1. Phase I clinical trial of BmrhGM-CSF To investigate single-dose safety of BmrhGM-CSF, twenty seven volunteers were selected and randomly divided into 5 dose groups (150μg,300μg,600μg,750μg, 900μg, orally administered once a day). During the test, the next dose trial didn't begin until there was no significant adverse drug reactions happened in the previous trial. Subjects had to undergo a medical examination involved various indicators such as urine, blood biochemistry, ECG and clinical observation. The results suggested:(1) the safety range of a single dose of BmrhGM-CSF was <900μg. (2) no adverse effect happened related to BmrhGM-CSF.Pharmacodynamics of BmrhGM-CSF was studied in two pilot program. On the one hand, fourteen cancer patients receiving chemotherapy were randomly divided into 2 dose groups (150μg,300μg, orally administered twice a day for consecutive 14 days). The results of laboratory examination indicated that subjects had a good tolerability for BmrhGM-CSF through oral administration in multiple doses. The oral administration of BmrhGM-CSF to patients receiving chemotherapy increased leukocyte and neutrophil production. On the other hand, forty eight tumor patients receiving chemotherapy or radiotherapy were randomly divided into 2 groups: BmrhGM-CSF group (39 subjects) and placebo control group (9 subjects).In BmrhGM-CSF group, subjects began to orally administer the test formulation (300μg, twice a day for consecutive 12 days) on 3rd day after patients received chemotherapy or radiotherapy. In placebo control group, subjects began to orally administer the placebo formulation (2 capsules, twice a day for consecutive 12 days) on 3rd day after patients received chemotherapy or radiotherapy. In the placebo control group, the leukocyte and neutrophil cells number of subjects began to decrease on 3rd day. The leukocyte and neutrophil cells number of subjects on 9th day decreased to 3.31×109/L and 1.53×109/L respectively. In the BmrhGM-CSF group, the leukocyte and neutrophil cells number of subjects treated with BmrhGM-CSF on 9th day was 4.61×109/L,2.32×109/L. The results suggested that BmrhGM-CSF can cure WBC and neutral neutropenia induced by chemotherapy or radiotherapy.Using a single-dose, randomized, open-label, two-period crossover clinical trial design,19 healthy volunteers were orally administered with BmrhGM-CSF (8 mg/kg) and subcutaneously injected with rhGM-CSF (3.75 mg/kg) expressed in E. coli respectively. Serum samples were drawn from the brachial vein at 0.0h,0.5h,0.75h, 1.0h,1.5h,2.0h,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The rhGM-CSF serum concentration was determined by ELISA. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%.2. Absorption mechanism of BmrhGM-CSF by oral administrationMice were orally administered with 125I-BmrhGM-CSF at 10μg/kg or 50μg/kg, and urine was collected at various times to measure radioactivity. Eight hours post administration, the accumulated excretion rate reached 18% of the total 125I administrated. The serum sample was collected at 30min after 125I-BmrhGM-CSF administration, and subjected to SDS-PAGE followed by autoradiography. An 18-20 kDa radioactive protein band was detected in high dose groups (50μg/kg), and undetected in low dose group (10μg/kg), which might be the intermediate metabolites of BmrhGM-CSF. This indicated that BmrhGM-CSF could be absorbed into blood through oral administration route.SD rats were fed with 50μg/kg, 10μg/kg of BmrhGM-CSF, respectively. After 15min, rhGM-CSF can be detected in the SD rats' serum. rhGM-CSF concentration in the 30min serum samples reached the highest values, 1010ng/L for 50μg/kg dose group and 212ng/L for 10μg/kg dose group respectively. rhGM-CSF concentration in serum gradually decreased and maintained up to 12h. rhGM-CSF was unable to be detected in 12h serum samples. The results suggested that oral administration of BmrhGM-CSF could be absorbed into blood.In the self-cross-over trial, the serum samples collected from 3 volunteers at the 0.0h, 1.0h,2.0h,3.0h, and 4.0h after the oral administration of BmrhGM-CSF or the subcutaneous injection of rhGM-CSF (expressed in E.Coli) were subjected to MS analysis (Q-Trap MS/MS TOF, ABI Co.). The 0.0h sample analysis spectrogram was used as control and compared with the analysis spectrogram of 1.0h,2.0h,3.0h and 4.0h hour sample in order to obtain peaks with different mass-to-electric charge ratios. Further analysis of the peaks through both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis concluded that the molecular weight and amino acid sequences of these peptides matched hGM-CSF fragments. Using the 0h MS data as control, the results of Enhanced Mass Spectrometry (EMS) and EPI analysis suggested that there were differential peaks of the mass-to-electric charge ratio in the serum samples after the oral administration of BmrhGM-CSF or injection of rhGM-CSF. Peptide mapping based on the molecular weights demonstrated the presence of hGM-CSF peptide fragments in the differential peaks. The molecular weights of those different polypeptides were ranged from 2,039 Da to 7,336Da, which indicated that orally administered BmrhGM-CSF could be absorbed into the blood after penetrated through the intestinal tract.Mice were orally administered with rhGM-CSF (expressed in E.Coli) which was mixed with freeze-dried pupa powder.The results proved that the serum rhGM-CSF concertration increased after mice treated with the mixture of rhGM-CSF and freeze-dried pupa powder. Pupa were homogenized and centrifuged to remove precipitate, then the solution was placed in the upper sub-funnel to separate protein-rich-in-lipid and protein layer. Both of protein-rich-in-lipid and protein layer were made into freeze-dried powder and used as the drug-loaded material to wrap rhGM-CSF expressed in E. coli. The wrapped rhGM-CSF were orally administered to mice. The results showed that the rhGM-CSF concentration in the serum of mice treated with rhGM-CSF wrapped by protein-rich-in-lipid were 98.73 ng/L and 106.28ng/L at 30min and 60min after administration respectively. While in the group treated rhGM-CSF wrapped by protein of pupa, the value of serum rhGM-CSF were 48.73ng/L and 76.25ng/L at 30min and 60min after administration respectively. Therefore, both protein-rich-in-lipid and protein of pupa may promote rhGM-CSF absorbability into blood by oral administration. For this, the effect of protein-rich-in-lipid may be better than that of protein.The serum samples were obtained from mice that treated with rhGM-CSF wrapped by pupa lipoprotein. The serum protein were purified by 10RI column using fast liquid chromatography (FPLC).Then the protein samples were automatically collected according to different peak and were analyzed by MALDI-TOF MS. The results showed that there was rhGM-CSF (MW 14.5kDa) in the serum of mice which orally administered rhGM-CSF. Its mass spectrum was similar to that of mice subcutaneously injected rhGM-CSF. In addition, the rhGM-CSF wrapped by protein-rich-in-lipid was fixed by 5% glutaraldehyde on the copper platform. Then the copper platform was placed under scanning electron microscope to observe its microscopic morphology. It was found that spherical microcapsules were formed in the sample of rhGM-CSF wrapped by protein-rich-in-lipid. These spherical microcapsules made of protein-rich-in-lipid were fat-soluble. They might be fused with membrane of the small intestine mucosa. The spherical microcapsules carrying rhGM-CSF could be transferred into blood from the small intestinal wall by endocytosis.Pupa proteins were purified and separated by Sephadex G-150 column chromatography. Thirty six tubes full of protein samples were collected from Sephadex G-150 column. These protein samples were analyzed by Biacore X-100 and found one of them could be combined with rhGM-CSF expressed in E. coli. The results indicated that there were some rhGM-CSF-binding factors in pupa proteins. The structure and composition of these rhGM-CSF-binding factors should be further analyzed.This paper not only provided some experimental evidence for Phase II clinical trial of BmrhGM-CSF but also laid a certain theoretical foundation for the development of other peptide and protein drugs produced by silkworm bioreactor.