Patent Application
20230277697
The present invention relates to a theragnostic method for treating a subject affected by a neoplasia by administering in both the diagnostic and therapeutic steps the same radioisotope, which is a 64Cu++ salt. Such method comprises a diagnostic step to select a subject, in which 64Cu++ cellular uptake is detectable. In case no uptake is detectable, the treatment is not advisable. In case radioactive copper uptake from cancer lesions is detectable, this has a predictive value of response to treatment and the subject is selected for the treatment. If the subject is selected for the treatment, the method further comprises a step of treating cancer in said subject by administering a therapeutically effective amount of the same 64Cu++ salt.
Copper, essential trace element with many physiological functions, plays an important role in tumor angiogenesis and can stimulate endothelial cell proliferation (Hu, 1998). Copper metabolism, that is tightly regulated through sophisticated homeostatic mechanism in physiological conditions, is profoundly altered in neoplastic disease together with its cellular deposition—from cytoplasm in normal tissue to intranuclear and perinuclear zones in tumors (Fuchs et al., 1989). In the past twenty years, many preclinical studies have demonstrated an effect of copper on cancer development; in particular, a copper's alteration has been observed in tumor bearing in mice and rats. Furthermore, a highly significant increase in both serum copper level and ceruloplasmin level was observed in patients with various types of tumors compared to healthy subjects (Fisher and Shifrine, 1978, Scanni et al., 1979). In particular, several authors reported an increased copper content in serum of patients affected by different tumor types such as lung cancer (Bal et al., 2019), cervical cancer (Zhang et al., 2018) and prostate cancer (Saleh et al., 2019). In addition, in glioma patients both serum copper and ceruloplasmin values showed a significantly higher concentration compared to healthy subjects or subjects affected by non-tumorous neurological diseases (Manjula et al., 1992).
Currently, the importance of copper in carcinogenesis and metastasis formation and in resistance to treatment has been explored. In addition, research work on copper deregulation in oncology and the recent understanding of copper metabolism have led to the development of numerous therapeutic strategies targeting this trace element. Despite numerous research studies and the improvement of knowledge on copper metabolism over the years, some shadow areas persist (Lelièvre et al., 2020).
The deregulated levels of copper in tumor patients can be observed thanks to the radioactive isotope Copper-64 (64Cu). This is one of the copper isotopes that is showing its potential in the nuclear medicine field for the PET (Positron Emission Tomography) imaging, which can be or PET/CT (Computed Tomography) or PET/MRI (Magnetic Resonance Imaging). Once injected to the patient, this isotope follows the abnormal distribution of copper in the human body that can be visualized by PET. Copper-64 is the only copper isotope that possesses three decay modalities and it has a half-life (12.7 h) that allows to obtain efficient clinical imaging at delayed scan times, exploiting better target-to-background signal; furthermore, it allows for the convenient distribution of the radiopharmaceuticals after its synthesis at centralized production sites, and for easier management of scheduled activities at the clinical nuclear medicine facilities. 64Cu can undergo β+ emission to nickel-64, β— emission to zinc-64. Both daughter nuclides are stable. In addition to beta minus, 64Cu decays also by Auger electron emission, these electrons have an average energy of 2 keV, 126 nm range in tissue (Howell, 1992) and are considered high-LET (Linear Energy Transfer) radiation. The capability to reach the cell nuclei and to emit Auger electrons in proximity of the DNA leads to tumor cell death and this mechanism may enhance cancer therapy.
The beta plus emission gives it the capability to be a diagnostic agent, indeed different literature evidences showed the capability of 64Cu in the form of Copper Chloride (64CuCl2) to be a good diagnostic imaging agent in different cancer types, such as breast, prostate or melanoma, both in preclinical studies (Peng et al., 2006; Cal et al., 2014, Qin et al., 2014), but also in clinical settings. In patients affected by prostate cancer, 64CuCl2 PET/CT seems to show superior characteristics comparing to the clinical standard 18F-FCH (Capasso et al., 2015; Piccardo et al., 2017) and also in glioblastoma patients 64CuCl2 showed the potential to become a diagnostic agent (Panichelli et al., 2016). Instead, as mentioned above, the potential promising therapeutic use is given by the auger electron emission, but still less evidences are present in literature.
During the last few years, in the field of nuclear medicine unprecedented advances have been shown: one of the main driving forces is the so-called “theragnostic” concept that combines the use of a diagnostic biomarker with a therapeutic option. Recent developments have significantly broadened the scope of radionuclide imaging and therapies that now extends to neuroendocrine tumors, prostate cancer, or hematologic malignancies (Lapa et al., 2019).
The crucial point for the application of a theragnostic agent is the dose that, systemically administered, should reach the target tissue in sufficient quantities. Ideally, theragnostic compounds involve chemically and biologically identical compounds. Actually, the market offers the so called “theragnostic pairs” that are combinations of a diagnostic agent and a therapeutic one (Table 1). Most of them are not chemically or biologically identical, but they are similar in biodistribution and the first injection of the diagnostic agent would predict the biodistribution of the therapeutic counterpart. The diagnostic radiometals 111In an 68Ga and their therapeutic counterparts 90Y and 177Lu, are all trivalent metals, which can be attached to a targeting moiety with the same chelator, but there are differences in their chemical structure, which can affect their biological properties and biodistribution (Ballinger et al., 2018).
123I-iodide
131I-iodide
123I-iobenguane
131I-iobenguane
99mTc-MDP
223Ra chloride
111In-ibritumomab
90Y- ibritumomab
68Ga-DOTATATE
68Lu-DOTATATE
68Ga-PSMA-11
177Lu-PSMA-617
However, still the dosage regimen and its use in a theragnostic approach is still not clear to the experts in the field and can't be simply deduced due to the poor clinical data available for this treatment. In Table 2 different studies are reported, which showed the potential therapeutic use of 64CuCl2 in different cancers such as prostate cancer, glioblastoma, melanoma, but none of them anticipates the proper theragnostic dosage regimen and approach.
64CuCl2
64CuCl2
64CuCl2
64CuCl2 is able to
64CuCl2
64CuCl2 is more
64CuCl2
64CuCl2 is able to
All these studies refers to a preclinical (in animals or in-vitro) application of 64CuCl2 and none of these report clinical data. Furthermore, literature data are vague and not clear on the theragnostic approach to be used with Cu-64, its best formulation, its best radioconcentration, its dose regimen, dosing intervals that can bring to a safe and effective use.
In the study of Ferrari et al. groups of tumor implanted nude mice were treated with 64CuCl2 to simulate single and multiple dose therapy protocols (333 MBq and 55.5 MBq/day×6 days respectively), and results were analysed to estimate therapeutic efficacy. Experiments were performed in animals bearing 3 weeks old tumours. In this study three groups of mice were compared: 30 no-treated animals, 30 animals treated with a single administration of 333 MBq of 64CuCl2, and 30 animals treated with a multiple-dose regimen of 55.5 MBq×6 days of 64CuCl2, one injection each day.
Moreover, the study of Qin et al., showed the therapeutic capability of copper chloride in melanoma tumor. Specifically, as soon as the tumor diameter size reached 0.5-0.8 cm, tumor-bearing mice were administered with one dose of 64CuCl2 (about 74 MBq) and tumor sizes were monitored over the treatment period. Results showed that the tumor growth in both the B16F10 and the A375M models under 64CuCl2 treatment were much slower than that of the control group. The therapeutic group showed significantly improved survival, compared with that of the control group. Taking into account this preclinical data is not possible to correlate these doses to humans. EP3071241B 1 discloses a formulation and an average dose, however only generic indications are provided, moreover there are no evidences of safety and efficacy of that dose, furthermore there is no explanation of the theragnostic approach and method to use it as a theragnostic tracer. A theragnostic approach with 64CuCl2 has not yet been elucidated. Despite many therapeutic advances, a number of common cancers, such as cancer of the breast, prostate, brain are still a frequent cause of death and new treatment approaches are needed. Furthermore, actually common treatments such as radiotherapy and chemotherapy are showing the development of resistance to treatment. Therefore, for cancer patients the necessity to develop new effective treatments is essential.
Furthermore, commercially available theragnostic pairs use different molecules for the diagnosis and therapy with the risk to do not have the exact biodistribution of the tracer, for the prediction of the therapeutic one, furthermore with the lack of monitoring the response to treatment with the same therapeutic dose.
In view of the drawbacks of the prior art, it would be highly advantageous to provide an efficacious theragnostic approach for the parenteral administration of 64CuCl2 to patients affected by tumors in both adults and pediatric population.
The issue of a safe and effective parenteral delivery of therapeutic doses of 64CuCl2 is solved by the present invention.
The present invention is based on the discovery that, differently from what has been reported until now, Copper-64 in the form of salt (eg. 64CuCl2) can be used as a real theragnostic agent, not only as a component of a theragnostic pair. In other words, the same chemical entity (copper) and the same isotope (64) can be used both as diagnostic and therapeutic agent. The exactly same product gives the dual action and the diagnostic injection can predict exactly the biodistribution of the following injections.
Therefore, the present invention relates a method for treating a neoplasm in a human subject, which comprises the following steps:
According to a aspecific embodiment of the invention, the above method is preceded by the following steps:
The purpose of step (ii) is to measure the content of copper in the blood, as it may act as a biomarker of tumor activity. In particular, increased levels of circulating copper over normal ranges indicate increased use of copper by tumor cells.
The purpose of steps (a) and (b) in the above method is to select a subject, in which there is copper-64 uptake, in case there is no uptake the treatment is not advisable. In case there is copper uptake from the tumor lesion, this can have a predictive value of response to treatment, in case there is no uptake the subject would not start the treatment.
Maximum serum copper levels in blood may vary depending on the analytical method used to measure them. In particular, maximum values of serum copper levels are from 60 to 150 μg/dL. Therefore, serum copper level over normal ranges are usually over 150 μg/dL.
According to another embodiment the present invention relates to a radiopharmaceutical composition comprising a 64Cu++ salt and its use in a theragnostic approach for selection, therapy and monitoring response to treatment of subjects affected by tumors.
In particular, the present invention relates to a radiopharmaceutical composition of 64Cu++ to be used with a specific therapeutic dosage and regimen in a human subject, wherein said subject has previously been selected for the treatment by PET/CT or PET/MRI imaging with the same 64Cu++ salt.
According to an embodiment of the invention, the radiopharmaceutical composition comprising as the active ingredient copper-64 in ionic form (64Cu++), in combination with suitable excipients, and/or diluents is a saline solution. In particular, counter-ions may be present in the solution, such as for example chloride, acetate, citrate ions etc., to make the solution at a pH suitable for injection.
For example, the formulation can be composed by 5% of 64CuCl2, 2% acetate buffer solution, 10-93% NaCl 0,9% or for example the formulation can be composed by 8% of 64Cu(OAc)2, 4% of acetate buffer solution, 12-88% of water for injection (WFI), 1% antioxidant (e.g. ascorbic acid). According to an embodiment of the invention, the radiopharmaceutical composition has radioconcentration of 2,775 MBq/ml at calibration time.
According to another embodiment of the invention, the specific activity of this radiopharmaceutical composition is equal to 10,000 MBq of Cu/micrograms of copper.
The radiopharmaceutical composition is preferably a sterile solution that can be used as a ready-to-use solution for intravenous use.
The radiopharmaceutical composition preferably has a pH suitable for direct injection and comprised between 4-8.
The value of radioconcentration of 2,775 MBq/ml at calibration time is quite high, compared to standard practices, but from a pharmaceutical point of view showed stability up to 48 h from the end of synthesis. Furthermore, the inventors showed the higher ability of 64CuCl2 at 2,775 MBq/ml, to induce a cell-killing effect compared to lower concentrations. This is the first time that the impact of the drug radioconcentration has been correlated to the cell-killing effect for the translation to humans.
The inventors performed internalization and survival studies on different tumor cell lines (U87MG [glioblastoma], PC-3 [prostate cancer], CAMA1 [breast cancer], C8161 [melanoma], HT-29[colorectal cancer]) applying the same amount (dose) of radioactivity (eg. 10 MBq), but at different radioconcentrations, e.g. 2,775 MBq/ml vs. 1,850 MBq/ml, vs. 925 MBq/ml.
The results showed that in all the cell lines there was a direct impact of radioconcentration on cell uptake 4 h after treatment: significantly higher with the concentration of 2,775 MBq/ml compared to the lowest concentration. The increased uptake with the concentration of 2,775 MBq/ml has brought also to an increased cell-killing effect at 24 h after treatment as shown, as an example, in the cell survival curve of U87MG line. This effect of the radioconcentration has a great impact on patients for the optimization of treatment.
In general, for radiopharmaceuticals the radioactive concentration influence the radiochemical purity and the decrease in radioactive concentration increase the radiochemical purity. Therefore, it would not be obvious for a skilled person that increasing the radioconcentration of 64Cu radiopharmaceuticals can bring to a benefit for the biological effect: theoretically the increasing of radioconcentration generally brings to a higher content of impurities including “non-radioactive” copper. However, contrary to what one may expect, in this invention the formulation with the radioconcentration of 2,775 MBq/ml is characterized by a high level of purity: a low content of the “non-radioactive” copper and an high content of “radioactive” copper-64 ions, that are able to release an higher content of Auger electron in the tumor cell. In comparison to the other formulations (1,850 MBq/ml, 925 MBq/ml) this give an improved therapeutic effect.
In consideration of our in-vitro tests, 64CuCl2 2,775 MBq/ml was chosen as the preferred radioconcentration to be injected to patients as the most suitable for the theragnostic approach showing advantages from both an efficacy point of view and taking into account a practical aspect. Indeed, this radioconcentration solve the problem to have a big amount of radioactivity necessary for the treatment in a reduced volume, considering that low radioconcentration values corresponds to high volumes, whereas high radioconcentrations values are associated to smaller values. The reduction of the volume represents an advantage from a radioprotection side, in fact let the nurse to inject directly to the patient, reducing the time of exposure compared to administering per continuous infusion, and growing the patient's compliance.
According to an embodiment of the invention, after the blood sample collection, the patient firstly receive one diagnostic administration, for example, a human patient receives a single dose between 5.3 MBq/kg-13 MBq/kg. As an example, a patient of 70 kg can receive a dose of 371 MBq of 64CuCl2, typically by intravenous injection. For a child of 25 kg the diagnostic dose is 132,5 MBq, preferably the dose should be in the range 150-200 MBq.
From 1 h to 4 h after injection a PET/CT or PET/MRI imaging is performed.
The PET imaging is evaluated to see the uptake of 64CuCl2 in the cancer lesion of the patient. The PET imaging analysis after the diagnostic dose would consider a lesion as positive determining the target to background ratio (TBR). TBR is the ratio between the maximum SUV (SUVmax) of each region of interest and the maximum SUV (Standardized Uptake Value) of the level of some normal tissues to be considered “background tissue”(BKG). The measurement of the SUVmax of each collection site corresponding to the lesion must be carried out by positioning a VOI (Volume Of Interest), which fully includes the collection site.
TBR will be calculated as follow:
The arithmetic average of the SUVmax values measured on the VOI drawn on the gluteal muscles of each side will be calculated and it will be referred to as the muscle background uptake value. For the other tissues, the maximum SUV value measured for the single VOI selected will be used. A lesion is determined as positive for 64CuCl2 uptake (i.e. evidencing an uptake of 64CuCl2), if TBR results equal or higher than 5.
If there is a lesion uptake in the PET/CT or PET/MRI and the value of copper in blood are above the normal threshold, the patient can be selected for copper chloride treatment and can proceed with the injection of treatment doses, otherwise the patient cannot proceed.
If the patient is selected for the treatment, the method then further comprises a step of treating cancer in said patient selected for a treatment by administering a therapeutically efficient amount of 64CuCl2. According to an embodiment of the invention, the dose that can be injected for therapeutic scope to have a safe and effective action is from a minimum of 13 MBq/kg to a maximum of 105 MBq/kg. The dose will be calculated on the basis of the weight of the subject. For example, a patient of 70 kg can receive a dose between 1,850 MBq and 4,810 MBq. For a child of 25 kg the dose can be between 325 MBq to 2,625 MBq. In specific embodiments, a therapeutically efficient amount of the composition is administered to said subject 1 to 7 times per treatment cycle. The cycle can be repeated more than one time, preferably from one cycle to seven. The frequency of administration can comprise from 1 to 3 administrations per week. For each treatment cycle, the dosage regimen can comprise the administration of an increasing dose or of a fixed dose to the patient.
If an increased dose regimen is used, typically the first dose is an initial therapeutic dose (X1) and the following doses may be calculated as follow:
An exemplary treatment regimen comprises the following administrations for each cycle to an adult patient: 1st dose=1,850 MBq, 2nd dose=2,460MBq, 3rd dose=2,922 MBq, 4th dose=3,292 MBq, 5th dose=3,606 MBq, 6th dose=3,865 MBq, 7th dose=4,068 MBq, where the total activity administered is equal to 22,063 MBq per cycle. A different exemplary treatment regimen comprises the following administrations for each cycle to an adult patient: 1st dose=4,810 MBq, 2nd dose=4,810 MBq, 3rd dose=4,810 MBq, 4th dose=4,810 MBq, 5th dose=4,810 MBq, 6th dose=4,810MBq, 7th dose=4,810MBq, where the total activity administered is equal to 33,670 MBq per cycle.
Before selecting patient for a further cycle of treatment, a PET/CT or PET/MRI with 64CuCl2 is performed at least after 30 days after the end of the last treatment. If the PET/CT or PET/MRI evaluation shows response to treatment (stable disease, partial reduction, complete reduction), the patient can proceed with a further cycle of treatment. The treatment and the dose calculation based on the weight can be applied both to an adult patient and to pediatric population.
These doses and dosage schemes have never been tested before for efficacy and safety and do not derive from the preclinical literature data. In fact, starting from all pre-clinical data, it is possible to extrapolate and estimate the injectable dose to the patient, the extrapolation takes into account the differences in organ weights between man and animal, assuming a similar kinetic trend. The inventors performed a simulation with OLINDA software taking into account the possible variation of the organs exposed to greater risks, which is kidney in mouse while liver in humans (Linder et al., 1998). However, the extrapolation cannot be translated to humans, because the converted dose for humans resulted very high. Therefore, the doses were defined starting from the diagnostic dose. Furthermore, this is the first time, in which an evaluation of the safety profile and the Dose Limiting Toxicity was performed. No treatment related toxicities were registered associated to liver/kidney and red marrow distribution.
The disclosure also relates to a pharmaceutical composition as described in the previous section for use as PET/CT or PET/MRI imaging agent in monitoring and determining whether a subject response or not to 64CuCl2 treatment.
After each therapeutic injection it is possible to perform a PET/CT or PET/MRI to the subject to monitor response to the treatment without the necessity to inject other tracers. The monitoring of response to treatment let to control the trend of the disease and the response of the body to the treatment, including monitoring possible toxicities evaluating the biodistribution. Therefore, for example after the 4th therapeutic dose of the first cycle of therapy the physician decide to perform a PET/CT scan to evaluate if continuing the treatment or interrupting, preventing the continuation of the treatment in patient that doesn't answer to the treatment.
The results of the PET imaging can be compared with a standard imaging performed before treatment beginning. Nowadays there are no PET techniques evaluated to monitor response to 64CuCl2 treatment.
The human subject that undergo the treatment can be male or female, can be an adult patient or a child.
The radiopharmaceutical composition and the method of the invention can bring a great contribution to patient care comparing to existing therapies. In particular, with respect to other treatments with radiopharmaceuticals defined as theragnostic, which contemplate the injection of two different doses and two different compounds, the method according to the invention envisions that the same radiopharmaceutical can be used as diagnostic, therapeutic and to monitor response to treatment; this brings the following advantages to patients:
“Theragnostics” (or “theragnostic” method) is here intended to be a treatment strategy that combines therapeutics with diagnostics. It associates both a diagnostic test that identifies patients most likely to be helped or harmed by a new medication, and targeted drug therapy based on the test results.
A “neoplasm” is here intended to be a type of abnormal and excessive growth of tissue. This abnormal growth usually forms a mass, when it may be called “tumor”. The process that occurs to form or produce a neoplasm is called “neoplasia”. According to ICD-10 (Neoplasms-International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) Version for 2010. World Health Organization. Retrieved 19 Jun. 2014) neoplasms are classified into four main groups: benign neoplasms, in situ neoplasms, malignant neoplasms, and neoplasms of uncertain or unknown behavior. Malignant neoplasms are also simply known as “cancers”.
According to the present invention, the expressions “treatment” or “treating” are intended to refer to activities designed to cure, mitigate the symptoms or delay the progression of a disease or a pathological condition.
The expression “radiopharmaceutical” composition is intended here to refer to a pharmaceutical composition comprising a radioactive isotope.
According to the present invention, the expression “radioconcentration” is intended to mean concentration of a radioisotope. It is measured in mCi/ml or MBq/ml at the time of calibration (TOC), generally at the end of synthesis (EOS).
With the term “excipient”, it is intended here to refer to conventional excipients, i.e. inert compounds towards the active ingredient of a composition.
A glioblastoma patient of 70 kg that belongs to the class of recurrent glioblastoma. The patient underwent surgery, radiotherapy and chemotherapy and after that experienced recurrence. Standard treatment are not effective in this setting therefore the patient receive a diagnostic dose of 64CuCl2 equal to 925 MBq. After 1 h from administration a PET/CT scan is performed. The patient resulted positive for copper lesion at the diagnostic dose and started treatment.
The treatment followed this scheme and the patient received once a week:
1st dose=1,850 MBq, 2nd dose=2,460MBq, 3rd dose=2,922 MBq, 4th dose=3,292 MBq, 5th dose=3,606 MBq, 6th dose=3,865 MBq, 7th dose=4,068 MBq.
Where the total activity administered is equal to 22,063 MBq per cycle.
The radiopharmaceutical composition tested with this dosage regimen showed the capability to induce a tumor volume reduction. In particular, standard imaging showed a tumor volume reduction after 3 months from treatment beginning up to 50%.
A prostate cancer patient of 70 kg that belongs to the class of metastatic prostate cancer. The patient at initial diagnosis had local tumor, and after surgery (radical prostatectomy) he underwent radiotherapy and chemotherapy however he experienced local recurrence plus a bone metastatic lesion. After receiving all standard treatments, the patient receive a diagnostic dose of 64CuCl2 equal to 925 MBq. After 1 h from administration a PET/CT scan is performed. The patient resulted positive for copper lesion at the diagnostic dose and started treatment.
The treatment followed this scheme and the patient received once a week:
1st dose=4,810 MBq, 2nd dose=4,810 MBq, 3rd dose=4,810 MBq, 4th dose=4,810 MBq, 5th dose=4,810 MBq, 6th dose=4,810MBq 7th dose=4,810MBq
Where the total activity administered is equal to 33,670 MBq x cycle.
A glioblastoma patient of 71 kg that belongs to the class of recurrent glioblastoma. The patient underwent surgery, radiotherapy and chemotherapy and after that experienced recurrence. Standard treatment are not effective in this setting therefore the patient receive a diagnostic dose of 64CuCl2 equal to 925 MBq. After 1 h from administration a PET/CT scan is performed. The patient resulted positive for copper lesion at the diagnostic dose and started treatment.
The treatment followed this scheme and the patient received once a week:
1st dose=4,810 MBq, 2nd dose=4,810 MBq, 3rd dose=4,810 MBq, 4th dose=4,810 MBq, 5th dose=4,810 MBq, 6th dose=4,810MBq 7th dose=4,810MBq
Where the total activity administered is equal to 33,670 MBq×cycle.
The patient resulted responder to the treatment as showed by the comparison between pre- and post-treatment MRI.
A paediatric patient of 25 kg affected by high grade glioma, can receive a diagnostic dose of 64CuCl2 equal to 132,5 MBq. After 1 h from administration a PET/CT scan is performed. If the child results positive for copper lesion at the diagnostic dose the treatment can start.
The patient receive the following treatment scheme with 3 treatments per week:
1st dose=1,700 MBq, 2nd dose=1,700 MBq, 3rd dose=1,700 MBq, 4th dose=1,700 MBq, 5th dose=1,700 MBq, 6th dose=1,700 MBq 7th dose=1,700 MBq
Where the total activity administered is equal to 11,900 MBq×cycle.
