Dosage plans and formulations for adenovirus type B.

Abstract

To provide methods for treating human patients with replicable oncolytic adenovirus.SOLUTION: Disclosed is a method comprising the steps of: systemically administering multiple doses of a parenteral formulation of a replicable oncolytic adenovirus of subgroup B in a single treatment cycle, where the total dose given in each dose is in the range of 1×1010-1×1014 viral particles, and where each dose of virus is administered over a period of 1 to 90 minutes, for example, at a rate of viral particle delivery in the range of 2×1010 particles per minute to 2×1012 particles per minute. The disclosure further extends to formulations of the oncolytic adenoviruses and combination therapies of the viruses and formulations with other therapeutic agents.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates, for example, to a method of treating a patient with a replicable oncolytic adenovirus, using a dosing regimen designed to give the virus an appropriate therapeutic effect and / or minimize in vivo adverse events. This disclosure also extends to the formulations described herein, methods for preparing such formulations, and the use of such formulations in particular in the treatment of cancer. [Background technology]

[0002] Cancer is a leading cause of death and a serious disease worldwide. There are more than 200 different types of cancer, and the type of treatment depends on the type of cancer. Typically, treatment involves surgery, chemotherapy, and / or radiation therapy. These treatments are often unsuccessful or only partially successful and have serious side effects. The five-year survival rate for cancer ranges from less than 5% to over 95%, depending on the type of cancer (CRUK statistics, 2000-2001). For example, in the UK, between 2005 and 2009, the five-year survival rate for patients with colorectal cancer, which accounts for 13% of all cancers in men and women in the UK, was approximately 55%. For patients with metastatic colorectal cancer, the five-year survival rate drops to 12%.

[0003] The management of metastatic cancer is primarily palliative, involving a combination of palliative surgery, chemotherapy, radiation, and supportive care. While clinical outcomes such as overall survival, response, and toxicity are important, alternative outcomes such as progression-free survival, quality of life, convenience, acceptability, and patient choice are also crucial. Clearly, new therapies are needed to improve these outcomes.

[0004] Cancer cells acquire specific mutations during alteration that make them more tolerant to viral infection. Cancer cells induce suppression of the host's antitumor activity. Changes within tumor cells and the local microenvironment create potential vulnerabilities that expose tumors to viral infection (Liu et al. 2007; Liu et al. 2008; Roberts, 2006).

[0005] There is a long history of using viruses to treat cancer, beginning with case reports of temporary remission of cancer after natural viral infection or viral vaccination. The earliest report is probably from 1912, describing the regression of cervical cancer in patients who received rabies vaccinations. Similar results were seen in cancer patients who received smallpox vaccinations or those who had naturally occurring viral infections such as mumps or measles. Based on these reports and animal data, the inoculation of patients with live viruses for cancer treatment began in the late 1940s and early 1950s.

[0006] However, the usual experience was that after a temporary tumor regression, the tumor would grow again, and the patient would die. Long-term complete remission rarely resulted from these vaccinations. Albert B. Sabin, MD, who developed the raw oral polio vaccine in 1957, commented: "The most unfortunate aspect is the fact that the virus is oncolytic, and even if we pierce the tumor, the individual's immune response to the virus is so rapid that the effect quickly disappears, and the tumor continues to grow."

[0007] Currently, while the number of oncolytic adenoviruses has been identified, the only virus approved for clinical use worldwide to date is Oncorine (H101), a subgroup C adenovirus modified by E1B-55KD that is conditioned to replicate in P53-deficient cancer cells (H101 is a close analogue of ONYX015, as described by Bischoff et al. 1996). Oncorine is administered by intratumoral injection for head and neck cancers.

[0008] Talimogene laherparepvec (Tvec) is an oncolytic virus based on herpes simplex virus type 1 lacking ICP34.5 and ICP47, expressing US11 as its initial gene and encoding GM-CSF. The OPTiM trial was a multinational, open-label, randomized trial designed to evaluate the efficacy and safety of treating patients with unresectable stage III (bc) and IV (M1a-c) disease with talimogene laherparepvec as an intratumoral therapy compared to subcutaneous administration of GM-CSF. In the interim analysis, the sustained response rate was 16% with talimogene laherparepvec compared to 2% with GM-CSF. Other oncolytic viruses currently under development for intratumoral administration are listed below (Sheridan 2013). • Reolysin, serotype 3 (Daring strain) of oncolytic reovirus • PV701, oncolytic Newcastle disease virus • CG0070, a conditionally replicating adenovirus encoding GM-CSF. • Pexastimogene devacirepvec (Pexa-Vec, JX-594), a thymidine kinase-deficient vaccinia virus encoding GM-CSF. • Cavatak, unmodified Coxsackievirus A21 Conditional herpes simplex virus type 1 lacking Seprehvir (HSV1716) and ICP34.5 • DNX-2401, a conditionally replicating adenovirus encoding an integrin-binding peptide. • CGTG-102, a conditionally replicating adenovirus encoding GM-CSF.

[0009] ColoAd1 is a chimeric (Ad11 / Ad3) serogroup B adenovirus developed using a directional evolutionary process and is considered suitable for the treatment of epithelial-derived cancers and metastatic forms, including colorectal cancer (Kuhn, I et al. 2008).

[0010] To date, clinical trials of oncolytic viruses have primarily focused on intratumoral injection of the virus. A review of clinical trials by Aghi & Martuza (2005) found that 25 out of 36 trials used intratumoral injection for viral administration. However, this method is only practical when treating easily accessible tumors and patients where tumor structures such as stromal tissue and necrotic areas within it do not restrict the delivery of viral nucleic acids within the tumor (Ries & Korn 2002).

[0011] Cancer deaths are often a result of inaccessible tumors or metastases. Oncolytic viruses administered intratumor rely on systemic dissemination from the tumor to these secondary tumors. However, dissemination is transient and often proves ineffective (Ferguson et al. 2012).

[0012] Therefore, intratumoral injection is suitable for only a limited number of cancers and is not suitable for treating many metastatic cancers, for example.

[0013] Intravenous administration of oncolytic viruses is commonly used in relation to acute toxicity and rapid clearance. For example, in the case of group C adenovirus Ad5, whose uptake is mediated by the ubiquitous sackievirus receptor (CAR), rapid hepatic clearance and immunological neutralization are well documented, while adverse effects including acute hepatotoxicity, influenza-like illness, and hematological changes are regularly reported.

[0014] The currently established view is that repeated administration is necessary to achieve and maintain effectiveness. For all oncolytic cancer treatments studied, treatment is generally expected to be chronic, involving repeated administrations over weeks, months, or even years. For example, in the case of PV701, treatment in at least one patient continued for approximately 10 months, with a 6-day interval between the end of one treatment cycle and the start of the next.

[0015] Neumanatis et al. (2001) reported the administration of intravenous infusion of ONYX-015 to cancer patients in cycles of up to 24 weeks. In an ongoing Phase III clinical trial (Clinicaltrials.gov identifier NCT01708993), Reolysin® (oncolytic reovirus) was infused for 1 hour from day 1 to day 3 until progression, and then every 3 weeks thereafter. In a recently reported Phase I clinical trial (Clinicaltrials.gov identifier NCT01380600), JX-594 was administered intravenously every 2 weeks over four cycles, and in a second ongoing Phase I / II clinical trial (Clinicaltrials.gov identifier NCT01394939), JX-594 was administered intravenously weekly for 5 weeks, followed by three intratumoral boosts for liver metastases in patients with metastatic colorectal cancer. In the ongoing OPTIM clinical trial, talimogene laherparepvec was administered intratumorally every two weeks for up to 18 months (Clinicaltrials.gov identifier NCT00769704). [Overview of the Initiative]

[0016] A first aspect of this disclosure provides a method for treating a human patient, the method including: Multiple doses of a parenteral formulation of subgroup B replicable oncolytic adenovirus are administered systemically in a single treatment cycle. The range of the total dose given for each administration is 1 × 10 per dose. 10 ~1 × 10 14 These are individual virus particles, Each dose of the virus has a viral particle delivery rate of 2 × 10⁻¹⁶ per minute. 10 ~2×10 12 It is administered so as to be within the range of particles.

[0017] In an independent aspect, this disclosure relates to ColoAd1 used to treat ovarian cancer, for example, by administering a therapeutically effective dose of ColoAd1 to a patient with ovarian cancer using, for example, the dosing regimen described herein.

[0018] In a further independent aspect, the present disclosure relates to a combination therapy comprising a chemotherapeutic agent that does not inhibit oncolytic adenovirus such as ColoAd1 and adenovirus activities such as in vivo virus replication.

[0019] In one embodiment, the combination therapy is used in the treatment of cancers particularly described herein, specifically colorectal cancer or ovarian cancer (including its metastatic forms).

[0020] In one embodiment, ColoAd1 in the combination therapy is administered according to the dosing schedule described herein.

[0021] Also provided is a parenteral formulation of a subgroup B replicable oncolytic adenovirus for use in the treatment described herein.

[0022] The present disclosure also extends to the use of a parenteral formulation of a subgroup B replicable oncolytic adenovirus for the manufacture of a medicament described herein and for use in the treatment described herein.

[0023] In one aspect, also, 1×10 10 ~1×10 14 A single dose of virus particles of subgroup B replicable oncolytic adenovirus in the range of, for example, 6×10 12 is provided.

[0024] Also provided is an infusion or infusion rate for administering 2×10 9 ~2×10 12 virus particles (VP) per minute, for example, 1.5×10 11 VP per minute.

Brief Description of the Drawings

[0025] [Figure 1] Shows the cytotoxicity profile of A549 cells of ColoAd1 in the presence of fresh human whole blood. [Figure 2] In vivo distribution of 1e11 (1×1011) particles of ColoAd1 in normal BalbC mice 24 hours after injection. [Figure 3] In vivo distribution of ColoAd1 and ColoAd1CJ132 in CD46 transgenic mice 1 hour and 72 hours after injection. [Figure 4] Clearance dynamics of ColoAd1 from major organs, liver, spleen, and lungs, in CD46-expressing mice after 65 days. [Figure 5] The dynamics of ColoAd1 in mice, regardless of whether serum neutralization was administered concurrently. [Figure 6] Cytokine levels at the beginning and after equivalent therapeutic doses in preclinical toxicity studies of CD-1 mice. [Figure 7A] Cytokine levels (ng / L) over time (TNF(A), gamma interferon(B), and IL6(C)) in human cancer patients with metastatic solid epithelial tumors after intravenous administration of ColoAd1. [Figure 7B] Same as above [Figure 7C] Same as above [Figure 8A] Systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients with metastatic solid epithelial tumors. [Figure 8B] Systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients with metastatic solid epithelial tumors. [Figure 9-1] A shows the systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients administered 1e10 (1 × 10¹⁰) ColoAd1 virus particles over a 5-minute period. B shows the systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human patients administered 1e11 (1 × 10¹¹) ColoAd1 virus particles over a 5-minute period. [Figure 9-2]C represents the systemic pharmacokinetics of ColoAd1 (genome copy number per mL of blood) in human patients administered 1e12 (1 × 10¹²) ColoAd1 virus particles over a 5-minute period. D represents the systemic pharmacokinetics of ColoAd1 (genome copy number per mL of blood) in human cancer patients administered 1e13 (1 × 10¹³) ColoAd1 virus particles over a 5-minute period. [Figure 9-3] E represents the systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients administered 3e12(3 × 10¹²) ColoAd1 virus particles over 5 minutes. F represents the systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients administered 3e12(3 × 10¹²) ColoAd1 virus particles over 20 minutes. [Figure 9-4] Systemic pharmacokinetics of ColoAd1 (genome copy number per 1 mL of blood) in human cancer patients administered 6e12 (6 × 10¹²) ColoAd1 virus particles over a 40-minute period. [Figure 10] The longer the infusion of the same dose, the lower the cMax level at the end of the infusion (Cohort 5 vs. Cohort 6). [Figure 11] Time-course MCP1 levels (ng / L) in human cancer patients with metastatic solid epithelial tumors after intravenous administration of ColoAd1. [Figure 12] A schematic diagram showing the ColoAd1 replication cycle in a cell. [Figure 13] ColoAd1 infection of cancer cells, as shown by nuclear staining in a colon cell line after in vitro viral infection. [Figure 14] A shows nuclear staining (Hexon staining) of ColoAd1 in colon tissue from a colorectal cancer patient after administration of ColoAd1 via intravenous injection (IT). B shows isotype control staining of A. C shows colon tissue without nuclear staining in stromal cells (Hexon staining) (after intravenous administration of ColoAd1 to a colorectal cancer patient). D shows isotype control of C. [Figure 15] 320 compounds (clinically approved or in development) whose effects on viral replication were analyzed. [Figure 16-1] An in vivo mouse model demonstrating the efficacy of paclitaxel and ColoAd1 combination therapy (and control). [Figure 16-2] Same as above [Figure 17] In vivo data on ColoAd1 and chemotherapy in mouse models [Figure 18] In vivo data on ColoAd1 and chemotherapy in mouse models [Modes for carrying out the invention]

[0026] In one embodiment, the dose is 1 × 10 10 ~1 × 10 13 The range, for example, 1 × 10 10 ~1 × 10 12 These are virus particles.

[0027] In one embodiment, the total dose administered in one treatment cycle is 1 × 10⁻⁶ 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 or 9 x 10 12 These are virus particles.

[0028] In one embodiment, the total dose administered in one treatment cycle is 6 × 10 12 These are virus particles.

[0029] The inventors hypothesize that the most crucial factor for effectiveness is establishing a productive infection within the tumor in the early stages, for example, before an antiviral immune response occurs.

[0030] Therefore, the administration plan needs to strike a balance between delivering sufficient virus and generating sufficient plasma levels of virus for a sufficiently long time to disseminate viral infection to cancer cells, while, for example, not causing (or minimizing) toxicity and serious adverse events in the patient.

[0031] The inventors were the first to demonstrate that tumor infection with adenovirus type B can be established by the dose of viral particles administered intravenously. Viral infection in the nuclei of cancer cells in patients with colorectal cancer treated with ColoAd1 intravenously is also shown when the hexon in the cells is stained and when independently analyzed by PCR. The presence of the virus in the nuclei indicates that the viral life cycle is in progress and that the increased viral load in the patient allows the virus to replicate.

[0032] When administering oncolytic adenovirus systemically to a patient, several administration variables must be considered. These administration variables include, but are not limited to, the route of viral administration, the dose of the virus, the rate of viral administration at each dose, the interval between individual viral doses in a given cycle, the number of viral doses per treatment cycle, the interval between treatment cycles, the number of treatment cycles, and finally, the use of any concomitant medications or other supportive therapies used to enhance efficacy and minimize adverse events. Each administration parameter, in turn, depends on the specific characteristics of the type of oncolytic virus being investigated.

[0033] Important parameters include, but are not limited to, the relative degree and binding affinity of the virus to tumor cells compared to non-tumor cells, the relative selectivity and capacity of the virus in tumor cells compared to non-tumor cells, and the rate of viral uptake and clearance by specific or nonspecific binding of the virus to reticuloendothelial cells (e.g., hepatic Kupffer cells) and blood components.

[0034] These key parameters are, in turn, driven by important physical and phenotypic characteristics of a particular viral type, but are not limited to, viral receptor specificity, the charge carried on the viral coat, the presence or absence of an envelope, viral particle size, viral particle immunogenicity, viral particle inflammatory capacity, viral tumor specificity, viral replication rate, and viral killing capacity.

[0035] Therefore, the appropriateness of any given administration regimen varies depending on the different types of viruses, and the most appropriate regimen may be specific to the type of virus being administered. For example, Zhang et al. (2012) described an Ad5-Ad48 chimeric virus created to reduce hexone binding to blood coagulation factor X in order to eliminate hepatic isolation, enhance circulation, and reduce toxicity while maintaining antitumor activity. Similarly, Shashkova et al. (2009) described significant differences among wild-type human adenovirus serotypes 5, 6, 11, and 35 when studied as potential anticancer agents. Therefore, since different viral types are expected to behave with significant differences when systemically administered to humans, the optimal drug administration strategy cannot be predicted a priori without in vivo data and preferably supporting clinical data.

[0036] The dosing regimens described herein may be particularly suitable for achieving this for group B adenoviruses compared to current practices such as more regular intervals and longer-term repeated administrations.

[0037] The goal of an optimized dosing plan for a given oncolytic adenovirus is to maximize viral delivery to tumor cells while minimizing both side effects (adverse events) and the induction of antiviral immunity. This is to create an appropriate risk-benefit therapy profile while allowing for repeated viral administrations as appropriate for treatment. Optimized dosing plans thus differ between viral types, particularly between adenovirus subtypes due to differences in the viral coat.

[0038] Prior art based on Ad5, a subgroup C adenovirus, has seen extensive research on coxsackie adenovirus receptor-mediated infectivity. When delivered systemically, over 90% of the delivered dose is taken up by the liver. Rapid and widespread loss to the liver reduces tumor uptake of the virus, thereby diminishing therapeutic efficacy. The majority of this dose is taken up by cytokines that produce innate immune cells, such as Kupffer cells, which specialize in macrophage residentization in the liver. Ad5 also exhibits hepatotoxicity, causing Kupffer cell necrosis and subsequent depletion.

[0039] Shoshkova et al. (2009) showed that Kupffer cell depletion by Ad5 increased the level of hepatocyte transmission upon subsequent delivery of the Ad5 vector, and suggested that the elucidated mechanism for Ad5 is not necessarily related to adenoviruses from subgroup B, such as Ad11 and Ad35. Their data suggest that while no subgroup B adenovirus is recognized by Kupffer cells to a significant extent, or does not cause death in these cells, subgroup C adenoviruses similarly interact with Kupffer cells. In particular, Shoshkova suggests that pre-administration of an Ad11-based virus does not have the same beneficial effect on Kupffer cells as Ad5. This paper concludes that while some binding may occur for subgroup B adenoviruses (including Ad11), this effect is actually minimal.

[0040] While not wanting to be constrained by theory, the inventor believes, contrary to prior art proposals, that cytokines that produce innate immune cells such as Kupffer cells may play a role in the clearance of subgroup B adenoviruses.

[0041] Furthermore, the binding of blood coagulation factor X to Ad5 hexone is a mechanism of hepatocyte infection, and this mechanism may also be related to other adenoviruses in vivo (see, for example, Molecular Therapy vol.17 no.10, 1683-1691 October 2009), but it is not generally a mechanism of hepatic uptake for adenoviruses from subgroup B.

[0042] The globally high seroprevalence of Ad5 (high Ad5 neutralizing antibody titers in the human population) and certain other adenovirus serotypes raise significant concerns about the systemic application of adeno-based therapies with high seroprevalence. This is because these bloodborne viruses may be neutralized by existing antibodies (Vogels et al., Journal of Virology, Aug 2003 Vol 77, No.15 pp 8263-8271).

[0043] Adenovirus subgroup B has certain inherent advantages, namely its association with low seroprevalence (Stone et al., Journal of Virology 2005 Vol 79 No.8, pp. 5090-5104) and its low inflammatory capacity. Therefore, the initial dose can be far more efficient than, for example, that of Ad5. However, its ability to evade the immune system after systemic delivery can be a concern for repeated administration. Thus, even with local suppression of the immune system by cancer, immune system evasion is likely the greatest obstacle to the long-term success of oncolytic virus therapy based on adenovirus subgroup B.

[0044] The data generated by the inventors support the position that the therapeutic effect of subgroup B oncolytic adenoviruses can be improved, and / or that the elimination of adenovirus neutralization by the immune system can be minimized by using an appropriate dosing regimen.

[0045] In one embodiment, the drug regimen of this specification can also minimize side effects such as flu-like symptoms and inflammatory responses.

[0046] In one embodiment, self-replicating adenovirus can be repeatedly administered during an initial "administration window" before a specific antiviral immune response occurs, and a later administration window can be utilized again when the specific antiviral immune response has declined once more. That is, it would consist of several treatments over a short period before initiating a subsequent treatment cycle.

[0047] An advantage of this approach is that by administering self-replicating adenovirus in this manner, the viral blood concentration establishes an auto-amplifying infection within the tumor (known to be an immunosuppressive environment), thereby potentially avoiding the need for chronic repeated administration of oncolytic viruses. To establish an auto-amplifying infection within the tumor, it is beneficial to maintain viral levels in the patient's bloodstream at a level exceeding the effective infection concentration as much as possible, without causing adverse events. This concept is similar to identifying the therapeutic window for the virus, i.e., the dose range or administration plan in which the therapeutic effect is optimized and side effects are minimized.

[0048] This can be achieved by optimizing both the dosage and the rate of viral infusion. In one embodiment, the rate of viral infusion is equal to or greater than the rate at which the body clears the virus.

[0049] Once infection is established within the tumor, the virus is relatively protected from neutralizing antibodies and is given a potentially tolerable environment that allows it to repeatedly produce therapeutic effects without dose-limiting toxicity.

[0050] In addition, the viral concentration C( max The inventors hypothesize that the peak in ) is the cause of side effects, and that a flat pharmacological profile is desirable.

[0051] In one embodiment, C max For example, 3 x 10 per 1 ml 8 It is maintained below a certain value, such as DNA copies. maxThe level is likely to induce serious adverse events or toxicity in some patients.

[0052] In one embodiment, the injection rate has a greater influence than the absolute amount of virus administered.

[0053] Based on data generated at clinics, the virus was found to be expelled at a rate exceeding its clearance rate, for extended periods of up to 72 hours or more, at a rate of up to 1.5-2 × 10⁶ per minute. 11 The virus particles (i.e., the total dose of the virus is 6 × 10) 12 We believe that we can deliver the virus particles to patients without inducing serious adverse events.

[0054] In one embodiment, the viral genome in the blood is C max This is 3 x 10 per 1 ml of blood. 8 It is maintained at a sub-genomic level.

[0055] We evaluated the initial rate of viral clearance under several scenarios and believe the estimated α-half-life is in the range of 18 minutes.

[0056] The use of prophylactic anti-inflammatory agents during oncolytic virus therapy is controversial. On the one hand, it has been suggested that their use can minimize adverse events and thus improve the tolerance of oncolytic Newcastle disease virus (Lorence et al., 2007). On the other hand, there are reports that fever may be associated with the enhanced oncolytic effect of adenovirus (Yu et al., 2007).

[0057] The inventors have found that the use of prophylactic or therapeutic agents (including anti-inflammatory drugs, steroids, antiemetics, antidiarrheals, or analgesics) administered during this treatment cycle can improve the tolerability of this dosing regimen, particularly by enabling higher doses or more frequent administrations.

[0058] In one embodiment, steroids are administered between treatment cycles.

[0059] Thus, we hypothesize that these six parameters, used individually or collectively, are crucial in achieving the goal of proper delivery of oncolytic subgroup B adenoviruses. a) The number of viral particles administered at each dose, b) The rate at which each virus dose is administered (number of virus particles delivered per minute) c) The number of individual doses of the virus in the treatment cycle, d) The interval between each individual dose within the treatment cycle, e) Use of prophylactic anti-inflammatory drugs between treatment cycles, and f) The period between treatment cycles. These parameters can be adjusted to each other; for example, if the dosage is increasing, it can be administered at a slower infusion rate to offset the adverse effects of the increase.

[0060] If the dose is too low, the level of viral particles may not be sufficient to establish effective infection of cancer cells. If the infusion rate is too slow, the viral particles may then be easily cleared by natural viral sinks (e.g., cytokines that produce innate immune cells such as hepatic Kupffer cells or blood components), and effective infection of cancer / tumor cells may not be achieved. If the viral dose is too high and / or the infusion rate is too fast, the high concentration of viral particles may increase the number of adverse events. The latter may induce an inflammatory cytokine response, increasing the side effects experienced by the patient. A moderate infusion rate can optimize the dose delivered.

[0061] On average, the clearance rate of type B adenoviruses such as ColoAd1 is approximately 18 minutes, with an α-half-life of about 18 minutes.

[0062] A single dose of the virus may not be able to establish infection, but it can adequately occupy or eliminate the viral sink (e.g., cytokines that produce innate immune cells such as hepatic Kupffer cells or blood components). If the viral sink is adequately occupyed or eliminated, and the next dose is administered immediately thereafter, the kinetics of the virus can be altered for subsequent doses, resulting in a longer circulating half-life and / or higher peak plasma levels. In this case, one or more doses administered immediately after the initial dose may more effectively establish effective infection of cancer cells.

[0063] However, if the interval between doses is too long (e.g., more than 14 days), the viral sink may have time to replenish, the benefits of the previous dose may be lost, and / or a specific antiviral immune response may occur. For example, depletion of cytokine-producing innate immune cells such as hepatic Kupffer cells in this dosing regimen can have a significant secondary benefit in that it can significantly reduce the release of Kupffer-mediated cytokines with respect to subsequent viral doses, allowing these doses to be well tolerated even when faced with higher plasma levels of the virus.

[0064] Thus, the inventor proposes managing a predetermined treatment cycle over a relatively short period, as described below, for example.

[0065] From the inventor's completed research, it appears that for group B adenovirus, multiple doses in a treatment cycle, with each dose administered over relatively short periods, infused at a moderately rapid rate, optionally in combination with prophylactic agents, and spaced out over relatively short intervals, is suitable for infecting cancer cells with oncolytic adenovirus type B with minimal toxicity.

[0066] The treatment cycle may be repeated as needed.

[0067] We monitor inflammatory cytokines TNF, gamma interferon, IL-6, and MCP-1 as markers of acute toxicity, and believe that the second or subsequent doses reduce toxicity and increase the virus's ability to infect cancer cells in each case. This is because, if these doses are administered at appropriate levels, rates, and frequencies, non-cancerous viral sinks are cleared or occupied by both the first and second doses.

[0068] In one embodiment, three doses are used in a treatment cycle, and in a further embodiment, three or more doses are used in a treatment cycle.

[0069] In one embodiment, a single dose is administered on any or all of the 1st, 3rd, 5th, 14th, and 21st days.

[0070] In another embodiment, follow-up doses are administered as maintenance or booster doses, for example, every other week, weekly, every two weeks, or every three weeks, weekly, or every three weeks, for an appropriate period, particularly while the treatment is beneficial to the patient as maintenance therapy, for example, while the patient remains in remission.

[0071] Those skilled in the art will understand that various modifications to the treatment cycle can be made according to the individual patient's needs.

[0072] This disclosure also extends to subgroup B replicable oncolytic adenoviruses used to treat human patients by administering at least one dose systemically, such as multiple doses of parenteral formulations containing adenovirus during a single therapeutic cycle, with a total dose of 1 × 10⁶ in each dose. 10 ~7×10 12 For example, 1 × 10 10 ~5×10 12 The virus particles are within range and are administered over a period of 1 to 90 minutes.

[0073] In a further embodiment, the disclosure extends to the use of subgroup B replicable oncolytic adenoviruses for the treatment of human patients by administering at least one dose systemically, such as multiple doses of a parenteral formulation containing adenovirus during a single therapeutic cycle, with the total dose in each dose being 1 × 10⁻⁶ 10 ~1 × 10 13 For example, 1 x 10 10 ~7×10 12 For example, 1 × 10 10 ~5×10 12 , or 6×10 12 The virus particles are within range and are administered over a period of 1 to 90 minutes.

[0074] In one embodiment, the first dose in a given cycle of treatment is lower than the dose administered in subsequent treatments of the cycle.

[0075] Contrary to Shoshkova's suggestion based on mouse studies, administration of Ad11-based viruses does not appear to have a beneficial priming effect on cytokine-producing innate immune cells such as Kupffer cells. In practice, optimizing the dose and timing of administration of group B oncolytic adenoviruses may be used to minimize side effects and therefore may be beneficial.

[0076] In one embodiment, the dosage is, for example, 6 × 10 over a period of 20 to 60 minutes, such as 40 minutes. 12 That is the case.

[0077] In one embodiment, higher first and second doses (i.e., corresponding to normal therapeutic doses) are desirable to completely occupy cytokine-producing innate immune cells such as Kupffer cells (and / or other viral sinks), and thus optimize the delivery of the next dose. In other words, the first and second doses may be equal.

[0078] In one embodiment, the total dose administered contains an equal number of virus particles. This can be particularly advantageous in that it simplifies the manufacture of the viral formulation, reduces the risk of drug administration errors, and can actually provide a very effective treatment plan.

[0079] In one embodiment, a follow-up treatment cycle is set up one to six months, for example, two, three, four, or five months after the completion of the previous treatment cycle, in order to allow the immune response to decay.

[0080] In one embodiment, the follow-up cycle may be a single dose administered weekly or bi-weekly, over a period of 1 month to 5 years, such as every 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, or 36 months.

[0081] In one embodiment, the follow-up treatment cycle is initiated approximately 14 days after the last dose of the initial treatment cycle.

[0082] The follow-up cycle may also act as a maintenance dose, thereby helping to maintain a level of viral load sufficient to provide a therapeutic effect.

[0083] In one embodiment, there may be one, two, three, four, five or more treatment cycles, for example, one or two.

[0084] In one embodiment, there is only one treatment cycle, and no subsequent treatment cycles.

[0085] In one embodiment, a liquid parenteral formulation for infusion or injection of a replicable oncolytic subgroup B adenovirus (e.g., ColoAd1) is provided, wherein the formulation comprises 6 × 10⁶ units per dose. 12 Virus particles, etc., 1 x 10⁶ per dose 10 ~1 × 10 14 A range of doses for viral particles is provided.

[0086] Also, for example, 6 x 10 per dose 12Also disclosed is a method of treating a patient by administering a parenteral formulation according to this disclosure, including viral particles, in the dosages described herein, that is capable of replicating oncolytic subgroup B adenovirus.

[0087] The disclosure also discloses a method of treating a patient by administering a parenteral formulation of the present disclosure containing a replicable oncolytic subgroup B adenovirus, the method comprising co-administering to the patient one or more substances or agents selected from the group including anti-inflammatory, steroid, antihistamine, antipyretic, and liquid for hydration.

[0088] Also disclosed is a method for determining an appropriate time to administer to a patient the next cycle of a parenteral formulation according to the Disclosure, which contains a replicable oncolytic subgroup B adenovirus, the method comprising the steps of determining the existing titer of patient-specific antiviral immunity prior to a first treatment cycle, successively determining patient-specific antiviral immunity after the first treatment cycle, and delaying the next treatment cycle until the patient-specific antiviral immunity has decreased to a pre-specified baseline percentage.

[0089] As used herein, the term “determine sequentially” means determining a patient’s antiviral immunity at multiple time points, which may be regularly or irregularly spaced. The multiple measurements obtained can be used, for example, to generate an average titer over a specific period.

[0090] As used herein, the term “pre-specified baseline percentage” means viral titer defined as a threshold or limit value for a particular patient, taking into account factors such as baseline measured before the initiation of treatment, patient prognosis, ongoing cancer treatment, and adverse side effects.

[0091] In one embodiment, the “pre-specified baseline percentage” is 90% or less of the patient’s baseline viral titer, for example, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.

[0092] In the alternative embodiment, no test is performed before administering the next treatment cycle.

[0093] One embodiment provides the use of a glass or plastic syringe having an internal volume in the range of 3 to 50 milliliters, wherein the syringe is 1 × 10 10 ~1 × 10 14 For example, 1 × 10 10 ~7×10 12 (Use 1 × 10 10 ~6×10 12 or 1 × 10 10 ~5×10 12 , or 1 × 10 10 ~4×10 12 , or 1 × 10 10 ~3×10 12 , or 1 × 10 10 ~2×10 12 , or 1 × 10 10 ~1 × 10 12 The formulation comprises a parenteral preparation containing viral particles of a subgroup B replicable oncolytic adenovirus, the preparation being sterile, filled into a syringe under sterile conditions, and used for therapeutic purposes, specifically to manufacture a drug that can be injected or intravenously administered to a human subject.

[0094] Those skilled in the art understand, for example, that the formulation may contain excess virus particles to compensate for virus particles that may be adhering to the surface of a syringe and that are not subsequently administered.

[0095] One advantage is that the pre-filled syringe is likely to significantly improve the usability and cost-effectiveness of the manufactured subgroup B oncolytic adenovirus by eliminating the need to prepare doses in a specialized pharmacy using specialized equipment (including extraction hoods) and trained personnel.

[0096] This disclosure also extends to pre-filled vials of the formulation, specifically, vials containing single doses, to the extent defined herein.

[0097] In one embodiment, the viral preparation is provided in a concentrated form, such as a concentrate, suitable for dilution with physiological saline, glucose, or a sterile isotonic diluent to resemble a topical application before administration to a patient.

[0098] One advantage is that the dosing regimen described herein is suitable for delivering a therapeutically effective dose of subgroup B oncolytic viruses to cancer targets. In particular, the dosing regimen described herein can minimize the neutralization and / or clearance of oncolytic viruses by cytokine-producing innate immune cells and the immune system, such as hematopoietic agents, synchrocellular and Kupffer cells. The latter leads to increased availability of therapeutic doses of oncolytic viruses and, overall, to improved prognosis and / or survival in patients. Another advantage is that this regimen can also provide an improved quality of life for patients by minimizing adverse events and / or side effects during treatment.

[0099] In one embodiment, patients receiving the treatment described herein have an increased survival rate compared to patients receiving current standard treatment at the time of filing, for example, showing a statistically significant increase in survival rate.

[0100] In one embodiment, patients receiving the treatment described herein experience a reduction in tumor burden compared to patients receiving standard treatment at the time of filing, for example, a statistically significant reduction in tumor burden.

[0101] In one embodiment, patients receiving the treatment described herein have an increased likelihood of entering remission compared to patients receiving standard treatment at the time of filing, for example, showing a statistically significant increase in remission.

[0102] In one embodiment, the amount or extent of metastasis is reduced, for example, patients receiving the treatment described herein show a statistically significant reduction in the amount or extent of metastasis compared to patients receiving standard treatment at the time of filing.

[0103] While we do not wish to be bound by theory, even if prior art suggests otherwise, it is conceivable that mononuclear phagocytic cells, particularly cytokine-producing innate immune cells such as Kupffer cells, may be involved in the clearance of type B oncolytic viruses from circulation.

[0104] Furthermore, studies of mice conducted according to the present invention suggest that, for example, when administered in a short time frame after the second dose, or when lower toxicity or both may be observed, cytokine-producing innate immune cells such as Kupffer cells may be depleted or occupied after the first or second dose of the treatment plan, preventing efficient clearing of the third and subsequent doses. When administered within a relatively short period, cytokine levels do not significantly increase after the second or third dose compared to the first dose, and the cytokine markers are assumed to indicate the latter. The inventors take the position that the mechanism for clearing the virus may be suppressed after the first and second doses.

[0105] While studies in mice, particularly with viruses, are not always equivalent to what is observed in human systems, in this example, human observations appear to correlate well with those in the mouse model developed by the inventors. The effects of administration regimens on cytokine responses and ColoAd1 pharmacokinetics are also illustrated by the inventors in human subjects.

[0106] As used herein, “method of treating a patient by systemic administration” means a method of administering a therapeutic agent to a human being in order to achieve the transfer of the entity into the patient’s circulatory system, and the treatment is intended to prevent, slow, improve or cure the progression of malignant tumors such as cancer or complications or related symptoms by direct administration into the circulatory system, for example, by intravenous administration.

[0107] In one embodiment, systemic delivery offers the opportunity to treat primary tumors, obviously inaccessible or undiagnosed tumors, and / or metastases. This is particularly advantageous as it leads to a better overall prognosis for the patient and / or improved survival rates.

[0108] Therefore, as used herein, systemic delivery does not refer to treatment localized within a tumor or body cavity such as the abdominal cavity. Examples of systemic delivery include intravenous infusion, intramuscular injection, and subcutaneous injection.

[0109] Parenteral formulations refer to formulations designed not to be delivered via the gastrointestinal tract or by local administration. Typical parenteral delivery routes include injection, implantation, or infusion. In one embodiment, the formulation is provided in a form for bolus delivery.

[0110] In one embodiment, the parenteral formulation is in the form of an injection. Injections include intravenous, subcutaneous, intratumoral, or intramuscular injections. As used herein, injection means the insertion of a fluid into the body via a syringe. In one embodiment, the method of this disclosure does not require intratumoral injection. Injections generally involve administering a fluid of 150 mL or less over a short period of time, for example, 1.5 minutes or less.

[0111] In one embodiment, the formulation is delivered into the peritoneal cavity.

[0112] Intracranial injections may be necessary for brain metastases from head and neck cancer or epithelial cancer.

[0113] In one embodiment, the parenteral formulation is in the form of an infusion.

[0114] As used herein, infusion means administering a fluid at a slower rate by drip infusion, infusion pump, syringe pump, or syringe pump or equivalent device. In one embodiment, the infusion is administered over a period ranging from 1.5 minutes to 90 minutes, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

[0115] In one embodiment, the dosage of the formulation is 100 mL or less, particularly 50 mL or less, such as 30 mL, 10 mL, 3 mL, or 5 mL or less, administered by a syringe pump, etc. The latter can be called a slow infusion.

[0116] In one embodiment, the injection is delivered at a rate within the range of 0.5 to 6 ml per minute, for example, 0.75 ml per minute.

[0117] In one embodiment, the injection is performed at a rate of 2 × 10⁻⁶ per minute. 9 ~2×10 12 Virus particles (VP), for example, 1.5 × 10⁻¹⁶ per minute 11 He is a VP (Voice of Professional).

[0118] In one embodiment, the injection is administered as a slow infusion, for example, over a period of 1.5 to 30 minutes or 1.5 to 40 minutes.

[0119] In one embodiment, the formulation is intended for intravenous administration. This route allows for rapid access to a large portion of organs and cells and is particularly effective for delivering oncolytic viruses because it is useful for treating metastases, such as established metastases located in highly angiogenic regions, particularly the liver and lungs.

[0120] In one embodiment, the combination of administration methods is used, for example, IV and intratumor, or intraperitoneal and intratumor, or IV and intraperitoneal.

[0121] Accordingly, in one embodiment, the systemic administration of the present disclosure may be used in combination with other administration routes, such as simultaneous or sequential intratumoral administration, for example, the first pre-treatment cycle may be intratumoral and the second treatment cycle may be systemic as described in the present disclosure. Alternatively, the first treatment cycle may follow the present disclosure, and subsequent cycles or boosts may be intratumoral as needed. The therapeutic formulations are typically sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for administration to humans, and in particular as a single dose, it may be formulated as a pre-filled device such as a syringe or vial.

[0122] In one embodiment, two or more doses may be used in a treatment cycle, for example, two, three, four, five, or six doses may be used in each treatment cycle, and may be provided, for example, as a kit.

[0123] Each dose administered in a given treatment cycle may be referred to as a treatment in this specification.

[0124] In one embodiment, a dose lower than the next dose administered in the cycle is used, for example, the lower dose may be in the range of 30-95% of the next single or multiple doses, e.g., 50, 60, 70, or 80%.

[0125] In one embodiment, it is desirable to use a first dose higher than the next dose administered in the cycle to completely occupy cytokine-producing innate immune cells such as Kupffer cells and to optimize the delivery of the next dose.

[0126] A higher dose means 100% or more of the next dose, for example, 110%, 115%, 120%, 125%, 130%, 135%, 140%, or 145% of the next dose, or for example, 105-150% of the next dose.

[0127] In one embodiment, one, two, three, or all doses contain a number equal to the number of viral particles. This can be particularly advantageous in that it simplifies the manufacture of the viral preparation and can actually provide a very effective treatment plan.

[0128] In one embodiment, the "same dose," i.e., the same number as the number of virus particles, is administered in one or more doses, such as the total dose of the treatment cycle. However, it may also be administered at different rates, for example, as described herein.

[0129] As used herein, a treatment cycle refers to a treatment period between rest periods in a treatment process that is repeated according to a schedule. A treatment cycle generally means multiple (at least two) treatments administered as part of a treatment program or schedule, typically lasting approximately 1 to 4 weeks, such as 3 weeks, 2 weeks, or 1 week. Generally, a given treatment cycle becomes part of a larger treatment plan.

[0130] In one embodiment, the treatment cycle is a period of 14 days or less, such as 10, 9, 8, 7, or 5 days, for example, 7 or 5 days.

[0131] In one embodiment, each additional single or multiple dose is administered at intervals of approximately 48 hours, such as every 40 to 56 hours. This is advantageous because it can be administered within a normal week of administration or within the setting of an outpatient.

[0132] In one embodiment, the first dose is administered on day 1, and the therapeutic dose is further administered every other day, such as on days 1, 3, 5, 7, 9, 11, and 13, or approximately every 48 hours, such as every 40 to 56 hours.

[0133] In one embodiment, the plasma viral level in the patient after administration (e.g., a second or subsequent administration) is at least 2 × 10⁶ per ml at a time of 15 minutes or more, for example, 20, 30, 40, 50, 60 minutes or more. 6 These are virus particles.

[0134] In in vitro tests conducted by the inventor (see Figure 1), virus particles in human whole blood at 37°C were killed, <2 × 10⁻⁶ 6 The particle size drops to less than 50% per milliliter. Furthermore, the inventors were able to demonstrate that when the viral genome level is, for example, 1.6e6 to 1e8 or higher, live viral particles are present in the patient's blood and can be consistently detected using a plaque assay.

[0135] In one embodiment, the interval between treatment cycles is at least 14 days.

[0136] The formulation generally includes a pharmaceutically acceptable diluent or carrier, such as a non-toxic, isotonic carrier compatible with the virus, where the virus is stable for the required period.

[0137] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, or polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained by the use of a dispersant or surfactant, such as lecithin or a nonionic surfactant such as polysorbate 80 or 40. The dispersion may also be aided in maintaining the required particle size by the presence of a surfactant. Examples of isotonic agents include sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0138] In one embodiment, a sterile isotonic diluent such as physiological saline or glucose (e.g., 5% glucose) is used.

[0139] In one embodiment, the parenteral formulation used in the method may contain one or more detergents, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, phosphate buffer and / or Tris buffer, sugars such as dextrose, mannose, sucrose, etc., salts such as sodium chloride, magnesium chloride, or potassium chloride, and nonionic surfactants such as briji®, PS-80, PS-40. The formulation may also contain a preservative such as EDTA ethanol or a combination of EDTA and ethanol, which is thought to prevent one or more possible decomposition pathways.

[0140] In one embodiment, the formulation comprises purified oncolytic virus, for example, 1 × 10⁶ per dose. 10 ~1 × 10 14 Virus particles, for example, 1 × 10⁶ per dose 10 ~7×10 12 Virus particles, especially 1 x 10⁶ per dose 10 ~1 × 10 12 Contains virus particles, and may contain excess amounts as needed.

[0141] In one embodiment, the formulation according to the present disclosure is 6 × 10 12 Contains virus particles.

[0142] In one embodiment, the virus concentration in the formulation is 2 × 10 8 ~2×10 14 It is in the range of vp / mL, for example, 2 × 10 12 It is vp / ml.

[0143] In one embodiment, the parenteral formulation contains glycerol.

[0144] In one embodiment, the formulation comprises an oncolytic adenovirus from subgroup B, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), glycerol, and a buffer.

[0145] In one embodiment, the parenteral formulation consists of a virus, for example 5 mM HEPES, for example 5-20% (v / v) glycerol, for example hydrochloric acid to adjust the pH to 7-8, and water for injection.

[0146] In one embodiment, 2 × 10 12 0.7 mL of ColoAd1 at a concentration of vp / mL is mixed in 5 mM HEPES and 20% glycerol with a final pH of 7.8.

[0147] Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents in the composition, such as monostearate and gelatin.

[0148] Therefore, the oncolytic adenoviruses used herein can be administered in time-release formulations, such as compositions containing sustained-release polymers. The oncolytic adenoviruses can be prepared with carriers that protect against neutralization and / or prevent rapid release, such as release-controlled formulations including implants and microencapsulated delivery systems.

[0149] For example, biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid / polyglycol copolymers (PLG) can be used. Biocompatible, non-degradable polymers such as polyethylene glycol and poly(N-(2-hydroxypropyl)methacrylamide) can also be used. Many methods for preparing such formulations are known to those skilled in the art.

[0150] Sterile injection solutions can be prepared, for example, by incorporating the oncolytic adenovirus in the required amount in a suitable solvent with one or a combination of the components described herein, and subsequently by sterilization by filtration. Generally, dispersions are prepared by incorporating the oncolytic adenovirus into a sterile vehicle containing a basic dispersion medium and other necessary components.

[0151] Generally, the parenteral formulations described herein are sterile liquid formulations, such as aqueous formulations, substantially free of particulate matter, and prepared and sterilized in a sterile manner, for example, by passing them through a 0.2 micron filter.

[0152] In one embodiment, the therapeutic parenteral formulation is administered, for example, using a sheath needle or cannula, to minimize contact of the formulation with the patient's epidermis. This precaution is thought to minimize the patient's immune response to the oncolytic virus, for example, by minimizing contact with Langerhans cells in the skin.

[0153] As used herein, replication ability refers to a virus that can replicate within a host cell. In one embodiment, replication ability encompasses both replication-capable and replication-selective viruses.

[0154] As used herein, replication ability refers to oncolytic adenoviruses that can replicate in human cells, such as cancer cells, without supplementing what is required by wild-type viruses, and without relying on, for example, defective cellular mechanisms. In other words, they are tumor-selective by infecting tumor cells preferentially over non-tumor cells. ColoAd1 is an example of a virus with replication ability.

[0155] As used herein, replication-selective or selective replication means an oncolytic adenovirus capable of replicating in cancer cells, using components that are specific to or upregulate defective cellular mechanisms within said cancer cells, such as p53 mutations, thereby achieving a degree of selectivity that exceeds that of healthy / normal cells.

[0156] As used herein, oncolytic subgroup B adenoviruses refer to adenoviruses from subgroup B that contain at least hexons and fibers (see Shenk et al. and Table 1) that preferentially infect and / or lyse tumor cells compared to normal cells. Thus, as used herein, oncolytic subgroup B adenoviruses include chimeric, mutant, or variant forms of group B adenoviruses, along with fibers and hexons, that retain oncolytic properties.

[0157] As used herein, adenoviruses or adenovirus serotypes refer to any currently known (51) or future isolated human adenovirus serotypes. See, for example, Strauss (1984) and Shenk (2001). Adenovirus serotypes are classified into subgroups, as shown in Table 1. Table 1 shows the classification of adenovirus serotypes.

[0158] [Table 1]

[0159] Examples of subgroup B viruses include Ad11 (wild type), such as Ad11a and Ad11p (Genbank deposit number: AF532578), and the chimeric adenovirus ColoAd1. The latter is disclosed in International Patent No. 2005 / 118825, and the complete sequence of the virus is provided in Sequence ID No. 1 of that patent.

[0160] Therefore, in one embodiment, the virus used in the method according to this disclosure is a chimeric virus.

[0161] As used herein, chimeric adenoviruses refer to adenoviruses having DNA from two or more different adenovirus serotypes, such as those produced using the method of International Patent No. 2005 / 118825, which is incorporated herein by reference.

[0162] In one embodiment, the chimeric adenovirus is ColoAd1. ColoAd1 is thought to kill tumor cells by a mechanism more closely similar to necrosis than to apoptosis (unpublished data produced at Oxford University). This has potential beneficial effects (Kirn et al. 2001; Small et al. 2006; Reid et al. 2002; Liu et al. 2007; Ferguson et al. 2012).

[0163] ColoAd1 has been shown to be potent in multidrug-resistant cancer cell lines and cancer stem cell-like cells that are known to be resistant to apoptosis. Inflammatory necrotic cell death may be appropriate due to the development of a specific anti-tumor immune response. ColoAd1 can enhance the ability of tumor cells to rapidly release and spread before target cells die.

[0164] ColoAd1 is a chimera of Ad11 and Ad3, but possesses an outer capsule that is completely homologous to that of Ad11. The viral dynamics of ColoAd1, such as its inflammatory capacity and immunological properties, are most closely similar to those of Ad11 and other subgroup B adenoviruses.

[0165] In one embodiment, the oncolytic virus used in the method of the present disclosure is deleted in or part of the E3 and / or E4 region. This may be beneficial because it allows the virus to replicate more rapidly in vivo.

[0166] Furthermore, the E3 deletion may contribute to the rapid clearance of viruses from non-cancer cells by encoding an E3 region that may be involved in evading host immunity.

[0167] In one embodiment, the virus used in the method of the present disclosure is based on Ad11 or derived therefrom such that the hexons and fibers are substantially similar to Ad11, such as Ad11p. Furthermore, since the designation of adenovirus serotypes is based on the external characteristics of the virus, i.e., the characteristics of the hexons and fibers, the present disclosure is useful in adenovirus type B having similar surface characteristics.

[0168] In one embodiment, the adenovirus type B is OvAd1 or OvAd2, disclosed in Sequence ID No. 1 and Sequence ID No. 2 of International Patent No. 2008 / 080003, which are incorporated herein by reference, respectively.

[0169] As used herein, substantially similar refers to the amino acid sequences of one or more related proteins that are at least 95% identical (e.g., 96, 97, 98, 99, or 100% identical) to the “whole” of a particular protein. The proteins being compared may be part of a larger entity, but the full length of the related fragment or component is compared.

[0170] Adenovirus type 5 (Ad5) generally enters cells via the coxsackievirus receptor (CAR). However, adenovirus serotype 11 (Ad11) is a subgroup B adenovirus that targets a different receptor (CD46) expressed at low levels on all nucleated cells. In normal cells, CD46 is often hidden on the basolateral surface of the cell and is not available for viral binding (Varela JC et al., Int J Cancer 2008 Sep 15;123(6):1357-63; Maisner et al., 1997). However, in tumor cells, surface expression is typically elevated, especially in more advanced and high-grade tumors (Kinugasa et al., 1999). Therefore, Ad11 efficiently infects cancer cells, from lung epithelial carcinoma (A549 cells), liver cancer cells (HepG2), prostate cancer (DU 145 and LNCaP), laryngeal cancer (Hep2), and breast cancer (CAMA and MG7), to, for example, glioblastoma, medulloblastoma, and neuroblastoma cells (Mei et al. 2003). Thus, Ad11 preferentially infects tumor cells and viruses derived therefrom, and is considered useful in the treatment of at least one of the above cancers. As a chimera of Ad11 and Ad3, ColoAd1 shares these properties with Ad11.

[0171] In one embodiment, the virus used in the method of this disclosure includes a transgene, for example, a therapeutic transgene (in particular one or more transgenes) for in vivo expression. As used herein, a transgene refers to a gene not found in the parent or wild-type virus. The gene can function as a marker or reporter for tracking the effect of viral infection; or the gene can play a role in improving the effectiveness of the virus; or the gene can deliver a cytotoxic agent to a cell.

[0172] Therapeutic transgenes can enable cells to express therapeutic agents such as siRNA, shRNA, polypeptides, tumor-associated antigens (TAAs), cytokines, antibodies, or anti-angiogenic factors.

[0173] Examples of therapeutic antibodies include anti-VGEF antibodies such as bevacizumab, anti-EGFR antibodies such as cetuximab, anti-CD20 antibodies such as rituximab, and immune system activation modulators such as anti-PD-1 and anti-PD-L1, and anti-CTLA4 (e.g., ipilimumab). Single-chain antibodies, antibody subunits, antibody fragments, and TRAPs can also encode, as can full-length antibodies. Important to this disclosure, the inclusion of these proteins does not alter the surface properties of the virus and can be readily incorporated into the genome without adverse effects on the administration described herein, while providing an additional therapeutic mechanism for attacking cancer cells.

[0174] Examples of cytokines include, in particular, interferon-alpha, interferon-gamma, and IL-2.

[0175] This alters the tumor microenvironment by causing RNA, antibodies, polypeptides, TAAs, or cytokines to be expressed in the tumor, but it is thought to offer an opportunity to avoid systemic side effects of the delivered drugs. For example, it may be possible to stimulate the local immune system to attack cancer. This local effect can be modulated by changing whether or not RNA, antibodies, polypeptides, TAAs, or cytokines are secreted from cells when expressed during the viral life cycle.

[0176] In one embodiment, the transgene encodes a thymidine kinase, for example, from a non-human origin or from cytosine deaminase, or from a bacterial origin or from yeast.

[0177] In one embodiment, the antibody, polypeptide, cytokine, or similar substance is of non-human origin and is not humanized. The latter has the advantage of being less likely to have a detrimental effect on the activity of entities within cancer cells and can evade cancer cells, as it locally attracts the attention of the immune system and is rapidly cleared.

[0178] In one embodiment, the virus encodes and expresses a protein that is visible or visible in vivo, such as GFP or a similar fluorescent protein. If the virus selectively infects cancer cells, the virus can express a visible or visible protein that can be used to highlight areas of cancerous tissue for excision or radiation.

[0179] In one embodiment, the virus may be armed with therapeutic genes capable of inducing anti-tumor immune function, inhibiting tumor angiogenesis, or activating prodrugs. As used herein, therapeutic dose refers to the amount of oncolytic adenovirus appropriate to achieve the intended therapeutic effect when used in an appropriate treatment plan, e.g., to improve the symptoms or condition of a disease. A dose can be considered a therapeutic dose in the treatment of cancer or metastasis when the number of viral particles is sufficient to slow or halt tumor or metastatic growth, reduce the size of the tumor or metastatic carcinoma, and / or extend the patient's lifespan. An appropriate therapeutic dose is generally a balance between therapeutic effect and tolerable toxicity, e.g., if the benefits achieved by the treatment are tolerable.

[0180] In one embodiment, the therapeutic dose range does not have dose-limiting toxicity.

[0181] As used herein, dose-limiting toxicity means a serious adverse event during treatment that prevents any further increase in dose, frequency, or intensity, or prevents the continuation of treatment at any dose level. Tolerable toxicity, for example, associated with high doses, means that the latter is unsuitable for use as a therapeutic dose in the context of this disclosure.

[0182] In one embodiment, pre-existing immunity to the Ad11 capsid is weak enough to allow for the effective administration of further therapeutic doses from day 7 onward.

[0183] In one embodiment, the poor immunostimulatory properties of the Ad11 capsid allow for the effective administration of further therapeutic doses from day 7 onward.

[0184] In one embodiment, intravenous delivery of the virus is less immunogenic than subcutaneous or intramuscular administration of the virus in terms of antiviral immunogenicity.

[0185] Generally, the toxicity of Ad11 is considered lower than certain other adenoviruses, such as Ad5. While this, along with its low seroprevalence, is beneficial, Ad11 may not be sufficient to evade the immune response. Although the literature suggests that subgroup B adenoviruses are not toxic to hepatocytes, macrophages in the lungs, liver (Kupffer cells), and spleen may clear oncolytic viruses after systemic delivery.

[0186] Rapid delivery of at least two doses of oncolytic virus may be beneficial in generating sufficient viral levels to be maintained for the duration necessary to infect target cells, i.e., cancer cells.

[0187] By delivering at least two doses in succession, a) the immune mechanism is occupied by the first dose and escapes a full-scale assault of the immune system to reach the target by the second dose, and / or b) the at least two consecutive doses can induce one or more beneficial events that allow the virus to reach sufficient levels in vivo for a sufficient period of time to reach the target cells, once it has reached and infected the target cells, in any way that it can replicate.

[0188] As used herein, in vivo distribution refers to distribution in vivo.

[0189] While not wanting to be constrained by theory, the inventors believe that a first dose of the virus, using cytokine-producing innate immune cells such as Kupffer cells, can downregulate clearance, thereby improving the bioavailability of further therapeutic doses. Thus, a first dose of the virus can "deplete" the phagocytic cell "sink" for circulating the virus, thereby achieving better delivery and / or increased efficacy. Depleting the phagocytic cell sink also reduces the tendency for subsequent doses to release cytokines, allowing for higher viral blood levels without excessive toxicity.

[0190] As used herein, bioavailability refers to the amount of virus available to perform the intended therapeutic function in vivo.

[0191] In one embodiment, the method herein, in which at least three doses are administered, minimizes adverse effects and / or toxicity to the patient.

[0192] In one embodiment, for example, the adenovirus is made stealthy by coating it with a polymer to at least partially evade the patient's immune system.

[0193] As used herein, "stealthy" means modifying the outer surface of an adenovirus to evade the patient's immune response, for example, by using a polymer. Examples of suitable polymers are disclosed in International Patent Nos. 98 / 19710, 00 / 74722, 2010 / 067041, 2010 / 067081, and 2006 / 008513, which are incorporated herein by reference.

[0194] In one embodiment, the oncolytic virus is conjugated with a cytotoxic or immunomodulatory agent.

[0195] In one embodiment, the oncolytic adenovirus is provided pegylated, for example, to reduce immunogenicity and / or increase its half-life.

[0196] In one embodiment, the treatment method is used to treat tumors.

[0197] As used herein, tumors also refer to abnormal masses of tissue resulting from uncontrolled and progressive excessive cell division, also known as neoplasms. These may be benign (not cancerous) or malignant. Tumors include all forms of cancer and metastasis.

[0198] In one embodiment, the tumor is a solid tumor. The solid tumor may be localized or metastatic.

[0199] In one embodiment, the tumor is of epithelial origin.

[0200] In one embodiment, the tumor is a solid tumor.

[0201] In one embodiment, the tumor is a malignant tumor such as colorectal cancer, hepatocellular carcinoma (liver cancer), prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, head and neck cancer, or lung cancer.

[0202] In one embodiment, the tumor is a malignant tumor of the colorectal colon.

[0203] As used herein, malignant tumors refer to cancer cells.

[0204] In one embodiment, the cancer is colorectal cancer and / or its metastatic form, such as liver metastases.

[0205] In one embodiment, the cancer is liver cancer and / or a metastatic form thereof.

[0206] In one embodiment, the cancer is lung cancer and / or a metastatic form thereof.

[0207] In one embodiment, the cancer is ovarian cancer and / or its metastatic form, such as lung metastases.

[0208] In one embodiment, the cancer is renal cancer and / or a metastatic form thereof.

[0209] In one embodiment, the cancer is bladder cancer and / or a metastatic form thereof.

[0210] In one embodiment, the cancer is throat cancer.

[0211] In one embodiment, the cancer is a skin cancer such as melanoma. In one embodiment, the cancer is leukemia. In one embodiment, the cancer is glioblastoma, medulloblastoma, or neuroblastoma. In one embodiment, the cancer is neuroendocrine carcinoma. In one embodiment, the cancer is Hodgkin's or non-Hodgkin's lymphoma.

[0212] In one embodiment, oncolytic adenoviruses are used to treat or prevent metastasis.

[0213] In one embodiment, the oncolytic adenovirus described herein is suitable for treating cancer cells that have migrated to the lymph nodes. The inventors have shown that an oncolytic virus administered to a colorectal cancer patient can infect cancer cells that have migrated to the lymph nodes.

[0214] In one embodiment, the virus, formulation and administration regimen according to this disclosure are suitable for treating abnormal precancerous cells.

[0215] In one embodiment, the methods or formulations described herein are used for the treatment of drug-resistant cancer.

[0216] In one embodiment, the method or formulation is used to sensitize cancer cells to drug resistance to the drug. More detailed types of cancer

[0217] lung cancer Lung cancer is classified by histological type and, microscopically, by the size and appearance of malignant cells seen by a histopathologist. For treatment purposes, it is distinguished into two broad classes. One is non-small cell lung cancer and the other is small cell lung cancer.

[0218] In one embodiment, carcinomas are lung cancers, such as small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

[0219] The three main subtypes of non-small cell lung carcinoma - NSCLC are adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0220] Approximately 40% of lung cancers are usually adenocarcinomas that originate from peripheral lung tissue. Bronchioloalveolar carcinoma, a subtype of adenocarcinoma, is commonly found in female non-smokers and can have excellent long-term survival.

[0221] Squamous cell carcinoma accounts for approximately 30% of lung cancers. Typically, it occurs near thick airways. Hollow cavities and associated cell death are generally seen at the center of the tumor. Approximately 9% of lung cancers are large cell carcinomas. They are named large cell carcinomas because the cancer cells are large and have excessive cytoplasm, large nuclei, and prominent nucleoli.

[0222] In small cell lung carcinoma - small cell lung cancer (SCLC), the cells contain dense neurosecretory granules (vesicles containing endocrine hormones), which gives this tumor an association with endocrine / tumor paraneoplastic syndromes. In most cases, it occurs in thicker airways (primary and secondary bronchi). These cancers grow rapidly and spread in the early stages of the disease. 60% - 70% have metastatic disease at the time of initial diagnosis.

[0223] In one embodiment, the cancer is non-small lung cancer.

[0224] Liver cancer In one embodiment, the cancer is liver cancer, for example, liver metastases from primary cancer, such as colon cancer that has spread to the liver. In one embodiment, the liver cancer is hepatocellular carcinoma (HCC).

[0225] Kidney cancer In one embodiment, for example, using the oncolytic adenovirus disclosed herein, treatment of kidney cancer such as renal cell carcinoma and / or urothelial cell carcinoma is provided. Other examples of kidney cancer include squamous cell carcinoma, paraganglioma (renal tumor), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear cell sarcoma of the kidney, mesoblastic nephroma, Wilms tumor, mixed epithelial stromal tumor, clear cell adenocarcinoma, transitional epithelial carcinoma, inverted papilloma, renal lymphoma, teratoma, carcinosarcoma, and carcinoid tumor of the renal pelvis.

[0226] Bladder cancer In one embodiment, the cancer is bladder cancer, for example, any of several types of malignant tumors that arise from the epithelial layer of the bladder (i.e., urothelium). Approximately 90% of bladder cancers are transitional epithelial carcinomas. The remaining 10% are squamous cell carcinomas, adenocarcinomas, sarcomas, small cell carcinomas, and secondary deposits from cancers in other parts of the body. Staging is as follows. T (primary tumor) · TX Primary tumor cannot be evaluated · T0 No evidence of primary tumor · Ta Non-invasive papillary carcinoma · Tis Carcinoma in situ ("flat tumor") · T1 Tumor infiltrates into the subepithelial connective tissue · T2a Tumor infiltrates into the superficial muscle (inner half) · T2b Tumor infiltrates into the deep muscle (outer half) · T3 Tumor infiltrates into the perivesical tissue · T3a Microscopic · T3b Macroscopic (mass outside the bladder) · T4a Tumor infiltrates into the prostate, uterus, or vagina · T4b Tumor infiltrates into the pelvic wall or abdominal wall N (lymph nodes) · NX Regional lymph nodes cannot be evaluated · N0 No regional lymph node metastasis · N1 Single regional lymph node metastasis with a maximum diameter of 2 cm or less • N2 Metastasis of a single lymph node with a maximum diameter of 2 cm or more but less than 5 cm, or multiple lymph nodes with a maximum diameter not greater than 5 cm. • N3: Metastasis of lymph nodes with a maximum diameter of 5 cm or more. M (Distant Metastasis) • MX cannot evaluate distant metastasis. • M0 No distant transfer M1 distant metastasis

[0227] This disclosure applies to all stages of bladder cancer.

[0228] Ovarian cancer In an independent aspect, this disclosure relates to ColoAd1, formulations of ColoAd1, or combination therapies containing ColoAd1, used in treating ovarian cancer, for example, by administering a therapeutically effective dose of ColoAd1 to a patient with ovarian cancer, for example, using the dosing regimen described herein.

[0229] Ovarian cancer has more than 30 different types, which are classified according to the type of cells in which treatment begins. Malignant ovarian tumors can start with three common cell types. • Surface epithelium - cells that cover the inner layer of the ovary • Germ cells - cells destined to form eggs • Interstitial cells – cells that release hormones and connect different structures in the ovary.

[0230] This disclosure relates to the treatment of ovarian cancer from any source in specific epithelial cells, as described herein, for example. Epithelial ovarian cancer (EOC) accounts for 85–90% of all ovarian cancers.

[0231] Common epithelial tumors—epithelial ovarian tumors—originate from the cells that cover the outer surface of the ovary. Most epithelial ovarian tumors are benign (non-cancerous). There are several types of benign epithelial tumors, including serous adenomas, mucinous adenomas, and Brenner tumors. Carcinomatous epithelial tumors are carcinomas—meaning they begin within the tissue that covers the ovary. These are the most common and most dangerous of all types of ovarian cancer. Unfortunately, about 70% of women with common epithelial ovarian cancer go undiagnosed until the disease has progressed to a later stage.

[0232] There are several epithelial ovarian tumors that are not clearly identified as cancerous under a microscope. These are called borderline tumors or low-grade (LMP) tumors. The methods described herein include treatment for the latter.

[0233] Germ cell tumors – Ovarian germ cell tumors arise from eggs or egg-producing cells. While some are cancerous and may be life-threatening, most germ cell tumors are benign (non-cancerous). The most common germ cell malignancies are mature teratomas, undifferentiated germ cell tumors, and endodermal sinus tumors. Germ cell malignancies occur most frequently in teenage and 20-year-old women. Today, 90% of patients with ovarian germ cell malignancies are cured and can retain their fertility.

[0234] Stromal tumors—ovarian stromal tumors are a rare class of tumors that arise from the connective tissue cells that together make up the ovaries and the cells that produce the female hormones, estrogen and progesterone. The most common types are granular, follicular, and Sertoli-Leydig cell tumors. These tumors are very rare, and usually about 70% are presented as stage I disease (cancer is limited to one or both ovaries).

[0235] Removal of the ovaries eliminates the risk of ovarian cancer but does not eliminate the risk of a less common cancer called primary peritoneal cancer. Primary peritoneal cancer is closely related to epithelial ovarian cancer (the most common type). It develops from cells in the peritoneum (the lining of the abdomen) and can be seen under a microscope. It is similar in terms of symptoms, spread, and treatment.

[0236] Stages of Ovarian Cancer Once diagnosed with ovarian cancer, the stage of the tumor can be determined when the doctor can tell during surgery whether the cancer has spread outside the ovaries. There are 4 stages of ovarian cancer - from stage I (early disease) to stage IV (advanced disease). The treatment plan and prognosis (the likely course and outcome of the disease) are determined by the stage of the cancer. Descriptions of the various stages of ovarian cancer are as follows.

[0237] Stage I - The growth of the cancer is limited to one ovary and in two ovaries. Stage IA - The growth is limited to one ovary and the tumor is confined inside the ovary. There is no cancer on the outer surface of the ovary. There is no ascites containing malignant cells. The capsule of the ovary remains intact. Stage IB - The growth is limited to both ovaries and there is no tumor on their outer surfaces. There is no ascites containing malignant cells. The capsule of the ovaries remains intact. Stage IC - The tumor is classified as either stage IA or IB and one or more of the following are present: (1) the tumor is present on the outer surface of one or both ovaries; (2) the capsule has ruptured; and (3) there is ascites with malignant cells or ascites with a positive peritoneal wash. Stage II - Growth of the cancer is seen in one or both ovaries with pelvic extension. Stage IIA - The cancer has extended and / or is present in the uterus or fallopian tubes or both. Stage IIB - The cancer has extended to other pelvic organs. Stage IIC - The tumor is classified as either Stage IIA or IIB and one or more of the following are present: (1) the tumor is located on the outer surface of one or both ovaries, (2) the capsule is ruptured, and (3) there is ascites with malignant cells or positive peritoneal lavage fluid. Stage III - Cancer growth is observed in one or both ovaries, and (1) the cancer has spread beyond the pelvis to the abdominal wall, or (2) the cancer has spread to the lymph nodes. The tumor is confined to the true pelvis, but there is histologically evidenced malignant dilation in the small intestine or omentum. During stage IIIA staging, the surgeon may be able to see cancer in one or both ovaries, but the cancer is not visible throughout the abdomen and has not spread to the lymph nodes. However, a microscopic biopsy check reveals very small deposits of cancer on the peritoneal surface of the abdomen. Stage IIIB - A tumor is present in one or both ovaries, and there is a cancerous deposit that is large enough to be seen by a surgeon but does not exceed 2 cm in diameter. The cancer has not spread to the lymph nodes. Stage IIIC - Tumors are present in one or both ovaries, and (1) the cancer has spread to the lymph nodes, and / or (2) the cancerous deposits are larger than 2 cm in diameter and are found in the abdomen, or both. Stage IV – The most advanced stage of ovarian cancer. The cancer has grown in one or both ovaries, and distant metastasis (the cancer has spread to organs located outside the peritoneal cavity) has occurred. Finding ovarian cancer cells in pleural effusion (from the cavity surrounding the lungs) is also evidence of stage IV disease.

[0238] In one embodiment, ovarian cancer is type I, e.g., IA, IB, or IC; type II, e.g., type IIA, IIB, or IIC; type III, e.g., type IIIA, IIIB, or IIIC; or type IV.

[0239] This disclosure relates to the treatment of any stage of ovarian cancer, particularly as described herein.

[0240] Combination therapy In one embodiment, the virus is administered in combination with the administration of cancer treatment or therapy.

[0241] As used herein, “in combination” includes administering an oncolytic virus before, concurrently with, and / or after cancer treatment or therapy.

[0242] In one embodiment, oncolytic adenovirus is used in combination with high-intensity focused ultrasound (HIFU) therapy.

[0243] Cancer treatments include surgery, radiation therapy, targeted therapy, and / or chemotherapy.

[0244] As used herein, cancer treatment refers to treatment with therapeutic compounds or biological agents, such as antibodies, that are intended to treat cancer and / or its maintenance therapy.

[0245] In one embodiment, cancer treatment is selected from any other anticancer therapy, including chemotherapeutic agents, targeted anticancer agents, radiotherapy, radioisotope therapy, or any combination thereof.

[0246] In further independent embodiments, the disclosure relates to combination therapies comprising, for example, an oncolytic adenovirus type B such as ColoAd1, and a chemotherapeutic agent that does not interfere with the activity of the adenovirus. Adenovirus type B such as ColoAd1 as used herein includes its formulations, for example, its pharmaceutical formulations.

[0247] As used herein, "activity" refers to the beneficial properties or characteristics of a virus, such as its ability to replicate in cancer cells, including oncolytic activity and / or in vivo viral replication.

[0248] In one embodiment, the combination therapy ColoAd1 is administered according to the regimen described herein.

[0249] Generally, combination therapy is provided as a formulation of adenovirus and a formulation of chemotherapeutic agent. Therefore, the administration of adenovirus and chemotherapeutic agent should be appropriately separate events. These administrations may be on the same day or on different days.

[0250] In one embodiment, the adenovirus is administered in the first week under appropriate control, and the chemotherapeutic agent is administered in the following week, for example, the next week.

[0251] In one or more embodiments, the chemotherapeutic agent and the adenovirus may have a synergistic therapeutic effect.

[0252] Oncolytic adenoviruses may be used as neoadjuvant therapy (preoperative treatment) to shrink tumors, treat metastases, and / or prevent metastases or further metastases. Oncolytic adenoviruses may also be used as adjuvant therapy (postoperative treatment) to treat metastases and / or prevent metastases or further metastases.

[0253] As used herein, "simultaneous" means administering an additional cancer treatment concurrently or nearly concurrently with a tumor-lytic adenovirus preparation. The treatment may be contained within the same preparation or administered as a separate preparation.

[0254] In one embodiment, the virus may be administered in combination with the administration of a chemotherapeutic agent such as paclitaxel, Abraxane, or a similar drug.

[0255] As used herein, chemotherapeutic agents refer to specific antitumor chemicals or drugs that selectively destroy malignant cells and tissues. Examples include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor alkylating agents. Other examples of chemotherapeutic agents include doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine, irinotecan, cisplatin, and oxaliplatin. The appropriate dose can be selected by the physician based on the nature of the cancer being treated.

[0256] Surprisingly, the inventors established that certain classes of therapeutic agents, such as topoisomerase or PARP inhibitors, can inhibit viral replication in vivo. If it is desirable to establish viral infection in cancer cells so that the virus can replicate, co-administration of compounds that inhibit viral replication may be undesirable.

[0257] In one embodiment, the chemotherapeutic agent is not an enzyme inhibitor. Therefore, in one embodiment, the combination therapy does not use a topoisomerase inhibitor.

[0258] In one embodiment, the chemotherapeutic agent is not a PARP inhibitor.

[0259] In one embodiment, the combination therapy uses platinum containing a chemotherapeutic agent such as cisplatin, carboplatin, or oxaliplatin.

[0260] In one embodiment, the combination uses a microtubule inhibitor, such as vincristine sulfate, epotilon A, N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide (ABT-751), an ataxol-derived chemotherapeutic agent, such as paclitaxel, abraxane, or docetaxel, or a combination thereof.

[0261] In one embodiment, the combination uses an mTor inhibitor. Examples of mTor inhibitors include everolimus (RAD001), WYE-354, KU-0063794, papamycin (sirolimus), temsirolimus, defololimus (MK-8669), AZD8055, and BEZ235 (NVP-BEZ235).

[0262] In one embodiment, the combination uses a Pi3 kinase inhibitor. Examples of Pi3 kinases include GDC-0941, ZSTK474, PIK-90, LY294002, TG100-115, XL147, AS-605240, PIK-293, AZD6482, PIK-93, TGX-221, IC-87114, and the following, as described in International Patent No. 2011 / 048111. The compounds disclosed (incorporated herein by reference), 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethinylquinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-in-1-yl)quinazoline-4(3H)- On; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-on; 6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)hex-5-ic acid; 2-((4-amino-3 -(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;3-((2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-(3-(2-(2-hydroxyethoxy)ethoxy)prop-1-in-1-yl)-4-oxoquinazoline-3(4H)-yl)methyl)benzonitrile;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-morpholinoethoxy)prop-1-inyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethinylquinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo [3,4-d]pyrimidine-1-yl)methyl)-3-(3-chlorobenzyl)-5-ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(3-chlorobenzyl)-5-ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(2-fluorobenzyl)quinazoline-4(3H)-one 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(2-fluorobenzyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(3-methoxybenzyl)quinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl) (Tyl)-5-ethynyl-3-(3-methoxybenzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(3-(tri-fluoromethyl)benzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(3-(trifluoromethyl)benzyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(4-chlorobenzyl)-5-ethynylquinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(4-(methylsulfonyl)benzyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5- Ethinyl-3-(4-(methyl-sulfonyl)benzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethinyl-3-(4-(trifluoromethyl)benzyl)quinazoline-4(3H)-one; 3-((2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethinyl-4-oxoquinazoline-3(4H)-yl)methyl)benzonitrile; 2-((4-A Mino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(3-(methyl-sulfonyl)benzyl)quinazoline-4(3H)-one;3-((2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-4-oxoquinazoline-3(4H)-yl)methyl)benzonitrile;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl) -3-(4-chlorobenzyl)-5-ethinylquinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(4-chlorobenzyl)-5-(3-methoxyprop-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(3-methoxybenzyl)-5-(3-methoxyprop-1-inyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-methoxyprop-1-inyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(4-(trifluoromethyl)benzyl)quinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl) -1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-methoxyethoxy)prop-1-inyl)quinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-((5-methylisoxazole-3-yl)methyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3, 4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-((5-methylisoxazole-3-yl)methyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(3-chloro-2-fluorobenzyl)-5-ethynylquinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2, 6-Difluorobenzyl)-5-Ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(4-chloro-2-fluorobenzyl)-5-Ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-Ethynylquinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-(3-methoxyprop-1-inyl)-3-(3-(trifluoromethyl)benzyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(4-fluorobenzyl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-cyclopentylprop-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-(3-(benzyloxy)prop-1-inyl)-3-(2-chlorobenzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(5 -Hydroxypent-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(2-fluoro-5-methoxybenzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(3,4-dichlorobenzyl)-5-ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl )-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-benzyl-5-ethynylquinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(2-trifluoromethylbenzyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(4-methoxybenzyl)quinazoline-4(3H)-one;4-((2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-4-oxoquinazoline-3(4H)-yl)methyl)benzonitrile; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-ethynyl-3-(2-fluoro-4-methoxybenzyl)quinazoline-4(3H)-one; 1-(3-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroxymethyl; Nazolin-5-yl)prop-2-inyl)urea; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-fluorobenzyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-inyl)quinazolin-4(3H)-one; 2-((4-amino-3-(4-fluoro-3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethinyl-quinazolin- 4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-phenoxyprop-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-fluorobenzyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one; 6-(2-( (4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-methoxyethyl)hex-5-inamide;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(7-morpholino-7-oxohepto-1-in-1-yl)quinazolin-4(3H)-one;2-( (4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(5-morpholino-5-oxopento-1-in-1-yl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-((5-methylpyrazine-2-yl)methyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-oxo-6-(piperidine-1-yl)hex-1-in-1-yl)quinazoline-4(3H)-one;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)-N,N-diethylhex-5-inamide;7-( 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)hept-6-ic acid;2-acetamide-N-(3-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)prop-2-in-1-yl)acetamide;2-((4-A Mino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(3-methoxy-5-(trifluoromethyl)benzyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-methoxyphenethyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;2-(( 4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(benzo[b]thiophen-2-ylmethyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-fluoro-3-methoxybenzyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;Methyl 3-((2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)-4-oxoquinazoline-3(4H)-yl)methyl)benzoic acid; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-((1-methyl-1H-pyrazole-4-yl)methyl)-5-(6-morpholino-6 -Oxohex-1-in-1-yl)quinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(benzofuran-5-ylmethyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-((2-methylthiazo (4-yl)methyl)-5-(6-morpholino-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methylpiperazine-1-yl)-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3, 4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-morpholinopiperidine-1-yl)-6-oxohex-1-in-1-yl)quinazoline-4(3H)-one; 5-(6-(4-acetylpiperazine-1-yl)-6-oxohex-1-in-1-yl)-2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)quinazoline-4(3H)-one;N-(4-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)buto-3-in-1-yl)morpholine-4-carboxamide;5-(6-(4-acetyl-piperazine-1-yl)-6-oxohex-1-in-1-yl)-2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine- 1-yl)methyl)-3-(2-chlorobenzyl)quinazoline-4(3H)-one; N-(4-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)buto-3-in-1-yl)morpholine-4-carboxamide; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl (Lu)-5-(5-(bis(2-methoxyethyl)amino)pento-1-inyl)-3-(2-chlorobenzyl)quinazoline-4(3H)-one;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)-N-cyclopentylhex-5-inamide;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo [3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(tetrahydro-2H-pyran-4-yl)hex-5-inamide; 6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-morpholinoethyl)hex-5-inamide;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-(2-methoxyethyl)piperazin-1-yl)-6-oxohex-1-inyl)quinazoline-4(3H)-one;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin N-5-yl)-N-(2-(dimethylamino)ethyl)hex-5-inamide; 6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyridine-4-yl)hex-5-inamide; 6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-( 2-Chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyridine-4-yl)hex-5-inamide;2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-(dimethylamino)piperidine-1-yl)-6-oxohex-1-inyl)quinazolin-4(3H)-one;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H- Pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-inamide; 6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-inamide;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-(4-methylpiperazine-1-yl)ethyl)hex-5-inamide; 6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-methyl-N-(2-(4-methylpiperazine-1-yl)ethyl )Hex-5-inamide;6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-isopropylhex-5-inamide;6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-isopropylhex-5-inamide;6-(2-((4-amino-3-(; 4-Hydroxyphenyl)-1H-Pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-5-yl)-N,N-dimethylhex-5-inamide;2-((4-amino-3-(4-hydroxyphenyl)-1H-Pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-oxo-6- (Pyrrolidine-1-yl)hex-1-in-1-yl)quinazolin-4(3H)-one; 6-(2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyrrolidine-3-yl)hex-5-inamide; 2-((4-amino-3-(4-hydroxy (Cyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(3-(dimethylamino)pyrrolidine-1-yl)-6-oxohex-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6 -(3-(dimethylamino)pyrroridine-1-yl)-6-oxohex-1-inyl)quinazoline-4(3H)-one; 2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methyl-1,4-diazepan-1-yl)-6-oxohex-1-inyl)quinazoline-4(3H)-one;Examples include 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methyl-1,4-diazepan-1-yl)-6-oxohex-1-inyl)quinazoline-4(3H)-one, 2-((4-amino-3-(4-hydroxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-inyl)quinazoline-4(3H)-one or its stereoisomers, tautomers and isotopic derivatives, and pharmaceutically acceptable salts thereof.

[0263] In one embodiment, the combination uses a MEK inhibitor. Examples of MEK inhibitors include AS703026, CI-1040 (PD184352), AZD6244 (selumetinib), PD318088, PD0325901, AZD8330, PD98059, U0126-EtOH, BIX02189, or BIX02188.

[0264] In one embodiment, the combination uses an AKT inhibitor. Examples of AKT inhibitors include MK-2206 and AT7867.

[0265] In one embodiment, the combination uses an aurora kinase inhibitor. Examples of aurora kinase inhibitors include aurora A inhibitor I, VX-680, AZD1152-HQPA (Barasertib), SNS-314 mesylate, PHA-680632, ZM-447439, CCT129202, and Hesperadin.

[0266] In one embodiment, the combination uses a p38 inhibitor, such as N-[4-({4-[3-(3-tert-butyl-1-p-tolyl-1H-pyrazole-5-yl)ureido]naphthalene-1-yloxy}methyl)pyridine-2-yl]-2-methoxyacetamide, as disclosed in International Patent No. 2010 / 038086.

[0267] In one embodiment, the combination uses a Bcl-2 inhibitor. Examples of Bcl-2 inhibitors include ovatoclax mesylate, ABT-737, ABT-263 (navitoclax), and TW-37.

[0268] In one embodiment, the combination uses an antimetabolite. Examples of antimetabolites include capecitabine (Xeloda), fludarabine phosphate, fludarabine (Fludara), decitabine, larcitrexed (Tomdex), gemcitabine hydrochloride, and cladribine.

[0269] In one embodiment, the therapeutic agent is ganciclovir, which can help control the immune response and / or tumor vasculation.

[0270] In one embodiment, one or more treatments used in this method are metronomic, often involving continuous or frequent treatment with low doses of anticancer drugs, and are frequently performed concurrently with other therapies.

[0271] Subgroup B oncolytic adenoviruses, particularly Ad11 and ColoAd1, and the adenoviruses derived from them, appear to have a mechanism of action that is largely independent of apoptosis via epidermal necrolysis and cancer cell death, and therefore may be particularly synergistic with chemotherapeutic agents. Furthermore, immunosuppression that occurs during chemotherapy allows oncolytic viruses to function with high efficiency.

[0272] In one embodiment, the chemotherapeutic agent is administered parenterally.

[0273] In one embodiment, the chemotherapeutic agent is administered separately from the virus, either by a transient or alternative administration method, or both. Treatment can be carried out simultaneously or sequentially.

[0274] In one embodiment, the treatment for cancer is a targeted agent, such as a monoclonal antibody like bevacizumab, cetuximab, or panitumumab, or an antibody conjugate, particularly one in which the antibody or binding fragment is bound to a toxin.

[0275] In one embodiment, the treatment of cancer is an immunotherapy agent, such as ipilimumab or other anti-CTLA4, anti-PD-1, anti-PD-L1, or other checkpoint inhibitors, or a cytokine or cytokine analog.

[0276] As used herein, checkpoint inhibitors refer to drugs that inhibit signaling from T cell membrane proteins, thereby inhibiting or downregulating the activation and function of T cells.

[0277] In one embodiment, the virus is administered in combination with radiotherapy.

[0278] As used herein, radiotherapy refers to the medical use of ionizing radiation.

[0279] Cancer cells are generally undifferentiated and resemble stem cells. They regenerate more than even the healthiest differentiated cells and have a reduced ability to repair sublethal damage. DNA damage is then passed on through cell division. Accumulation of DNA damage in cancer cells can lead to death or more slow regeneration.

[0280] In one embodiment, radiotherapy is administered simultaneously.

[0281] In one embodiment, radiotherapy is administered continuously.

[0282] In one embodiment, the virus is administered in combination with complementary therapies for cancer treatment, such as the treatment of cachexia, including cancer cachexia, using S-pindolol, S-mepindolol, or S-bopindolol. An appropriate dose may range from 2.5 mg to 100 mg, administered as a single dose of 2.5 mg to 50 mg per day, or as multiple doses per day.

[0283] In one embodiment, the virus is administered in combination with the administration of one or more prophylactic agents selected from, for example, antipyretics, antihistamines, antiemetics, antidiarrheals, steroids, and analgesics.

[0284] Antipyretics include aspirin and nonsteroidal anti-inflammatory drugs, such as ibuprofen, naproxen, and ketoprofen.

[0285] Examples of antihistamines include acribastine, azalastine, brompheniramine, buclidine, bromodifenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, chlorodiphenhydramine, clemastine, cyproheptadine, desloratadine, dexbromophenylamine, deschlorpheniramine, dexchlorpheniramine, dimenhydrinate, dimethindene, diphenhydramine, doxylamine, ebstine, embramin, fexofenadine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadrine, pheninidamine, pheniramine, phenyltroxamine, promethazine, pyriramine, quetiapine, rupatadine, triperenamine, and triprolidine.

[0286] Examples of antiemetics include drasetron, granietron, ondansetron, tropisetron, palonoestron, mirtazapine, domperidone, olanzapine, droperidol, metoclopramide, arizaprid, and prochloperazine. In some cases, antihistamines can be used as antiemetics.

[0287] Antidiarrheal agents include methylcellulose, attapulgite, bismuth subsalicylate, atropine / diphenoxylate, loperamide, and other opioids such as codeine and morphine.

[0288] Examples of analgesics include nonsteroidal anti-inflammatory drugs such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine, and tramadol, as well as paracetamol, Cox-2 inhibitors, and opioid and morphine-like agents.

[0289] In one embodiment, the treatment of the virus is used in combination with a series of steroids.

[0290] Examples of steroids include hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, and dexamethasone.

[0291] As used herein, "preventive medicine" refers to preventive drugs or care consisting of measures taken to prevent or mitigate side effects, for example, during or after the administration of a virus.

[0292] In one embodiment, the prophylactic agent is administered separately from the virus, either by a temporary or alternative method of administration, or both. Treatment can be carried out simultaneously or sequentially.

[0293] In one embodiment, additional hydration is provided simultaneously or sequentially in combination with the administration of the virus.

[0294] As used herein, additional hydration means supplying the patient with more fluid than that contained in the formulation. This may be by injecting any form of a suitable liquid, such as saline or glucose.

[0295] In one embodiment, the viral therapy described herein is administered in combination with an anti-inflammatory agent, such as a steroid or a non-steroidal anti-inflammatory agent.

[0296] In one embodiment, the viral therapy according to this disclosure is administered in combination with an antipyretic.

[0297] In one embodiment, viral therapy is administered in combination with hydration therapy, such as intravenous administration of a fluid, including a specific isotonic saline or glucose.

[0298] In one embodiment, this method is suitable for treating outpatients.

[0299] As used herein, outpatients are patients who are not hospitalized during the treatment phase, but instead visit a doctor's office, clinic, or day surgery for treatment.

[0300] One embodiment provides a method for treating a patient with a pharmaceutical formulation described herein, including ColoAd1, comprising the step of administering to the patient intravenously a dose for day 1, then a dose for day 3, and a third dose for day 5.

[0301] In one embodiment, a parenteral formulation of a replicable oncolytic subgroup B adenovirus is provided for use in the treatment of tumors and / or malignant tumors and / or cancer, etc., by administering a first dose of the formulation described herein, followed by one or more further therapeutic agents thereof. Here, the first dose and further doses are administered within a period of 14 days, in particular as described above.

[0302] In one embodiment, the use of multiple cycles of therapy with a replicable oncolytic subgroup B adenovirus is provided. A therapy cycle should be interpreted herein as a series of viral doses administered to a patient over a relatively short period of time after the patient's response has been evaluated. A therapy cycle can be repeated multiple times if the risk-benefit ratio is determined to be in the patient's best interest.

[0303] One embodiment provides a method for evaluating the suitability of repeated cycles of treatment with a replicable oncolytic subgroup B adenovirus by determining a specific level of viral titer and comparing it to the pre-treatment titer, for example, a titer below a certain percentage of the pre-treatment titer exhibits positive risk-benefit properties for retreatment.

[0304] In one embodiment, the use of a parenteral formulation of a replicable oncolytic subgroup B adenovirus in the manufacture of a pharmaceutical product for the treatment of tumors and / or malignant tumors and / or cancer is provided by using the treatment plan described herein.

[0305] In one embodiment, the formulation is used to treat or prevent metastasis.

[0306] In one embodiment, a formulation is provided that, under sterile conditions, is presented as a sterile, pre-filled, and packaged syringe of the appropriate dose and volume, using appropriate air control such as a ventilation hood before administration to the patient, in order to avoid the need for complex and expensive dosage formulations.

[0307] In the context of this specification, "comprising" shall be interpreted as "including."

[0308] Aspects of the present invention that include specific elements are intended to extend to alternative embodiments that "consist of" or "essentially consist of" the relevant elements.

[0309] Any positive embodiment or combination described herein may be based on negative exclusion, i.e., denial. [Examples]

[0310] Preclinical competence and selectivity Table 2 shows the IC of ColoAd1 on various epithelial cell lines. 50 This indicates.

[0311] [Table 2]

[0312] ColoAd1 IC in a range of epithelial-derived cancer cells and normal cells 50 The number of ColoAd1 particles (IC) needed to kill 50% of cells. 50 ) was measured in vitro using a standard 6-day MTS assay. 1 Results from a study conducted by Schering AG and published in Kuhn et al., 2008. 2. Repetitions and additional studies performed by Oxford University (unpublished).

[0313] Table 3 shows the IC50 of ColoAd1 in various non-epithelial cell lines. 50 This indicates.

[0314] [Table 3]

[0315] IC for ColoAd1 in the range of non-epithelial cancers 50 The number of particles (IC) needed to kill 50% of cells. 50 ) was measured in vitro using a standard 5-day MTS assay. 1 Results from a study conducted by Schering AG and published in Kuhn et al., 2008. 2. Repetitions and additional studies performed by Oxford University (unpublished).

[0316] Table 4 shows ColoAd1 replication in various normal, non-cancerous human cell lines.

[0317] [Table 4]

[0318] Human cells growing in an in vitro monolayer were exposed to ColoAd1 for 72 hours. The total number of ColoAd1 genomic copies was then measured by qPCR. The data are presented as total genomic copies and as relative % to a cancer cell positive control (HT29). The viability of ColoAd1 material derived from these normal human cells on HT29 cancer cells was tested. In all cases, the ColoAd1 material recovered as such was unable to show replication within HT29 cells.

[0319] Preclinical pharmacokinetics ColoAd1 pharmacokinetics were obtained in CD-1 mice. Mice (3 per group) were administered virus particles via the tail vein, and the circulating genome in whole blood samples was measured by quantitative PCR (qPCR). The ColoAd1 half-life in this model is dose-dependent. At low input doses (1×10 9 ~2×10 10 ) on multiple dosing days, the mean alpha half-life was 1.8 ± 0.5 minutes, which is consistent with values previously reported for other adenoviruses (Green 2004). At high doses (2×10 11 or greater), saturation of clearance appears to occur, resulting in longer circulating levels (meaning an alpha half-life of 7.8 ± 2 minutes). The saturation of the ColoAd1 tests described herein is reflected via multiple pharmacokinetic parameters.

[0320] Table 5 shows a significant increase in the area under the curve (AUC), with the ColoAd1 dose being 2×10 11When administered in high doses, the particle half-life and percentage were retained at the 30-minute time point. It was particularly noted that optimal kinetics (AUC, percentage of retained particles at 30 minutes, and mean alpha t1 / 2) were all achieved when three equal high doses were administered, in contrast to the case of high doses after a low priming dose. From these data, it was predicted that if blood circulation time is significantly longer in humans than in mice, the half-life in humans would be considerably longer, and that low priming doses would be less likely to be valuable for subgroup B adenoviruses. Furthermore, in tumors present in human patients, replication of the virus in tumor cells was expected to lead to further amplification of the virus at later time points with subsequent release. Clinical trials were planned accordingly. Table 5: Hemodynamics of multiple intratumor injections in CD-1 mice (3 mice per group)

[0321] [Table 5]

[0322] Preclinical interaction studies Viral particles can interact with components of human blood, including antibodies, complement, and blood cells, leading to rapid neutralization (Lyons 2005, Carlisle 2009). These events are species-specific and cannot be effectively modeled in animals.

[0323] To evaluate neutralization in human blood, ColoAd1 may be incubated in freshly isolated human whole blood and applied to tolerant cells (HT29 colorectal tumor cells). The range of viral concentrations can be selected to cover the target clinical dose range (2 × 10⁶ per 1 ml of human blood). 6 ~2×10 9 (The particle size is assumed to be within the range of human blood volume.) Residual viral titer is determined by cytotoxicity and can be compared to viral infection in the absence of incubation in human blood (culture medium alone), with a viral particle concentration range of 2 × 10⁶ per ml. 6 ~2×109 The titer level is desirable.

[0324] The data in FIG. 1 further shows that ColoAd1 was slightly affected by human blood. Fresh human blood was collected from 9 subjects (A - I) using lithium heparin tubes. ColoAd1 virus particles were added to the blood samples at 2×10 9 VP / mL and diluted 10 - fold, and assuming the dose is completely diluted into the total blood volume (assuming 5 L of blood), it reflects a potentially equivalent human dose of 1×10 13 . After incubation at 37°C for 20 minutes, the virus / blood mixture was added to A549 tumor cells growing in a 96 - well plate. The percentage of remaining viable A549 cells was measured 5 days later and plotted as a percentage. The IC 50 occurs at a level approximately equivalent to a virus blood concentration of 2×10 6 VP / mL. This level of the virus was thus determined as the minimum target level for achieving in human clinical trials.

[0325] In several in vitro studies, the interaction between human blood cells and ColoAd1 was also carried out. Fresh blood was obtained from 4 individuals, and red blood cells, platelets, and white blood cells were washed and resuspended in PBS at physiological cell concentrations for use in individual experiments (5×10 9 , 2×10 8 and 6×10 6). qPCR analysis revealed that over 80% (82% ± 8%) of ColoAd1 was associated with human blood cells, primarily erythrocytes and leukocytes. There was no significant difference in the percentage of ColoAd1 bound to blood cells after 5-minute or 30-minute incubation. Ad5 showed relatively higher levels of binding to human blood cells (95.5 ± 1.2%) than ColoAd1. Based on relative fluorescence, 30-minute pre-incubation of ColoAd1-GFP with human blood cells predominantly inhibited infection of SW480 tumor cells (>90%) and only resulted in low levels of expression of CD46, the cell receptor for ColoAd1. Infection of HT29 cells, which express higher levels of CD46, was inhibited to a much lower degree (approximately 41%), presumably because these tumor cells express higher levels of the ColoAd1 receptor. Finally, infection of leukocytes expressing high levels of CD46 and therefore capable of acting as a "sink" for ColoAd1 was assessed using ColoAd1-GFP to determine the degree of transgene expression. After 24 hours, no evidence of transgene expression was observed in leukocytes. In contrast, previous studies had shown that Ad5 could efficiently infect monocytes in vitro under the same conditions. Taken together, these studies suggest that the interaction of ColoAd1 with cellular blood components is limited and significantly different from that of Ad5. Again, this was taken into consideration when designing the clinical trial.

[0326] Preclinical biodistribution of ColoAd1 The biodistribution and clearance of ColoAd1 were determined in normal mice and transgenic mice expressing the primary viral receptor CD46 (a receptor for group B adenoviruses not expressed in normal mice). 11Following tail vein administration of viral particles, viral particles were predominantly found in the liver, spleen, and lungs 24 hours later (Figure 2). The viral copy number per mg is shown, indicating that these large organs represent the main distribution sites of the entire virus, based on percentages. A similar distribution was observed in CD46 transgenic mice (Figure 3), demonstrating that the CD46 receptor is not a key determinant of distribution. The distribution of the non-replicating mutant (ColoAd1CJ132) was identical to that of ColoAd1, indicating that replication does not contribute to this distribution. However, in tumors present in human patients, subsequent release suggests that replication of the virus in cancer cells leads to further viral amplification at later time points, which is why clinical trials were likely planned accordingly.

[0327] Preclinical viral clearance To determine the time required for viral clearance to be completed, a long-term particle clearance study was performed in normal Balbc mice. The organs predominantly affected by viral distribution—liver, spleen, and lungs—were selected for analysis. Here, the total viral particles per organ were recorded as a percentage of the input dose at each time point, ensuring that results were not normalized to organ weight. At 1 hour, the majority of the input virus had already been isolated within the liver, less than 5% in the spleen, and less than 0.1% in the lungs. 24 hours post-injection, viral particles were rapidly cleared from these organs, leaving less than 1% of the input viral genome. Beyond 65 days post-injection, no significant levels of virus were detected in any tissue, and levels did not significantly exceed the background. Viral particles could not be recovered from tissues at 65 days post-administration. The dynamics of viral clearance (data presented as a percentage of input per organ) are summarized in Figure 4.

[0328] CD46 transgenic mice, 1 × 10⁶ times on a single occasion. 10ColoAd1 was administered via the tail vein of n=3 animals per time point at a dose of vp / mouse. Genomic copies (measured by qPCR) are presented as a percentage of genomic copy input.

[0329] Preclinical immunogenicity The development of a specific antiviral immune response may have a significant impact on hemodynamics. To investigate this possibility, a group of mice was repeatedly administered ColoAd1 over several months to generate a pool of superimmune serum. A second group of mice was passively immunized to ColoAd1 administered by intravenous injection of 10 or 20 μl using superimmune serum. These mice were then rested for 10 minutes and 5 × 10⁻⁶ 10 ColoAd1 was administered intravenously. Blood samples were collected from each mouse at 2, 10, and 30 minutes after ColoAd1 administration and analyzed by qPCR. The results are shown in Figure 5, demonstrating that the immune response to ColoAd1 significantly affects its kinetics and delivery, thus highlighting the importance of administering the drug before this response occurs.

[0330] Preclinical safety and toxicity Several safety and toxicity studies were conducted with ColoAd1, including pilot studies in CD-1 and Balb / c mice, and CD46 transgenic mice. In the final toxicity study in male and female CD-1 mice, ColoAd1 was administered in three doses over five days (days 1, 3, and 5) to model the intended clinical dosing regimen. Male and female CD-1 mice were administered ColoAd1 or formulation buffer as shown in Table 6 via intravenous bolus injection (dose volume = 100 μL), and the final study design involved lowering the dose in a specific group after unplanned deaths of four males in two groups on day 1. Table 6: Toxicity test design

[0331] [Table 6]

[0332] A standard set of safety endpoints, including clinical signs, body weight, plasma cytokine levels, clinicopathology, and macroscopic and microscopic examinations, was performed regularly. A standard list of tissues and organs was collected at autopsy on days 6 and 17.

[0333] No significant clinical signs were observed in males and females in groups 1, 2, or 3 on any treatment day. Clinical signs of adverse effects were observed in group 4, 2.2 × 10⁴ after the first dose on day 1. 11 and 6.96 × 10 10 While observed in both VP and animals, no further clinical signs of adverse effects were seen except in one male in group 4 on day 3. On day 2, dose-related weight loss was observed in all ColoAd1 treatment groups except for a female in group 2, but thereafter, no weight was affected in any treatment group. Hematological and liver function changes occurred over a longer period at the time of recording, but returned to normal ranges by the end of the recovery period. In summary, the most important clinical signs were observed after the initial dose, and subsequent doses were better tolerated.

[0334] Figure 6 shows the cytokine response over time in this study. The most significant increase in the cytokine MCP-1 was observed in groups 3 and 4 on day 1, 6 and 24 hours after the first treatment, and 6 hours after the treatment on day 5. No consistent increases were observed in animals in group 2 for other cytokines. Smaller, dose-dependent increases in IL-6, IFNγ, and TNFα were observed only in animals in groups 3 and 4, and were most frequent at lower concentrations compared to MCP-1, particularly in only some animals in each dose group of group 3.

[0335] The cytokine patterns observed in this study were consistent with the observed clinical signs, indicating that subsequent doses were well-tolerated after the initial administration, even at the same dosage.

[0336] Clinical trials At the time of filing the application, two clinical trials were conducted to investigate the safety and efficacy of ColoAd1 when delivered intravenously to human subjects with metastatic cancer.

[0337] The evolutionary study (ColoAd1-1001) is a Phase I / II clinical trial with a Phase I dose-escalation component conducted in patients with epithelial metastatic tumors and other patients with no further treatment options. In the Phase I dose-escalation portion of the study, patients received three equal doses of ColoAd1 intravenously on days 1, 3, and 5 (at 48-hour intervals). Using slow intravenous infusion, in the initial cohort, each patient received 30 ml of viral suspension (6 ml / min) over 5 minutes. Initially, in each cohort of three patients, 1 × 10⁶ doses were administered until dose-limiting toxicity due to adverse events was suggested. 10 The dose was increased by one log at a time, starting with viral particles. Each patient also received a symptom prophylaxis regimen, including a set regimen of supplemental fluids and anti-inflammatory agents (acetaminophen / paracetamol and ibuprofen). The safety and tolerability of this dosing regimen at each dose level were assessed by using physical examinations (including blood pressure, pulse, and body temperature), by inducing all adverse events, and by evaluating changes in hematological, biochemical, and cytokine characteristics. Viral dynamics and excretion were assessed using normal blood, urine, stool, and sputum samples. Efficacy was assessed by serial CT imaging according to objective criteria. In later stages of this study, the safety and efficacy of the maximum intravenous tolerated dose (MTD) of ColoAd1 in patients with metastatic colorectal cancer will be further investigated.

[0338] The second clinical trial (ColoAd1-1002) is a Phase 0 "window of opportunity" trial comparing intratumoral direct delivery versus intravenous delivery of ColoAd1 in patients with newly diagnosed primary (non-metastatic) colorectal tumors. Patients in this trial will be pre-operatively administered ColoAd1 surgically, and the resected tumors will be examined post-operatively to investigate the extent of viral delivery, replication, and spread after two different delivery and administration regimens. Safety and virological measures in this trial are broadly similar to those for ColoAd1-1001.

[0339] In the Phase I dose-escalation study of the ColoAd1-1001 trial, patients in seven patient cohorts (i.e., cohorts 1-7) received 1 × 10⁶ doses, as shown in Table 7 below. 13 ColoAd1 was administered intravenously at a dose level of viral particles including up to [specific level]. Table 7: Dosage plans for cohorts 1-7 in the Phase I dose escalation component of the ColoAd1-1001 clinical trial.

[0340] [Table 7]

[0341] The characteristic side effects of ColoAd1 in this study include fever, illness-like influenza, hypertransaminasemia, thrombocytopenia, neutropenia, diarrhea, and vomiting. However, 1 × 10 13 When the viral particle dose level was infused over 5 minutes, the dose was not well tolerated. In particular, two patients suffered dose-limiting toxicity (DLT) at this dose, including cytokine-mediated acute lung injury, and were less tolerable than with a single dose. One patient required steroids to treat this condition. Patients at this dose level also suffered from chills, hypertension, pain, hypertransaminasemia, prolonged PPT, and elevated D-dimer levels, although all of these symptoms resolved over time. As a result of these toxic effects at this poorly tolerated dose, the dose of ColoAd1 was reduced and then escalated again using a slow infusion rate. Using this strategy, 3 × 10⁶ doses were administered.12 When VP was administered over 5 minutes (Cohort 5) or 20 minutes (Cohort 6), the results were different. 12 The study showed that all patients tolerated VP well when administered over 40 minutes (Cohort 7).

[0342] These safety data, while preliminary at the time of writing, support characteristics very similar to those observed in mice. However, some patients persisted with fever and asthenia in the second week despite no ongoing administration, a phenomenon consistent with ongoing viral replication in human tumors (a phenomenon not likely to be seen in non-tumor-bearing mice). The final maximum tolerated dose for humans was thus 6 × 10⁶ on days 1, 3, and 5. 11 1 × 10⁻¹⁶ administered at an infusion rate of VP / min 12 ~1 × 10 13 As the virus particles are suspected to be present, each patient is also receiving prophylactic anti-inflammatory drugs and intravenous fluids according to the ColoAd1-1001 protocol. The final optimal dose plan is subject to further confirmation studies.

[0343] Table 8 summarizes the key viral pharmacokinetic parameters measured by qPCR for each patient in the Phase I dose-escalation component of the ColoAd1-1001 clinical trial. These results are largely consistent with preclinical data. In short, although there are dose-dependent cMAX and AUC and indicators of possible saturation rates at higher doses, the mean alpha half-life was approximately 18 minutes. Table 8: ColoAd1 pharmacokinetics in cohorts 1-7

[0344] [Table 8] *Instruction number for each patient nd: Not measured.

[0345] Figure 7 shows the cytokine patterns observed in cancer patients administered ColoAd1-1001 in clinical trials during the initial dose escalation phase (including confirmation of dose-limiting toxicity). Similar to mouse studies, the peak of the inflammatory cytokine response in humans was observed after the initial administration of ColoAd1 and decreased with subsequent administrations. Interestingly, this initial priming effect of ColoAd1 is not reliably observed at low doses but is clearly observed at higher doses, supporting the claim that high-dose regimens repeated at the same dose level may be optimal for intravenous administration of subgroup B adenovirus to human cancer patients.

[0346] Figure 7 specifically shows the time-series cytokine levels (μg / L) in human cancer patients with metastatic solid epithelial tumors after intravenous administration of ColoAd1, with 30 ml of viral suspension infusion over 5 minutes at four different dose levels (1e10, 1e11, 1e12, and 1e13 viral particles, respectively) on days 1, 3, and 5 (dose points indicated by arrows). Each patient also received prophylactic anti-inflammatory drugs and intravenous fluids. Patients tolerated doses up to 1e12 well, but two of the four patients administered the 1e13 dose experienced cytokine-mediated dose-limiting toxicity and could not receive more than one dose. For individual patients, elevated TNF and gamma interferon levels correlated well with tolerance, but elevated IL-6 did not (data not shown). Therefore, a rate of up to 2e11 viral particles per minute could be considered a well-acceptable infusion rate. Panel A: TNF; Panel B: Gamma interferon; Panel C: IL-6.

[0347] Figure 8 shows the systemic pharmacokinetics (genomic copy number per 1 mL of blood) of ColoAd1 in human cancer patients with metastatic solid epithelial tumors. Genomic copy number measured by qPCR.

[0348] In particular, Figure 8A shows the mean plasma levels in three patients from a well-tolerated dose (1e12 VP per dose) of ColoAd1-1001 administered at 2e11 VP / min, which is equivalent to an intravenous dose of ColoAd1 administered by infusing 30 ml of viral suspension over 5 minutes on days 1, 3, and 5 (time points for the doses indicated by arrows). A typical trend is observed where each subsequent dose increases the peak viral concentration.

[0349] This clearly demonstrates the beneficial effect of the claimed dosing regimen on the pharmacokinetics of the virus, as the peak viral levels after the second and third doses increased more significantly than those after the first dose. This demonstrates the benefit of occupying or clearing the sink of non-cancer viruses at the initial dose. This dose was well-tolerated in three patients.

[0350] Figure 8B shows patient cohorts 1-4 (1 x 10⁶ each) of the ColoAd1-1001 clinical trial. 10 , 1 x 10 11 , 1 x 10 12 and 1 × 10 13 The mean initial pharmacokinetics (viral DNA copies / ml) after the first administration are shown for each viral particle at four different dose levels. In each case, the administration is carried out over 5 minutes, so the viral infusion rate is 2 × 10⁶ per minute at the lowest dose. 9 From the virus particles, at the highest dose, 2 x 10⁶ times per minute 12 The viral particles increase. With the two doses above, the viral concentration in the blood remains high for a long time, at 2 × 10⁶ per mL. 6 The virus remains above the particle size. This is the lowest blood concentration and is effective in establishing infection within the tumor (as expected from preclinical studies, as shown in Figure 1). 1 × 10 12 Regarding the dosage, the target level is achieved in 1-2 hours, 1 x 10 13 Regarding the dosage, the target level is maintained for more than 6 hours. However, 2 x 10 per minute 12 1 x 10⁻¹⁰ Administered as viral particles 13The dose of viral particles was poorly tolerable, and two patients suffered acute cytokine-mediated dose-limiting toxicity; therefore, this administration regimen is inappropriate. A dose regimen (2 × 10⁶ / min) should be used with a well-tolerated infusion rate. 11 The maximum dose, perhaps 1 × 10⁶, is administered by viral particles or slower-moving particles. 13 It can be made higher than the viral particle. Using this data, a pharmacokinetic model can be used to determine the value of 1 × 10⁶ units injected over 1 hour. 13 Virus particles (1.67 x 10⁻¹⁰ per minute) 11 In most patients, the viral blood concentration remains at 2 × 10⁶ for more than 3 hours due to the viral particles. 6 This can be shown to maintain the above level.

[0351] Figures 9A–9H show the pharmacokinetics of patients in the ColoAd1-1001 clinical trial. Patients were administered a first dose of ColoAd1, and viral load was assessed by serial blood sampling using qPCR. The following treatment plans were tested: Figure 9A: 1e10 (1 × 10) administered over 5 minutes 10 ) Virus particles (Cohort 1). Figure 9B: 1e11 (1 × 10) administered over 5 minutes. 11 ) Virus particles (Cohort 2). Figure 9C: 1e12 (1 × 10) administered over 5 minutes. 12 ) Virus particles (Cohort 3). Figure 9D: 1e13 (1 × 10) administered over 5 minutes. 13 ) Virus particles (Cohort 4). Figure 9E: 3e12 (3 × 10) administered over 5 minutes. 12 ) Virus particles (Cohort 5). Figure 9F: 3e12 (3 × 10) administered over 20 minutes. 12 ) Virus particles (Cohort 6). Figure 9G: 6e12 (6 × 10) administered over 40 minutes. 12 ) Virus particles (Cohort 7).

[0352] Each curve represents specific values ​​of viral blood levels in subjects per unit time, before administration of ColoAd1 and up to approximately 6 hours after treatment.

[0353] Adverse side effects were first observed in patients when the viral blood concentration exceeded a threshold of approximately 3e8 viral genomes per mL.

[0354] Therefore, it was determined that a range of approximately 3e7-3e8 viral genomes per ML is the ideal therapeutic range, and regimens that maintain viral blood concentration within this range as much as possible are likely to maximize viral blood levels while minimizing toxic side effects.

[0355] As can be seen from the pharmacokinetic curve, Figure 9G (6e12 particles administered over 40 minutes) exhibits characteristics particularly well-suited to maintaining viral blood concentrations within the longest therapeutic range.

[0356] Figure 10 shows the C25 levels when the same dose was administered to patients via either slow or fast infusion in the ColoAd1-1001 trial. Max This shows a comparison of levels. Both cohorts 5 and 6 had 3 × 10⁻¹⁰ 12 The full dose of viral particles was administered, but for cohort 5, it was infused over 5 minutes (rapid infusion), and for cohort 6, it was infused over 20 minutes (slow infusion). This suggests that slowing the infusion rate is more effective in controlling C Max Less variability and lower average C Max This leads to limiting the injection rate of group B adenoviruses, resulting in higher C Max It is relevant when the level is related to toxicity.

[0357] Figure 11 shows the time course of MCP1 levels (μg / L) in human cancer patients after intravenous administration of ColoAd1 on days 1, 3, and 5 (dose points indicated by arrows) in the ColoAd1-1001 clinical trial. The graph shows a comparison between different patient cohorts (1-7) administered under different dosing regimens, as shown in Table 7.

[0358] MCP1 level concentrations (μg / L) were measured at the following time points: 0 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 96 hours, 102 hours, 108 hours, 120 hours, 168 hours, and 336 hours. These human data reflect a pattern similar to that observed after each dose of all doses tested in mice for MCP1 reduction levels, and therefore support the specific benefits of the claimed dosing regimen.

[0359] The cytokine pattern shown in Figure 12 is consistent with the cytokine pattern previously observed in mouse studies (see Figure 6), indicating that each subsequent dose is well tolerated after the first dose, even if each of the three doses is equal. Studies demonstrating tumor infection by adenovirus type B can be established by the dose of viral particles administered intravenously.

[0360] Figure 12 shows a typical replication cycle for adenovirus. Adenovirus structural proteins, such as hexone, make up 90% of the viral capsid and are only expressed at the end of infection after replication development. The proteins are then sent back to the nucleus for construction. The nucleus thus has the highest concentrations of hexone and other structural proteins during replication. Therefore, hexone staining of the nucleus can be used as a marker for cells successfully infected with ColoAd1 for the quantification of adenovirus.

[0361] In the ColoAd1-1002 clinical trial, patients with primary (non-metastatic) colorectal tumors received ColoAd1 either via intratumoral (IT) or intravenous (IV) delivery. In the IT group, the virus was administered via colonoscopy in multiple injections, up to a maximum dose of 1e8VP (actual dose depended on tumor size). In the IV group, a dose of 1e12VP was administered on days 1, 3, and 5 by infusion over 5 minutes. Subsequently, 7–14 days after the initial administration of ColoAd1, the primary tumor was resected and sent for pathological examination, including immunohistochemical (IHC) staining for ColoAd1 hexone.

[0362] Sections of formalin-fixed, paraffin-embedded human tumor samples were analyzed for the presence of the virus using an anti-hexone antibody (ab8251).

[0363] Staining was performed under assay conditions confirmed by the Ultraventana benchmark. Strong nuclear staining indicates the presence of capsid construction.

[0364] Isotype controls were processed simultaneously under the same conditions.

[0365] Figure 13 shows a transmissive EM image of a colorectal cancer cell line infected in vitro with ColoAd1.

[0366] Figure 14A shows images of cell staining in tumor samples (IT) infected with ColoAd1 after intratumor injection (IT) and subsequently stained with hexone. As can be seen from the figure, stromal cells lack nuclear staining, while cancer cells have substantial nuclear staining. Figure 14B shows the corresponding isotype controls. Together, these slides demonstrate that ColoAd1 selectively infects tumor cells without infecting normal cells after direct intratumor delivery.

[0367] Figure 14C shows images of cell staining in tumor samples infected with ColoAd1 after intravenous (IV) administration and subsequently stained with hexone. As can be seen from the figure, stromal cells lack nuclear staining, while cancer cells have substantial nuclear staining. Figure 14D shows the corresponding isotype controls.

[0368] Therefore, these images provide clear evidence that ColoAd1 can be selectively delivered to tumor cells in a manner equivalent to intratumoral delivery when the claimed intravenous administration plan is used.

[0369] Example 2: Combination of drugs ColoAd1 virus replication was evaluated in the colon cancer cell line HT-29 in the presence of 320 clinically approved compounds under investigation. HT-29 cells were seeded in 96-well plates at a density of 3.0e4 cells per well and incubated at 37°C in 5% CO2. After 4–6 hours of incubation, a mixture of virus and drug compound was prepared in cell medium and diluted to the cells to obtain a final dose of 10 ColoAd1 virus particles (ppc) and 0.1 μM of the drug compound per cell. Cells were incubated for 18 hours, and the entire viral genome within the cells was then evaluated by qPCR. The relative multiplicity changes in ColoAd1 replication compared to ColoAd1 virus alone are plotted for all 15 compounds in Figure 15. Insets show the increase in viral replication after 18 hours in the presence of a microtubule inhibitor and the decrease in viral replication in the presence of a topoisomerase inhibitor.

[0370] The effect of paclitaxel or cisplatin therapy on the efficacy of ColoAd1 in tumor models was evaluated in an ovarian cancer IP model. SCID mice were transplanted with SKOV-3 human ovarian cancer cells expressing 2.5e6 luciferase. Tumor burden was assessed by luciferase expression. Mice were imaged on day 5, the day before each treatment set, and at least every 5–7 days during the study period. All ColoAd1 treatments were administered using 5e9 viral particles delivered by intraperitoneal injection, and in the combination therapy group, paclitaxel (0.4 mg) or cisplatin (0.04 mg) was delivered after viral therapy. Disease progression was assessed by luciferase imaging using the IVIS imaging system. Figure 16 shows images of relative luminescence in mice administered via IP injection with PBS (A), paclitaxel (B), ColoAd1 (C), or paclitaxel and ColoAd1 (D), and Figure 17 shows the relative luminescence tracked over time for each administration group. Figure 18 shows the relative luminescence in mice administered via IP injection with PBS (group 1), ColoAd1 followed by cisplatin (group 2), cisplatin followed by ColoAd1 (group 3), or paclitaxel followed by ColoAd1 (group 4). The administration schedule is detailed in Figures 17 and 18.

Claims

1. Parenteral formulation of a replicable oncolytic adenovirus of subgroup B, for use in the treatment of cancer in human patients by intravenous administration of the parenteral formulation in multiple doses in a treatment cycle, each dose being administered over 5 to 60 minutes in the range of 1 × 10¹¹ to 1 × 10¹³ virus particles, with a single treatment cycle lasting no more than 14 days, the treatment cycle comprising administering a first dose on day 1, and subsequent doses every other day thereafter, the human patient receiving at least 2 × 10¹¹ per ml of plasma for a period of 15 minutes or more. ６ A parenteral formulation having a plasma level of viral particles, wherein the oncolytic adenovirus has fibers and hexones of wild-type Ad11.

2. The parenteral formulation according to claim 1, wherein further doses are administered on the third and fifth days.

3. The parenteral formulation according to claim 1 or 2, wherein the first dose is lower than the next dose.

4. The parenteral formulation according to claim 1 or 2, comprising an equal number of virus particles in the total dose administered.

5. The parenteral formulation according to any one of claims 1 to 4, wherein the adenovirus has replication ability.

6. The parenteral formulation according to any one of claims 1 to 5, wherein the adenovirus is ColOd1.

7. The parenteral formulation according to any one of claims 1 to 6, wherein the virus comprises a transgene.

8. The parenteral formulation according to claim 7, wherein the introduced gene causes a therapeutic agent to be expressed in cells.

9. The virus concentration in the aforementioned preparation is 2 × 10 ８ ~2 x 10 １４ A parenteral formulation according to any one of claims 1 to 8, wherein the virus particles / ml range.

10. A parenteral formulation according to any one of claims 1 to 9, wherein the interval between treatment cycles is at least 14 days.

11. The parenteral formulation according to any one of claims 1 to 10, wherein the viral formulation is administered in combination with the administration of further cancer treatment or therapy.

12. The parenteral formulation according to claim 11, wherein the further cancer therapy is a chemotherapeutic agent.

13. The parenteral formulation according to claim 12, wherein the chemotherapeutic agent is a checkpoint inhibitor.

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