RU2464030C2 - Introduction of anticoagulative system reg1 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and may be used for introducing an anticoagulative system in a subject wherein the system involves an aptamer which binds factor IX/IXa, and an antidote which binds the aptamer. That is ensured by measuring subject weight in kilograms. A dose of the aptamer effective to inhibit coagulation in the subject is introduced wherein the aptamer contains the sequence SEQ ID No:1, and wherein the dose of the aptamer makes approximately 0.1 mg/kg to approximately 2.0 mg/kg, or approximately 5 mg/kg to 10 mg/kg. It is followed by introducing a dose of the aptamer antidote in the subject wherein the antidote contains the sequence SEQ ID No:2, and the dose of the antidote is exclusively based on the relation of weight/weight with the dose of the aptamer, and wherein the relation of weight/weight of the dose of the antidote with the dose of the aptamer makes approximately 0.1:1 to approximately 20:1.

EFFECT: invention provides a lower risk of developing haemorrhage in the subject, an immediate therapeutic effect, easy dosage, predictable reversibility of anticoagulative system action.

24 dwg, 7 tbl

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority in accordance with American provisional application No. 60/808987, filed May 26, 2006, American provisional application No. 60/847809, filed September 27, 2006, and US provisional application No. 60/865352, filed November 10, 2006 all of which are entitled "Introduction of REG1 Anticoagulation System", the disclosures of which are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY

A method for introducing an aptamer and antidote system for regulating blood coagulation in a subject based on a dosage regimen of system components normalized to weight or body mass index is presented.

BACKGROUND

Anticoagulant Therapy in Emergency Care

Given the central role of thrombosis in the pathology of acute coronary heart disease, injectable anticoagulants have become the basis for the treatment of patients with acute coronary syndromes, such as unstable angina and myocardial infarction, and those undergoing coronary revascularization procedures (Harrington et al., 2004; Popma et al., 2004). Currently available anticoagulants include unfractionated heparin (UFH), low molecular weight heparins (LMWH) and direct thrombin inhibitors (DTI) such as recombinant hirudin, bivalirudin and argatroban. The existing paradigm for both the use of an anticoagulant and the creation of a long-acting antithrombotic drug is to strike a balance between efficacy, which means reducing the risk of ischemic complications, and safety, which means minimizing the risk of bleeding (Harrington et al., 2004). Each of the available drugs carries an increased risk of bleeding compared to placebo.

The main side effect associated with anticoagulant and antithrombotic drugs is bleeding, which can cause persistent disability and death (Ebbesen et al., 2001; Levine et al., 2004). As a rule, cardiologists-clinicians were inclined to compromise with an increased risk of bleeding if the drug could reduce the risk of ischemic complications or acute coronary syndromes or coronary revascularization procedures. However, recent data suggest that bleeding, especially those that require blood transfusion, have a significant impact on the outcome and cost of treatment for patients with ACS. The frequency of blood transfusions in patients who undergo elective coronary artery bypass grafting (CABG) surgery is 30-60%, and transfusion in these patients is associated with increased fast, medium and long-term mortality (Bracey et al., 1999; Engoren et al., 2002; Hebert et al., 1999). Bleeding is also the most frequent and costly complication associated with percutaneous coronary interventions (PCI), with transfusions performed in 5-10% of patients with an additional cost of $ 8,000- $ 12,000 (Moscucci, 2002). In addition, the frequency of significant bleeding in patients undergoing treatment for ACS is also high, ranging from 5% to 10% (excluding patients who undergo CABG), with bleeding and transfusion, independently associated with a significant increase in instant mortality (Moscucci and et al., 2003; Rao et al., 2004). Therefore, despite the continued development of new antithrombotic agents, there is a pronounced clinical need for safer anticoagulants.

Rapid cessation of the drug can be achieved passively by means of the composition of the drug, for example, a means for infusion with a short half-life with completion of the infusion as a method of cessation, or actively by administering a second drug, an antidote, which can neutralize the effect of the drug.

For patients hospitalized with acute coronary heart disease, the ideal anticoagulant should be administered by intravenous or subcutaneous injection, be immediately effective, easily dosed, and do not require frequent monitoring and be immediately and reliably reversible.

Current Approaches to Solving the Problem

UFH is currently the only antidote anti-clotting agent that is approved for use. However, UFH has significant limitations. First, heparin has complex pharmacokinetics, which makes the reliability of its use difficult (Granger et al., 1996). Secondly, the predictability of the dose of its antidote, protamine, is difficult, and there are serious side effects associated with its use (Carr and Silverman, 1999; Welsby et al., 2005). Finally, heparin can cause thrombocytopenia (HIT) and thrombocytopenia with thrombosis (HITT) (Warkentin, 2005; Warkentin and Greinacher, 2004).

Despite these limitations, heparin remains the most widely used anticoagulant for hospitalized patients, primarily because it is “reversible”. Newer generation anticoagulants, such as LMWH, have improved predictability of UFH dosing and do not require monitoring as a practice in their normal laboratory-based applications. HIT and HITT are less likely to occur with LMWH than with UFH, but this risk has not been eliminated with their use. Two of the three commercially available DTIs, lepirudin and argatroban, in particular, are approved for use in patients who have developed or had a history of HIT. Bivalirudin is approved for use as an anticoagulant during PCI and therefore represents an attractive alternative to UFH in patients with HIT. However, there are no direct and effective antidotes to terminate the anticoagulant effects for either LMWH or DTI, which is especially risky for patients who undergo surgical or percutaneous coronary vascular revascularization procedures (Jones et al., 2002). Bleeding in patients who received LMWH or DTI is stopped by the administration of blood products, including coagulation factors.

Blood coagulation and FIX

The cell coagulation model (FIG. 1) today provides the clearest explanation of how physiological coagulation in vivo coagulation occurs (Hoffman et al., 1995; Kjalke et al., 1998; Monroe et al., 1996).

According to this model, the coagulation reaction takes place in three stages: initiation, amplification and propagation. Coagulation is initiated on cells that carry tissue factor, such as activated monocytes, macrophages and endothelial cells. Coagulation factor VIIa, which forms a complex with tissue factor, catalyzes the activation of blood coagulation factors IX (FIX) and X (FX), which, in turn, produces a small amount of thrombin from prothrombin. In the amplification phase (also referred to as the priming phase), a small amount of thrombin produced in the initiation phase activates blood coagulation factors V, VIII and XI, and also activates platelets, which represent the surface on which the subsequent coagulation reactions undergo. In vivo, the small amounts of thrombin generated during the amplification phase are not enough to convert fibrinogen to fibrin due to the presence of endogenous thrombin inhibitors called serpins, such as antithrombin III, α-2-macroglobulin and heparin cofactor II. The final phase of the coagulation reaction, spread, occurs exclusively on the surface of activated platelets. In the propagation phase, significant amounts of FIXa are formed through FXIa-catalyzed activation of FIX. FIXa forms a complex with its mandatory cofactor FVIIIa, which activates FX. Subsequently, FXa forms a complex with its mandatory cofactor FVa. The FXa-FVa complex activates prothrombin, which leads to "explosive" thrombin formation and fibrin deposition. The end result is the formation of a stable clot.

Based on the specified model, FIXa plays two roles when folding. In the initiation phase, FIXa plays an important role in the formation of a small amount of thrombin through activation of FX in FXa and subsequent activation of prothrombin. However, this role of FIXa at least partially overlaps with the conversion of FX to FXa, which is catalyzed by tissue factor and FVIIa. The more critical function of FIXa is carried out in the propagation phase, in which the enzymatic complex FVIIIa / FIXa serves as the sole catalyst for the formation of FXa on the surface of the activated platelet. Therefore, a decrease in FIXa activity, either due to genetic FIX deficiency (i.e. hemophilia B) or as a result of pharmacological inhibition of FIX / IXa, is expected to have several effects on coagulation. First, inhibition or loss of action of FIXa should partially inhibit the initiation of coagulation. Secondly, inhibition or loss of action of FIXa should have a significant effect on the propagation phase of coagulation, leading to a significant reduction or elimination of the formation of thrombin. Finally, limiting the formation of thrombin during the propagation phase will at least partially suppress the amplification of coagulation by a feedback mechanism by reducing the activation of platelets and blood coagulation factors involved at earlier stages, such as factors V, VII, and XI.

Assessment of FIXa inhibitors in animals and humans in the prior art

Inhibitors of FIX activity, such as factor IXa with an inactivated active site (FIXai) or monoclonal antibodies against FIX (e.g., BC2 antibody), have potent anticoagulant and antithrombotic activity in several animal models, including various animal models of arterial thrombosis and stroke (Benedict and et al., 1991; Choudhri et al., 1999; Feuerstein et al., 1999; Spanier et al., 1998a; Spanier et al., 1997; Spanier et al., 1998b; Toomey et al., 2000). In general, these studies have shown that FIXa inhibitors have a higher ratio of antithrombotic activity to the risk of bleeding than unfractionated heparin in animals. However, in these studies, at doses slightly higher than the effective dose, the animals treated with these agents had bleeding profiles that did not differ from those for heparin. The indicated experience of controlled animal studies suggests that under clinical conditions the ability to control the activity of a FIXa inhibitor would increase its safety and facilitate its medical use. In addition, FIXai has been shown to be safe and effective as a heparin substitute in several surgical animal models that require anticoagulant therapy, including synthetic flap angioplasty models in rabbits, as well as CAPG models with extrapulmonary circulation in dogs and non-human primates (Spanier et al., 1998a; Spanier et al., 1997; Spanier et al., 1998b). FIXai has also been used successfully in several critically ill patients who need extra-pulmonary circulation and other extracorporeal circulation methods, such as extracorporeal membrane oxygenation (Spanier et al., 1998a), as part of charity-compassionate care at Columbia College of Physicians and Surgeons. . Thus, FIXa is a valid target for anticoagulant therapy in coronary vessel revascularization procedures (both CABG and PCI) and for the treatment and prevention of thrombosis in patients with acute coronary syndromes.

Development of an aptameric drug, drug-antidote pair and REG1

One approach to presenting controlled anticoagulation is to use an anticoagulant with an average long and lasting effect of ~ 12 hours or more, which can achieve a clinically adequate effect at relatively low doses, in combination with a second agent capable of specifically binding and neutralizing the primary anticoagulant. Such a drug-antidote combination can guarantee predictable and safe neutralization and termination of the anticoagulant activity of the drug (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8; Rusconi et al., 2002, Nature 419 ( 6902): 90-4).

When opening the REG1 anticoagulant system based on aptamer (see FIG. 2), the applicants applied the antidote drug technology. Aptamers are single-stranded nucleic acids that bind to target proteins with high affinity and specificity (Nimjee et al., 2005) like monoclonal antibodies. However, in order to bind and inhibit the target protein, the aptamer must adopt a specific globular tertiary structure. The formation of this globular tertiary structure requires that the aptamer adopt the correct secondary structure (i.e., the correct areas of paired and unpaired bases).

As shown in the schematic form in figure 2, the introduction of an oligonucleotide, a complementary part of the aptamer, can change the structure of the aptamer so that it can no longer bind to its target protein and thus effectively stops or neutralizes the pharmacological activity of the aptamer drug (Rusconi and et al., 2004, Nat Biotechnol. 22 (11): 1423-8; Rusconi et al., 2002, Nature 419 (6902): 90-4).

RB006 (40 kDa ester PL-guggaCUaUaCCgCgUaaUgCuGcCUccacT, in which P = mPEG2-NHS; L = C6 NH ₂ linker; G = 2'-OH G; g = 2'-O-Me G; C = 2'-FC; c = 2'-O-Me C; U = 2'-O-Me; u = 2'-O-Me U; a = 2'-O-Me A; and T = inverted 2'-H T (SEQ ID NO 1); see FIG. 2), the REG1 drug component is a direct inhibitor of FIXa that binds to the coagulation factor FIXa with high affinity and specificity (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8; Rusconi et al., 2002, Nature 419 (6902): 90-4; see also WO 05/106042 in the name of Duke University). RB006 exhibits anticoagulant activity by blocking the FVIIIa / FIXa-catalyzed conversion of FX to FXa. RB006 is a 31-nucleotide long modified RNA aptamer that is moderately stabilized against degradation by endonucleases by the presence of 2'-fluoro and 2'-O-methyl-sugar groups and stabilized against degradation by exonucleases by a 3'-inverted deoxythymidine cap. The nucleic part of the aptamer is conjugated to a 40-kilodalt polyethylene glycol (PEG) carrier to increase its half-life in the blood. With a bolus intravenous injection, the half-life of RB006 in mice is approximately 8 hours and in monkeys approximately 12 hours. Also, in order to maintain the anti-clotting condition, RB006 may be administered as a single bolus injection for more than a few hours, rather than by intravenous infusion.

As shown in FIG. 2, RB007 (cgcgguauaguccac, in which g = 2'-O-Me G; c = 2'-O-Me C; u = 2'-O-Me U; and a = 2'-O -Me A (SEQ ID NO 2); see FIG. 2), the antidote component of REG1 is an oligonucleotide that is complementary to a portion of RB006 that can effectively bind to RB006 and thus neutralize its anti-FIXa effect. RB007 is a 2'-O-methyl RNA oligonucleotide, 15 nucleotides in length, which is complementary to a portion of the REG1 drug component. The 2'-O-methyl modification confers antidote with moderate resistance to nucleases, which provides sufficient in vivo stability to allow it to find and contact RB006, but does not support long-term in vivo persistence.

Non-clinical development phase REG1

Applicants have obtained pharmacological data demonstrating the specificity of the RB006 aptamer for FIXa and the affinity of the RB007 antidote for the aptamer. The results of non-clinical pharmacological studies can be summarized as follows: the drug component REG1 (RB006 and / or related precursor compounds) can: (1) effectively inhibit the activation of coagulation factor X in vitro; (2) to extend the coagulation time of blood plasma in vitro in the plasma of humans and other animal species; (3) to provide a systemic anticoagulant effect in animals upon intravenous bolus administration; (4) prevent the formation of a blood clot in a model of thrombosis in case of damage to an artery in animals; (5) replace heparin in an animal extrapulmonary circulation model; and (6) be effective when re-injected into an animal within 30 minutes after neutralization with the antidote component of REG1.

Non-clinical pharmacological studies have so far shown that the antidote component of REG1 (RB007 and / or antidotes specific to the precursors of the drug component of REG1) can: (1) quickly and for a long time neutralize the anticoagulant activity of the drug component of REG1 (RB006) in vitro in the plasma of humans and other animal species; (2) to quickly and for a long time neutralize the anticoagulant activity of the REG1 drug component in vivo after intravenous bolus administration to animals that underwent systemic anticoagulation therapy with the indicated agent; (3) to prevent bleeding caused by a combination of super-therapeutic doses of the drug component REG1 and surgical trauma; and (4) to neutralize the anticoagulant activity of the drug component REG1 in animals after extrapulmonary circulation. In addition, the antidote did not show any anticoagulant or other pharmacological activity in vitro in human plasma or in animals after intravenous bolus administration.

There remains a need for a reliable route of administration that takes into account the predictable and reproducible effect of the aptamer-antidote system.

SUMMARY OF THE PRESENT INVENTION

It was found that there is a clear relationship between the dose normalized by weight and, importantly, the dose of aptamer normalized to the body mass index and its pharmacodynamic response. In addition, it was surprisingly found that the dose of the antidote of the aptamer should be normalized only by the amount of the aptamer introduced into the body, and not by any additional criteria, to inhibit the activity of the aptamer to the desired level. This new understanding provides support for certain routes of administration that take into account the predictable and repeated dosing regimen for clinical use.

In one embodiment, the present invention provides an improved method for administering an aptamer anticoagulation system, comprising: 1) measuring a body mass index (BMI) of a subject; 2) determination of the desired pharmacodynamic response; and 3) administering to the subject a dose of an aptamer anticoagulant to achieve the desired pharmacodynamic response based on comparing the dose to BMI with the pharmacodynamic response. In private embodiments, the implementation of the antidote of the aptamer is subsequently administered to the subject, wherein the dose of the antidote is presented based on the ratio of the previously administered dose of the aptamer to the desired decrease in the activity of the aptamer. In special cases, the indicated dose of the antidote is normalized depending on the time that has elapsed after the administration of the aptamer. In special cases, the ratio of antidote to aptamer is halved if the aptamer was introduced earlier than 24 hours.

In particular embodiments, a maximum level of anticoagulant effect is desired. In these cases, the aptamer may be provided in an amount of 4 mg / BMI or more. In other cases, an anticoagulation level of approximately 75% of the maximum is desirable. In such cases, a dose of about 0.75-1.5 mg / BMI is administered to the subject. In other cases, an anticoagulation level of approximately 50% of the maximum is desirable. In these cases, the dose presented is approximately 0.25-0.5 mg / BMI.

In particular general embodiments, the dosage of the anticoagulant used is from 0.1 to 10 mg / BMI. In another embodiment, the dosage is from 0.2 to 8 mg / BMI, or from 0.2 to 6 mg / BMI, from 0.2 to 5 mg / BMI, from 0.2 to 4 mg / BMI, from 0, 2 to 3 mg / BMI, 0.2 to 2 mg / BMI, or 0.2 to 1 mg / BMI. In some embodiments, the dose of anticoagulant is about 0.1 mg / BMI, or about 0.2 mg / BMI, or about 0.5 mg / BMI, or about 0.75 mg / BMI, or about 1 mg / BMI, or about 2 mg / BMI, or about 3 mg / BMI, or about 4 mg / BMI, or about 5 mg / BMI, or about 6 mg / BMI, or about 7 mg / BMI, or about 8 mg / BMI, or approximately 9 mg / BMI, or approximately 10 mg / BMI.

In another embodiment, the present invention provides an improved method for administering an aptameric anticoagulant system, comprising: 1) measuring the weight of a subject; 2) determination of the desired pharmacodynamic response; and 3) administering to the subject a dose of an aptameric anticoagulant to achieve the desired pharmacodynamic response based on comparing the dose per kilogram of the subject's weight with the pharmacodynamic response. In certain embodiments, the aptamer antidote is subsequently administered to a subject, wherein the administered dose of the antidote is based on the ratio to the previously administered dose of the aptamer, normalized to desirably reduce the activity of the aptamer. In special cases, the specified dose of the antidote is calculated based on the time that has elapsed after the administration of the aptamer. In special cases, the ratio of antidote to aptamer is doubled if more than 24 hours have passed since the introduction of the aptamer.

In particular embodiments, a maximum level of anticoagulant effect is desired. In these cases, the aptamer may be administered at a dose of 1.4 mg / kg or more. In other cases, an anticoagulation level of approximately 75% of the maximum is desirable. In such cases, a dose of from 0.5 to 0.75 mg / kg is administered to the subject. In other cases, an anticoagulation level of approximately 50% of the maximum is desired. In these cases, a dose of about 0.2 to 0.4 mg / kg is administered.

In particular general embodiments, the dose used is from 0.1 to 2 mg / kg, from 0.1 to 1.8 mg / kg, from 0.1 to 1.6 mg / kg, from 0.1 to 1.5 mg / kg, from 0.1 to 1.4 mg / kg, from 0.1 to 1.3 mg / kg, from 0.1 to 1.2 mg / kg, from 0.1 to 1.1 mg / kg, from 0.1 to 1.0 mg / kg, from 0.1 to 0.9 mg / kg, from 0.1 to 0.8 mg / kg, from 0.1 to 0.7 mg / kg, from 0.1 to 0.6 mg / kg, from 0.1 to 0.5 mg / kg, from 0.1 to 0.4 mg / kg, from 0.1 to 0.3 mg / kg or from 0 , 1 to 0.2 mg / kg. In other embodiments, the dose is 1 to 20 mg / kg, 1 to 18 mg / kg, 1 to 15 mg / kg, 2 to 15 mg / kg, 3 to 15 mg / kg, 4 to 15 mg / kg, 5 to 20 mg / kg, 5 to 15 mg / kg or 1 to 10 mg / kg, or 5 to 10 mg / kg, or approximately 1 mg / kg, approximately 2 mg / kg approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, or approximately 10 mg / kg. In a preferred embodiment, the aptamer anticoagulation system is a REG1 system that includes an anticoagulant aptamer and an oligonucleotide antidote. In particular non-limiting embodiments, the aptamer is RB006 (SEQ ID NO 1) and the antidote is RB007 (SEQ ID NO 2). In one embodiment, the pharmacodynamic response is measured in coagulating activity assays, such as APTT (plasma or whole blood) or active coagulation time (BAC), and can be expressed as absolute value, percent of effect, percent change, time-weighted average value, or the area under the curve for a certain period of time.

The level of pharmacodynamic response can be any that is necessary for a particular task. For example, in special cases, if the patient has a low risk of thrombotic complications, a low level of response may be desirable. In particular cases, it may not be desirable to achieve maximum inhibition of coagulation factor, and in particular, inhibition of FIX or FIXa by using an excessive amount of an anticoagulant, in particular an aptamer for FIXa, such as RB006. In other cases where the patient has a high risk of thrombotic complication or develops a thrombotic complication, a high level of response may be desirable. In such cases, it may be desirable to maximize the inhibition of coagulation factor and, in particular, FIX or inhibition of FIXa when using an excess amount of an anticoagulant, in particular an aptamer to FIXa, such as RB006.

In one embodiment, an anticoagulant aptamer, such as RB006, is for intravenous bolus administration. In another embodiment, an anticoagulant aptamer is provided for subcutaneous injection. In another embodiment, after an intravenous or subcutaneous bolus administration of the aptamer, an antidote is injected.

The procedures described herein allow the step-by-step administration of both an anticoagulant and an antidote, which allows you to titrate either one or both of the compounds to the desired level of inhibition and neutralization of the target activity.

The ratio of antidote to aptamer was calculated based on the desired level of inhibition of the aptamer. It was found that the dose of antidote should be calculated only on the basis of the dose of aptamer, and there is no need to further normalize the factors related to the subject. In one embodiment, the ratio of the aptamer to the antidote is 1: 1. In other embodiments, the ratio of the aptamer to the antidote is greater than 1: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or more. These ratios can also be calculated based on the ratio of antidote to aptamer, which may, for example, be less than about 1: 1, such as 0.9: 1, or about 0.9: 1, 0.8: 1, or about 0 , 8: 1, 0.7: 1, or approximately 0.7: 1, 0.6: 1, or approximately 0.6: 1, 0.5: 1, or approximately 0.5: 1, 0.45 : 1, or approximately 0.45: 1, 0.4: 1, or approximately 0.4: 1, 0.35: 1, or approximately 0.35: 1, 0.3: 1, or approximately 0.3 : 1, 0.25: 1, or approximately 0.25: 1, 0.2: 1, or approximately 0.2: 1, 0.15: 1, or approximately 0.15: 1, 0.1: 1 , or about 0.1: 1, or less than 0.1: 1, such as about 0.00 5: 1 or less. In some embodiments, the ratio is from 0.5: 1 to 0.1: 1, or from 0.5: 1 to 0.2: 1, or from 0.5: 1 to 0.3: 1. In other embodiments, the ratio is from 1: 1 to 5: 1, or from 1: 1 to 10: 1, or from 1: 1 to 20: 1.

In some embodiments, only partial neutralization of the aptamer activity occurs. For example, in some embodiments, the activity of the aptamer is neutralized by 90% or less than 90%, for example, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 30%, 20%, approximately 10% or less. The ratio of antidote to aptamer can be calculated either by comparing weight with weight, or based on moles.

In particular embodiments of the present invention, the organism or subject to which the dosage system is applied is a human. In certain embodiments, the body is a person who needs anticoagulant therapy. In certain embodiments, the body is a person who is undergoing vascular surgery, such as CABG surgery.

Brief Description of the Drawings

Figure 1 shows a cell model of coagulation. TF - tissue factor; vWF - von Willebrand factor; II - prothrombin; IIa - thrombin; Va, VIIa, VIIIa, IXa, Xa, XIa are the activated forms of coagulation factors V, VII, VIII, IX, X, and XI.

Figure 2 shows the anticoagulation system REG1. The system consists of a FIXa RB006 inhibitor and a suitable antidote RB007 for it. Recognition of a drug by an antidote occurs as shown by base pairing according to the Watson-Crick rule. RB006 is a modified RNA aptamer consisting of 2'-fluorine residues (upper case), 2'-O-methyl residues (lower case) and one 2'-hydroxyl residue (underlined). RB006 is conjugated to a 40-kDa polyethylene glycol carrier (P) via a 6-carbon aminolinker (L) and protected from degradation by nucleases via the inverted deoxythymidine at the 3'-end (idT). RB007 (antidote) is a 2'-O-methyl-modified RNA oligonucleotide.

Figure 3 is an in vitro APTT dose response curve diagram of RBT, showing that RB006 exhibits a concentration-dependent increase in APTT in normal pooled human plasma. The average value, in seconds, is the average APTT. The data approached the four-parameter logistic equation, which makes it possible to determine the IC ₅₀ from the curve.

Figure 4 presents a diagram of the anticoagulant effect of RB006 in the plasma of patients. The anticoagulant activity of RB006 was measured in 4 patients: two women and two men. Plasma samples were obtained from George King Biomedical (Overland Park, KS). Patients were screened and confirmed normal for coagulation factor levels. M / 55 means that the donor was a man, 55 years old; F / 49 means that the donor was a woman, 49 years old. As the APTT reagent, MDA Platelin L (Biomeriux) was used, which is relatively more sensitive to FIX levels than the APTT reagent used in the study, which is presented in Fig.3.

Figure 5 is a graph showing the neutralizing activity of a drug with the RB007 antidote. A small molar excess of the antidote RB007 with respect to the aptamer RB006 completely neutralizes the anticoagulant activity of RB006 for 10 minutes. The data shown are presented as mean ± standard error of the mean for three independent measurements. The molar ratio is based on moles of oligonucleotide aptamer and antidote (AD).

Figure 6 presents a graph of the re-introduction of the aptamer RB006 after neutralizing the antidote with a previous dose of the drug. Pigs were injected with 2.5 mg / kg RB006 aptamer and after 15 minutes, 3 mg / kg RB007 antidote (n = 2) was administered to neutralize the indicated initial dose. Further, 30 minutes after administration of the antidote RB007 (after 45 minutes of initial administration of the aptamer), pigs were re-injected with 2.5 mg / kg aptamer RB006. The change in clotting time was measured by (A) BAC (O) in whole blood assays; or by (B) APTT (O) in plasma coagulation assays. The data shown are presented as mean ± range for repeated measurements for each animal. The bold line (in A and B) is a simple interpolation line for the experimental points.

FIG. 7 is an in vitro RB006 diagram of the APTT dose-response curve in plasma of cynomolgus monkeys and humans. RB006 exhibits a dose-dependent prolongation of APTT in plasma from monkeys, which is very similar to that observed in human plasma. The experiments were performed using a reagent for APTT of the same brand, APTT-LS, as used to analyze plasma samples in non-clinical toxicity studies performed on monkeys (REG1-TOX001 and REG1-TOX003). Therefore, the obtained data serve as the basis for the interpretation of the APTT results REG1-TOX001 and REG1-TOX003 presented in sections 8.4. According to the manufacturer (Pacific Homeostasis, Middletown, VA), this reagent determines APTT ~ 87.3 seconds in plasma samples of people containing <1% FIX levels, 36.1 seconds in samples containing ~ 20% of normal FIX activity, and 27 , 5 seconds in samples containing 100% FIX activity. Javanese macaque citrate pooled plasma was provided by Charles River Laboratories, Sierra Division.

On Fig presents a graph of systemic anticoagulation in monkeys with the introduction of RB006. The level of anticoagulation in monkeys was monitored by APTT. For animals that were administered 15 mg / kg, RB006 data are presented as mean ± standard error of the mean (s.o.s.). For animals with dosage levels of 5 and 30 mg / kg, the data are presented as the average value ± range, since in each of the indicated groups there were only 2 animals in terms of dosage level.

Figure 9 presents a graph of systemic anticoagulation in monkeys RB006 and neutralization with the antidote RB007. The level of anticoagulation in monkeys was monitored by APTT. RB007 was administered at t = 3 hours after administration of RB006. Data are presented as mean ± SD

Figure 10 presents a diagram of the pharmacodynamic activity of RB006 in humans.

Figure 11 presents a diagram of the neutralization of the pharmacological activity of RB006 in humans by RB007.

11 is a diagram comparing the pharmacodynamic activity of RB006 with and without RB007.

12 is a diagram comparing the pharmacodynamic response of subjects who were given 60 mg of RB006, after which RB007 or placebo was administered after 3 hours.

On Fig presents a more detailed analysis of the relative increase in APTT relative to the initial value for 0-3 hours in all subjects who received RB006.

On Fig presents a graph of AUC _0-3 for each subject, depending on the dose level of RB006 (15, 30, 60 or 90 mg). Since the relative effect is measured for more than 3 hours, a value of "3" represents the absence of an answer to RB006, a value of 6 indicates an average 2-fold increase in the relative baseline, etc.

On Fig presents a chart normalized to the weight of the dose of RB006 as a function of the dose level of RB006.

On Fig presents a diagram of AUC _0-3 in comparison with the weight-normalized dose of RB006.

On Fig presents a chart normalized to BMI dose of subjects who were injected RB006, as a function of the dose level of RB006.

On Fig presents a diagram of AUC _0-3 for RB006 depending on the normalized dose of BMI.

On Fig presents a diagram of APTT compared with the initial level depending on% activity of FIX, showing APTT at various doses of RB006 (15, 30, 60 and 90 mg).

On Fig presents a diagram of the APTT response, depending on the use of four doses of aptamer RB006 and antidote RB007, used intravenously in patients with coronary artery disease.

On Fig presents a chart showing the dependence of APTT on time after administration of RB006 (0.75 mg / kg) for 1, 3 and 5 days in all treatment groups. Group 1: subjects who received a single dose of aptamer (0.75 mg / kg RB006) for 1, 3, and 5 days, after which one hour should be followed by the introduction of a fixed dose of antidote (1.5 mg / kg RB007); groups 2 and 3: subjects who received a single dose of RB006 aptamer (0.75 mg / kg) on days 1, 3, and 5, after which various single doses of RB007 were administered after one hour.

On Fig presents a graph of the dependence of the average APTT from time to time in the groups that were administered RB006 (0.75 mg / kg) and RB007 in various proportions compared to RB006.

On Fig presents a chart showing the percentage recovery of the time-average APTT after the introduction of RB006 after an hour of RB007 in the above ratios compared to RB006.

DETAILED DESCRIPTION

It was found that between the dosage normalized by weight and, what is important, the dosage normalized by the body mass index, there is a clear connection, in particular, for the aptamer anticoagulant and its pharmacodynamic response. In addition, it was surprisingly found that in order to inhibit the activity of the aptamer to the desired level, the dose of the antidote to the aptamer should be calculated only on the basis of the amount of the aptamer introduced into the body and not on any additional criteria. This new understanding provides support for certain modes of administration that allow for a predictable and multiple dosing regimen for clinical use.

Aptamer Development

Nucleic acid aptamers are isolated using an exponential enrichment method through the systemic evolution of ligands called SELEX. This method allows for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. The SELEX method is described, for example, in US patent No. 7087735, US patent No. 5475096 and US patent No. 5270163 (see also WO 91/19813).

The SELEX method involves the selection of a mixture of candidate oligonucleotides and stepwise repetition of binding, separation and amplification using the same general selection scheme to achieve virtually any desired criteria for binding affinity and selectivity. Starting with a mixture of nucleic acids, for example mixtures containing fragments of a randomized sequence, the SELEX method includes the steps of contacting the mixture with the target under conditions favorable for binding, separating unbound nucleic acids from those nucleic acids that specifically bind to the target molecules, destruction target nucleic acid complexes; amplification of nucleic acids dissociated from target nucleic acid complexes in order to obtain a ligand-enriched mixture nucleic acids, and then repeating the steps of binding, separation, dissociation and amplification as many times as necessary to obtain highly specific, high-affinity aptamers to the target molecule.

The basic SELEX method has been modified to achieve several particular goals. For example, US Pat. No. 5,707,796 describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural properties, such as bent DNA. US patent No. 5763177 describes a SELEX-based method for selecting aptamers containing photoreactive groups that are able to bind and / or crosslink when exposed to light and / or photoactivate the target molecule. US patent No. 5580737 describes a method for identifying highly specific aptamers that are able to distinguish closely related molecules, called Counter-SELEX. US patents No. 5567588 and No. 5861254 describe methods based on SELEX, which achieve highly efficient separation of oligonucleotides that have high and low affinity for the target molecule. US patent No. 5496938 describes methods for producing improved aptamers after carrying out the SELEX method. US Patent No. 5,705,337 describes methods for covalently linking a ligand to its target.

The feasibility of identifying aptamers to small peptides in solution has been demonstrated in US Pat. No. 5,648,214. The ability to use affinity ligand elution to produce aptamers that target a specific site on a target molecule is illustrated in US Pat. No. 5,780,228, which relates to the production of high affinity aptamers that bind with certain lectins. Methods for preparing tissue specific aptamers that include groups of cell types are described in US Pat. No. 6,127,119. The preparation of certain modified high affinity alkaline phosphatase ligands from calf intestines is described in US Pat. No. 6673553. US Pat. No. 6,716,580 describes an automated method for identifying aptamers, which includes the use of robotic manipulators.

In its simplest form, the SELEX method can be determined by the following series of steps.

1) Get a mixture of candidate nucleic acids with a different sequence. A candidate mixture typically includes regions of fixed sequences (that is, each member of a mixture of candidates contains the same sequences in the same position) and regions of randomized sequences. Regions of fixed sequences are selected either: (a) to assist during the amplification steps described below, (b) to simulate a sequence known to bind to a target, or (c) to increase the concentration of a given structural organization of nucleic acids in a mixture candidates. Randomized sequences can be completely randomized (that is, the probability of detecting the base at any position is one-fourth) or only partially randomized (for example, the probability of detecting the base at any position can be set at any level from 0 to 100 percent).

2) The candidate mixture is contacted with the selected target under conditions conducive to binding of the target and members of the candidate mixture. Under these conditions, the interaction between the target and the nucleic acids of the candidate mixture can be considered as the formation of target nucleic acid pairs between the target and those nucleic acids that have the strongest affinity for the target.

3) Nucleic acids with the highest affinity for the target are separated from nucleic acids with a lower affinity for the target. Since there is only an extremely small number of sequences (and possibly only one nucleic acid molecule) in the candidate mixture that correspond to nucleic acids with the highest affinity, it is usually desirable to set separation criteria so that a significant amount of nucleic acids in the candidate mixture (approximately 5-50% ) were saved during the separation.

4) Those nucleic acids that were selected during the separation as having a relatively higher affinity for the target are then amplified to create a new candidate mixture that is enriched in nucleic acids that have a relatively higher affinity for the target.

5) Repeating the above steps for separation and amplification, the newly formed mixture of candidates contains less and less weakly binding sequences, and the average degree of affinity of nucleic acids to the target usually increases. To the extreme extent, the SELEX method will result in a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids of the original candidate mixture that have the highest affinity for the target molecule.

Chemical modifications

One of the problems encountered in the therapeutic use of nucleic acids is that oligonucleotides in their phosphodiester form can rapidly decompose in body fluids by intracellular and extracellular enzymes, such as endonucleases and exonucleases, until the desired effect is manifested. Certain chemical modifications of the aptamer can be made in order to increase the in vivo stability of the aptamer or to enhance or deliver the aptamer.

Modifications to aptamers include, but are not limited to, those that provide other chemical groups that add additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and flexibility to the base of the aptamer or to the aptamer as a whole. Such modifications include, but are not limited to, modifications of the 2'-position of sugar, modifications of pyrimidine at the 5-position, modifications of purine at the 8-position, modifications of off-cyclic amines, a substitution in 4-thiouridine, a substitution in 5-bromo or 5-iodouracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylation, unusual base pairing combinations, such as isocitidine and isoguanidine isobases, etc. Modifications may also include 3'- and 5'-modifications, such as capping.

The SELEX method involves the identification of high affinity aptamers containing modified nucleotides giving the ligand improved characteristics, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the positions of the ribose and / or base and / or phosphate. The SELEX identified aptamers containing modified nucleotides are described in US Pat. No. 5,660,985, which describes oligonucleotides containing derivatives of nucleotide compounds chemically modified at the 5'- and 2'-positions of pyrimidines. US Patent No. 5,580,737 describes specific aptamers containing one or more nucleotides modified with 2'-amino (2'-NH ₂ ), 2'-fluoro (2'-F) and / or 2'-O-methyl (2 ' -OMe). US patent No. 5756703 describes oligonucleotides containing various 2'-modified pyrimidines.

The SELEX method involves combining selected oligonucleotides with other selected oligonucleotides and neoligonucleotide functional groups, as described in US Pat. Nos. 5,637,459 and 5,683,867. U.S. Pat. NH ₂ ), 2'-fluoro (2'-F) and / or 2'-O-methyl (2'-OMe). The SELEX method further includes combining selected aptamers with lipophilic or non-immunogenic high molecular weight compounds in a diagnostic or therapeutic complex, as described in US Pat. No. 6,011,020.

If the aptamers are obtained using the SELEX method, the modifications can be pre- or post-SELEX modifications. Pre-SELEX modifications can lead to aptamers with both specificity for the respective target and improved in vivo stability. Post-SELEX modifications targeting 2'-OH aptamers can result in improved in vivo stability without adversely affecting the binding ability of aptamers. In one embodiment, aptamer modifications include 3'-3'-inverted phosphodiester bonds at the 3'-end of the molecule and 2'-fluoro (2'-F) and / or 2'-amino (2'-NH ₂ ), and / or 2'-O-methyl (2'-OMe) modification of some or all of the nucleotides.

In one embodiment, the aptamer or its regulator can be covalently attached to a lipophilic compound, such as cholesterol, dialkylglycerol, diacylglycerol, or a non-immunogenic high molecular weight compound or polymer, such as polyethylene glycol (PEG). In these cases, the pharmacokinetic properties of the aptamer or modulator can be enhanced. In other embodiments, the aptamer or modulator may be encapsulated in the liposome. The lipophilic compound or non-immunogenic macromolecular compound can be covalently linked or bound via non-covalent interactions with the aptamer or modulator (s). In embodiments using a covalent compound, a lipophilic compound or a non-immunogenic macromolecular compound may be covalently linked to an aptamer or modulator at various positions, such as an acyclic amino group at the base, at the 5-position of the pyrimidine nucleotide, at the 8-position of the purine nucleotide, with a phosphate hydroxyl group or a hydroxyl group or another group at the 5'- or 3'-end. In one embodiment, covalent attachment is to a 5'- or 3'-hydroxyl group. The attachment of the oligonucleotide modulator to other components of the complex can be done directly or using linkers or spacers.

The oligonucleotides of the present invention can be modified at the base, sugar group or phosphate backbone, for example, to improve the stability of the molecule, hybridization, etc. The oligonucleotide may contain other attached groups. For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a crosslinking agent, activated by hybridization, a transport agent, a cleavage agent, activated by hybridization, etc. The oligonucleotide may contain at least one modified group in the base, which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- ( carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethylthiouridine, 5-carboxymethylaminomethyluracil, dihydrowracil, beta-D-galactosylquueuosin, inosine, N6-isopentyladenine, 1-methylguanine, 1-methylinyl-2-enyl, 2-methyl-2-enyl-2-enyl-2-enyl-2-enyl-2-enyl-2-enyl-2-enigin-2-methyl-2-enigin-2-methyl-2-enigin-2-methyl-2-enigin-2-methyl-2-enigin-2-methyl-2-enigin-2-methyl-2-eniginene methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-meth laminomethyluracil, 5-methoxyaminomethyl-2α-thiouracil, β-D-mannosylquesuosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-isopentenyladenine, uracyloxyacetic acid, pivutuoxiozin-2, 5-methynoacetincyl, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracyloxyacetic acid (v), 5-methylthiouracil, 3- (3-amino-3-N-carboxypropyl) and 2,6-diaminopurin .

The aptamer or modulator of the present invention may also contain at least one modified sugar group selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylose, and hexose. The aptamer or modulator may contain at least one modified phosphate backbone selected from the group consisting of, but not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, alkyl phosphotriethether and formal or their analogues.

Any of the oligonucleotides of the present invention can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (that is, which are commercially available, for example, from Biosearch, Applied Biosystems).

Modulators

The modulators of the present invention can be oligonucleotides, small molecules, peptides, oligosaccharides, for example aminoglycosides or other molecules that can bind to the aptamer or otherwise modulate the action of the aptamer, or chimera, or fusion, or related product of any of them.

In one embodiment, the modulator is an oligonucleotide complementary to at least a portion of the aptamer. In another embodiment, the modulator may be a ribozyme or deoxyribozyme that is directed to the aptamer. In a further embodiment, the modulator may be a peptide nucleic acid (PNA), morpholino nucleic acid (MNA), closed nucleic acid (LNA), or pseudocyclic oligonucleic bases (PCO), which include a sequence that is complementary or hybridizes with at least part of the aptamer.

The aptamer has an active tertiary structure, which depends on the formation of the correct stable secondary structure. Therefore, while the mechanism of duplex formation between the complementary oligonucleotide modulator of the present invention and the aptamer is the same as between two short linear oligoribonucleotides, the rules for predicting such interactions and the kinetics of formation of such a product depend on the intramolecular structure of the aptamer. The nucleation rate is important for the formation of the final stable duplex, and the speed of this step is significantly increased by directing the oligonucleotide modulator to single-stranded loops and / or single-stranded 3'- or 5'-tails present in the aptamer. For the formation of an intermolecular duplex, it is necessary that the free energy of the formation of an intermolecular duplex is favorable with respect to the formation of existing intramolecular duplexes inside the targeted aptamer.

Modulators can be designed to bind a particular aptamer with high specificity and desired affinity. Modulators can also be designed in such a way that upon binding, the structure of the aptamer changes in the direction of a more or less active form. For example, the modulator can be designed so that when it binds to the target aptamer, the three-dimensional structure of the aptamer changes so that the aptamer can no longer bind to its target molecule or binds to its target molecule with less affinity.

Alternatively, the modulator can be designed so that upon binding the three-dimensional structure of the aptamer changes so that the affinity of the aptamer to its target molecule increases. That is, the modulator can be designed so that upon binding in the aptamer a structural motif is formed in such a way that the aptamer can communicate with its target molecule.

In an alternative embodiment of the present invention, the modulator itself is an aptamer. In said embodiment, an aptamer is first obtained that binds to the desired therapeutic target. In a second step, a second aptamer is obtained that binds to the first aptamer using the SELEX method described in this application or another method and modulates the interaction between the therapeutic aptamer and the target. In one embodiment, the second aptamer inactivates the effect of the first aptamer.

In other alternative embodiments, the aptamer that binds to the target may be PNA, MNA, LNA, or PCO, and the modulator is an aptamer. Alternatively, the aptamer that binds to the target is PNA, MNA, LNA or PCO, and the modulator is PNA. Alternatively, the aptamer that binds to the target is PNA, MNA, LNA or PCO, and the modulator is MNA. Alternatively, the aptamer that binds to the target is PNA, MNA, LNA or PCO, and the modulator is LNA. Alternatively, the aptamer that binds to the target is PNA, MNA, LNA or PCO, and the modulator is PCO. If desired, any of them can be used in stereochemistry, which is found in nature, or in stereochemistry, which is not found in nature. For example, in a preferred embodiment, the aptamer is in configuration D, and in an alternative embodiment, the aptamer is in configuration L.

In one embodiment, the modulator of the present invention is an oligonucleotide that contains a sequence complementary to at least a portion of the targeted aptamer sequence. For example, an oligonucleotide modulator may contain a sequence complementary to 6-25 nucleotides of the target aptamer, typically 8-20 nucleotides, usually 10-15 nucleotides. Advantageously, the oligonucleotide modulator is complementary to 6-25 consecutive nucleotides of the aptamer, or 8-20 or 10-15 consecutive nucleotides. The length of the oligonucleotide modulator can be optimized, taking into account the aptamer target and the desired effect. Typically, an oligonucleotide modulator consists of 5-80 nucleotides in length, most often 10-30 and most often 15-20 nucleotides (e.g. 15-17). The oligonucleotide can be obtained from nucleotides having D or L stereochemistry, or mixtures thereof. Naturally occurring nucleic bases have a D configuration.

Various strategies can be used to determine the optimal site for binding of an oligonucleotide to a target aptamer. You can use the empirical strategy according to which complementary oligonucleotides "pass" through the aptamer. The passage experiment may include two experiments that are performed sequentially.

You can get a new mixture of candidates, in which each member of the mixture of candidates has a fixed region of nucleic acid, which corresponds to the oligonucleotide modulator of interest. Each member of the candidate mixture also contains a region of randomized sequences. According to this method, it is possible to identify what is referred to as “extended” aptamers that contain regions that can bind to more than one aptamer binding domain. According to this approach, 2'-O-methyl oligonucleotides (e.g., 2'-O-methyl oligonucleotides) of approximately 15 nucleotides in length, which are coordinated by about 5 nucleotides of the aptamer (e.g., oligonucleotides complementary to nucleotides 1-15, 6-, can be used) 20, 11-25, etc. aptamer). An empirical strategy can be particularly effective because the effect of the tertiary structure of the aptamer on hybridization efficiency can be difficult to predict. To assess the ability of various oligonucleotides to hybridize with a specific aptamer, in particular to determine the molar excess of the oligonucleotide, which is necessary to achieve binding of the entire amount of aptamer, the assays described in the examples that follow will be used. The ability of various oligonucleotide modulators to increase the rate of dissociation of the aptamer or the association of the aptamer with its target molecule can also be determined by conducting standard kinetic studies, for example, BIACORE assays. Modulators of oligonucleotides can be selected so that a 5-50-fold molar excess of the oligonucleotide or less is necessary to change the interaction between the aptamer and its target molecule in the desired manner.

Alternatively, the target aptamer may be modified to contain a single chain end (3 'or 5') to improve association with the oligonucleotide modulator. Suitable ends may contain from 1 to 20 nucleotides, preferably 1-10 nucleotides, more preferably 1-5 nucleotides and most preferably 3-5 nucleotides (for example, modified nucleotides such as 2'-O-methyl sequences). Single-stranded aptamers can be tested for binding and biological activity (for example, as described in the examples that follow) to verify that the addition of a single-stranded end does not violate the active structure of the aptamer. A number of oligonucleotides (for example, 2'-O-methyl oligonucleotides), which can form, for example, 1, 3 or 5 base pairs in the sequence of a single chain end, can be developed and tested for their ability to bind only to an aptamer containing a single chain end, as well , as well as their ability to increase the rate of dissociation of the aptamer or the association of the aptamer with its target molecule. To verify that the effects are due to the formation of a duplex, and not as a result of non-specific effects, you can use controls with randomized sequences.

Modulators of the oligonucleotides of the present invention contain a sequence complementary to at least part of the aptamer. However, absolute complementarity is optional. The sequence "complementary to at least part of the aptamer" referred to in the present description, refers to a sequence having sufficient complementarity to be able to hybridize with the specified aptamer. The ability to hybridize may depend on both the degree of complementarity and the length of the antisense nucleic acid. As a rule, the larger the hybridizing oligonucleotide, the more erroneous pairings between the bases of the target aptamer it can contain and still form a stable duplex (or triplex, depending on the circumstances). One of skill in the art can establish a tolerable degree of mismatch using standard procedures for determining the melting point of a hybrid complex. In particular aspects, the oligonucleotide may consist of at least 5 or at least 10 nucleotides, at least 15 or 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides. The oligonucleotides of the present invention can be DNA or RNA, or hybrid mixtures, or derivative compounds, or their modified versions, single-stranded.

In one embodiment, the modulator is a ribozyme or deoxyribozyme. There are at least five classes of ribozymes, and each has a different type of specificity. For example, group I introns have a size from about 300 to> 1000 nucleotides, and they need U in the target sequence directly from the 5'-side of the cleavage site, and they bind 4-6 nucleotides from the 5'-end of the cleavage site. Another class is RNaseP RNA (M1 RNA), which have a size of approximately 290 to 400 nucleotides. A third example is hammerhead ribozymes that have a size of about 30 to 40 nucleotides. For them, it is necessary that the UH sequence is located on the 5'-side of the cleavage site, and they bind to sequences with different numbers of nucleotides on both sides of the cleavage site. The fourth class comprises hairpin ribozymes, which are approximately 50 nucleotides in size. They need a GUC target sequence directly from the 3'end of the cleavage site, and they bind to 4 nucleotides at the 5'end of the cleavage site and a variable amount at the 3'end of the cleavage site. The fifth group is represented by hepatitis delta virus (HDV) ribozymes, which are approximately 60 nucleotides in size.

Another class of catalytic molecules is called "deoxyribozymes." Deoxyribozymes are single-stranded and cleave both RNA and DNA. A general model for deoxyribozymes, known as the 10-23 model, was proposed. According to the 10-23 model, deoxyribozymes possess a catalytic domain of 15 deoxyribonucleotides, which is flanked by two domains recognizing the substrate, each consisting of seven to nine deoxyribonucleotides.

The nucleic bases of the oligonucleotide modulators of the present invention can be connected via bonds between nucleic bases, for example peptidyl bonds (as in the case of Peptide Nucleic Acids (PNA); Nielsen et al. (1991) Science 254, 1497 and US Patent No. 5539082) and morpholine relationships (Qin et al., Antisense Nucleic Acid Drug Dev. 10, 11 (2000); Summerton, Antisense Nucleic Acid Drug Dev. 7, 187 (1997); Summerton et al., Antisense Nucleic Acid Drug Dev. 7, 63 ( 1997); Taylor et al., J Biol Chem. 271, 17445 (1996); Partridge et al., Antisense Nucleic Acid Drug Dev. 6, 169 (1996)), or by any other natural or modified association. Oligonucleic bases may also be closed nucleic acids (LNAs). Nielsen et al., J Biomol Struct Dyn 17, 175 (1999); Petersen et al., J Mol Recognit 13, 44 (2000); Nielsen et al., Bioconjug Chem 11, 228 (2000).

PNA are compounds that are similar to oligonucleotides, but differ in composition. In PNA, the deoxyribose backbone of the oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally occurring or non-naturally occurring nucleic acid base. PNA often has an achiral polyamide backbone consisting of N- (2-aminoethyl) glycine groups. To represent a complementary nucleic acid, purine or pyrimidine bases are linked to each subunit via a methylene carbonyl linker (1-3). PNA binds to complementary RNA or DNA in parallel or antiparallel orientation according to Watson-Crick base pairing rules. The uncharged PNA oligomers enhances the stability of hybrid PNA / DNA (RNA) duplexes compared to natural homoduplexes.

Morpholino-nucleic acids got this name because they consist of subunits of morpholine, each of which contains one of four genetic bases (adenine, cytosine, guanine and thymine) connected to a 6-membered morpholine ring. Between eighteen and twenty-five subunits of these four types of subunits are connected in a specific order by nonionic phosphorodiamidate intersubunit bonds to form a morpholine oligomer. These morpholine oligomers with their 6-membered morpholine groups constituting the backbone connected by non-ionic bonds represent substantially better antisense properties than RNA, DNA and their analogues having 5-membered ribose or deoxyribose backbones joined by ionic bonds (see www. gene-tools.com/Morphol-inos/body_morpholinos.HTML).

LNA is a class of DNA analogs that has some properties that make it a prime candidate as a modulator of the present invention. LNA monomers are bicyclic compounds, similar in structure to RNA monomers. LNA with DNA and RNA share the majority of chemical properties, namely they are soluble in water, can be separated by gel electrophoresis, precipitated with ethyl alcohol, etc. (Tetrahedron, 54, 3607-3630 (1998)). However, the introduction of LNA monomers in either DNA or RNA oligomers leads to high thermal stability of duplexes with complementary DNA or RNA, while obeying the Watson-Crick base pairing rules. Such high thermal stability of duplexes formed with LNA oligomers, together with the discovery that the primers containing at the 3'-end of LNA are substrates for enzymatic extension, for example, PCR reactions, are used in the present invention to significantly increase the specificity of detection of various nucleic acids in vitro assays described in this application. Amplification methods for individual alleles are highly differential (cross-reactions are not displayed), and several reactions can occur in one tube. See, for example, US patent No. 6316198.

Pseudocyclic oligomeric nucleic bases (PCO) can also be used as a modulator in the present invention (see US patent No. 6383752). PCOs contain two oligonucleotide segments that are connected through their 3'-3 'or 5'-5' ends. One of the PCO segments (“functional segment”) has some functionality (for example, an antisense oligonucleotide complementary to an mRNA target). Another segment (“protective segment”) is complementary to the 3 ′ or 5 ′ ends of the functional segment (depending on the end through which it is attached to the functional segment). As a result of the complementarity between the functional and protective segments, in the absence of target nucleic acids (for example, RNA), intramolecular pseudocyclic structures are formed in PCO. PCOs are more stable than conventional antisense oligonucleotides due to the presence of 3'-3 'or 5'-5' bonds and the formation of intramolecular pseudocyclic structures. Studies of pharmacokinetics, tissue distribution and stability in mice suggest that PCOs have higher in vivo stability and pharmacokinetics and tissue distribution, similar to that for sugar-phosphate oligonucleotides in general, but quick elimination from a number of tissues. If the fluorophore and quencher molecules are properly attached to the PCO of the present invention, the molecule will fluoresce if it is in a linear configuration and the fluorescence will be quenched in a cyclic conformation.

Peptide-based aptamer modulators represent an alternative class of oligonucleotide modulator molecules or their analogues. Modulators of this class proved to be especially useful when sufficiently active oligonucleotide modulators of the target aptamer cannot be isolated due to the lack of sufficient single-stranded regions to ensure nucleation between the target and the oligonucleotide modulator. In addition, peptide modulators provide bioavailability and pharmacokinetics other than oligonucleotide modulators.

Oligosaccharides, like aminoglycosides, can bind to nucleic acids and can be used to modulate the activity of aptamers. A synthetic drug that is introduced between the aptamer and the target and either disrupts or modifies the binding of the aptamer to the target can also be used as a therapeutic regulator. Such synthetic preparations can be identified by screening candidates in an assay that measures changes in the binding of the aptamer to the target in the presence and absence of a synthetic preparation, or using an in vivo or in vitro assay that measures the difference in the biological effect of the aptamer on the target in the presence and in the absence of a synthetic preparation. Once a synthetic preparation has been identified that exhibits the desired effect, techniques such as combinatorial approaches can be used to optimize the chemical structure to achieve the desired regulatory effect.

Standard binding assays can be used to identify and select modulators of the present invention. Non-limiting examples are gel retardation assays and BIACORE assays. That is, the tested modulators can be brought into contact with the aptamers for binding under test conditions or normal physiological conditions and a determination can be made whether the tested modulator actually binds to the aptamer. Test modulators, for which it is determined that they bind to the aptamer, can be analyzed in adequate biological activity analyzes (which will vary depending on the aptamer and its target molecule, for example, coagulation assays) to determine if the test modulator can affect The biological effect shown by the aptamer in relation to its target molecule.

Gel motility retardation analysis is a technique that is used to evaluate binding ability. For example, a DNA fragment containing a test sequence is first incubated with a test protein or a mixture containing putative binding proteins, and then separated in a gel by electrophoresis. If a DNA fragment binds to a protein, then it will be larger in size, and therefore its movement will be delayed in comparison with the free fragment. For example, one method for electrophoretic analysis of gel retardation may be (a) contacting in a mixture of a protein that binds a nucleic acid to a non-radioactive or radioactively labeled nucleic acid molecule containing a molecular probe under suitable conditions to provide specific binding interactions between the protein and the probe to form a complex in which said probe is selected from the group consisting of dsDNA, ssDNA and RNA; (b) electrophoresis of the mixture; (c) transfer, using transfer to a blot at positive pressure or capillary transfer, of a complex to a membrane, where the membrane is positively charged nylon; and (d) detecting a membrane-bound complex by detecting a non-radioactive or radioactive label in the complex.

Biacore technology measures the binding events on the surface of the sensor chip, so the interacting substance attached to the surface determines the specificity of the analysis. Determining the specificity of the interaction involves simply analyzing whether various molecules can bind to the attached interacting substance. Binding leads to an immediate change in the surface plasmon resonance (SPR) signal in such a way that it is immediately apparent whether the interaction occurs or not. SPR-based biosensors monitor the interaction by measuring the mass concentration of biomolecules located close to the surface. Surfaces give specificity by attaching one of the interacting partners. The sample containing the other partner (s) flows on the surface: if molecules from the sample bind to an interacting substance attached to the surface, the local concentration changes and the SPR response is measured. The indicated answer is directly proportional to the mass of molecules that are bound to the surface.

SPR occurs when, under certain conditions, light is reflected from a conductive film on the interface between two media with different refractive indices. In Biacore technology, the medium is a sample and glass of a sensor chip, and a conductive film is a thin layer of gold on the surface of the chip. SPR causes a decrease in the intensity of reflected light at a certain angle of reflection. The indicated angle varies depending on the refractive index near the surface on the opposite side of the reflected light. When the molecules in the sample bind to the sensory surface, the concentration and, therefore, the refractive index at the surface change, and the SPR response is detected. The construction of a graph of the dependence of the response on time during the interaction is a quantitative measure of the progress of interaction. Biacore technology measures the minimum intensity of the angle of reflected light. Light is not absorbed by the sample: instead, light energy is scattered through an SPR in a gold film. SPR response values are expressed in resonance units (RU). One RU represents a minimum angle change of 0.0001 °. For most proteins, this is approximately equivalent to a change in concentration of approximately 1 pg / mm ² on the sensory surface. The exact conversion factor RU to surface concentration depends on the properties of the sensor surface and the nature of the molecule responsible for the change in concentration.

There are a number of other assays that can determine if an oligonucleotide or its analog, peptide, polypeptide, oligosaccharide or synthetic perat is capable of binding to the aptamer in such a way that the interaction with the target is modified. For example, electrophoretic mobility retardation analyzes (EMSA), calorimetric titration, proximity scintillation analyzes, sedimentation equilibrium analyzes using analytical ultracentrifugation (see, for example, www.cores.utah.edu/interaction), fluorescence polarization analyzes, fluorescence anisotropy analyzes fluorescence intensities, fluorescence resonance energy transfer analysis (FRET), nitrocellulose filter binding assays, ELISA, ELONA (see, for example, US Pat. No. 5,789,163), RIA assays or analyzes equal to -equilibrium dialysis can be used to assess the ability of an agent to bind to the aptamer. Direct analyzes can be performed that directly determine the interaction between the agent and the aptamer, or competition or exclusion tests can be performed that determine the ability of the agent to displace the aptamer from its target (e.g. see Green, Bell and Janjic, Biotechniques 30 ( 5), 2001, p. 1094 and U.S. Patent No. 6,306,598). Once a candidate modulating agent has been identified, its ability to modulate the activity of the aptamer against its target can be confirmed in biological activity analyzes. Alternatively, if an agent is identified that can modulate the interaction of the aptamer with its target, such binding assays can be used to verify that the agent interacts with the aptamer directly, and to measure the affinity of said interaction.

In another embodiment, mass spectrometry can be used to identify the regulator that binds to the aptamer, the site of interaction of the regulator and the aptamer, and the relative binding affinity of the agents to the aptamer (see, for example, US Pat. No. 6,329,146, Crooke and others). Such mass spectral methods can also be used to screen chemical mixtures or libraries, in particular combinatorial libraries, for individual compounds that bind to the selected target aptamer, which can be used as aptamer modulators. In addition, mass spectroscopic methods can be used to simultaneously screen multiple target aptamers against, for example, a combinatorial library of compounds. In addition, mass spectroscopic methods can be used to identify the interaction between several types of molecules, in particular, “synthetic drugs” and the intermolecular interaction site in the aptamer-target complex.

In vivo or in vitro assays that evaluate the effectiveness of the regulator with respect to changes in the interaction between the aptamer and the target are specific for the disease being treated. There is a wide range of standard assays for determining biological properties that are known and can be used. Examples of assays for biological activity are provided in the patents cited in this application, which describe specific aptamers for specific applications.

The present invention also provides methods for identifying aptamer modulators. Modulators can be identified generally by binding assays, molecular modeling, or in vivo or in vitro assays that measure changes in biological function. In one embodiment, the binding of the modulator to the nucleic acid is determined by gel mobility retardation assay. In another embodiment, the binding of the modulator to the aptamer is determined by Biacore analysis.

In one embodiment, the modulator has the ability to substantially bind to the aptamer in solution with a concentration of less than one (1.0) micromole (μM), preferably less than 0.1 μM, and more preferably less than 0.01 μM. By "substantially" is meant that at least a 50 percent decrease in the biological activity of the target is observed by modulation in the presence of the target, and a 50% decrease is indicated here as an IC ₅₀ value.

Pharmaceutical Compositions

The aptamers or modulators of the present invention can be formulated into pharmaceutical compositions, which may include a pharmaceutically acceptable carrier, diluent or excipient. The exact nature of the composition will depend, at least in part, on the nature of the aptamer and / or modulator, including any stabilizing modifications, and route of administration. Typically, an aptamer or modulator is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, orally or topically, as needed.

Pharmaceutically useful compositions comprising an aptamer or modulator of the present invention can be formulated according to known methods, such as by preparing a mixture with a pharmaceutically acceptable carrier. Examples of such carriers and formulation methods can be found in Remington's Pharmaceutical Sciences. To formulate a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of an aptamer or modulator. Such compositions may contain mixtures of more than one compound.

In the methods of the present invention, the compounds can form an active ingredient and are generally used in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) selected appropriately with respect to the intended form of administration, that is, oral tablets , capsules, elixirs, syrups, suppositories, gels and the like, and are consistent with normal pharmaceutical practice.

For oral administration in the form of a tablet or capsule, the active component of the drug may be combined with an oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerin, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents may also be included in the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, sweeteners from corn, natural and synthetic resins such as gum, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, wax, etc. P. Lubricants used in such dosage forms include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar-agar, bentonite, xanthan gum, and the like.

In the case of liquid forms, the active component of the drug can be combined with suitable flavored suspending or dispersing agents, such as synthetic and natural gums, for example tragacanth, gum, methyl cellulose and the like. Other dispersing agents that may be used include glycerol and the like. For parenteral administration, sterile suspensions and solutions are desirable. Isotonic compositions, which typically contain suitable preservatives, are used if intravenous administration is desired.

Topical compositions containing the active ingredient of the drug can be mixed with a variety of carrier materials known in the art, such as, for example, alcohols, aloe gel, allantoin, glycerin, oils with vitamins A and E, mineral oil PPG2 myristyl propionate and the like, for example, for preparing alcohol solutions, topical disinfectants, disinfecting creams, skin gels, skin lotions and shampoos in gel or cream compositions.

The compounds of the present invention can also be used in the form of liposome delivery systems, for example, small unilamellar vesicles, large unilamellar vesicles and multilayer vesicles. Liposomes can be composed of a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The compounds of the present invention can also be combined with soluble polymers as inducible drug carriers. Such polymers may include polyvinyl pyrrolidone, pyran copolymer, polyhydroxypropyl methacrylamide phenol, polyhydroxyethyl aspartamide phenol or polyethylene oxide polylysine substituted with palmitoyl residues. In addition, the compounds of the present invention can be coupled (preferably via a covalent bond) to a polymer from the class of biodegradable polymers useful for achieving controlled release of the drug, for example polyethylene glycol (PEG), polylactic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyhydroetetamines, poly , polycyanoacrylates and crosslinked or amphiphilic hydrogel block copolymers. Cholesterol and similar molecules can be associated with aptamers to increase and prolong bioavailability.

The compounds can be used directly (for example, alone or in a liposome composition or in combination with a carrier (for example, PEG)) (see, for example, US patent No. 6147204, US patent No. 6011020). In one embodiment, a variety of modulators may be associated with a single PEG molecule. The modulator can be designed for one or different aptamers. In those embodiments in which different modulators are applied to the same aptamer, there is an increase in avidity due to multiple binding interactions with the aptamer. In a further embodiment, several PEG molecules may be attached to each other. In said embodiment, one or more modulators to one aptamer or different aptamers may be associated with each PEG molecule. This also leads to an increase in the avidity of each modulator to its target.

Lipophilic compounds and non-immunogenic macromolecular compounds with which modulators of the present invention can be formulated for use in the present invention and can be prepared by any of various methods now known in the art or subsequently developed. They are typically derived from a phospholipid, for example distearoylphosphatidylcholine, and may include other materials, such as neutral lipids, for example cholesterol, and also surface modifiers, such as positively charged (e.g. sterilamine or aminomannose or derivatives of aminomannitol cholesterol compounds) or negatively charged (e.g. diacetyl phosphate, phosphatidyl glycerol) compounds. Multilayer liposomes can be formulated by conventional techniques, i.e. by depositing a selected lipid on the inner wall of a suitable container or vessel, dissolving the lipid in a suitable solvent, and then evaporating the solvent to obtain a thin film on the inner surface of the vessel or by spray drying. Then, the aqueous phase is added to the vessel with a swirling or vigorously stirring movement, which leads to the formation of MLV. UV can be further obtained by homogenization, sonication or by forcing (through filters) MLV. In addition, UV can be obtained through detergent removal techniques. In certain embodiments, the complex comprises a liposome with targeted aptamer (s) associated with the surface of the liposome and an encapsulated therapeutic or diagnostic agent. Preformed liposomes can be modified to bind to aptamers. For example, a cationic liposome binds to a nucleic acid via electrostatic interactions. Alternatively, a nucleic acid attached to a lipophilic compound such as cholesterol can be added to preformed liposomes, with cholesterol becoming bound to the liposome membrane. Alternatively, the nucleic acid may bind to the liposome during the preparation of the liposome.

Administration Methods

Preferred routes for administering the agents of the present invention to a mammal are parenteral, intravenous, intradermal, articular, intra synovial, intrathecal, intraarterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, local, transdermal or suppository, peritoneal, transdermal, nasal nebulizer, surgical implant, via internal its application to the surgical fabric, by infusomats or via catheter. In one embodiment, the agent and carrier are incorporated into the slow release compositions, such as an implant, bolus, microparticle, microsphere, nanoparticle or nanosphere. For standard information on pharmaceutical compositions, see Ansel et al., Pharmaceutical dosage Forms and Drug Delivery Systems, Sixth Edition, Williams & Wilkins (1995).

The aptamers or modulators of the present invention can be administered parenterally by injection or by continuous infusion. Although the tissue to be treated can usually be accessed in the body through systemic administration, and therefore treatment is most often carried out by the intravenous administration of therapeutic compositions, other tissues and delivery techniques are presented where it is likely that the target tissue contains target molecule.

Thus, the aptamers and modulators of the present invention are typically applied orally, topically to vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, into the cavity, transdermally, and can be delivered by peristaltic methods. As noted above, pharmaceutical compositions can be administered to a patient in a variety of ways, for example, orally, topically to vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, into the cavity, transdermally, and can be delivered by peristaltic methods. Representative non-limiting approaches for topical administration to vascular tissue include (1) coating or soaking the blood vessel tissue with a gel containing a nucleic acid ligand for in vivo delivery, for example, by introducing a coated or impregnated vessel in place of a damaged or pathologically altered vascular tissue segment that has been deleted or bypassed; (2) delivery via a catheter to a vessel to which delivery is desired; (3) pumping a ligand nucleic acid composition into a vessel to be implanted in a patient. Alternatively, a nucleic acid ligand can be introduced into cells by microinjection or by encapsulating liposomes. Advantageously, the nucleic acid ligands of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in several separate doses. Accordingly, a modulator is provided by any suitable means for modifying the effect of a nucleic acid ligand by introducing a modulator.

Therapeutic compositions containing modulators of the polypeptides of the present invention are conventionally administered intravenously, for example, by single dose injection. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosages for a subject, each unit containing a predetermined amount of active substance that has been selected to achieve the desired therapeutic effect while being in the required diluent, i.e. carrier or medium.

The compositions are administered in a manner that is compatible with the drug composition and in a therapeutically effective amount as described herein. Suitable administration regimens are variable, but in general terms represent the first administration, followed by repeated doses at intervals of one or more hours as a subsequent injection or via another route of administration. Alternatively, continuous intravenous infusion is considered, which is sufficient to maintain blood concentration in the ranges defined for in vivo treatment methods.

As used herein, the terms “pharmaceutically acceptable”, “physiologically tolerable” and their grammatical variations, as they relate to compositions, carriers, diluents and reagents, are used interchangeably and represent that materials can be administered without significant or debilitating toxic side effects.

Pharmaceutically useful compositions comprising a modulator of the present invention can be formulated according to known methods, such as by admixing with a pharmaceutically acceptable carrier. Examples of such carriers and formulation methods can be found in Remington's Pharmaceutical Sciences. To obtain a pharmaceutically acceptable composition suitable for effective administration, such compositions must contain an effective amount of aptamer. Such compositions may contain mixtures of more than one modulator.

EXAMPLES

Coagulation Measurement Methods

Standard coagulation metrics include plasma-based prothrombin time (PV) and activated partial thromboplastin time (APTT) in both plasma and whole blood, and analysis of whole-blood active clotting time (BAC). While the activators used to start coagulation in each of these analyzes are different, they all have a common characteristic - clot formation is the end point of the analysis. It is important that in the indicated in vitro assays, for the formation of a sufficient amount of fibrin necessary to reach the end point, low levels of thrombin, ~ 10-30 nM, are sufficient. The indicated thrombin level represents the conversion of only 3-5% prothrombin to thrombin and is comparable to the amount of thrombin produced during the initiating phase of the coagulation reaction (Butenas et al., 2003; Mann et al., 2003). Thus, these analyzes report to a large extent the initiating phase of the coagulation reaction and do not fully reflect the significance of insufficiency or inhibition of blood coagulation factors, primarily those involved in the propagation phase of coagulation.

The method in which standard clotting-based assays reflect FIX / IXa activity is illustrated by their ability to detect or not detect clotting abnormalities in a patient with severe hemophilia A (FVIII deficiency) or B (FIX deficiency). A sign of hemophilia is an isolated extension of APTT, since a patient with hemophilia has pathological APTT but normal PV (Bolton-Maggs and Pasi, 2003). The coagulation cell model explains the paradox as to why normal PT is determined in patients with FVIII or FIX deficiency. PV analysis begins with superphysiological levels of tissue factor, sufficient to lead to the formation of a clot after 11-15 seconds. Therefore, high levels of the tissue factor-FVIIa complex used to initiate the reaction quickly lead to the formation of FXa in an amount sufficient to produce enough thrombin to achieve clot formation as an endpoint, even in the absence of FVIII or FIX. Thus, even a significant inhibition of the activity of FIX / FIXa will not affect the value of the PV assay, since the role of FIX in initiating coagulation in this assay is masked or bypassed. Thus, pharmacological inhibitors of FIXa, such as the anti-FIXa aptamer RB006, are not expected to increase the PV value.

Both APTT analyzes of plasma or whole blood are triggered by a charged participant such as celite or kaolin, phospholipid surface and calcium in quantities sufficient to form a clot after ~ 28-35 seconds. In patients with hemophilia B (and A), pathological APTT values are recorded; however, the magnitude of the increase in APTT in these patients is finite (that is, a limited value), since the initiation phase of coagulation is largely determined in the analysis. There is no strict correlation between the severity of hemophilia B in a patient and the value of his APTT, since APTT depends on other coagulation factors in addition to FIX. Therefore, the best criterion for interpreting how the pharmacological inhibition of FIXa will affect the analysis of APTT is plasma FIX analysis. Plasma FIX analysis is a variation of the standard APTT method in which, before performing APTT, the test plasma is diluted in a buffer and mixed with a FIX-deficient plasma so that the FIX level in the test plasma is the primary determinant of clot formation time. This analysis is typically used to determine the severity of hemophilia B (i.e., to determine FIX levels), or to diagnose acquired FIX inhibitors. The results of the FIX analysis are interpreted by comparing the time of clot formation in the test sample with a standard curve of the FIX level, which is obtained by serial dilution of normal plasma in a buffer before mixing with a FIX-deficient plasma. Table 1 presents a typical standard curve of the FIX level obtained for normal human plasma. [Please note: Absolute APTT times in this assay are reagent dependent]. As can be seen from table 1, with FIX levels of 25% of normal (that is, a decrease of 75%), APTT, clot formation times exceed the norm by 1.4 times. At FIX levels of ~ 3% of the norm (that is, a decrease of 97%), APTT times of clot formation exceed the norm by 2 times and at FIX levels of <1% of normal (that is, a decrease of> 99%), APTT times clot formation exceed the norm by 2.5 times. In carriers of hemophilia B (i.e., FIX levels of ~ 50% of normal), the normal APTT values are determined (Bolton-Maggs and Pasi, 2003), which is consistent with the standard FIX level curve. Taken together, the observations indicate that a significant percentage of FIX activity must be inhibited before the APTT lengthens.

Table 1
Standard curve for the analysis of FIX activity in human plasma % FIX level APTT convolution time Convolution time overrun factor one hundred* 48.0 1,0 fifty 58.6 1,2 25 65,4 1.4 12.5 75.1 1,6 6.25 85.1 1.8 3.13 97.0 2.0 1,56 105.8 2.2 0.78 119.7 2.5 * 100% FIX level represents dilution of normal pooled human plasma in 1: 5 buffer

Since BAC tests are used primarily in operating rooms and laboratories where catheterization is performed to monitor anticoagulation during interventions, there is little data on how BAC decreases FIX / FIXa activity, as hemophilia patients tend to treated with replacement factor therapy (or similar therapy) before undergoing such procedures. However, since BAC is an analysis whose end point is the formation of a blood clot that is triggered by charged particles, the pharmacological inhibition effect of FIXa in BAC analysis is likely to reflect that observed in the APTT analysis. Thus, it is expected that BAC lengthening will not be observed until a significant degree of inhibition of FIXa (> 50%) has been achieved. Therefore, by analogy with the APTT analysis, the BAC lengthening is likely to be modest compared to the lengthening observed with unfractionated heparin. Finally, in response to inhibition of FIXa, analysis is likely to reach saturation. This similarity in the APTT and BAC reactions has been demonstrated in monkeys treated with various doses of RB006 in non-clinical toxicity studies.

The effects of the REG1 anticoagulation system on coagulation

Previous data indicate that anti-FIXa aptamers do not lengthen PV both in vitro and after intravenous administration to animals (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8; Rusconi et al., 2002, Nature 419 (6902): 90-4; Dyke, 2006, Circulation. 114 (23): 2490-7). As shown in FIG. 3, RB006 exhibits a dose-dependent increase in APTT in pooled normal human plasma in vitro. The data obtained indicate that for the RB006 APTT, the dose-response curve is the most sensitive from 0 to 30-50 μg / ml, and then goes to a plateau. These features, including the increase phase and the plateau phase of the APTT dose-response curve, are consistent in plasmas from all species in which RB006 or previous anti-FIX aptamers exhibit cross-reactivity, including humans, pigs, mice, and monkeys (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8). The maximum APTT value achieved in response to in vitro plasma treatment with the anti-FIXa aptamer depends on the APTT reagent used and the species. It is important, however, that this APTT maximum is consistent with complete or nearly complete inhibition of FIXa activity. This is confirmed by the fact that the maximum APTT in response to anti-FIXa aptamers is equivalent to APTT in human plasma containing a FIX level <1% of normal (but normal in relation to the levels of all other coagulation factors) and APTT in FIX-knockout plasma Mice Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8). Thus, the APTT plateau in response to RB006 probably reflects the saturation of FIX / FIXa inhibition with the aptamer.

In addition, comparing the data in FIG. 3 with the standard plasma FIX analysis curve in Table 1 provides an understanding of RB006 activity. APTT increases ~ 1.4 times in response to RB006 at a concentration of RB006 ~ 5 μg / ml, indicating that this concentration of RB006 is sufficient to inhibit plasma FIX activity by ~ 75%. In addition, based on an analysis of plasma FIX, almost 95% inhibition of plasma FIX (2.0-fold increase in APTT) is achieved at a concentration of RB006 from 10 to 15 μg / ml.

In order to evaluate the individual variability of the effect of the anticoagulant RB006, in vitro studies were performed by measuring the concentration of the APTT in the plasma of patients depending on the RB006 concentration. A comparison of the in vitro RB006 APTT dose-response curve in pooled normal human plasma vs. plasma from patients is shown in FIG. 4.

As shown in FIG. 4, the concentration-dependent increase in RB006 APTT is very similar for each subject's plasma. In addition, the concentration-dependent increase in RB006 APTT in patients' plasma is very similar to that in pooled normal human plasma (20 donors in the pool). RB006 also increases blood coagulation time as measured by BAC analysis (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8). However, the interpretation of BAC changes as a function of RB006 concentration is currently limited due to the difficulty of performing in vitro dose-response studies with BAC, as this analysis requires fresh whole blood, and is time sensitive.

The neutralization of the anticoagulant activity of RB006 with the RB007 antidote was measured in vitro using APTT analysis. As shown in FIG. 5, since the concentration of RB007 is increased relative to a fixed concentration of RB006 in pooled human plasma, the change in the value of APTT returns to its initial levels, indicating complete neutralization of anticoagulation activity by RB007. The minimum molar excess of RB007, which is necessary to completely neutralize RB006 in vitro in human plasma, is approximately 3 to 4-fold (i.e., the molar ratio of antidote to oligonucleotide portion of the aptamer). This is consistent with the measured thermodynamic stability of the duplex RB006-RB007 (T _pl. ~ 90 ° C).

The data presented in figure 5 also serve as the basis for choosing the ratio of the dose of the antidote RB007 to the dose of the drug RB006 used in non-clinical pharmacological safety studies and toxicity studies and clinical trials. The minimum molar excess of RB007 relative to RB006, which is necessary to achieve complete neutralization of RB006 in vitro in human plasma, is from 3 to 4 times. Given the difference in molecular weight of RB007 (5269 Da, sodium salt) and RB006 (~ 50964 Yes, sodium salt), this translates to a weight ratio of antidote: drug of 0.5: 1. Since this is an in vitro result and therefore does not predict how the pharmacokinetics of any of the components will influence the neutralization of the drug in vivo, the antidote: drug weight ratio of 0.5: 1 reflects the minimum antidote ratio that would be expected to effectively neutralize medicine. Therefore, as a starting dosage in non-clinical and clinical studies, a small multiple of the minimum in vitro effective dose ratio, the weight ratio of antidote: drug, was 2: 1.

Summarizing the above, the anti-FIXa aptamer RB006 is a potent inhibitor of the coagulation factor FIXa, capable of completely or almost completely inhibiting the activity of FIXa in vitro. The anticoagulant activity of RB006, as well as the neutralization of aptamer activity by RB007, can be effectively monitored through APTT and BAC assays. From in vitro studies, the relationships between percent inhibition of FIX and changes in APTT were well defined for RB006. The corresponding molar ratio of antidote to aptamer sufficient to achieve complete inhibition of aptamer activity was also determined in in vitro studies, from which the dose / mg / kg antidote: aptamer 2: 1 dose ratio selected for the REG1 anticoagulation system was derived.

Non-clinical pharmacology, drug distribution and toxicity

The pharmacological activity of the REG1 anticoagulation system and its individual components, drug and antidote (or less active prototypes of the drug and antidote, called RB002 and RB004, respectively), has been demonstrated in vitro and in clinically relevant animal models.

The anticoagulant activity of anti-FIXa aptamer was evaluated in systemic studies of anticoagulation in pigs (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8), in models of extrapulmonary blood circulation in sheep, and in a pharmacological safety study on cynomolgus monkeys. The antithrombotic activity of the anti-FIXa aptamer was also demonstrated in a model of artery damage in mice (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8). The neutralizing activity of the antidote in relation to the drug was demonstrated in vitro in human plasma (Rusconi et al., 2002, Nature 419 (6902): 90-4), in systemic models of anticoagulation in pigs, in models of surgical trauma in mice (i.e. intersection tail in animals against the background of pronounced anticoagulation) (Rusconi et al., 2004, Nat Biotechnol. 22 (11): 1423-8), in models of extrapulmonary blood circulation in sheep and in a pharmacological safety study on cynomolgus monkeys. In addition, in studies of systemic anticoagulation in pigs, the ability of the drug to be re-administered shortly after the antidote was neutralized with a previous dose of the drug was demonstrated.

The pharmacokinetic characterization of the REG1 anticoagulation system needed a bioanalytical strategy that relied on a new methodology for quantifying aptamer, antidote, and aptamer / antidote levels in plasma samples. These methods were applied to samples collected after in vivo toxicity studies, which allowed the determination of the pharmacokinetics of all three molecular components under conditions of single and multiple dosing on monkeys and mice.

A comprehensive safety study of the REG1 anticoagulation system was conducted. Initial toxicity studies were performed on monkeys and mice under dosing conditions, which simulated the intended use of the drug in the initial clinical trials (i.e., with the sequential administration of the aptamer, 3 hours after which the antidote was administered). Clinically multiple dosages of each component were tested from lower to higher in the same dose ratio as intended for clinical use, and for both species the effects of aptamer and antidote were tested separately. In both studies on monkeys, several treatment groups were formed that received single doses of aptamer, antidote, or both test positions according to the scheme, which imitated the intended introduction in the initial clinical trials. In addition, groups that were given repeated doses for two weeks (14 daily doses for mice and 7 doses every other day for two weeks, were included in a 14-day study in mice and in a single and multiple toxicity studies in monkeys). monkeys). Specialized endpoints were included in toxicity studies to evaluate pharmacodynamic responses, exposure to REG1 components, and the effects of oligonucleotides as a class of compounds. Basic toxicity studies were supplemented by monkey pharmacological safety assessments (using radio telemetry), a list of genetic toxicity tests, and a blood compatibility test.

Studies of anticoagulant and drug neutralizing activity in pigs

The ability to re-enter the RB006 aptamer after neutralizing the initial dose of the aptamer with the RB007 antidote was evaluated in a pig systemic anticoagulation model. In these studies, a second dose of the drug was administered 30 minutes after administration of the antidote. A 30-minute window between the administration of the antidote and the repeated administration of the aptamer was chosen in order to obtain a clear experimental demonstration of the neutralization of the anticoagulant activity of the first dose of the aptamer. As shown in FIG. 6, the peak of anticoagulant activity and the time to reach the peak of anticoagulant activity after the second dose of the aptamer were essentially the same for the initial dose of the aptamer, indicating that repeated administration of the aptamer after neutralization with the antidote of the first dose of the aptamer is feasible. The data obtained are consistent with the observed pharmacokinetics of RB007 in both mice and monkeys, which indicate that RB007 has a very short plasma half-life (i.e. several minutes) and does not accumulate in plasma to a detectable concentration even at significantly higher doses. than used in this study. Given the half-life of the antidote, it is likely that the aptamer can be effectively reintroduced at a shorter time interval than 30 minutes after administration of the antidote.

The effectiveness of the REG1 anticoagulation system in aorto-coronary artery bypass grafting (CABG) with extrapulmonary circulation in sheep

REG1 can be used as a reversible antidote for anticoagulant during interventions for revascularization of the coronary bed [aorto-coronary artery bypass grafting (CABG) and percutaneous intervention in the heart (PCI)] as a reversible antidote for anticoagulant for use in patients, including people with acute coronary syndromes , and as an anticoagulant for other indications in which it would be advantageous to use a reversible antidote for antithrombotic or anticoagulation therapy. The studies described in this application are intended to determine the dose range of the anticoagulant component REG1, RB006 necessary to maintain the extrapulmonary shunt (CPB) in the open state in the animal undergoing CABG surgery with CPB, and determine the appropriate dose of the antidote component REG1, RB007, necessary to neutralize RB006 in this model.

RB006 (anticoagulant) was administered intravenously to 10 sheep at the beginning of the coronary artery bypass grafting operation. At the end of the operation, RB007 (an RB006 neutralizing agent) was intravenously administered to completely neutralize the effects of RB006. After 28 ± 3 days, all animals were euthanized.

Representative samples of the right and left kidneys, liver, lung, and entire brain were collected. Hearts were washed with a stream of Ringer's solution with lactate or physiological saline until the blood was purified and fixed by pressure perfusion at ~ 100 mm Hg. 10% neutral buffered formalin (NBF) for at least 6 hours. After completion of fixation, hearts were placed in 10% NBF. Representative tissue samples collected during autopsy were fixed with 10% NBF immersion.

Hearts were cut across approximately every 1 cm (like a loaf of bread) and examined for pathological changes. Ten slices of each heart were collected and embedded in paraffin. Three of the ten sections included: LCX anastomosis, aortic anastomosis, and mid-graft. The remaining seven sections included: the wall of the right atrium, the wall of the left atrium, the interatrial septum, the wall of the right ventricle, the wall of the left ventricle, the interventricular septum, and the apex. All paraffin blocks containing myocardial tissue were cut into two parts, one for staining with hematoxylin and eosin (H&E) and the other for staining according to Masson's Verhoeff Elastin (MVE). Samples of the kidneys, liver, lung, and brain were embedded in paraffin and cut as follows: one slice of each kidney, one slice of the liver, one slice of the lung, and one slice of each of the four brain tissue samples, for a total of eight slices. All micropreparations obtained were stained with H&E.

The correlations between macroscopic and histological observations in this study indicate that most pathological changes relate to either a surgical procedure (e.g., adhesions) or euthanasia (e.g., foam in the trachea and bronchi). Adhesions are common consequences of this type of procedure, and were not considered excessive in this study.

One animal was found to have a small, minimally attached thrombus in an aortic anastomosis. The thrombus, as it seems, did not impede the blood flow in the graft. There were no characteristic microscopic signs indicating this. The microscopy data at the site of the anastomosis were similar in type and amplitude to those for other experimental animals in both groups. With one exception: no experimental animal had any macroscopic sign of thrombosis or occlusion within any part of the coronary artery bypass graft. Accidental thrombus formation is very common in this model; therefore, the association with the introduction of RB006 is regarded as doubtful.

Pharmacodynamic activity of the REG1 anticoagulation system in cynomolgus monkeys

In vitro, the anticoagulant activity of RB006 in the plasma of cynomolgus monkeys is reflected as a concentration-dependent prolongation of clot formation time in the APTT assay. As can be seen in FIG. 7, the APTT RB006 dose-response curve is most sensitive in the range from 0 to 50 μg / ml, and then reaches a plateau, as was noted for other species. Dose response curves for monkeys and humans are similar except that humans have a wider response range. In human plasma, a concentration-dependent increase in APTT to approximately 200 μg / ml is observed, while in the monkey plasma, the concentration-response curve reaches a plateau at approximately 50 μg / ml. The plateau of the curve for human plasma is reached at a value of APTT, which is equivalent to that determined for human plasma containing <1% FIX activity in plasma, and is likely due to saturation of the target, FIXa. Plasma FIX analyzes were performed to facilitate the interpretation of the RB006 APTT dose-response curve for monkey plasma. As shown in table 2, APTT in monkey plasma is sensitive to FIX. However, the magnitude of the response to lower FIX levels is small. A 75% reduction in FIX leads to a 1.4-fold increase in APTT, a> 95% decrease in FIX leads to a doubling of APTT, and a 99.9% decrease in FIX in plasma leads to a 2.5-fold increase APTT.

table 2
Standard curve for the analysis of the activity of FIX in the plasma of cynomolgus monkeys % FIX level APTT coagulation time Coagulation time increase coefficient one hundred* 35.1 1,0 fifty 41.9 1,2 25 49.4 1.4 12.5 55.9 1,6 6.25 62,2 1.8 3.13 68.0 1.9 1,56 74.7 2.1 0.78 77.7 2.2 0.39 83.8 2,4 0,098 88.1 2.5 * 100% FIX level represents 1: 5 dilution of normal pooled plasma of Javanese macaques in the buffer. FIX-deficient human plasma (George King Biomedical) was used as a source of FIX-deficient plasma.

A comparison of the data in Fig. 7 with the data presented in Table 2 indicates that ~ 6 μg / ml RB006 is necessary to inhibit approximately 90% of plasma FIX activity in monkeys (i.e., a 1.6-fold increase in APTT occurs at the indicated concentration), and that> 95% inhibition of plasma FIX activity occurs at concentrations of RB006 of 10-12 μg / ml. In vitro RB006 APTT, the monkey plasma dose-response curve reaches a plateau with an approximately 2.5-fold increase from the initial value (initial value ~ 24 seconds, maximum APTT ~ 60 seconds), which is consistent with the amplitude of the increase in APTT observed in the FIX analysis plasma at a FIX level <0.1% of the normal FIX level (see table 2). Therefore, the plateau for the RB006 APTT dose-response curve probably represents saturation of the target in monkey plasma (i.e., complete inhibition of FIX activity). In conclusion, the% inhibition of FIX against the concentration of RB006 in monkey plasma in vitro in general is similar to that observed in vitro in human plasma, the main differences being that the range of RB006 concentration from initial to maximum APTT in humans is wider, and the dose increase the answer is smoother for human plasma than for monkey plasma.

In vivo activity of RB006 and RB007 for cynomolgus monkeys

The relationship between the anticoagulant properties of RB006 and the RB006 / RB007 complex and plasma levels of these compounds was evaluated in a monkey pharmacological safety study REG1-TOX001. Briefly, 12 monkeys were divided into three treatment groups. Group 1 received the anti-FIXa aptamer RB006, group 2 received the antidote RB006, RB007, and group 3 was administered the REG1 anticoagulation system, i.e. RB006, after which RB007 (after three hours). Doses were doubled, with the first dose being administered on day 4 of the study and the second dose being administered on day 13. To better understand the dose-response relationship for RB006, the four monkeys enrolled in group 1 (RB006, aptamer only) were divided into two groups on day 13, where two animals received a low dose (group 1a, 5 mg / kg RB006) and two animals received a high dose (group 1b, 30 mg / kg RB006).

As shown in FIG. 8, administration of RB006 in doses ranging from 5 to 30 mg / kg resulted in severe anticoagulation in monkeys. The average APTT for each dose level exceeded 60 seconds within 0.25 to 24 hours after administration of RB006, which is equivalent to a plasma FIX level in monkeys <0.1% of normal. In response to the administration of RB006, a dose-dependent increase in APTT occurs.

However, the dose-response relationship is not immediately obvious due to the fact that up to a 6-hour period after administration of RB006, the plasma RB006 level exceeds the concentration at which the in vitro APTT dose-response curve reaches a plateau (~ 40-50 μg / ml; cm table 3 and Fig.7). At time points beyond 6 hours after administration of RB006, when the concentration of RB006 decreases below this level, the dose-response relationship is more obvious. APTT in monkeys receiving doses of 5 and 15 mg / kg RB006 was determined until it returned to its original value. The average value of APTT returned to its initial level after 120 hours at a dose level of 5 mg / kg and after 192 hours at a dose level of 15 mg / kg, which is consistent with the in vitro APTT dose-response curve (Fig. 7), and with the observed period the half-life constituting for RB006 in monkeys is approximately 12 hours (see table 3). Whole blood active clotting time (BAC) data reflected APTT data (data not shown).

Toxicokinetic data were collected at several time points during the first 24 hours after administration of RB006 using an ELISA with hybridization of double oligomers. As shown in table 3, the concentration of RB006 increased as a function of the administered dose, and the half-life of RB006 was in the 12-hour range. Consistent with the data presented in Fig. 8, a comparison of plasma RB006 levels (Table 3) with the in vitro dose-response curve shown in Fig. 7 indicates that the animals were in a state of deep anticoagulation for the first 24 hours after the introduction of RB006 at all dose levels. These dose levels significantly exceed the proposed clinical range. There is an excellent match between the average RB006 concentration 24 hours after administration in animals in group 1a and the average APTT in these animals. The average concentration of RB006 in animals that received 5 mg / kg RB006 after 24 hours was 15.9 μg / ml, and the average value of APTT was 61.1 seconds. This is very favorably combined with the expected result based on the in vitro monkey dose response curve RB006 (see FIG. 7). Therefore, this study confirms the practical relevance of APTT for monitoring the level of anticoagulation in monkeys that received RB006, and the data support the use of APTT for monitoring the status of anticoagulation in people receiving RB006 in initial clinical trials.

Table 3
Plasma RB006 levels in animals of group 1, REG1-TOX001 (μg / ml) Time after injection (hours) Dose levels in group 1 (animals / dose level) 5 mg / kg (n = 2) * 15 mg / kg (n = 4) 30 mg / kg (n = 2) * Before introduction 0.2 <0.04 0.2 0.25 59.8 179.8 ± 28.9 465.5 3 66.6 145.6 ± 32.5 328.9 6 42.1 101.5 ± 13.4 275.3 24 15.9 51.1 ± 11.2 164.6 * On day 13 of the experiment, the animals were divided into groups 1a (5 mg / kg) and 1b (30 mg / kg). For these dose levels, the average plasma level of two animals per dose level is presented. RB006 in animals of groups 1a and 1b at the time point before administration is residual from a dose of 15 mg / kg per day 4. The LLOQ of this analysis is <0.04 μg / ml.

In animals of group 2, which were administered only the RB007 antidote, the mean values of APTT and BAC did not change in response to the administration of RB007 at all test dose levels (30 and 60 mg / kg). These toxicokinetics were fixed at various time points during the first 24 hours after administration of RB007 using an ELISA with hybridization of double oligomers. As shown in table 4, low but detectable antidote levels were present in the plasma of animals receiving RB007 0.25 hours after injection of 30 mg / kg per day 4 or 60 mg / kg per day 13. The levels obtained were very variable, but in were generally dose dependent. The level of antidote after administration was very low compared to the concentration of aptamer (in group 1) after intravenous injection. Thus, it becomes clear that with the introduction of only the antidote, it has a very short half-life in plasma, and is largely eliminated from circulation 15 minutes after the injection.

Table 4
Plasma RB007 levels in animals of group 2, REG1-TOX001 (μg / ml) Time after injection RB007 (hours) Dose Levels in Group 2 (4 animals / dose) 30 mg / kg 60 mg / kg Before introduction <0.01 <0.01 3.25 0.4 ± 0.1 0.6 ± 0.5 6 0.02 ± 0.01 * <0.02 *** 24 0.01 ± 0.01 ** <0.01 *** * 1 animal with <LLOQ 0.01 was included in the calculation
** 3 animals with <LLOQ 0.01 were included in the calculation
*** Average among LLOQ

APTT data in animals treated with RB006, 3 hours after which RB007 was administered (group 3), are shown in FIG. 9. Consistent with data from animals that were administered only RB006, administration of RB006 at the indicated dose levels results in severe anticoagulation, with an average APTT of 0.25 and 3 hours after administration, which is consistent with substantially complete inhibition of FIX at both dose levels. Subsequent administration of RB007 quickly and completely neutralized the anticoagulant effects of RB006 in monkeys, with the average APTT returning to its original value within 15 minutes after administration of RB007 (first time point) for both tested dose levels of RB006 / RB007. In animals of group 3, which were introduced 30/60 mg / kg, RB006 / RB007, APTT was determined within 5 days after administration of RB006. APTT data collected over this time period indicate that the anticoagulant effects of RB006 were neutralized for a long time without a sign of the resumption of anticoagulation for more than 120 hours, or approximately 10 half-lives of RB006 in the monkey (Fig. 9). The duration of neutralization of the anticoagulant activity of RB006 with the RB007 antidote is fully consistent with the observed thermodynamic stability of the present drug-antidote complex.

Toxicokinetic data were collected within 24 hours after administration of RB006 in animals of group 3 (table 5). For group 3 animals, plasma concentrations of both free RB006 (i.e., RB006 not bound to RB007) and in combination with RB006 (i.e. RB006 bound to RB007) were measured. In agreement with the APTT data presented in FIG. 9, the mean plasma concentrations of RB006 at 0.25 and 3 hours after administration were very high. Within 15 minutes after the introduction of RB007, the average concentration of free RB006 decreased 5,000 to 10,000 times to levels below the lower measurement limit (LLOQ) of the analysis used. Along with a decrease in the levels of free RB006, the average plasma concentration of RB006 in the complex increased from less LLOQ analysis to ~ 125 to 220 μg / ml at dose levels of 15/30 and 30/60 mg / kg, respectively, indicating that fast the decrease in the concentration of free RB006 was due to the binding of RB007 to RB006. The concentration of free RB006 remained below the LLOQ analysis for at least 3 hours after administration of RB007, which is consistent with the results of APTT. At 21 hours after administration of RB007 (24 hours after administration of RB006), very low levels of RB006 were determined in several animals (average value of only 0.17 μg / ml or lower). However, the obtained RB006 levels were too low to exhibit a measurable anticoagulant effect, consistent with the lack of aPTT extension after 24 hours or longer in animals that were given the REG1 anticoagulation system.

Table 5
Group 3 REG1-TOX001: plasma levels of free and in the complex RB006 (mg / ml) Time since injection RB006 (hours) Dose Levels in Group 3 15/30 mg / kg RB006 + RB007 30/60 mg / kg RB006 + RB007 Free RB006 RB006 as part of the complex Free RB006 RB006 as part of the complex Before introduction <0.04 but. 0.05 ± 0.01 but. 0.25 280.2 ± 64.3 but. 467.6 ± 67 but. 3.0 214.6 ± 31.8 <0.04 488.4 ± 68.6 <0.04 3.25 <0.04 125.1 ± 7.9 <0.04 218.2 ± 27.2 6 <0.04 98.7 ± 20.5 <0.04 184.8 ± 28.9 24 0.14 ± 0.08 * 8.3 ± 4.5 <0.04 ± 0.01 ** 22.3 ± 12 * 1 animal with LLOQ <0.04 μg / ml was included in the calculation
** 3 animals with LLOQ <0.04 μg / ml were included in the calculation
RB007 was administered at t = 3 hours immediately after a 3-hour blood sampling, (n.o.) not determined.

The essence of non-clinical pharmacological studies in monkeys

Presented studies demonstrate that RB006 is a potent anticoagulant in monkeys that is able to achieve substantially complete inhibition of FIX activity within 24 hours or longer after a single intravenous injection of a drug at doses higher than clinical. Comparison of in vitro studies of anticoagulant activity of RB006 in monkeys with APTT and toxicokinetic data from a pharmacological safety study demonstrates a good agreement between the expected and observed prolongation of APTT depending on plasma concentration of RB006. Therefore, APTT analysis will serve as a useful tool for monitoring anticoagulation caused by the administration of RB006. The similarity between the in vitro RB006-APTT dose-response curves for humans and monkeys suggests that the data from this study on monkeys (REG1-TOX001) are the same as the large general toxicity study on monkeys (REG1-TOX003 ) will provide useful guidance in predicting a person’s response to the introduction of RB006. Finally, APTT and the toxicokinetics of REG1-TOX001 demonstrate that RB007 is a very effective RB006 antidote. Within 15 minutes after intravenous bolus administration of RB007 to animals that were injected with RB006, the average APTT values returned to the levels prior to administration of RB006 and remained at this initial level for the entire monitoring period (up to 120 hours). The observed neutralization of the anticoagulant activity of RB006 by RB007 was fully confirmed by toxicokinetic data and is consistent with the measured thermodynamic stability of the RB006-RB007 complex. Toxicokinetic studies have shown that free RB006 levels decreased to below LLOQ analysis within 15 minutes after administration of RB007, accompanied by a significant increase in RB006 concentration in the complex, and without a significant increase in free RB006 levels during toxicokinetic analysis (24 hours after administration of RB006). Therefore, the data obtained in studies on monkeys showed that the REG1 anticoagulation system behaves as expected with respect to achieving stable, long-term and affordable for monitoring anticoagulation after a single intravenous injection of the aptamer, after which there is a quick, complete and long-acting neutralization of the activity of the aptamer during intravenous bolus injection of an antidote. Such characteristics of the REG1 anticoagulation system were achieved at low to high multiples of the intended clinical dose range (i.e., suitable doses for toxicity studies), but without adverse effects in animals.

Toxicokinetics REG1

To perform a quantitative analysis of the concentrations of free aptamer (RB006), free antidote (RB007) and aptamer / antidote complex (RB006 / RB007) in plasma in monkeys and mice, bioanalytical methods were developed and approved. These methods were used to analyze samples obtained during the pharmacological safety study on monkeys (study No. REG1-TOX001), a 14-day study in mice (study No. REG1-TOX002) and single / multiple administration studies on monkeys (study No. REG1- TOX003). For all three studies, separate groups of animals were formed, which were administered either only an aptamer, or only an antidote, or an aptamer, 3 hours after which an antidote followed. Multiple dose levels for each administration condition were tested in all studies and two of the present studies (a 14-day study in mice and a single / multiple administration study in monkeys) also included re-administration of test agents. The dose levels of aptamer tested in these studies ranged from 0.25 to 45 mg / kg in monkeys and from 2.5 to 22.5 mg / kg in mice. Doses of the tested antidote were two times higher than those for the aptamer (i.e. up to 90 mg / kg in monkeys and 45 mg / kg in mice). This ratio is similar to that intended to be used in clinical trials.

For all three studies, the results of toxicokinetics were similar in describing the following properties of the REG1 anticoagulation system.

- Plasma aptamer concentrations after intravenous injection were dose proportional over a wide dose range with a modest degree of variation between animals. No gender differences were evident in either monkeys or mice.

- The clearance of the aptamer from plasma was relatively slow (that is, the estimated half-life was at least 12 hours in monkeys and ~ 8 hours in mice). Such a slow clearance was expected based on the structure of the PEGylated aptamer and is consistent with published reports regarding the pharmacokinetics of other PEGylated oligonucleotides. The minimum clearance of the aptamer, in combination with its high degree of inhibition of factor IX, is presented for a relatively stable degree of anticoagulation for 6 hours, based on the measurement of pharmacodynamic markers, i.e., activated partial thromboplastin time and active clotting time. The specified profile is a desirable property of the aptamer component of the REG1 anticoagulation system.

- An intravenous injection of only an antidote (without prior administration of the aptamer) led to very low plasma levels, even for the first time sampling after injection (10-15 minutes). The antidote levels measured at the indicated early time points were orders of magnitude lower than those for the aptamer (i.e., compared to the aptamer levels in those groups that received the aptamer alone), despite the fact that the antidote dose levels were twice as high. In aggregate, antidote data indicates that he has a very short plasma half-life if taken separately. The use of a relatively high dose level (30 mg / kg) in monkeys every other day for 7 doses (14 days) did not lead to accumulation of the antidote in the plasma.

- For groups that received an aptamer and received an antidote 3 hours later (that is, the complete REG1 anticoagulation system), the concentration of free aptamer decreased sharply within a few minutes after administration of the antidote to a level below or slightly above the limits of quantification (using a very sensitive analysis based on hybridization), which indicates the complete binding of the circulating aptamer to an antidote. As noted in the treatment with only one antidote, under these conditions, the levels of free antidote were very low. Antidote binding of the aptamer was associated with virtually complete neutralization of the aptamer activity (i.e., normalization of coagulation parameters), which is consistent with the anticipated action of the REG1 anticoagulation system.

- In parallel with the elimination of the free aptamer, the aptamer / antidote complex was detected in plasma at levels comparable to the complete binding of the aptamer to the antidote. The complex was eliminated from plasma at a rate slightly faster than that for the free aptamer (that is, comparable to the speed of clearance of the aptamer in groups administered only with the aptamer), but much lower than the free antidote, as was expected due to the presence of polyethylene glycol groups in the complex (obtained from aptamer). Extensive elimination of the aptamer / antidote complex from plasma was observed within 21 hours after administration of the antidote. With the repeated administration of the aptamer and antidote (REG1 coagulation system) to the monkeys every day for two weeks there was no accumulation of the complex or free aptamer in the blood, no change in the pharmacokinetics of the aptamer (that is, during the period before the antidote was introduced) and no signs of cumulative anticoagulation carried out by the aptamer.

- The only difference between the pharmacokinetics in mice and monkeys was a slightly longer half-life of the aptamer in monkeys (at least 12 hours, compared to ~ 8 hours in mice).

Clinical use of REG1 in humans

When choosing a method of anticoagulation for use in an individual patient or patient population, clinicians take into account the features of various pharmacological strategies. Given that the main adverse effect of anticoagulation is bleeding (that is, an abnormally increased pharmacological activity), for an intensive care indication, the ideal anticoagulant would be 1) delivered intravenously or subcutaneously, 2) immediately therapeutic, 3) easily dosed, so as not to require frequent monitoring, and most importantly, 4) immediately and predictably reversible. The REG1 anticoagulation system was developed in response to the unmet medical need for an effective, safe, and rapidly reversible anticoagulation agent.

REG1 can be used in many clinical situations to treat people and other animals in need of such treatment. For example, REG1 can be used in coronary and peripheral vascular revascularization procedures associated with arterial disease and occlusions as an antidote neutralizing anticoagulant. In particular, REG1 can be used as an antidote-neutralizing anticoagulant in coronary revascularization procedures (aortic-coronary artery bypass grafting (CABG) and percutaneous heart surgery (PCI)), as an antidote-neutralizing anticoagulant for use in patients with acute coronary syndromes and as an anticoagulant, for other indications, in which it would be advantageous to use an antidote neutralizable antithrombotic or anticoagulant yvayuschey therapy. Diseases and procedures for which the methods of the present invention can be used include, but are not limited to, peripheral vascular transplant procedures, including those associated with iliac, carotid, brachial, aorta, renal, mesenteric, femoral, popliteal, tibial, and peritoneal vessels; prevention of deep vein thrombosis; prevention of pulmonary embolism after orthopedic surgery or in patients with cancer; prevention of atrial fibrillation; prevention of thrombotic stroke; and for indications requiring extracorporeal circulation of blood, including, without limitation, hemodialysis and extracorporeal membrane oxygenation. Additional examples of potential diseases and procedures in which the methods of the present invention may be used include, but are not limited to, patients undergoing cardiac surgery with extrapulmonary circulation; patients who form blood clots in the heart, or with peripheral embolization; and patients who have other hypercoagulable conditions. The methods of the present invention may also be useful for the prevention of DVT and pulmonary embolism in immobilized patients and for maintaining the functioning of permanent intravenous catheters and systems for intra-arterial or intravenous administration.

The dose range of the anticoagulant component REG1, RB006, will depend on the indication. For example, a human dose of RB006 may be from about 0.1 mg / kg to about 10 mg / kg. For certain indications, the dose range will be from about 0.5 mg / kg to about 9 mg / kg, from about 0.75 mg / kg to about 8 mg / kg, from about 1 mg / kg to about 7 mg / kg, about from 1.5 mg / kg to about 6.0 mg / kg, from about 2.0 mg / kg to about 5.0 mg / kg, from about 2.5 mg / kg to about 4.0 mg / kg. For certain indications, the drug component will be used in the dose necessary to maintain the open state during the procedure. For certain indications, only RB006 will be administered, without subsequent administration of a neutralizing antidote.

The appropriate dose of the antidote component REG1, RB007, necessary to neutralize or partially neutralize RB006, depends on the amount of RB006 administered. The dose of antidote may vary in the mass ratio of antidote: drug (milligrams of antidote: milligrams of drug), from about 0.1: 1 to about 20: 1, from about 0.25: 1 to about 15: 1, from about 0, 5: 1 to about 12: 1, from about 0.75 to about 10: 1, from about 1: 1 to about 9: 1, from about 1.5: 1 to about 8: 1, from about 2: 1 to about 7.5: 1; from about 2.5: 1 to about 6: 1; from about 3: 1 to about 5: 1.

The most important property of the REG1 anticoagulation system, which contributes to the development of confidence in its safe clinical use, is the well-established ability of the antidote to predictably neutralize the pharmacological activity of the aptamer in a dose-dependent manner.

Human REG1 Coagulation Evaluation

This study was the first in which the REG1 anticoagulation system was evaluated in humans. Studies of single intravenous increasing doses (IV) of the REG1 anticoagulation system were performed in healthy volunteers. The subjects in this study were randomly assigned to either receive the test drug or placebo in one of three regimens with one of four (4) different dose levels. In each regimen at each dose level, subjects were randomized to 7: 1 anti-placebo treatment receiving REG1 or placebo. For all placebo injections, 0.9% sodium chloride USP injections were used. To obtain REG1 or placebo, subjects were randomized at each dose level.

To reduce risks to the minimum and the safety of the subjects involved in this study, to the maximum in the following order, three administration schemes were formed:

Scheme 1: after the placebo of the drug, the active antidote component RB007 followed, or after the placebo of the drug, the placebo of the antidote followed.

Scheme 2: after the active drug RB006, the active component RB007 followed, or after the placebo of the drug, the placebo antidote followed.

Scheme 3: the placebo antidote followed the active drug RB006, or the placebo antidote followed the drug placebo.

In Scheme 1, the antidote component of the REG1 anticoagulation system (RB007) was evaluated. Each subject received a placebo injection at time 0 according to this pattern (i.e., the time when the first bolus injection was given). After three (3) hours, subjects received an intravenous injection of the active antidote component (RB007), while one (1) subject received a placebo.

In Scheme 2, the combination of the active drug component of the REG1 anticoagulation system (RB006) was evaluated, followed by the active antidote component of the REG1 anticoagulation system (RB007). Subjects under this scheme received an injection of the active drug component (RB006) at time 0, and one (1) received a placebo. Three (3) hours later, subjects who received the active drug component received an injection of the active antidote component (RB007), while one (1) subject who received a placebo instead of the drug component received a placebo instead of the antidote.

In Scheme 3, the active drug component of the REG1 anticoagulation system (RB006) was evaluated. Subjects under this scheme received an injection of the active drug (RB006) at time 0, and one (1) received a placebo instead of an antidote. After three (3) hours, all subjects received a placebo instead of an antidote.

The test active drug component (RB006) was administered in four (4) dose levels: (1) low dose (15 mg RB006); (2) intermediate low dose (30 mg RB006); (3) intermediate high dose (60 mg RB006); and (4) a high dose (90 mg RB006). The starting dose and subsequent increasing doses were chosen to achieve maximum plasma concentrations, which determine three (3) key aspects of the in vitro APTT dose-response curve for RB006 in pooled normal human plasma: low dose to achieve the maximum plasma concentration at which APTT begins to increase in the RB006 in vitro dose-response curve (~ 4 μg / ml); two (2) intermediate doses to achieve plasma concentrations that limit the IC ₅₀ in the in vitro RB006 APTT dose-response curve (~ 8-16 μg / ml); and a high dose to achieve a plasma concentration at which the in vitro RB006 APTT dose-response curve begins to plateau (~ 25 μg / ml).

The test active antidote component (RB007) was administered in four (4) corresponding dose levels equivalent to a double dose level of the drug (RB006) based on mg / kg: (1) low dose (30 mg RB007); (2) intermediate low dose (60 mg RB007); (3) intermediate high dose (120 mg RB007); and (4) a high dose (180 mg RB007).

Table 6 outlines the doses for each phase 1A group of this study.

The test drug component (RB006), the test antidote component (RB007), and the corresponding placebo were administered as an injection for one (1) minute. The test drug component REG1 or placebo was administered at time 0 and the antidote component or placebo was administered at three (3) hours.

Table 6
Planned doses for three phase 1a regimens Group Scheme 1: placebo + antidote (RB007), mg Scheme 2: drug (RB006), mg + antidote (RB007), mg Scheme 3: drug (RB006), mg + placebo Dose Level 1: Low Dose thirty fifteen thirty fifteen Dose Level 2: Intermediate Low Dose 60 thirty 60 thirty Dose Level 3: Intermediate High Dose 120 60 120 60 Dose Level 4: High Dose 180 90 180 90

REG1 was evaluated in healthy volunteers to determine the safety profile and describe the PK and PD response of the REG1 anticoagulation system. The present study was the first in which an anticoagulation system using an aptamer and an aptamer oligonucleotide antidote was used in humans. The results indicate that after an intravenous bolus injection of the drug by APTT, a dose-response reaction was detected with a quick and prolonged return to the initial level of APTT after a bolus intravenous injection of the antidote. A similar pattern was observed for YOU, as for APTT. PV remained unchanged compared to baseline.

Subjects were injected with RB006 or 0.9% saline saline as an intravenous bolus injection at time zero and the anticoagulant effect was evaluated over time by measuring the plasma APTT (FIG. 10). APTT values for each treatment group are presented as mean values ± s.o.s. specific APTT. Specific APTT is the APTT value for an individual subject at a given time divided by the initial APTT value before the introduction of RB006 for a given subject. A value of 1 indicates no response to RB006, and a value> 1 indicates an anticoagulant effect. A clear response to the dose in terms of specific APTT is observed with an increase in the dose of RB006 from 15 mg to 60 mg. The half-life as the pharmacodynamic activity of RB006, as determined by APTT analysis, seems to be at least 12 to 18 hours, since this is the time during which the average specific APTT of subjects who were injected with 60 mg of RB006 reaches the maximum specific APTT determined by in subjects who were administered 30 mg of RB006.

Subjects were given RB006 or 0.9% physiological saline (placebo) by intravenous bolus injection at time zero and then either RB007 or placebo by intravenous bolus injection 3 hours after administration of RB006. The anticoagulant effect after administration of RB006 was detected for a long time by measuring the plasma APTT (FIG. 11). APTT values for each treatment group are presented as mean values ± s.o.s. specific APTT. Specific APTT is the APTT value for an individual subject at a given time divided by the initial APTT value before the introduction of RB006 for a given subject. A clear response to the dose in terms of specific APTT is observed with an increase in the dose of RB006 from 15 mg to 90 mg. The introduction of RB007 led to a complete, fast (within 5 minutes) and long-term neutralization of the pharmacological activity of RB006, which is confirmed by the return of specific APTT to the initial values after administration of RB007.

Treatment regimens were carried out as described in the above figures 10 and 11. Comparison of the pharmacodynamic response in subjects who were injected with 60 mg of RB006, followed by administration of RB007 or placebo after 3 hours, showed a quick and long-term neutralizing activity of RB007 (Fig. 12) . The introduction of RB007 effectively eliminates the state of subsequent anticoagulation in subjects, as shown by comparing the area under the APTT response curve from 3 to 24 hours in the presence or absence of RB007 administration.

The possibility of introducing the REG1 anticoagulation system in the form of an intravenous bolus injection that does not lead to complement activation in primates is unexpected, given the association of complement activation and, therefore, toxicity observed upon administration, previously observed with similar bolus injections of other types of oligonucleotide molecules. See, for example, Galbraith et al. (1994) "Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey," Antisense Research and Development 4: 201-206; and Levin, A. A., Monteith, D. K., Leeds, J. M., Nicklin, P. L., Geary, R. S., Butler, M., Templin, M. V., and Henry, S. P. (1998). Toxicity of oligonucleotide therapeutic agents, In Handbook of Experimental Pharmacology, G. V. R. e. a. Born, ed. (Berlin: Springer-Verlag), pp. 169-215.

Strategic analysis of dosing parameters

On Fig shows a more detailed analysis of the relative increase in APTT compared with the original after 0-3 hours for all subjects who received RB006. Consistent with the data from tests on monkeys, the APTT level reaches a maximum and reaches a plateau within a few hours. The data were analyzed by determining the area under the specific APTT curve compared to the baseline measured over the first three hours after administration. 19 shows how the response to RB006 relates to% inhibition of FIX. The data obtained demonstrate that it is possible to inhibit> 99% of FIX activity in a step-wise manner using an anticoagulant.

On Fig presents AUC _0-3 for each subject, which are shown in order of dose level RB006 (15, 30, 60 or 90 mg). Since the relative effect is measured for more than 3 hours, a value of "3" indicates a lack of response, a value of 6 indicates an average of 2-fold increase relative to the original, etc.

15 shows a weighted dose of RB006 as a function of the dose level of RB006. Figure 16 shows the relationship between the pharmacodynamic effect of RB006 (AUC _0-3 ) and the "weight-normalized" dose of RB006. The weight-normalized dose is in the range of 0.2 mg / kg to 1.6 mg / kg with an AUC range of _0-3 from about 3 to 10 units. The diagram demonstrates that there is a clear relationship between the response and the dose of anticoagulant, weight-normalized, with fairly low variability between subjects.

As shown in FIGS. 20 and 21, there is a clear association of body mass index (BMI) of registered subjects with a dose level of RB006. BMI 19-25 corresponds to the norm, 25-30 corresponds to overweight and> 30 corresponds to obesity. Subjects in this study ranged from a BMI of about 16 to a BMI of more than 35. Body mass index (BMI) is an indicator calculated by a person’s weight and height. BMI is a reliable indicator of obesity in humans. BMI does not measure body fat directly, but studies have shown that BMI correlates with direct methods for measuring body fat, such as underwater weighting and dual energy X-ray absorptiometry (DXA). BMI can be considered an alternative to direct methods for measuring body fat. BMI is the same for adults and children. The calculation is based on the following formulas:

Units Formula and calculation method Kilograms and meters (or centimeters) Formula: Weight (kg) / [Height (m)] ² Calculation Method:
[weight (kg) / height (m) / height (m)] In the metric system, the formula for calculating the BMI is the weight in kilograms divided by the square of growth in meters. Since growth is often measured in centimeters, to express the growth in meters, you need to divide the growth in centimeters by 100. Pounds and inches Formula: Weight (lb) / [Height (in)] ² × 703 Calculation Method:
[weight (lb) / height (in) / height (in)] × 703 To calculate BMI, you need to divide the weight in pounds (lb) by the square of the growth in inches (in) and multiply by the conversion factor 703.

On Fig shows the dosage, normalized to BMI, for subjects receiving RB006, as a function of the dose level of RB006. On Fig shows the relationship between AUC _0-3 for RB006 with a dose normalized to BMI. Dosages ranged from 0.5 mg / BMI to about 4.5 mg / BMI. The range of AUC _0-3 ranged from approximately 3 to 10 units. As can be seen in the diagram, there is a clear connection between the pharmacodynamic parameters and the dosage normalized to BMI. This relationship is even more pronounced than the relationship with the dose, normalized to weight, with less variability. The association of BMI with specific AUC _0-3 indicates that the drug is probably mainly distributed in the central part of the body, and not on the periphery or in the corresponding tissues. This distribution provides additional support for the use of the REG1 system as an anticoagulant for parenteral administration.

Evaluation of the REG1 system in patients with stable angina pectoris

Studies were performed on 50 patients with stable angina pectoris taking aspirin and / or clopidogrel. Patients were randomly assigned to three groups (only RB006, RB006, followed by RB007, or only placebo) with 4 dosage levels of RB006 and RB007.

Initial characteristics included an average age of 61 years (interquartile range (IQR) 56-68), 20% of women, 80% had a history of percutaneous intervention on coronary vessels and 34% had a history of aorto-coronary artery bypass grafting. The APTT median 10 minutes after a single intravenous (IV) bolus injection of low, intermediate low, intermediate high and high doses of RB006 was 29.2 seconds (IQR 28.1-29.8), 34.6 seconds (IQR 30.9- 40.0), 46.9 seconds (IQR 40.3-51.1) and 52.2 seconds (IQR 46.3-58.6), p <0.0001, (APTT normal range 27-40 seconds) . RB007 returned APTT to <10% above the upper limit of normal with a median of 1 minute (IQR 1-2) (FIG. 1) without a return increase up to 7 days. Despite dual antiplatelet therapy, 38% of subjects had no significant bleeding or other serious undesirable complications.

On Fig presents the results of a comparison of the response of the APTT at four aptamer / antidote dosages in comparison with placebo. Group 1 "low dose" was administered 15 mg of RB006 at time 0 and 30 mg of antidote RB007 at time 3 hours by intravenous bolus injection. Group 2 "intermediate low dose" was administered 30 mg of RB006 at time 0 and 60 mg of RB007 antidote at time 3 hours as an intravenous bolus injection. Group 3 "intermediate high dose" was administered 50 mg of RB006 at time 0 and 100 mg of antidote RB007 at time 3 hours as an intravenous bolus injection. Group 4 "high dose" was administered 75 mg of RB006 at time 0 and 150 mg of antidote RB007 at time 3 hours as an intravenous bolus injection. At both 50 and 75 mg / kg RB006, a pronounced increase in APTT was determined, which was completely neutralized when RB007 was administered at a 2-fold aptamer concentration.

Repeated dosing system REG1

Studies were performed on 38 patients with overall good health. Three treatment groups were formed: group 1, in which subjects received a single dose of aptamer (0.75 mg / kg RB006) on days 1, 3, and 5, followed by a fixed dose of antidote (1.5 mg / kg RB007) through one hour; and groups 2 and 3, in which subjects received a single dose of RB006 aptamer (0.75 mg / kg) on days 1, 3, and 5, followed by single different doses of RB007 administered after one hour. Titration of a dose of RB007 in subjects in groups 2 and 3 is presented in table A below.

Table a
The dose ratio of antidote (RB007) and drug (RB006) for groups 2 and 3 Day Antidote: Drug Ratio RB007 (mg / kg): RB006 (mg / kg) Group 2 one 2: 1 1.5: 0.75 3 1: 1 0.75: 0.75 5 0.5: 1 0.375: 0.75 Group 3 one 0.2: 1 0.15: 0.75 3 1: 1 0.75: 0.75 5 TBD ¹ TBD: 0.75 ¹ Antidote ratio: drug tested on day 5 was between 0.1: 1 and 1: 1, and was based on the results of APTT days 1 and 3.
^{2 The} dose of antidote ranged from 0.075 mg / kg to 0.75 mg / kg.

The dose of RB006 (0.75 mg / kg) was selected based on the weight-normalized response to RB006. On average, the indicated weight-normalized dose of RB006 increased APTT of subjects by 2 times. Aptamer RB006, an antidote, and their corresponding placebo were administered as an injection for one (1) minute. On Fig shows the time-weighted APTT after administration of RB006 (0.75 mg / kg) on days 1, 3 and 5 at various dosages of the antidote.

On Fig shows the percentage effect on APTT from the introduction of RB006 in the respective groups. An approximately 270% increase in APTT was noted after administration of 0.75 mg / kg aptamer in all three groups, and it did not significantly differ during the three days of treatment.

Figure 23 shows the average APTT in the groups administered RB006 (0.75 mg / kg) and RB007 in various ratios to RB006. RB006 was administered at time 0, and RB007 in the above ratios was administered after one hour. As can be noted from the diagram, RB007 changed the anticoagulant dose of the antidote to the aptamer. In addition, as can be seen from FIG. 23, the inversion effect of RB007 for each of the tested ratios was relatively stable for a long time with a gradual decrease in the pharmacodynamic activity of RB006 over a long time, as expected for the present compound.

On Fig shows the percentage recovery of time-weighted APTT in the groups that were administered RB006 (0.75 mg / kg) and RB007 in various ratios with RB006. RB006 was administered at time 0, and RB007 in the above ratios was administered after one hour. With the lowest ratio tested, 0.125: 1, RB007 neutralized the effect of RB006 by approximately 40%. At 0.2: 1, RB007 neutralized the effect of RB006 by approximately 50%. At 0.3: 1, RB007 neutralized the effect of RB006 by approximately 75%. At 0.5: 1, RB007 neutralized the effect of RB00 by approximately 85%. And at higher ratios, 1: 1 or 2: 1, RB007 effectively completely neutralized the effect of RB006.

Claims (14)

1. The method of introducing an anticoagulant system to a subject, where the system includes an aptamer that binds factor IX / IXa, and an antidote that
connects the aptamer,
including:
a. measurement of the weight of the subject in kilograms;
b. administering to the subject a dose of an aptamer effective to achieve coagulation inhibition in a subject where the aptamer comprises the sequence of SEQ ID No: 1 and where the dose of the aptamer is from about 0.1 mg / kg to about 2.0 mg / kg, or from about 5 mg / kg to about 10 mg / kg; and
c. administering an aptamer antidote dose to a subject where the antidote includes the sequence of SEQ ID No: 2, and the antidote dose is based only on the weight / weight ratio with the dose of the aptamer, and where the weight / weight ratio of the antidote dose of the aptamer is from about 0.1: 1 to approximately 20: 1. 2. The method according to claim 1, wherein the dose of the aptamer is from about 0.1 mg / kg to about 1.5 mg / kg. 3. The method according to claim 1, in which the dose of the aptamer is approximately 1.0 mg / kg 4. The method according to claim 1, wherein the dose of the aptamer is from about 0.6 mg / kg to about 0.8 mg / kg. 5. The method according to claim 1, in which the dose of the aptamer is from 0.1 to 2 mg / kg 6. The method according to claim 1, in which the dose of the aptamer is from 5 to 10 mg / kg 7. The method according to claim 1, in which the aptamer is administered by intravenous bolus administration. 8. The method according to claim 1, in which the aptamer is administered by subcutaneous injection. 9. The method according to claim 1, in which the aptamer and antidote are administered in a weight / weight ratio of from about 0.25: 1 to about 15: 1. 10. The method according to claim 1, in which the aptamer and antidote are administered in a weight / weight ratio of from about 0.5: 1 to about 12: 1. 11. The method according to claim 1, in which the aptamer and antidote are administered in a weight / weight ratio of from about 1: 1 to about 9: 1. 12. The method according to claim 1, in which the inhibition of coagulation after administration of a dose of antidote is reduced by less than 90%. 13. The method according to claim 1, in which the activity of the aptamer is reduced by about 50%. 14. The method according to claim 1, in which the inhibition of coagulation after administration of a dose of the antidote is completely neutralized.

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