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Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

Molecular and cellular effects of radiotherapy on tumor microenvironment (TME) can help prime and propagate antitumor immunity. We hypothesized that delivering radiation to all tumor sites could augment response to immunotherapies. We tested an approach to enhance response to immune checkpoint inhibitors (ICIs) by using targeted radionuclide therapy (TRT) to deliver radiation semiselectively to tumors. NM600, an alkylphosphocholine analog that preferentially accumulates in most tumor types, chelates a radioisotope and semiselectively delivers it to the TME for therapeutic or diagnostic applications. Using serial 86Y-NM600 positron emission tomography (PET) imaging, we estimated the dosimetry of 90Y-NM600 in immunologically cold syngeneic murine models that do not respond to ICIs alone. We observed strong therapeutic efficacy and reported optimal dose (2.5 to 5 gray) and sequence for 90Y-NM600 in combination with ICIs. After combined treatment, 45 to 66% of mice exhibited complete response and tumor-specific T cell memory, compared to 0% with 90Y-NM600 or ICI alone. This required expression of STING in tumor cells. Combined TRT and ICI activated production of proinflammatory cytokines in the TME, promoted tumor infiltration by and clonal expansion of CD8+ T cells, and reduced metastases. In mice bearing multiple tumors, combining TRT with moderate-dose (12 gray) external beam radiotherapy (EBRT) targeting a single tumor augmented response to ICIs compared to combination of ICIs with either TRT or EBRT alone. The safety of TRT was confirmed in a companion canine study. Low-dose TRT represents a translatable approach to promote response to ICIs for many tumor types, regardless of location.

BackgroundCurrent clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically ‘cold’ tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease.MethodsMice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry.ResultsA cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm3tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm3B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival.ConclusionThe combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor + anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2(+), immunologically “cold”) melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50–150 mm(3)) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 lg daily, treatment days 6–10), and + anti-CTLA-4 (100 lg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8(+) and CD4(+)) and monocytes/macrophages (F4/80(+)) but no changes in FOXP3(+) regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 ((86)Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (~12:1 brain tumor/brain and ~8:1 flank tumor/muscle). When NM600 was chelated to therapeutic β-particle-emitting (90)Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed.

BackgroundImmune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model.MethodsB78 (GD2+) melanoma ‘primary’ tumors were engrafted on the right flank of C57BL/6 mice. After 3–4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6–10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay.ResultsISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8+ and CD4+ T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8+ infiltration and CD8+:FOXP3+ ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1. Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts.ConclusionISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases.

Background: Radiation (RT) activates a type 1 interferon (IFN1) response and this is critical to the effect of RT in priming a response to immune checkpoint blockade (ie anti-CTLA4). However, little is known about the time course of this effect. Clinical interest in utilizing systemically administered targeted radionuclide therapy agents (TRT) is growing as these agents can be used to target multiple sites of disease including micro-metastases. It is unclear how IFN1 activation induced by continuous delivery of RT during exponential decay of a TRT source will compare to that induced following instantaneous external beam RT (EBRT). Here we report the time course of IFN1 response following RT in vitro and in vivo. Methods: For in vitro studies, we utilized murine models of melanoma (B16, B16 STING knockout, B78), and head and neck squamous cell carcinoma (MOC2). EBRT was prescribed to 2.5 Gy, 12 Gy, or 3 fractions of 8 Gy. For in vivo studies, syngeneic C57BL/6 mice were engrafted with either B78 or MOC2 cells on the flank and RT was delivered when mean tumor size was 100-150 mm3. EBRT was prescribed to 2.5 Gy or 12 Gy. For TRT, we used 90Y conjugated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells of nearly any type, including B78 and MOC2. Tumor-specific dosimetry for 90Y-NM600 was determined using sequential 86Y-NM600 PET/CT imaging (3h, 24h, 48h, 72h) and a Monte Carlo based dose calculation platform. TRT was prescribed to a cumulative absorbed dose of 2.5 Gy or 12 Gy. Following delivery of RT, cells or tumors were harvested at 1d, 7d, and 14d post RT and RNA was isolated. Gene expression of Ifn-β and IFN response elements was quantified by qPCR and normalized to untreated controls. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and anti-CTLA-4 therapy reduced tumor growth and prolonged survival compared to single agent therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following RT in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT of equivalent cumulative dose. Further evaluation of the timing and magnitude of IFN1 response following EBRT and TRT may be critical to the optimal integration with immunotherapies. Citation Format: Justin C. Jagodinsky, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Ishan Chakravarty, Ian S. Arthur, Luke M. Zangl, Ryan J. Brown, Erin J. Nystuen, Sarah E. Emma, Caroline Kerr, Won Jong Jin, Peter M. Carlson, Jonathan W. Engle, Eduardo Aluicio-Sarduy, Todd E. Barnhart, Trang Le, KyungMann Kim, Bryan P. Bednarz, Jamey P. Weichert, Ravi B. Patel, Zachary S. Morris. Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3060.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by uncontrolled joint inflammation and destruction of bone and cartilage. Previous studies have shown that C-X-C motif chemokine 10 (CXCL10) has important roles in RA development and that blocking CXCL10 expression effectively inhibits arthritis progression in animal models. However, clinical study using anti-CXCL10 monoclonal antibody (MDX-1100) to block CXCL10 expression in patients with RA did not show significant effectiveness. Therefore, we turned our attention to C-X-C motif chemokine receptor 3 (CXCR3), which is a receptor for CXCL9, CXCL10, and CXCL11, to treat RA. In the present study, administration of JN-2, our newly developed CXCR3 antagonist, ameliorated the progression of arthritis in a collagen-induced arthritis animal model. JN-2 also inhibited CXCR3-induced cell migration and pro-inflammatory cytokine expression of bone marrow–derived macrophages and CD4+ T cells in vitro. In addition, we found that CXCL10 formed an auto-amplification loop through activation of NFκB. Furthermore, Phosphorylation of p65 at serine 536 played an important role in the auto-amplification of CXCL10. Overall, the present results demonstrated that JN-2 decreased inflammation by inhibiting CXCR3-enhanced cell migration and pro-inflammatory cytokine expression, which then ameliorated arthritis progression.

Amplification of the chemokines CXCL10 and RANKL has been suggested to promote osteoclast differentiation and osteolytic bone metastasis, but a function for endogenous CXCL10 in these processes is not well established. In this study, we show that endogenous CXCL10 is critical to recruit cancer cells to bone, support osteoclast differentiation and promote for the formation of osteolytic bone metastases. Neutralizing CXCL10 antibody reduced migration of cancer cells expressing the CXCL10 receptor CXCR3, and loss of CXCR3 or CXCL10 decreased bone tumor burden in vivo. Bone colonization augmented host production of CXCL10, which was required for cancer growth and subsequent osteolysis. Direct interactions between cancer cells and macrophages further stimulated CXCL10 production from macrophages. Growth of bone metastases required CXCL10-stimulated adhesion of cancer cells to type I collagen as well as RANKL-mediated osteoclast formation. Together, our findings show that CXCL10 facilitates trafficking of CXCR3-expressing cancer cells to bone, which augments its own production and promotes osteoclastic differentiation. CXCL10 therefore may represent a therapeutic target for osteolytic bone metastasis. Cancer Res; 72(13); 3175–86. ©2012 AACR.

Objective Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage during normal and pathological bone turnover. Recently, several studies revealed that alpha-tocopheryl succinate (αTP-suc) have demonstrated potent anti-cancer activities in vitro and in vivo. However, the effects of αTP-suc on osteoclast formation and bone resorption remain unknown. Thus, in this study, we examined the effects of αTP-suc on osteoclast differentiation and bone resorbing activity in inflammatory bone loss model. Methods Osteoclast differentiation assay was performed by cocultures of mouse bone marrow cells and calvarial osteoblasts in culture media including interleukin-1 (IL-1). Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP). The level of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). ICR mice were administered an intraperitoneal injections of αTP-suc or dimethyl sulfoxide (DMSO) 1 day before the implantation of a freeze-dried collagen sponge loaded with phosphate-buffered saline (PBS) or IL-1 over the calvariae and every other day for 7 days. The whole calvariae were obtained and analyzed by micro-computed tomography (CT) scanning, and stained for TRAP. Results αTP-suc inhibits osteoclast formation in cocultures stimulated by IL-1 and decreased the level of expression of RANKL mRNA in osteoblasts. In addition, administered intraperitoneal injections of αTP-suc prevented IL-1-mediated osteoclast formation and bone loss in vivo. Conclusion Our findings suggest that αTP-suc may have therapeutic value for treating and preventing bone-resorptive diseases, such as osteoporosis.

Background: It has been known that melatonin is involved in the modulation of nociceptive transmission. However, the effect of melatonin administered spinally has not been examined. Therefore, we examined the effect of melatonin on the formalin-induced or thermal-induced nociception at the spinal level. Methods: Intrathecal catheter was inserted into the subarachnoid space of male Sprague-Dawley rats. Pain was assessed by formalin test (induced by injection of 50μl of a 5% formalin solution to the hindpaw) or Hot-Box test (induced by radiant heat application to the hindpaw). The effect of intrathecal melatonin was examined on flinching behavior in the formalin test or withdrawal response in Hot-Box test. Results: Intrathecal melatonin produced a limited, but dose-dependent reduction of the flinching response during phase 1 and 2 in the formalin test. In addition, melatonin delivered at evening also decreased the flinching response in both phases of the formalin test. Melatonin restrictively increased the withdrawal latency in Hot-Box test. Conclusions: These results suggest that melatonin is active against the formalinand thermal-induced nocicpetion at the spinal level, but the effect is limited. (Korean J Pain 2006; 19: 137-141)

Background: Serotonin 3 receptor is involved in the modulation of nociceptive transmission in the spinal cord. The serotonin 3 receptor antagonist has been used for the management of opioid-induced nausea and vomiting. The aim of this study was to examine whether the analgesic effect of morphine is antagonized by serotonin 3 receptor antagonists at the spinal level. Methods: Rats were implanted with lumbar intrathecal catheters. For nociception, a formalin solution (5%, 50) was injected into the hind paw of male Sprague-Dawley rats. To determine whether the effect of intrathecal morphine was mediated via serotonin 3 receptors, serotonin 3 receptor antagonists were intrathecally administered 10 min prior to the morphine delivery. Following the formalin injection, formalin-induced nociceptive behavior (flinching response) was observed for 60 min. Results: Intrathecal morphine produced a dose-dependent suppression of the flinches in both phases during the formalin test. The analgesic action of morphine was not reversed by serotonin 3 receptor antagonists (LY-278,584, ondansetron), which had little per se effect on the formalin-induced nociception. Conclusions: Spinal serotonin 3 receptors may not be involved in the analgesia of morphine on a nociceptive state evoked by a formalin stimulus.

Background: Cannabinoids have shown antinociceptive action. The aims of this study were to examine the effect of chronic infusion of a cannabinoids receptors agonist (WIN 55,212-2) for thermal nociception at the spinal level, and to also observe the development of toxicity. Methods: Male Sprague-Dawley rats were implanted with lumbar intrathecal catheters with the nociceptive response (withdrawal response latency) determined by exposing the plantar surface of the hindpaw to radiant heat. Initially, the effect of intrathecal WIN 55,212-2 was evaluated followed by the change in the effect at 1, 2, 3 and 4 weeks after repeated infusion. Finally, the histopathological findings were assessed 1 and 4 weeks following the infusion of WIN 55,212-2. Results: Intrathecal WIN 55,212-2 was found to produce a limited antinociception during the thermal test. %MPE of WIN 55,212-2 at 1, 2, 3, and 4 weeks after infusion was not different from each other. No abnormal pathological findings were observed following a chronic intrathecal infusion of WIN 55,212-2. Conclusions: WIN 55,212-2, a cannabinoids receptors agonist, may be useful in the management of thermal nociception, without changing the effectiveness or causing the toxicity following a chronic infusion at the spinal level.

Background: Inhalation anesthetics are known to depress ventilatory response to hypercapnea. Doxapram hydrochloride is an analeptic drug, which acts as a respiratory stimulant via peripheral and central chemoreceptors. Although the postoperarive infusion of doxapram hydrochloride is known to attenuate the impairment of respiratory function, no report is available on respiratory response to this drug when applied during anesthesia. Therefore, the present study aimed to evaluate the effect of doxapram hydrochloride on respiratory function during anesthesia. Methods: Sixty adult patients undergoing operation under spontaneous ventilation via laryngeal mask airway (LMA) were randomly categorized into 3 groups: A control group, which received 5% dextrous infusion, and two groups in which patients were infused with doxapram hydrochloride (0.5 or 2 mg/kg/hr) starting 15 min after commencement operation. Anesthesia was maintained with 1 MAC sevoflurane - 4 L - 2 L under spontaneous ventilation via LMA. Tidal volume (), respiratory rate (RR), and arterial carbon dioxide tension () were measured just before and 15 min after the induction of anesthesia, 15 min after the start of operation and 15, 30, 45, and 60 min after the start of doxapram hydrochloride infusion. Results: Measured values of RR and were significantly elevated during anesthesia venous those measured just before the induction of anesthesia in all groups. was significantly reduced during anesthesia venous just before the induction of anesthesia in all groups. All percent changes of , RR and were similar all any measurement times, and showed no significant changes after the infusion of doxapram hydrochloride in all groups. Conclusions: Intraoperative doxapram hydrochloride treatment did not produce any significant respiratory response improvement during 1 MAC sevoflurane anesthesia.