Method for alleviating radiation injury by geranylgeranyl transferase inhibitor

Abstract

A method for treating a patient having intestinal radiation injury is disclosed. The method includes administering to the patient having intestinal radiation injury an effective amount of a geranylgeranyl transferase inhibitor. In one embodiment, the geranylgeranyl transferase inhibitor is GGTi-298. In another embodiment, the geranylgeranyl transferase inhibitor is GGTi-2133.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 354,299, filed on June 22, 2022, entitled "Mitigation of Radiation Injury By Geranylgeranyl Transferase Inhibitors". The complete disclosure of the provisional application is incorporated herein by reference.

[0002] Description of Research and Development Funded by the Federal Government Not applicable

Background Art

[0003] In the event of a radiation emergency, people may be exposed to ionizing radiation, and therapeutic interventions (i.e., radiation mitigators) are needed to reduce radiation injury when administered more than 24 hours after radiation exposure. The U.S. Food and Drug Administration (FDA) has approved several treatment strategies to mitigate hematopoietic radiation injury, but no mitigators for radiation injury in other organ systems, including the intestine, have been approved yet.

[0004] Therefore, it is desirable to develop a radioprotective agent for radiation injury in other organ systems. Geranylgeranyl transferase inhibitors (GGTi) are currently in clinical trials for treating patients with advanced malignancies. However, their effectiveness as a radioprotective agent for intestinal radiation injury has not yet been investigated. In a mouse model, the inventors found that when GGTi was intraperitoneally administered starting 24 hours after partial body irradiation (PBI, 12 Gy of γ-rays with both hindlimbs shielded from radiation), and GGTi was further administered every 48 hours thereafter, intestinal injury measured on days 3.5 and 14 was alleviated. Furthermore, when GGTi was intraperitoneally administered starting 24 hours after 12 Gy of PBI, followed by a second dose of GGTi at 72 hours, the composition of the intestinal microbiota in irradiated mice changed. Finally, GGTi inhibits the growth rate of human pancreatic and prostate cancer cell lines in a time-dependent and dose-dependent manner.

Summary of the Invention

[0005] The present invention relates to a method of using a geranylgeranyl transferase inhibitor for alleviating or treating intestinal radiation injury, including in the context of radiation accidents / terrorism and radiation therapy. In one aspect, the method of the present invention includes administering an effective amount of a geranylgeranyl transferase inhibitor to a patient having intestinal radiation injury. As an example, the geranylgeranyl transferase inhibitor can be GGTi-298 or GGTi-2133.

[0006] In adult male C57BL / 6J mice, the inventors found that intraperitoneal administration of GGTi-2133 (commercially available, Sigma Aldrich), three times a week for a total of two weeks starting 24 hours after 12 Gy of γ-ray PBI, significantly attenuated weight loss. In the small intestine, compared to intraperitoneal administration of the vehicle (90% sterile saline, 5% DMSO, and 5% Kolliphor® EL), it was observed that GGTi-2133 prevented the reduction in villus height, mucosal surface area, goblet cell number, immune cell number (neutrophils and T lymphocytes), and the number of proliferating cells in the crypts. Furthermore, GGTi-298 (commercially available, MedChemExpress, Monmouth Junction, NJ) alleviated the harmful effects of 12 Gy of γ-ray PBI on villus height, crypt depth, and crypt width. Additionally, when GGTi-2133 was intraperitoneally administered 24 hours after 12 Gy of PBI, followed by a second dose of GGTi-2133 at 72 hours, the number of surviving crypts in the small intestine on day 3.5 was significantly enhanced compared to irradiated mice treated with the vehicle. Moreover, microbial community analysis of cecal contents on day 3.5 revealed that GGTi-2133 increased the percentage of Akkermansia muciniphila in irradiated mice. This bacterium is inversely proportional to obesity, diabetes, inflammation, and metabolic disorders. Finally, the PBI-induced increase in the percentage of Turicibacter and Clostridium species was alleviated by GGTi-2133. Although some Clostridium species are known to be pathogenic, the increase in Turicibacter depletes CD8+ lymphocytes in the intestine, which can have an adverse effect on intestinal immune function. These data indicate that GGTi is a potent alleviator of intestinal radiation injury.

[0007] Human pancreatic cancer cell lines (PANC-1, BxPC-3, PSN-1) and human prostate cancer cell line (LNCaP) were cultured in vitro at 37°C and 5% CO2 and incubated with three different concentrations of GGTi-2133. The cell proliferation and metabolic rates were measured once a day for a total of 4 days using the MTT assay. GGTi-2133 reduced the cell proliferation rate in all four cell lines in a dose-dependent manner. Furthermore, in the clonogenic assay, GGTi-2133 reduced the number of surviving colonies in the PANC-1 and PSN-1 cell lines in a dose-dependent manner. Finally, GGTi-2133 reduced the metabolic activity of LNCaP cells exposed to ionizing radiation. These data indicate that GGTi reduces tumor cell proliferation and enhances the effect of ionizing radiation on tumor cells.

[0008] These and other features, objects, and advantages of the present invention will be better understood by considering the following detailed description of the preferred embodiments and the appended claims in conjunction with the drawings described below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0010] With reference to Figures 1A to 10F, preferred embodiments of the present invention can be described. The present invention relates to a method of using a geranylgeranyl transferase inhibitor for alleviating or treating intestinal radiation injury, including in the context of radiation accidents / terrorism and radiation therapy. In one aspect, the method of the present invention includes administering an effective amount of a geranylgeranyl transferase inhibitor to a patient having intestinal radiation injury.

[0011] In the event of a radiation / nuclear attack or accident, partial body irradiation (PBI) can cause damage to vital organs including the intestine. Etiologies and treatment strategies for alleviating gastrointestinal radiation toxicity are not well established. Damage to endothelial cells (ECs) that form the inner lining of blood vessels contributes to the etiology of intestinal toxicity. The present inventors and others have shown that loss of endothelial thrombomodulin (eTM) and impaired production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) are mechanistically related to the etiology of intestinal radiation toxicity. In particular, both TM and eNOS are under positive transcriptional regulation by Kruppel-like factor 2 (KLF2).

[0012] The mevalonate pathway regulates multiple cellular processes by synthesizing sterol isoprenoids (e.g., cholesterol) and non-sterol isoprenoids (e.g., dolichol, heme-A, isopentenyl tRNA, and ubiquinone). Geranylgeranyl transferase (GGT) is an important enzyme in the non-sterol isoprenoid pathway and is responsible for the post-translational modification of various proteins. The inventors have found that a pharmacological inhibitor of GGT (hereinafter, GGTi) enhances endothelial function and prevents radiation-induced suppression of eTM, eNOS, and KLF2 in primary human ECs in culture. And most importantly, the inventors have found that co-culture of ECs and crypts promotes the growth of intestinal organoids and that ECs require KLF2 to promote the growth of organoids. The inventors hypothesized that GGTi treatment alleviates radiation injury in the mouse intestine by enhancing endothelial function after PBI. The inventors also hypothesized that alleviation of intestinal radiation injury promotes mouse survival within 30 days after whole-body irradiation. The inventors tested the hypothesis under two specific aims: Aim 1 - To identify the dose of GGTi that provides maximum alleviation of acute radiation toxicity; and Aim 2 - To determine whether the effect of GGTi on delayed gastrointestinal tract is KLF2-dependent.

[0013] Data generated from this study indicate that intraperitoneal administration of GGTi-2133 (Sigma) three times a week starting 24 hours after 12 Gy of γ-ray PBI significantly alleviates intestinal radiation toxicity and attenuates weight loss in male C57BL / 6J mice. Furthermore, the inventors show that GGTi-298 also alleviates intestinal injury after 12 Gy of PBI. GGTi-2133 treatment partially restores the number of surviving crypts and changes the microbial community composition of the mouse cecum on day 3.5 post-irradiation. Additionally, the inventors observed that EC promotes the growth of intestinal organoids ex vivo in a KLF2-dependent manner. Finally, GGTi inhibits the proliferation rate and colony-forming ability of human pancreatic and / or prostate cancer cell lines in culture and also enhances the radiation-induced inhibition of prostate cancer cell proliferation in culture. The alleviation of various parameters of intestinal injury after PBI and the efficacy of GGTi in reducing cancer cell line proliferation are described below.

[0014] GGTi-2133 treatment attenuates intestinal structural damage and weight loss after PBI: As shown in FIGS. 1A - 1E, in male adult C57BL / 6J mice, 12 Gy of PBI significantly reduced villus height, increased crypt depth, decreased mucosal surface area (MSA), and increased enterocyte length and enterocyte nuclear length compared to the sham-irradiated group, but GGTi treatment attenuated this reduction in villus height, increase in crypt depth, decrease in MSA, and increase in enterocyte length and their nuclear lengths in irradiated mice. For each mouse, villus height and crypt depth were measured in 30 randomly selected villi and crypts (magnification 20x), MSA was measured in 5 randomly selected areas (magnification 20x), and enterocyte length and the length of their nuclei were measured in 3 enterocytes / villus in 10 villi (magnification 100x). As shown in FIG. 1F, mice treated with GGTi (2.5 mg / kg body weight) had significantly less PBI-induced weight loss than vehicle-treated mice.

[0015] GGTi-2133 treatment accelerates the recovery of intestinal immune cells after PBI: As shown in Figures 2A - 2H, PBI significantly reduced the numbers of neutrophils and lymphocytes and increased the numbers of interstitial and intraepithelial macrophages in the intestine compared with sham - irradiated mice. However, significant recovery of the numbers of neutrophils and lymphocytes and decrease in the numbers of interstitial and intraepithelial macrophages were observed in GGTi - treated irradiated mice. Neutrophils and lymphocytes were scored at 40× magnification. The average numbers of interstitial and intraepithelial macrophages per villus are shown in Figures 2G - 2H. Macrophages were scored in 30 villi at 100× magnification.

[0016] GGTi-2133 treatment increases the number of intestinal proliferating cells after PBI: Two proliferation markers (PCNA and Ki - 67) were used. As shown in Figures 3A - 3F, PBI significantly decreased the number of proliferating cells per crypt compared with the sham - irradiated group, while treatment with 2.5 mg / kg body weight of GGTi significantly increased the number of proliferating cells in the crypts of PBI - exposed mice. The average numbers of PCNA - and Ki - 67 - positive cells in 25 randomly selected crypts are shown in Figures 3E - 3F. Cells were scored at 40× magnification.

[0017] GGTi-2133 treatment enhances the number of intestinal mucus - secreting cells after PBI: As shown in Figures 4A - 4E, a significant decrease in the number of mucus - secreting goblet cells in the intestine after PBI was observed compared with the sham - irradiated group, while GGTi treatment increased the number of goblet cells in irradiated mice. Notably, as shown in Figure 4F, PBI also reduced the staining intensity of goblet cells, suggesting that radiation inhibits mucus production, while GGTi treatment significantly enhanced the staining intensity. The average number of MSCs / 20 villi and the average staining intensity of alcian blue / 25 villi are shown in Figures 4E - 4F. Cells were scored at 20× magnification.

[0018] GGTi-2133 increases the expression of olfactomedin 4 (OLFM4), an intestinal stem cell marker, after PBI: We assayed OLFM4 protein using immunohistochemistry and computer image analysis and classified immunoreactive regions as "dark", "medium", or "weak" based on staining intensity. PBI significantly decreased the area of dark OLFM4 staining, suggesting a decrease in stem cell number compared to sham irradiation. GGTi-2133 treatment restored OLFM4 staining in irradiated animals (Figs. 5A-5E). Figs. 5A-5E show that the expression of the stem cell marker olfactomedin 4 (OLFM4), investigated by immunohistochemistry in the small intestine, was reduced 2 weeks after 12 Gy of γ-ray PBI and increased after GGTi treatment.

[0019] GGTi-298 treatment attenuates intestinal structural damage after PBI: To ensure that another formulation of GGTi provides a similar alleviation of intestinal damage after PBI, we used GGTi-298 (MedChemExpress). As shown in Figs. 6A-6D, in male adult C57BL / 6J mice, 12 Gy of PBI significantly reduced villus height and increased crypt depth and width compared to the sham-irradiated group, but GGTi treatment (5 or 10 mg / kg body weight) attenuated this reduction in villus height and the increase in crypt depth and width in irradiated mice. For each mouse, villus height and crypt depth and width were measured in 30 randomly selected villi and crypts (magnification 20x). Thus, Figs. 6B-6D show that another GGTi formulation, GGTi-298, is also effective in alleviating intestinal radiation injury in our mouse model.

[0020] ECs lacking the KLF2 gene do not promote the growth of intestinal organoids: The inventors hypothesized that GGTi at least partially alleviates radiation injury by improving the function of endothelial cells (ECs). Figures 7A-7D show that ECs promote organoid growth in a KLF2-dependent manner. Mouse small intestinal crypts (250 crypts / well in a 24-well plate) were used to grow organoids in vitro for 4 days with Kruppel-like factor 2 (KLF2) wild-type or KLF2 knockdown human microvascular ECs (NFκB-TIME; ATCC; CRL-4049, 100,000 cells / well). The growth of intestinal crypt organoids was significantly induced by the presence of ECs, and ECs required KLF2 to promote organoid growth. Mouse intestinal organoids grown in the presence of human microvascular ECs (NFκB-TIME; ATCC; CRL-4049) significantly increased the number of organoids (p = 0.0001). Incubation of crypts with ECs lacking the KLF2 gene did not promote the number of intestinal organoids when compared to the number of organoids grown without ECs, as shown in Figures 7A-7D.

[0021] GGTi-2133 treatment enhances the number of surviving crypts on day 3.5 after PBI: Male C57BL / 6J mice were exposed to 12 Gy of PBI and administered GGTi-2133 (2.5 mg / kg) 24 hours and 72 hours after irradiation. Specimens of the jejunum were collected on day 3.5 after irradiation. The presence of more than 5 PCNA-positive cells aggregated within a crypt was recorded as a surviving crypt. As shown in Figures 8A-8D, a significant decrease in the number of surviving crypts (p < 0.0001) was observed after irradiation compared to the sham-irradiated group, but treatment with GGTi-2133 (2.5 mg / kg) significantly (p = 0.002) increased the number of surviving crypts. Figures 8A-8D show that GGTi-2133 treatment significantly increases the number of surviving crypts in the small intestine on day 3.5 after 12 Gy of PBI in a mouse model. Specimens of the small intestine were stained for proliferating cell nuclear antigen (PCNA) to identify proliferating cells. As described above, intestinal crypts with at least 5 PCNA-positive cells were considered surviving crypts.

[0022] GGTi-2133 alters the percentages of three bacterial species in the cecal contents of mice on day 3.5 post-PBI: Male C57BL / 6J mice were exposed to 12 Gy of PBI and administered GGTi-2133 (2.5 mg / kg) 24 and 72 hours after irradiation. Samples of cecal contents were aseptically collected 3.5 days after PBI. Microbiome analysis shows that the GTTi treatment enhances Akkermansia muciniphila while suppressing Turicibacter species and Clostridium species in irradiated mice, as shown in FIGS. 9A - 9C. There were 8 mice in each group. In particular, FIGS. 9A - 9C show that treatment with GGTi-2133 (2.5 mg / kg) 24 and 72 hours after 12 Gy of PBI in mice changes the percentages of three bacterial strains in the cecal contents. GGTi-2133 increased the percentage of Akkermansia muciniphila in irradiated mice. In PBI + vehicle-treated mice, the percentages of Turicibacter and Clostridium species increased compared to sham-irradiated vehicle-treated mice. The percentages of Turicibacter and Clostridium species were reduced in PBI + GGTi-2133-treated mice compared to PBI + vehicle-treated mice.

[0023] GGTi-2133 treatment suppresses the proliferation rate and colony formation ability of human pancreatic and / or prostate cancer cells, and further enhances the radiation-induced decrease in the proliferation rate: As shown in FIGS. 10A-10F, GGTi-2133 treatment suppresses the proliferation rate and metabolic activity of three pancreatic cancer cell lines (PANC-1, BxPC-3, and PSN-1) and one prostate cancer cell line (LNCaP) in a time- and dose-dependent manner, suppresses the colony formation ability of PANC-1 and PSN-1 cell lines in a dose-dependent manner, and enhances the radiation-induced decrease in the proliferation rate and metabolic activity of LNCaP cells in culture. These findings suggest that GGTi has potent antitumor activity against various cancer types. Therefore, when used as a mitigating agent for intestinal radiation injury by radiotherapy, GGTi may not have an adverse effect on tumor control by radiation.

[0024] Based on scientific literature, it is understood that GGTi-2418 can be safely administered to humans at doses up to 2,060 mg / m 2 (54 mg / kg body weight) on days 1-5 of each 21-day cycle. By using the body surface area conversion factor, 54 mg / kg in humans is comparable to 660 mg / kg in mice. In humans, GGTi can generally be administered at an effective dose with a dose escalation of approximately 200 mg / m 2 ~2060 mg / m 2 per day on days 1-5 of each 21-day cycle. In rats, the maximum tolerated dose of GGTi is 200 mg / kg for single dosing or 150 mg / kg / day for 7 consecutive days. In addition to intraperitoneal administration, enteral tube feeding, intravenous infusion, and subcutaneous administration are other acceptable modes of administration. In the method of the present invention, GGTi-298 and GGTi-2133 are preferred GGTis, but the use of other GGTis is expected to show the same effect, although the magnitude of the effect may not be very large.

[0025] The method of using GGTi as a mitigating agent for intestinal radiation injury described herein can be utilized in the situation of radiation exposure due to accidents or malicious activities, or in the situation of abdominal radiotherapy for cancer patients.

[0026] Achieved milestones: The inventors have completed a study to investigate the acute toxicity of the intestine after PBI using two different formulations of GGTi (Figures 1A - 6D and Figures 8A - 9C). The delayed toxicity study under specific objective 2 is ongoing. The inventors developed KLF2 knockdown stable ECs and used them to show that endothelial KLF2 is required for ECs to promote the growth of intestinal crypt organoids in vitro (Figures 7A - 7D), highlighting the importance of KLF2 in ECs for intestinal recovery. The inventors showed that GGTi reduces the growth rate and enhances the cytotoxic effect of radiation in human cancer cell lines (Figures 10A - 10F), suggesting that GGTi does not promote cancer cell growth.

[0027] Experimental plan: Data indicate that three - times - weekly intraperitoneal administration of GGTi starting 24 hours after 12 Gy of γ - ray PBI significantly reduced weight loss, decreased villus height, decreased mucosal surface area, decreased goblet cell number, decreased immune cells (neutrophils and T lymphocytes), decreased number of proliferating cells in the crypt, increased crypt depth, and increased number of stromal and intraepithelial macrophages. These data indicate that GGTi is a potent alleviator of intestinal radiation injury. However, in contrast to the inventors' previous hypothesis that GGTi acts mainly through improving EC function, GGTi may also mediate a direct effect on epithelial or intestinal epithelial stem cells (ISCs). The inventors hypothesize that after irradiation, GGTi treatment promotes survival and alleviates intestinal injury as a result of the direct effect of GGTi on ISCs and the indirect outcome through promoting EC function. Furthermore, although not yet tested, the inventors expect that the significant alleviation of intestinal radiation injury by GGTi provides a survival benefit after irradiation. The inventors propose two objectives to test these hypotheses.

[0028] Objective 1 - Determine the dose of GGTi that provides maximum mitigation against radiation lethality: To further investigate GGTi as a radiation mitigator, the inventors establish that GGTi provides a survival benefit after irradiation. The inventors optimize the GGTi dose that provides maximum survival benefit in adult male and female C57BL / 6J mice exposed to total body irradiation (TBI, 7 - 9 Gy of γ-rays) and partial body irradiation (PBI, 17 - 18.5 Gy of γ-rays). The inventors have extensive experience with these radiation models. Twenty-four hours after irradiation, the inventors administer GGTi-2133 (Sigma) or vehicle by intraperitoneal injection. The inventors test four doses of GGTi-2133 administered three times a week until 30 days after irradiation.

[0029] Study plan for Objective 1: Mouse TBI and PBI models: The inventors use male and female 8 - 12-week-old C57BL / 6J mice (The Jackson Laboratory, stock number: 000664). After a two-week quarantine, the radiation experiments are initiated. The inventors expose unanesthetized mice to a single dose of 7, 7.5, 8, 8.5 or 9 Gy of γ-ray TBI or 17, 17.5, 18 or 18.5 Gy of γ-ray PBI (shielding both hindlimbs) using a cesium 137 source (Mark 1, Model 68A, JL Shepherd). In the inventors' experience, these radiation doses cause lethality in the range of 30 - 100% in C57BL / 6J mice. To generate survival curves for 30 days, the mice (n = 10 mice / group) are monitored twice daily for 30 days after irradiation.

[0030] GGTi treatment: GGTi-2133 (Millipore Sigma) is dissolved in a vehicle of 90% sterile saline, 5% DMSO and 5% Kolliphor® EL (Sigma-Aldrich) and administered by intraperitoneal injection three times a week. GGTi-2133 treatment is initiated 24 hours after TBI or PBI and continued until euthanasia. Based on the previous results above, four doses (0, 2.5, 5 and 10 mg / kg body weight) are tested.

[0031] Objective 2 - To define whether the GGTi-mediated mitigation of epithelial radiation injury is a direct effect on ISCs or an endothelium-dependent outcome. Rationale and hypothesis: Radiation-induced loss of ISCs impairs epithelial regeneration and leads to breakdown of the mucosal barrier. Several previous studies have shown that molecules that directly protect ISCs reduce intestinal radiation injury. We have shown that protection of stromal cells promotes ISC regeneration after radiation. In this regard, our studies have shown that intestinal stromal ECs play an important role in suppressing radiation injury. We found that ECs with wild-type KLF2, but not KLF2 knockdown ECs, promote intestinal organoid growth when co-cultured with primary crypts. We use this organoid model to test the hypothesis that GGTi alleviates radiation injury to the mucosal epithelial layer by directly enhancing ISC regeneration and promoting endothelial function.

[0032] In previous studies, we showed that co-culturing intestinal organoids with human microvascular ECs promotes organoid growth. We use this co-culture model to determine whether the GGTi effect on intestinal epithelium is endothelium-dependent. Crypts are irradiated and grown in organoid culture media with and without GGTi in the presence and absence of mouse intestinal microvascular ECs (MIMEC; Cell Biologics). Furthermore, to determine whether GGTi-mediated protection is endothelium KLF2-dependent, we co-culture crypts with wild-type or KLF2 knockdown MIMEC. Impact: The proposed studies provide important mechanistic insights for developing intestinal radiation mitigation strategies using GGTi.

[0033] Research plan for Objective 2: Crypt isolation and organoid culture: Collect small intestine fragments from unirradiated adult male and female C57BL / 6J mice (n = 5), and isolate intestinal crypts by treatment with Gentle Cell Dissociation Reagent (StemCell Technologies). Mix the suspension containing the same number of isolated crypts with Matrigel (BD Biosciences) at a ratio of 1:1 (vol / vol) and plate it in a 48-well plate. Add Intesticult™ OGM mouse basal medium (StemCell Technologies) and change it every 2 - 3 days. We collect intestines from 5 unirradiated mice to isolate crypts, and seed the crypts in duplicates per mouse for each treatment group.

[0034] Organoid irradiation and GGTi treatment: On day 5, expose the organoids to 3, 4, 5, and 6 Gy of γ-rays and further incubate them for 3 more days in Intesticult™ OGM mouse basal medium containing various concentrations of GGTi (0, 2.5, 5, and 10 μM). Determine the number of organoids, budding, and size on day 8.

[0035] Generation of KLF2 knockdown stable cell line: Achieve KLF2 knockdown using small hairpin RNA (shRNA), whereby we can generate and maintain stable KLF2-deficient MIMEC cell lines. Use Lipofectin (Invitrogen) to stably transfect either the shRNA KLF2 plasmid (Santa Cruz) or the control shRNA into MIMEC (Cell Biologics).

[0036] Irradiation of mice, isolation of crypts, and co-culture of crypts with ECs: Expose mice (n = 5) to 6, 7, and 8 Gy of γ-ray TBI, and isolate crypts on day 4 after TBI as described above. Co-culture the crypts with KLF2 wild-type or knockdown ECs for 8 days with or without GGTi (0, 2.5, 5, and 10 μM), and measure organoid growth as described above.

[0037] Expected Results, Interpretations, Pitfalls, and Alternative Approaches: The inventors predict that GGTi will mitigate TBI- and PBI-induced radiation lethality and enhance ex vivo organoid growth. Since the inventors expect GGTi to act in a KLF2-dependent manner, they predict that GGTi will not promote organoid growth when crypts are co-cultured with KLF2 knockdown ECs.

[0038] Pitfalls and Alternative Approaches: After transfection, the growth rate of MIMECs may be impaired, in which case the inventors will optimize the growth medium with a higher percentage of FBS, etc. TBI-induced hematopoietic injury is an important determinant of mouse survival. If the inventors do not observe significant lethal protection by GGTi, they will inject the FDA-approved radioprotectant G-CSF (10 μg / kg body weight / day, 3 times / week, intraperitoneal injection), an inducer of hematopoietic stem cells, in combination with GGTi after TBI.

[0039] The present invention has been described with reference to specific preferred alternative embodiments that are merely exemplary and do not limit the full scope of the invention as set forth in the appended claims.

Claims

**Claim 1** A method for treating a patient having intestinal radiation injury, the method comprising administering to the patient having intestinal radiation injury an effective amount of a geranylgeranyl transferase inhibitor. **Claim 2** The method according to claim 1, wherein the geranylgeranyl transferase inhibitor is GGTi-298. **Claim 3** The method according to claim 1, wherein the geranylgeranyl transferase inhibitor is GGTi-2133. **Claim 4** The method according to claim 1, wherein the effective amount is 200 mg / kg or less as a single dose. **Claim 5** The method according to claim 1, wherein the effective amount is 150 mg / kg / day or less for 7 consecutive days. **Claim 6** The effective amount is 200 mg / m ２ / day to 2060 mg / m ２ / day, and the method according to claim 1. **Claim 7** The method according to claim 1, wherein the administration is by intraperitoneal injection. **Claim 8** The method according to claim 1, wherein the administration is by intravenous infusion. **Claim 9** The method according to claim 1, wherein the administration is by subcutaneous injection.

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