UA94948C2 - Administration of the reg1 anticoagulation system - Google Patents

Abstract

An improved method of administration of an aptamer and antidote system to regulate blood coagulation in a host is provided based on weight adjusted or body mass index-adjusted dosing of the components of the system to provide a desired pharmacodynamic response. In addition, a method of reversing activity of the aptamer to a desired extent is provided where an antidote dose is based solely on its relationship to the aptamer dose.

Description

This application claims priority to US Provisional Application Me 60/808987, filed May 26, 2006, US Provisional Application Me 60/847809, filed September 27, 2006, and US Provisional Application Me 60/865352, filed November 10, 2006, that all named "KES1 anti-seize system introduction", the disclosures of which are incorporated herein in their entirety.

The method of administration of the aptamer and antidote system for the regulation of blood coagulation in a subject based on the dosage regimen of system components normalized by weight or body mass index is presented.

Anti-erosive therapy in emergency care

Given the central role of thrombosis in the pathology of acute ischemic heart disease, injectable anticoagulants have become the mainstay of treatment for patients with acute coronary syndromes such as unstable angina and myocardial infarction and those undergoing coronary revascularization procedures (Nagtipdiop et al. 2004; Rorta et al. 2004). Currently available anticoagulants include unfractionated heparin (UFH), low-molecular-weight heparins (LMWH), and direct thrombin inhibitors (DTIs), such as recombinant hirudin, bivalirudin, and argatroban. The existing paradigm for both anticoagulant use and the development 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 (Nagtipdiop et al., 2004). Each of the available agents carries an increased risk of bleeding compared with placebo.

The main side effect associated with anticoagulant and antithrombotic drugs is bleeding, which can cause permanent disability and death (Errezep et al., 2001; Iemipe et al., 2004). As a general 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, recently obtained data suggest that bleeding, especially those that require blood transfusion, significantly affects the outcome and cost of treatment of patients with ACS.

The frequency of blood transfusions in patients undergoing elective coronary artery bypass surgery (CABG) is 30-6095, and transfusion in these patients is associated with increased immediate, medium-term and long-term mortality (Vgaseu et al., 1999; Epdogep et al. ., 2002; Nebegi et al., 1999). Bleeding is also the most frequent and costly complication of percutaneous coronary interventions (PCI), with transfusions performed in 5-1095 patients with an additional cost of 58000-512000 (Mozsissi, 2002).

In addition, the rate of major bleeding in patients treated for ACS is also high, ranging from 595 to 1095 (excluding patients undergoing CABG), with bleeding and transfusion independently associated with a significant increase in immediate mortality (Mo5sissi et al., 2003; Kao et al., 2004). Therefore, despite the ongoing development of new antithrombotic agents, there is a pronounced clinical need for safer anticoagulants.

Rapid termination of the drug may be achieved passively by a drug composition, such as an infusion agent with a short half-life ending the infusion as a means of termination, or actively by administration of a second agent, an antidote, that can neutralize the effect of the drug.

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

Current approaches to solving the problem

CEN is currently the only reversible antidote approved for use. However, OEN has significant limitations. First, heparin has complex pharmacokinetics, which makes the reliability of its use difficult (Sgapdeg et al., 1996). Second, the dose predictability of its antidote, protamine, is complicating, and there are serious side effects associated with its use (Saighi

Biimeptap, 1999; Muei5ru et al., 2005). Finally, heparin can cause thrombocytopenia (NIT) and thrombocytopenia with thrombosis (TTT) (Umagkepiip, 2005; Umagkepiip and Ogeipaspeg, 2004).

Despite these limitations, heparin remains the most widely used anticoagulant for hospitalized patients primarily because it is "reversible." Newer generation anti-erosive agents, such as HMUMN, have improved SHEN dosing predictability and do not require monitoring as a practice in their routine use based on laboratory studies. NIT and NITT are less common when using HMUMN than OEN, but when they are used, the indicated risk is not eliminated. Two of the three commercially available OTIs, lepirudin and argatroban in particular, are approved for use in patients who have developed or had a history of NIT. Bivalirudin is approved for use as an anticonvulsant agent during RSI and therefore represents an attractive alternative to SENS in patients with NIT. However, there are no direct and effective antidotes to stop the anticoagulant effects either for I MUMN or for OTI, which represents a special risk when they are used in patients undergoing surgical or percutaneous coronary revascularization procedures (Chope5 et al., 2002). Bleeding in receiving patients

HMMUN or STI, buy by introducing blood products, including coagulation factors.

Blood clotting and SIN

To date, the cellular model of coagulation (Fig. 1) represents the clearest explanation of how physiological coagulation and coagulation of blood vessels occurs (Noitap et al., 1995; KiaiKe et al., 1998; Mopgoe et al., 1996).

According to this model, the coagulation reaction takes place in three stages: initiation, amplification, and propagation. Initiation of coagulation occurs on cells that carry tissue factor, such as activated monocytes, macrophages, and endothelial cells. Coagulation factor Ma, which forms a complex with tissue factor, catalyzes the activation of blood coagulation factors IX (PIX) and X (EX), which, in turn, produces a small amount of thrombin from prothrombin. In the amplification phase (the phase also referred to as priming), a small amount of thrombin produced in the initiation phase activates the coagulation factors M, MY! and XI, and also activates platelets, which represent the surface on which subsequent coagulation reactions take place. However, the small amounts of thrombin generated during the amplification phase are not sufficient to convert fibrinogen to fibrin due to the presence of endogenous thrombin inhibitors called serpins, such as antithrombin I, α-2-macroglobulin, and heparin cofactor II. The final phase of the coagulation reaction, spreading, occurs exclusively on the surface of activated platelets. In the propagation phase, significant amounts of RIX are formed with the help of

EHia-catalyzed activation of the RHC. RIKha forms a complex with its obligatory activating cofactor EMPA

EX. Subsequently, EC forms a complex with its obligatory cofactor EM. The EXa-RMa complex activates prothrombin, which leads to the "explosive" formation of thrombin and the deposition of fibrin. The end result is the formation of a stable clot.

Based on the specified model, during folding, EiHa plays two roles. In the initiation phase, RIXa plays an important role in the formation of a small amount of thrombin with the help of activation of EX in EXa and subsequent activation of prothrombin. However, the specified role of RIXa overlaps at least partially with the conversion of EX to EXA, which is catalyzed by tissue factor and EMIIIa. A more critical function of RiXa is carried out in the propagation phase, in which the enzyme complex EMIP/RIXa serves as the only catalyst for the formation of EXa on the surface of an activated platelet. Therefore, a reduction in RiX activity, either due to a genetic deficiency of RiX (ie, hemophilia B) or as a result of pharmacological inhibition of RiX/Xa, is expected to have several effects on coagulation. First, the inhibition or loss of action of RiIX should partially inhibit the initiation of coagulation.

Second, inhibition or loss of action of RIX should have a significant effect on the propagation phase of coagulation, leading to a significant reduction or elimination of thrombin formation. Finally, limiting thrombin generation during the propagation phase at least partially inhibits amplification of coagulation in a feedback mechanism by reducing platelet activation and coagulation factors involved in earlier stages, such as factors M, MI! and XI.

Prior art animal and human evaluation of RIX inhibitors

Inhibitors of PIH activity, such as factor IXa with an active site inactivation (PiHai) or monoclonal antibodies against PIH (eg, BC2 antibody), have potent antiplatelet and antithrombotic activity in several animal models, including various animal models of arterial thrombosis and stroke (Vepedisi et al. et al., 1991; Spotsangi et al., 1999; Recegvivip et al., 1999; Zrapieg et al., 199va; Zrapieg et al., 1997; Zrapieg et al., 199860; Tooteu et al., 2000). In general, in these studies, it was shown that RiIXa 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, animals treated with these agents had bleeding profiles that did not differ from those for heparin. The indicated experience of controlled studies on animals suggests that in clinical conditions, the ability to control the activity of the RIX inhibitor would increase its safety and facilitate its medical use. In addition, RiIChai has been shown to be safe and effective as a replacement for heparin in several animal surgical models requiring anticoagulant therapy, including synthetic patch angioplasty models in rabbits, as well as canine extrapulmonary vasculature models of CABG and nonhuman primates (zZrapieg et al., 1998a; Zrapieg et al., 1997; Zrapieg et al., 199860). RiHai has also been successfully used in several critically ill patients requiring extrapulmonary circulation, and in the creation of other extracorporeal circulation methods, such as extracorporeal membrane oxygenation (Zrapieg et al., 1998a), by doctors Soishtbia SoiIede oi Rpuzisiap5 apa Zygdeop5 within charity care. Thus, RiXa is a reasonable target for therapy with anticoagulants during coronary revascularization procedures (both CABG and PCI) and for the treatment and prevention of thrombosis in patients with acute coronary syndromes.

Development of aptamer drug, drug-antidote pair and KES1

One approach to providing controlled anticoagulation is the use of an anticoagulant with an intermediate to long-acting duration of 12 hours or more, which can achieve clinically adequate action 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 a predictable and safe neutralization and termination of the anticoagulant activity of the drug (Kizsopi et al., 2004, May Violations. 22 (11):1423-8; Kizsopi et al., 2002, May 419 ( 6902):90-4).

In the discovery of the aptamer-based KES1 anti-coagulant system (see Fig. 2), the applicants applied drug-antidote technology. Aptamers are single-stranded nucleic acids that bind with high affinity and specificity to target proteins (Mitte et al., 2005) similar to monoclonal antibodies. However, in order to bind and inhibit the target protein, the aptamer must adopt a defined globular tertiary structure. Formation of the indicated globular tertiary structure requires that the aptamer adopt the correct secondary structure (ie, the correct regions of paired and unpaired bases).

As shown in schematic form in fig. 2, the introduction of an oligonucleotide complementary to the aptamer part can change the structure of the aptamer in such a way that it can no longer bind to its target protein and thus effectively terminates or neutralizes the pharmacological activity of the aptamer drug (Kiv5sopi et al., 2004 , May Violations. 22 (11):1423-8; Ki5sopi et al., 2002, Mashge 419 (6902):90-4).

KVOO06 (40 kDa complex ester P-I -dnoddasoaasSSoyaaSdOaadSysssSOssast, in which P-tRES2-MH5; I -06

MNe»g linker; 9-2-O0 0; 9-2-O-Me 0; Sat-2-E C; c-2'-O-Mi C; O52-E OO; ts-2-0O-Me Sh; a-2-0-Me A; and Teinverted 2-H T (5EO IT MO 1); see fig. 2), the component of the drug BES1 is a direct inhibitor of RiXa, which binds to the coagulation factor of RiXa with high affinity and specificity (Ki5sopi et al., 2004, May

VioyesNnpoi. 22 (11):1423-8; Kyvsopi et al., 2002, Maishge 419 (6902):90-4; see also M/O05/106042 in the name of bike

Opimegzpyu). KVO0b exhibits anti-inflammatory activity by blocking the EMpa/RIXa-catalyzed conversion of EX to EX. KVO06b is a modified RNA aptamer, 31 nucleotides in length, moderately stabilized against degradation by endonucleases by the presence of 2'-fluoro and 2-O-methyl containing sugar groups, and stabilized against degradation by exonucleases by a 3'-inverted deoxythymidine cap. The nucleic part of the aptamer is conjugated with a 40-kilodalton polyethylene glycol (PEG) carrier to increase its half-life in the blood. With a bolus intravenous injection, the half-life of ВО0О06 in mice is approximately 8 hours and in monkeys approximately 12 hours. Also, to maintain an anti-coagulant state, it can be administered for more than a few hours

EVO06 in the form of a single bolus injection, and not by intravenous infusion.

As shown in fig. 2, EB007 (sdsddsatsadissas, in which d-2-0O-Me o; c-2-O-Mi C; i-2'-0O-Me Sh; and ah2-O0-

Me A (ZEO IOT MO 2); see fig. 2), the antidote component of KES1, is an oligonucleotide that is complementary to the KVO0Ob part, which can effectively bind to KVOOb and, thus, neutralize its anti-RiXa effect.

KVO07 is a 2'-O-methyl RNA oligonucleotide, 15 nucleotides in length, that is complementary to the KES1 drug component. The 2'-O-methyl modification endows the antidote with moderate nuclease resistance, providing sufficient IP stability to allow to find and contact KVOOb, but does not support long ip pass persistence.

Non-clinical stage of KES1 development

The applicants obtained pharmacological data demonstrating the specificity of the KVO0b aptamer to RihXa and the affinity of the KVO007 antidote to the aptamer. The results of non-clinical pharmacological studies can be briefly formulated as follows: medicinal component of KESI! (BO06 and/or related precursor compounds) can: (1) effectively inhibit the activation of the coagulation factor X hip miigo; (2) to prolong the coagulation time of blood plasma and migo in the plasma of humans and other animal species; (3) to have a systemic anticoagulant effect in animals with bolus intravenous administration; (4) prevent thrombus formation in an animal model of arterial injury thrombosis; (5) replace heparin in an animal model of extrapulmonary circulation, and (6) be effective when re-administered to the animal within 30 minutes of neutralization by the antidote component of KESI1.

In non-clinical pharmacological studies to date, it has been shown that the antidote component

EES1 (KVO07 and/or antidotes specific to the precursors of the drug component KES1) can: (1) quickly and for a long time neutralize the anti-inflammatory activity of the drug component KES1 (KVOO6b) and migo in the plasma of humans and other animal species; (2) to quickly and for a long time neutralize the anticoagulant activity of the drug component RECI1 ip immediately after intravenous bolus administration to an animal that received systemic anticoagulant therapy with the specified agent; (3) prevent bleeding caused by the combination of supratherapeutic doses of the drug component of KESI1 and surgical trauma and (4) neutralize the anticoagulation activity of the drug component of KESI1 in animals after extrapulmonary circulation. In addition, the antidote did not show any anticonvulsant or other pharmacological activity of ip migo in human plasma or in animals after intravenous bolus administration.

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

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

In one embodiment, the present invention represents an improved method of administration of an aptamer anti-inflammatory system, which includes: 1) measurement of the body mass index (BMI) of the subject; 2) determination of the desired pharmacodynamic response; and 3) administering to the subject a dose of the aptamer anticoagulant to achieve the desired pharmacodynamic response based on comparing the dose to BMI with the pharmacodynamic response. In particular embodiments, the aptamer antidote is subsequently administered to the subject, and the antidote dose is presented based on the ratio of the previously administered aptamer dose to the desired decrease in aptamer activity. In some cases, the indicated dose of the antidote is normalized depending on the time that has passed since the introduction of the aptamer. In some cases, the ratio of antidote to aptamer was reduced by two times if the aptamer was administered earlier than 24 hours.

In private implementation options, the maximum level of anti-inflammatory effect is desired. In these cases, the aptamer can be given in an amount of 4 mg/MT or more. In other cases, the desired level of anticoagulation is approximately 7595 of the maximum. In such cases, the subject is administered a dose of approximately 0.75-1.5 mg/MT. In other cases, an anticoagulation level of approximately 5095 of the maximum is desirable. In these cases, the recommended dose is approximately 0.25-0.5 mg/BMI.

In private general variants of implementation, the dosage of the used anticoagulant is from 0.1 to 10 mg/MT. In another embodiment, the dosage is from 0.2 to 8 mg/BMI, or from 02 to 6

MG/L MT, from 0.2 to 5 mg/BMI, from 0.2 to 4 mg/BMI, from 0.2 to 3 mg/MT, from 0.2 to 2 mg/BMI, or from 0, 2 to 1 mg//MTt. In some embodiments, the dose of the antihypertensive agent is about 0.1 mg/MT, or about 0.2

MG/M, or about 0.5 mg/MT, or about 0.75 mg/MT, or about 1 mg/MT, or about 2

MG/ MT, or about 3 mg//MT, or about 4 mg/MT, or about 5 mg//MT, or about 6 mg//MTt, or about 7 mg//MT, or about 8 mg/BMI, or about 9 mg/BMI, or about 10 mg/MTt.

In another embodiment, the present invention represents an improved method of introducing an aptamer anti-inflammatory system, which includes: 1) measuring the subject's weight; 2) determination of the desired pharmacodynamic response; and C) administering to the subject a dose of the aptamer 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 the subject, with the dose of the administered antidote based on the ratio of the previously administered aptamer dose, normalized for the desired decrease in aptamer activity. In some cases, the indicated dose of the antidote is calculated based on the time that has passed since the introduction of the aptamer. In some cases, the ratio of the antidote to the aptamer is doubled if more than 24 hours have passed since the aptamer was administered.

In private implementation options, the maximum level of anti-inflammatory effect is desired. In these cases, the aptamer can be administered at a dose of 1.4 mg/kg or more. In other cases, the desired level of anticoagulation is approximately 7595 of the maximum. In such cases, the subject is administered a dose of 0.5 to 0.75 mg/kg. In other cases, the desired level of anticoagulation is approximately 5095 of the maximum. In these cases, a dose of approximately 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 from 1 to 20 mg/kg, from 1 to 18 mg/kg, from 1 to 15 mg/kg, from 2 to 15 mg/kg, from C to 15 mg/kg, from 4 to 15 mg/kg, from 5 to 20 mg/kg, from 5 to 15 mg/kg, or from 1 to 10 mg/kg, or from 5 to 10 mg/kg, or is about 1 mg/kg, about 2 mg/ kg, about C mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg . In a preferred embodiment, the aptamer anticoagulation system is the KES1 system, which includes an anticoagulant aptamer and an oligonucleotide antidote. In private, non-limiting embodiments, the aptamer is KV006 (5EO IT

MO 1) and the antidote is KVO07 (5ZEO IST MO 2). In one embodiment, the pharmacodynamic response is measured in coagulation activity assays, such as aPTT (plasma or whole blood) or active clotting time (ACT), and may be expressed as an absolute value, percent effect, percent change, time-weighted average, or value area under the curve for a certain period of time.

The level of pharmacodynamic response can be whatever is required for the particular task.

For example, in some cases, if the patient is at low risk of a thrombotic complication, then a low response rate may be desirable. In some cases, it may not be desirable to achieve maximal inhibition of the coagulation factor, and in particular, inhibition of SIN or Rih by using an excess amount of an anticoagulant, especially a Rih aptamer such as KVO06. In other cases, when the patient is at high risk for or developing a thrombotic complication, a high response rate may be desirable. In such cases, it may be desirable to maximize the inhibition of the coagulation factor, and in particular, RICH or inhibition of Rich when using an excess amount of an anti-coagulant agent, in particular, an aptamer to Rich, such as KVOO6.

In one embodiment, the anticoagulant aptamer, such as KVO0b, is intended for intravenous bolus administration. In another embodiment, the anti-caking aptamer is presented for subcutaneous injection. In another embodiment, after intravenous or subcutaneous bolus administration of the aptamer, an antidote is injected.

The procedures described herein allow for the stepwise administration of both an antidote and an antidote, allowing one or both compounds to be titrated to the desired level of inhibition and neutralization of target activity.

The ratio of antidote to aptamer was calculated based on the desired level of aptamer inhibition.

It was found that the dose of the antidote should be calculated only based on the aptamer dose, and should not be additionally normalized for factors related to the subject. In one embodiment, the ratio of aptamer to antidote is 1:1. In other embodiments, the ratio of aptamer to antidote is greater than 1:1, such as 2:11, 3:11, 41, 5:1, 6:1, 7:1, 8:1, 9:1, 1011, or greater. Said ratios may also be calculated based on the ratio of antidote to aptamer, which may, for example, be less than about 11, such as 0.9:1 or about 0.9:1, 0.8:1 or about 0.8:1 , 0.7:1 or about 0.7:1, 0.6:1 or about 0.6:1, 0.5:1 or about 0.5:1, 0.45:1 or about 0.45 :1, 0.41 or about 0.471, 0.35:1 or about 0.35:1, 0.3:1 or about 0.3:1, 0.25:1 or about 0.251, 0.2:1 or about 0.2:1, 0.15:1 or about 0.15:1, 0.111 or about 0.111 or less than 0.1:1, such as about 0.005:1 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 0.31. 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 aptamer activity occurs.

For example, in some embodiments, the activity of the aptamer is neutralized by 9095, or less than 9095, such as by about 8095, by about 7095, by about 6095, by about 5095, by about 4095, by about 3095, by about 2095, by about 1095, or Less. The ratio of antidote to aptamer can either be calculated using a weight-to-weight comparison or on a mole basis.

In private variants of the implementation of this invention, the organism or subject to which the dosing system is applied is a person. In certain embodiments, the organism is a human in need of anticoagulant therapy. In certain embodiments, the organism is a person undergoing vascular surgery, such as CABG surgery.

Brief description of the drawings

In fig. 1 shows the cellular model of coagulation. TE - tissue factor; UM/E - von Willebrand factor; AND! - prothrombin; Ia - thrombin; Ma, Ma, Miia, IXa, Xa, Xia - activated forms of coagulation factors M, MII, MP, IX, X and

XI.

In fig. 2 shows the KESI1 anti-seize system. The system consists of the RIX inhibitor KVO06 and the antidote KVO07 suitable for it. Recognition of the drug by the antidote occurs as shown by base pairing according to the Watson-Crick rule. KVO06b is a modified RNA aptamer consisting of 2'-fluoro residues (upper case), 2'-O-methyl residues (lower case) and one 2"-hydroxyl residue (underlined). KVO06 is conjugated to a 40-kDa polyethylene glycol carrier (P) through the b-carbon amino linker ()U, which is protected from degradation by nucleases with the help of an inverted deoxythymidine at the 3'-end (at).

KVOO07 (antidote) is a 2'-O-methyl-modified RNA oligonucleotide.

Fig. C is a plot of the dose-response curve of the APTT of KWOO6b ip miigo, showing that KWOOob exhibits a concentration-dependent increase in the APTT of normal pooled human plasma. The average value, in seconds, is the average AHTCHUCH. The data were fit to a four-parameter logistic equation, which allows you to determine the ISvo by the curve.

In fig. 4 presents a diagram of the anticoagulant effect of KVOOb in the plasma of patients. Anti-erosion activity

EVO006 was measured in 4 patients: two women and two men. Plasma samples were obtained from seogde Kipd

Viotedis! (Omepapa Ragk, K5). Patients were screened and confirmed to have normal levels of coagulation factors. M/55 means that the donor was a man, age 55; P/49 means that the donor was a woman, age 49. As a reagent of АЦТЧ, MOA Rialeyip H. (Vioteghih) was used, which is relatively more sensitive to the levels of ХХ than the reagent of АЦЦХ, used in the study presented in fig. 3.

In fig. 5 presents a graph showing the neutralizing activity of the drug by the antidote

KVO07. A small molar excess of the KVO07 antidote relative to the KVOOb aptamer completely neutralizes the anti-inflammatory activity of KVO0Ob within 10 minutes. Data shown are the mean and standard error of the mean for three independent measurements. The molar ratio is based on moles of oligonucleotide aptamer and antidote (AB).

In fig. b presents the schedule of re-introduction of the KVO0b aptamer after neutralization of the antidote by the previous dose of the drug. Pigs were injected with 2.5 mg/kg of aptamer KVO0b, and after 15 minutes, 3 mg/kg of antidote KVOO7 (p-2) was injected to neutralize the specified starting dose. Next, 30 minutes after the administration of the antidote KVOO07 (after 45 minutes of initial aptamer administration), pigs were re-injected with 2.5 mg/kg of aptamer KVOO06. The change in coagulation time was measured by (A) CHAZ (C) in whole blood analyses; or by (B) AChT (0) in plasma coagulation assays. Data shown are presented as mean value and range for repeated measurements for each animal. The bold line (in A and B) is a simple line of interpolation of the experimental points.

In fig. 7 presents a diagram of the KVOO06 ip migo dose-response curve of AChT in the plasma of Javan macaques and humans. KVO0Ob reveals a dose-dependent prolongation of AChT in plasma from monkeys, which is very similar to that observed in human plasma. Experiments were performed using the reagent for APTC of the same brand, ARTT-I 5, as used for the analysis of plasma samples in non-clinical toxicity studies performed on monkeys (KES1-TOKHO01 and KES1-TOKHO003). Therefore, the obtained data serve as the basis for the interpretation of the results of АХТЧКЕС1-ТОХО001 and КЕС1-ТОХО03, presented in sections 8.4. According to the manufacturer (Rasiis Noteobviavziz, Miadieim/p, MA), the specified reagent determines an AChT of -87.3 seconds in human plasma samples containing "195 levels of SIN, 36.1 seconds in samples containing 72095 of normal activity

RICH, and 27.5 seconds in samples containing 10095 RICH activity. Citrate pooled plasma of Javan macaques was provided by SpPagiez Kimeg I arogaiyugie5, bie!ta Oimiviop.

In fig. 8 shows the schedule of systemic anticoagulation in monkeys with the introduction of KVOO0b. The level of anticoagulation in monkeys was monitored by AChTUCH. For animals injected with 15 mg/kg, data from KVO0Ob are presented as mean value and standard error of the mean (s.p.s.). For animals with dosage levels of 5 and 30 mg/kg, the data are presented as the mean value and the range, since there were only 2 animals in each of the indicated dosage level groups.

In fig. 9 presents a schedule of systemic anticoagulation in monkeys KVOO06 and neutralization with the antidote KVOO7.

The level of anticoagulation in monkeys was monitored by APT. KVO07 was administered 1-3 hours after the introduction of KVO0b. The data are presented as the mean value x s.p.s.

In fig. 10 presents a diagram of the pharmacodynamic activity of KVOO06 in humans.

In fig. 11 presents a diagram of the neutralization of the pharmacological activity of KVO06 in humans with the help of vvo07.

In fig. 11 presents a diagram comparing the pharmacodynamic activity of KVO06 with and without the introduction of KVOO07.

In fig. 12 presents a graph comparing the pharmacodynamic response in subjects who were administered 60 mg of EVO06 followed by 3 hours of KVOO07 or placebo.

In fig. 13 presents a more detailed analysis of the relative increase in AChT with respect to the initial value during 0-3 hours in all subjects receiving ЕВОО06.

In fig. 14 presents a diagram of ACo-z for each subject depending on the dose level of EVO006 (15, 30, 60 or 90 mg). Since the relative effect is measured over 3 hours, a value of "3" represents no response to KVOO06, a value of 6 indicates an average 2-fold increase relative to baseline, etc.

In fig. 15 presents a diagram of the weight-normalized dose of KVOOb as a function of the level of the KVOOb dose.

In fig. 16 shows the diagram of Aso-z in comparison with the weight-normalized dose of KVOO06.

In fig. 17 presents a diagram of the BMI-normalized dose of subjects who were administered ЕВО06 as a function of the ЕВООb dose level.

In fig. 18 shows the diagram of ACo-z for KVO06 depending on the BMI-normalized dose.

In fig. 19 presents a diagram of AChT in comparison with the initial level depending on the 9o activity of GRIH, which shows AChT at different doses of KVOOb (15, 30, 60 and 90 mg).

In fig. 20 presents a diagram of the APTC response depending on the use of four doses of the aptamer KVOOb and the antidote KVO07 administered intravenously in patients with coronary artery disease.

In fig. 21 presents a diagram showing the dependence of AChT on time after the introduction of KVOOb (0.75 mg/kg) on days 1, 3 and 5 in all treatment groups. Group 1: subjects who received a single dose of aptamer (0.75 mg/kg

KVOO6) on days 1, 3 and 5, after which one hour later the administration of a fixed dose of antidote (1.5 mg/kg

KVO07); groups 2 and 3: subjects who received a single dose of aptamer KWOO06 (0.75 mg/kg) on days 1, 3 and 5, after which, one hour later, various single doses of KWOO7 were administered.

In fig. 23 presents a diagram of the dependence of the average AChT on time in the groups that were administered KVOOb (0.75 mg/kg) and KVOO07 in different ratios compared to KVOO06.

In fig. 24 presents a diagram showing the percentage recovery of the time-weighted average of АХТЧУХ after the introduction of КВОО06б after an hour of КВОО07 in the listed ratios in comparison with КВООб.

It was found that there is a clear relationship between weight-normalized dosing and, importantly, body mass index-normalized dosing, particularly for the aptamer anticoagulant and its pharmacodynamic response. In addition, surprisingly, it was 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 aptamer introduced into the body, and not on any additional criteria. This new understanding provides support for defined routes of administration that allow for predictable and multiple dosing regimens in clinical use.

Development of aptamers

Nucleic acid aptamers are isolated using the method of exponential enrichment by systematic evolution of ligands, called ZE EX. This method allows for rapid evolution of nucleic acid molecules with highly specific binding to target molecules. Method 5EG EX is described, for example, in US patent Mo 7087735, US patent Me 5475096 and US patent Me 5270163, (see also UUO 91/19813).

Method 5EG EX includes selection of a mixture of candidate oligonucleotides and stepwise repetitions of binding, separation and amplification, using the same general selection scheme to achieve virtually any desired criterion of binding affinity and selectivity. Starting with a mixture of nucleic acids, for example, mixtures containing random sequence fragments, the 5EGEX method includes the steps of contacting the mixture with a target under conditions favorable for binding, separating unbound nucleic acids from those nucleic acids that specifically bind bound to target molecules, disruption of target nucleic acid complexes, amplification of nucleic acids dissociated from target nucleic acid complexes to obtain a ligand-enriched mixture of nucleic acids, and then repeating the steps of binding, separation, dissociation, and amplification the number of times necessary to obtain highly specific, high-affinity aptamers to the target molecule.

The basic 5EGEH method has been modified to achieve several private goals. For example, a patent

US Mo 5707796 describes the use of ZE EX in combination with gel electrophoresis for the selection of nucleic acid molecules with defined structural properties, for example, bent DNA. US Patent No. 5,763,177 describes a 5EG EX-based method for selecting aptamers containing photoreactive groups that are capable of binding and/or crosslinking upon exposure to light and/or photoinactivating a target molecule. Patent

US Me 5580737 describes a method for identifying highly specific aptamers that are able to distinguish between closely related molecules, called Socipie-5EGEH. US patents Mo 5567588 and Me 5861254 describe methods based on ZE EX, which achieve highly efficient separation of oligonucleotides with high and low affinity to the target molecule. US patent Mo 5496938 describes methods of obtaining improved aptamers after carrying out the BE EX method. US Patent No. 5,705,337 describes methods of covalently linking a ligand to its target.

The feasibility of identifying aptamers to small peptides in solution was demonstrated in the patent

US Pat. No. 5,648,214. The ability to use ligand affinity elution to produce aptamers directed to a specific site on a target molecule is illustrated in US Pat. No. 5,780,228, which relates to the preparation of high-affinity aptamers that bind to specific lectins. Methods for producing aptamers for specific tissues including groups of cell types are described in US Pat. No. 6,127,119. Production of specific modified high-affinity ligands for calf intestinal alkaline phosphatase is described in US Pat. No. 6,673,553. US Pat. No. 6,716,580 describes an automated method for aptamer identification, including application of robotic manipulators.

In its simplest form, the ZE EX method can be determined using the following series of steps: 1) A mixture of candidate nucleic acids with a different sequence is obtained. A candidate mixture typically includes regions of fixed sequences (ie, each member of the candidate mixture contains the same sequence at the same position), and regions of randomized sequences. Regions of fixed sequences are chosen either: (a) to aid in the amplification steps described below, (b) to mimic a sequence known to bind to a target, or (c) to increase the concentration of a given nucleic acid construct in the mix of candidates. Randomized sequences can be completely randomized (ie, the probability of finding a base at any position is one in four), or only partially randomized (eg, the probability of finding a base at any position can be set anywhere from 0 to 100 percent). 2) The candidate mixture is brought into contact with the selected target under conditions favorable for binding of the target and candidate mixture members. Under these conditions, the interaction between the target and the nucleic acids of the candidate mixture can be considered as the formation of nucleic acid-target pairs between the target and those nucleic acids that have the strongest affinity to the target.

C) Nucleic acids with the highest affinity to the target are separated from nucleic acids with lower affinity to the target. Since there are only an extremely small number of sequences (and possibly only one nucleic acid molecule) in the candidate mixture that correspond to the nucleic acids with the highest affinity, it is usually desirable to set the separation criteria so that a significant number of nucleic acids in the candidate mixture (approximately 5-50905) are retained during separation. 4) Those nucleic acids selected during separation as having a relatively higher affinity to the target are further amplified to create a new candidate mixture enriched in nucleic acids having a relatively higher affinity to the target. 5) By repeating the above separation and amplification steps, the resulting mixture of candidates contains fewer and fewer weakly binding sequences, and the average affinity of the nucleic acids for the target tends to increase. At the most extreme, the ZEGEH 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 be rapidly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is detected. Certain chemical modifications of the aptamer can be made in order to increase the ip beyond the stability of the aptamer or to enhance or effect the delivery of the aptamer.

Aptamer modifications include, but are not limited to, those that provide other chemical groups that impart additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and flexibility to the aptamer bases or to the aptamer as a whole. Such modifications include, but are not limited to, 2-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, extracyclic amine modifications, 4-thiouridine substitution, 5-bromo- or 5-ioduracil substitution; skeletal modifications, phosphorothioate or alkyl phosphate modifications, methylation, unusual base-pairing combinations, such as iso-bases isocytidine and isoguanidine, etc. Modifications may also include 3'- and 5'-modifications, such as capping.

The ZEG EX method involves the identification of high-affinity aptamers containing modified nucleotides that add improved characteristics to the ligand, such as improved IP mimo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at ribose and/or base and/or phosphate positions. Identified by the method of 5EGEH aptamers containing modified nucleotides are described in US patent Me 5660985, which describes oligonucleotides that contain derivative compounds of nucleotides, chemically modified in the 5- and 2'-positions of pyrimidines. US patent Me 5580737 describes specific aptamers containing one or more nucleotides modified with 2'-amino (2'-MH»2), 2-fluoro (2-E) and/or 2'-O-methyl (2-

OMe). US patent Me 5756703 describes oligonucleotides containing various 2'-modified pyrimidines.

The 5EGEH method includes combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional groups, as described in US patents Mo 5637459 and Mo 5683867.

US Patent No. 5,637,459 describes highly specific aptamers containing one or more nucleotides modified with 2'-amino (2'-MH), 2'-fluoro (2-PE), and/or 2-O-methyl (2- OMe). The 5EGEX method additionally 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 ZEGE method, the modifications can be pre- or post-5EI EX modifications. Modifications of pre-ZEGEH can lead to aptamers with both specificity for the respective target and improved IP mimo stability. Post-ZEG EX modifications directed to 2-OH aptamers can lead to improved IP stability without adversely affecting the binding capacity of the aptamers. In one embodiment, aptamer modifications include 3'-3'-inverted phosphodiester bonds at the 3'-end of the molecule and 2'-fluoro (2'-PE) and/or 2'-amino (2'-MH"), and/or 2'-O-methyl (2-OMe) modification of some or all 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 macromolecular compound or polymer, such as polyethylene glycol (PEG). In these cases, the pharmacokinetic properties of the aptamer or modulator may be enhanced. In other embodiments, the aptamer or modulator can be encapsulated in a liposome. A lipophilic compound or a non-immunogenic high molecular weight compound can be covalently linked or linked through non-covalent interactions with the aptamer or modulator(s). In embodiments using a covalent compound, the lipophilic compound or non-immunogenic high molecular weight compound can be covalently linked to the aptamer or modulator at various positions, such as the 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 other group at the 5- or 3-terminus In one embodiment, the covalent attachment is to the 5'- or 3'-hydroxyl group. Connection of the oligonucleotide modulator to other components of the complex can be done directly or with the use of linkers or spacers.

Oligonucleotides according to the present invention can be modified by the base, sugar group or phosphate skeleton, for example, to improve the stability of the molecule, hybridization, etc. Oligonucleotide can contain other attached groups. For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a hybridization-activated crosslinking agent, a transport agent, a hybridization-activated cleavage agent, etc. The oligonucleotide can contain at least one modified group in the base selected from a group that includes, but is not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethylthiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-O-galactosylqueuosine, inosine, Mb-isopentyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, Mb-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2a-thiouracil, B-O-mannosylqueuosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-M-isopentenyladenine, uraciloxyacetic acid, vibutoxocin, pseudouracil, queuosine, 2-thiocytosine, 5-m ethylthiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uraciloxyacetic acid(m), 5-methylthiouracil, 3-(3-amino-3-M-carboxypropyl) and 2,6 -diaminopurine.

An 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 including, but not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, complex alkylphosphotriester, and formacetal or analogs thereof.

Any of the oligonucleotides of the present invention can be synthesized using standard methods known in the art, for example, using an automated DNA synthesizer (ie, which are commercially available, for example, from Viozeags, Arriei Viozuvietv).

Modulators

Modulators according to the present invention can be oligonucleotides, small molecules, peptides, oligosaccharides, for example, aminoglycosides, or other molecules that can bind to an aptamer or otherwise modulate the action of an aptamer, or a chimera or a fusion or a linked product of each of them.

In one embodiment, the modulator is an oligonucleotide complementary to at least part of the aptamer. In another embodiment, the modulator can be a ribozyme or a deoxyribozyme that targets the aptamer. In an additional embodiment, the modulator can be a peptide nucleic acid (PMA), a morpholino nucleic acid (MMA), a closed nucleic acid (IMA), or a pseudocyclic oligonucleotide (PSO) comprising a sequence that is complementary to or hybridizes to at least part of aptamer

An aptamer has an active tertiary structure that depends on the formation of the correct stable secondary structure. Therefore, while the mechanism of duplex formation between a complementary oligonucleotide modulator of the present invention and an aptamer is the same as between two short linear oligoribonucleotides, both the rules for predicting such interactions and the kinetics of formation of such a product depend on the intramolecular structure of the aptamer. The rate of nucleation is important for the formation of a final stable duplex, and the rate of this step is greatly increased by targeting the oligonucleotide modulator to single-stranded loops and/or single-stranded 3'- or 5'-tails present in the aptamer. Intermolecular duplex formation requires that the free energy of intermolecular duplex formation be favorable relative to the formation of existing intramolecular duplexes within the targeting aptamer.

Modulators can be designed to bind a specific 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 to the side of a more or less active form. For example, a modulator can be designed so that upon binding to a target aptamer, the three-dimensional structure of said aptamer changes such that the aptamer can no longer bind to its target 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 in such a way that the affinity of the aptamer for its target molecule increases. That is, the modulator can be designed so that upon binding, a structural motif is formed in the aptamer in such a way that the aptamer can bind to its target molecule.

In an alternative embodiment of the present invention, the modulator itself is an aptamer. In this embodiment, an aptamer that binds to the desired therapeutic target is first obtained. In the second step, a second aptamer is obtained, which binds to the first aptamer using the 5EGEX 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 can be PMA, MMA,

Both the MA or RSO and the modulator are aptamers. Alternatively, the aptamer that binds to the target is PMA, MMA,

IIMA or RSO, and the modulator is RMA. Alternatively, the aptamer that binds to the target is PMA, MMA, (MA or

RSO, and the modulator is MMA. Alternatively, the aptamer that binds to the target is PMA, MMA, MA or PSO, and the modulator is MA. Alternatively, the aptamer that binds to the target is PMA, MMA, MA, or PSO, and the modulator is PSO. Each can be used in either a naturally occurring stereochemistry or a non-naturally occurring stereochemistry if desired. For example, in a preferred embodiment, the aptamer is in the O configuration, and in an alternative embodiment, the aptamer is in the H configuration.

In one embodiment, the modulator of the present invention is an oligonucleotide containing a sequence complementary to at least part of the target sequence of the aptamer. For example, an oligonucleotide modulator can contain a sequence complementary to 6-25 nucleotides of the target aptamer, usually 8-20 nucleotides, more often 10-15 nucleotides. Preferably, 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. As a rule, an oligonucleotide modulator consists of 5-80 nucleotides in length, more often 10-30 and most often 15-20 nucleotides (for example, 15-17). Oligonucleotide can be obtained from nucleotides having O or G/. stereochemistry, or their mixture. Nucleic bases found in nature have the O configuration.

Different strategies can be used to determine the optimal site for binding an oligonucleotide to a target aptamer. An empirical strategy can be used, according to which complementary oligonucleotides "walk" along the aptamer. A run-through experiment may include two experiments performed sequentially.

A new candidate mixture can be obtained in which each member of the candidate mixture has a fixed nucleic acid region corresponding 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 are referred to as "extended" aptamers containing regions that can bind to more than one binding domain of the aptamer. According to this approach, 2-O-methyl oligonucleotides (eg, 2-O-methyl oligonucleotides) of about 15 nucleotides in length coordinated by about 5 aptamer nucleotides (eg, oligonucleotides complementary to nucleotides 1-15, 6-20, 11 -25, etc. aptamer). An empirical strategy can be particularly effective because the effect of aptamer tertiary structure on hybridization efficiency can be difficult to predict. To assess the ability of different oligonucleotides to hybridize with a given aptamer, especially to determine the molar excess of oligonucleotide required to achieve binding of the entire amount of aptamer, you can use the assays described in the examples that follow. The ability of different oligonucleotide modulators to increase the rate of aptamer dissociation or aptamer association with its target molecule can also be determined by performing standard kinetic studies, e.g., assays

VIASOKE. Oligonucleotide modulators can be selected in such a way that a 5- to 50-fold molar excess of the oligonucleotide is less or necessary to change the interaction between the aptamer and its molecule-

target as desired.

Alternatively, the aptamer target can be modified to contain a single-stranded 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). Aptamers with single-stranded ends 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 disrupt the active structure of the aptamer. A number of oligonucleotides (eg, 2'-O-methyl oligonucleotides) that can form, for example, 1, 3, or 5 base pairs in the single-stranded end sequence can be designed and tested for their ability to bind only to the aptamer containing the single-stranded end. , as well as their ability to increase the rate of aptamer dissociation or aptamer association with its target molecule. Controls with randomized sequences can be used to verify that effects are due to duplex formation and not non-specific effects.

Oligonucleotide modulators according to the present invention contain a sequence complementary to at least parts of the aptamer. However, absolute complementarity is optional. A sequence "complementary to at least part of the aptamer" referred to herein means a sequence having sufficient complementarity to be able to hybridize with said aptamer. The ability to hybridize can depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the more hybridized an oligonucleotide is, the more mispairs between aptamer-target bases it can contain and still form a stable duplex (or triplex as the case may be). A person skilled in the art can establish a tolerable degree of mispairing using standard procedures for determining the melting point of a hybrid complex. In particular aspects, an 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. Oligonucleotides according to 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 a deoxyribozyme. There are at least five classes of ribozymes, and each has a different type of specificity. For example, group I introns are approximately 300 to »1000 nucleotides in size, and they require O in the target sequence immediately 5-side from the cleavage site, and they bind 4-6 nucleotides 5-end from the cleavage site. Another class is represented

EMaseR RNA (M1 RNA), having a size of approximately 290 to 400 nucleotides. A third example is the hammerhead ribozyme, which is approximately 30 to 40 nucleotides in size. For them, it is necessary that there is an OH sequence on the 5' side of the cleavage site, and they bind to sequences of different numbers of nucleotides on both sides of the cleavage site. The fourth class consists of hairpin ribozymes, which are approximately 50 nucleotides in size. They require a SIS target sequence immediately 3'-end from 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 the ribozymes of the hepatitis delta virus (DHV), the size of which is approximately 60 nucleotides.

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, has been proposed. According to the "10-23" model, deoxyribozymes have a catalytic domain of 15 deoxyribonucleotides flanked by two substrate recognition domains, each consisting of seven to nine deoxyribonucleotides.

Nucleic bases of oligonucleotide modulators according to the present invention can be connected by means of bonds between nucleic bases, for example, peptidyl bonds (as in the case of peptide-nucleic acids (PMA); MielyIzep et al. (1991) 5Zsiepse 254, 1497 and US patent Me 5539082) and morpholine bonds (Oip et al., Apiizepze Mysiyis Asii Ogid Oem. 10, 11 (2000); Zittepop, Apiizepzee Mysiwis Asii Ogyd Oem. 7, 187 (1997); zZuttepgop et al., Apiizepse Mysieis Asius Ogyd Oem. 7, 63 (1997); Tauiog et al., U Vioi Spec. 271, 17445 (1996);

Rapgidde and others, Apiizepze Mysieis Asii Ogod Oemu. 6, 169 (1996)), or by any other natural or modified bond. Oligonucleic bases can also be closed nucleic acids (MA). MieiIzep et al., In Viotoi 5igisi JuOp 17, 175 (1999); Reyegzep et al., In My Kesodpiia 13, 44 (2000); Mivizep et al., Viosopitsd Speth 11, 228 (2000).

PMAs are compounds that are similar to oligonucleotides, but differ in composition. In PMA, 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.

PMA most often has an achiral polyamide skeleton consisting of M-(2-aminoethyl)glycine groups. To represent a complementary nucleic acid, purine or pyrimidine bases are linked to each subunit through a methylene carbonyl linker (1-3). PMA binds to complementary RNA or DNA in parallel or antiparallel orientation according to the Watson-Crick base pairing rules. The uncharged nature of PMA oligomers enhances the stability of hybrid PMA/DNA (RNA) duplexes compared to natural homoduplexes.

Morpholino-nucleic acids received this name because they consist of morpholin subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and thymine) connected to a β-hell morpholin ring. From eighteen to twenty-five subunits of the specified four types of subunits are connected in a certain order by neon phosphorodiamidate misubunit bonds to form a morpholine oligomer. The specified morpholine oligomers with their b--len morpholine groups that make up the backbone, connected by nonionic bonds, actually present superior antisense properties than RNA, DNA and their analogues having 5-membered ribose or deoxyribose skeletons, with connected by ionic bonds (see Ulym.depe-yu0iI5.sot/Mogrpo!-ipoz/rodu togrpoiipo5.NTMI).

IMAs are a class of DNA analogs that have several properties that make them prime candidates as modulators of the present invention. Monomers (MA are bicyclic compounds, structurally similar to RNA monomers. IMA from DNA and RNA share most chemical properties, namely, they are soluble in water, can be separated by gel electrophoresis, precipitated with ethyl alcohol, etc. ( Teigapeagop, 54, 3607-3630 (1998)).However, the introduction of I MA monomers into either DNA or RNA oligomers leads to high thermal stability of duplexes with complementary DNA or RNA, while obeying the rules of Watson-Crick base pairing. Such high thermal stability of duplexes, formed with I MA oligomers, together with the discovery that primers containing SMA at the 3'-end are substrates for enzymatic elongation, for example, the RSC reaction, are used in the present invention to significantly increase the specificity of detection of various nucleic acids in IP migo assays, described in this application.The methods of amplification of individual alleles are highly differential (cross-reactions are not shown), and in a single tube can flow and several reactions. See, for example, US Pat. No. 6,316,198.

Pseudocyclic oligomeric nucleic acid bases (CSOs) can also be used as a modulator in the present invention (see US Pat. No. 6,383,752). RSOs contain two oligonucleotide segments connected by their 3-3 or 5-5 ends. One of the RSO segments ("functional segment") has some functionality (for example, an antisense oligonucleotide, complementary to an mRNA target). Another segment (the "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 RSO. PSOs 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. Pharmacokinetics, tissue distribution and stability studies in mice suggest that

PSOs have a higher IP, mimo stability and pharmacokinetics and distribution in tissues, similar to that of sugar-phosphate oligonucleotides in general, but rapid elimination from a number of tissues. If the fluorophore and quencher molecules are correctly attached to the RCO of the present invention, the molecule will fluoresce if it is in the linear configuration, its fluorescence will be quenched in the cyclic conformation.

Peptide-based aptamer modulators represent an alternative class of oligonucleotide modulator molecules or their analogs. Modulators of this class have proven to be particularly useful when sufficiently active oligonucleotide modulators of the aptamer-target cannot be isolated due to a lack of sufficient single-stranded regions to allow nucleation between the target and the oligonucleotide modulator. In addition, peptide modulators provide bioavailability and pharmacokinetics that differ from oligonucleotide modulators.

Oligosaccharides, like aminoglycosides, can bind to nucleic acids and can be used to modulate the activity of aptamers. A synthetic drug that penetrates 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 drugs can be identified by candidate screening in an assay that measures changes in aptamer binding to the target in the presence and absence of the synthetic drug, or by using an ip mimo or ip mi assay that measures differences in the biological effect of the aptamer on target in the presence and absence of the synthetic drug. Once a synthetic drug that exhibits the desired effect is identified, 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 include gel retardation assays and VIASORE assays. That is, tested modulators can be brought into contact with aptamers for binding under test conditions or normal physiological conditions, and determine whether the tested modulator actually binds to the aptamer.

Tested modulators that are found to bind to the aptamer can be analyzed in appropriate biological activity assays (which will vary depending on the aptamer and its target molecule, e.g. folding assays) to determine whether the tested modulator can affect on the biological effect revealed by the aptamer relative to its target molecule.

The gel retardation assay is a technique used to assess binding capacity.

For example, a DNA fragment containing a test sequence is first incubated with a test protein or a mixture containing the expected proteins that bind, and then separated in a gel by electrophoresis. If a DNA fragment binds to a protein, it will be larger in size, and therefore its movement will be delayed compared to a free fragment. For example, one method for electrophoretic gel retardation analysis may be to (a) contact a mixture of nucleic acid-binding protein with a non-radioactive or radiolabeled nucleic acid molecule containing a molecular probe under conditions suitable to provide specific binding interactions between the protein and the probe with the formation of a complex in which the indicated probe is selected from the group consisting of dlDNA, olDNA and

RNA; (B) electrophoresis of the mixture; (c) transfer, using positive pressure blot transfer or capillary transfer, of the complex to a membrane, wherein the membrane is positively charged nylon; and (j) detection of the membrane-bound complex by detecting a non-radioactive or radioactive label in the complex.

Viasoge technology measures binding events on the surface of the sensor chip, thus the interacting substance attached to the surface determines the specificity of the analysis. Determining the specificity of the interaction involves simply analyzing whether different molecules can bind to the attached interacting substance.

Binding results in an immediate change in the surface plasmon resonance (SPR) signal, so that it is immediately obvious whether an interaction is occurring or not. Biosensors based on 5RK observe the interaction by measuring the mass concentrations of biomolecules located close to the surface.

Surfaces add specificity by attaching one of the interacting partners. A sample containing another partner(s) flows over the surface: if molecules from the sample bind to an interacting substance attached to the surface, the local concentration changes and the SAM response is measured.

The specified response is directly proportional to the mass of molecules bound to the surface.

ORE occurs when, under certain conditions, light is reflected from a conductive film on the surface of the separation of two media with different refractive indices. In Viasoge technology, the medium is the sample and the glass of the sensor chip, and the conductive film is a thin layer of gold on the surface of the chip. SAM causes a decrease in the intensity of reflected light at a certain reflection angle. The specified angle varies depending on the index of refraction near the surface on the opposite side from the reflected light. When molecules in the sample bind to the sensing surface, the concentration and thus the refractive index of the surface changes, and a 5REK response is detected. Plotting the response versus time during the interaction provides a quantitative measure of the progress of the interaction. Viasoge technology measures the minimum intensity of the reflected light angle. The light is not absorbed by the sample: instead, the light energy is scattered through the 5PE in the gold film. The values of the SAM response are expressed in resonance units (CO). One KO represents a change in the angle of minimum intensity by 0.00017. For most proteins, this is roughly equivalent to a concentration change of about 1 pg/mm? on the touch surface. The exact coefficient of conversion of RI into concentration at the surface 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 whether an oligonucleotide or its analog, peptide, polypeptide, oligosaccharide or synthetic drug is able to bind to an aptamer in such a way that the interaction with the target is modified. For example, electrophoretic mobility retention (EM5A) assays, calorimetric titration, scintillation convergence assays, sedimentation equilibrium assays using analytical ultracentrifugation (see, e.g., mulu.soge5.shap.edi/piegasiop), fluorescence polarization assays, fluorescence anisotropy assays, fluorescence intensity assays, fluorescence resonance energy transfer (GKET) assays, nitrocellulose filter binding assays,

ELISA, EGOMA (see, for example, US patent Mo 5789163), RIA assays or equilibrium dialysis assays can be used to assess the ability of the agent to bind to the aptamer. Direct assays can be performed in which the interaction between the agent and the aptamer is directly determined, or competition or displacement assays can be performed in which the ability of the agent to displace the aptamer from its target can be performed (eg, see Sgeep, Wei! and Dapiis, Viotesppidnez 30 (5), 2001, page 1094 and US Patent Mo 6306598). Once a candidate modulating agent is identified, its ability to modulate aptamer activity against its target can be confirmed in biological activity assays. 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(s) of interaction between the regulator and the aptamer, and the relative binding affinity of the agents to the aptamer (see, e.g., US Pat. No. 6,329,146 to SgookKe other). Such mass spectral methods can also be used to screen chemical mixtures or libraries, in particular, combinatorial libraries, for individual compounds that bind to a selected aptamer-target that can be used as aptamer modulators. In addition, mass spectroscopic methods can be used to simultaneously screen multiple aptamer targets 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 site of intermolecular interaction in the aptamer-target complex.

IP mimo or IP migo assays that assess the effectiveness of the regulator in terms of changing 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 biological activity assays are provided in the patents cited in this application that describe certain aptamers for specific applications.

The present invention also provides methods for identifying aptamer modulators. Modulators can be identified in general using binding assays, molecular modeling, or ip mimo or ip migo assays in which a change in biological function is measured. In one embodiment, the binding of the modulator to the nucleic acid is determined using a gel retardation assay.

In another embodiment, binding of the modulator to the aptamer is determined using the Viasogue assay.

In one embodiment, the modulator has the ability to actually bind to the aptamer in solution at 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 "properly speaking" is meant that at least a 50 percent reduction in the biological activity of the target is observed by modulation in the presence of the target, and the 5095 reduction is denoted here as the ISvo value.

Pharmaceutical compositions

Aptamers or modulators of the present invention may be formulated into pharmaceutical compositions that 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 the route of administration. Typically, the aptamer or modulator is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, orally, or topically, as needed.

Pharmaceutically useful compositions containing an aptamer or modulator of the present invention may be formulated according to known methods, such as by admixture with a pharmaceutically acceptable carrier. Examples of such media and assembly methods can be found in Ketipdiopye Rpaptasecshiisai zZsiepse5. To formulate a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of aptamer or modulator. Such compositions may contain mixtures of more than one compound.

In the methods of the present invention, the compounds may form the active ingredient, and are typically used in admixture with suitable pharmaceutical diluents, excipients or carriers (all materials referred to collectively herein as "carriers") selected appropriately with respect to the intended form of administration, i.e. oral tablets, capsules, elixirs, syrups, suppositories, gels, etc., and which are consistent with normal pharmaceutical practice.

For oral administration in the form of a tablet or capsule, the active ingredient 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, disintegrants 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, sugars from corn, natural and synthetic resins such as gum, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, wax and etc. Lubricants used in such dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar-agar, bentonite, xanthan gum, and the like.

In the case of liquid forms, the active ingredient of the drug may be combined with suitable flavored suspending or dispersing agents, such as synthetic and natural gums, for example tragacanth, gum, methylcellulose, etc. Other dispersants that may be used include glycerin and the like. Sterile suspensions and solutions are preferred for parenteral administration.

Isotonic compositions, which usually contain suitable preservatives, are used if intravenous administration is desired.

Topical compositions containing the active drug component 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, PPO2 myristyl propionate, etc., to obtain, for example, alcohol solutions, disinfectants for topical application, disinfectant creams, skin gels, skin lotions, and shampoos in gel or cream based compositions.

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

The compounds of the present invention can also be combined with soluble polymers as carriers of the drug to be administered. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethylene oxide polylysine substituted with palmitoyl residues. In addition, the compounds of the present invention may be linked (preferably via a covalent bond) to a polymer from the class of biodegradable polymers useful for achieving controlled drug release, e.g., polyethylene glycol (PEG), polylactic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphiphilic block copolymers of hydrogels. Cholesterol and similar molecules can be linked to aptamers to increase and prolong bioavailability.

The compounds can be applied directly (eg, alone or in a liposomal composition or in a complex with a carrier (eg, PEG)) (see, for example, US patent Mo 6147204, US patent Me 6011020). In one embodiment, various modulators can be linked to a single PEG molecule.

The modulator can be assigned to one or different aptamers. In those variants of implementation in which different modulators are applied to one aptamer, there is an increase in avidity due to multiple binding interactions with the aptamer. In an additional embodiment, several PEG molecules can be attached to each other. In this embodiment, one or more modulators to one aptamer or different aptamers can be linked to each PEG molecule. This also leads to an increase in the affinity of each modulator for its target.

Lipophilic compounds and non-immunogenic high molecular weight compounds with which the modulators of the present invention can be formulated for use in the present invention and can be prepared by any of a variety of methods now known in the art or subsequently developed. They are generally derived from a phospholipid such as distearoylphosphatidylcholine and may include other materials such as neutral lipids such as cholesterol and also surface modifiers such as positively charged ones (such as sterylamine or aminomannose or aminomannitol derivatives of cholesterol) or negatively charged (for example, diadetyl phosphate, phosphatidylglycerol) compounds. Multilayered liposomes can be assembled using conventional techniques, i.e. by depositing the selected lipid on the inner wall of a suitable container or vessel by dissolving the lipid in a suitable solvent, and then evaporating the solvent to form 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 intensive stirring motion, which leads to the formation of MIM. OM can be further obtained by homogenization, sonication or pressing (through filters) MI M. In addition, OM can be obtained using detergent removal techniques. In certain embodiments of the present invention, the complex includes a liposome with targeting aptamer(s) bound to 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 using electrostatic interactions. Alternatively, a nucleic acid attached to a lipophilic compound, such as cholesterol, can be added to preformed liposomes, whereby the cholesterol becomes bound to the liposomal membrane. Alternatively, the nucleic acid can bind to the liposome during liposome assembly.

Methods of introduction

Preferred routes of administration of the agents of this invention into the mammalian body are parenteral, intravenous, intradermal, intraarticular, intrasynovial, intrathecal, intraarterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, local, transdermal patch, by rectal, vaginal or urethral suppository, peritoneal, transdermal, nasal spray, surgical implant, by internal surgical application to tissue, by infusomate or through a catheter. In one embodiment, the agent and carrier are administered in a slow release formulation such as an implant, bolus, microparticle, microsphere, nanoparticle, or nanosphere. For standard information on pharmaceutical compositions, see Apzei, etc., Rpagppaseshisa!| dozade Rogt5 apa

Oga Oeiiimegu Zuvietv, 5ihii Edibop, M/Shiatv 8 UMiKip5 (1995).

Aptamers or modulators of the present invention can be administered parenterally by injection or by continuous infusion. Although the tissue to be treated can generally be accessed in the body by systemic administration, and therefore treatment is most often performed by intravenous administration of therapeutic compositions, other tissues and delivery techniques are presented where there is a possibility that the tissue -the target contains the target molecule.

Thus, the aptamers and modulators of the present invention are typically administered orally, topically to vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavityally, transdermally, and may be delivered by peristaltic methods. As noted above, pharmaceutical compositions can be administered to a patient by various routes, for example, orally, topically to vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavityally, transdermally, and can be delivered by peristaltic methods. Representative non-limiting approaches for local delivery to vascular tissue include (1) coating or permeating the blood vessel tissue with a gel containing a nucleic acid ligand for i.p. mimo delivery, e.g., by introducing a coated or perfused vessel in place of an injured or pathologically altered segment of vascular tissue, that was removed or bypassed; (2) delivery via a catheter to a vessel to which delivery is desired; (3) injection of the nucleic acid-ligand composition into the vessel to be implanted in the patient. Alternatively, the nucleic acid-ligand can be introduced into cells by microinjection or by liposome encapsulation. Preferably, the nucleic acid ligands of the present invention can be administered in a single daily dose, or the total daily dosage can be administered in several separate doses. Accordingly, the modulator is represented by any suitable means for altering the effect of the nucleic acid-ligand by introducing the modulator.

Therapeutic compositions containing polypeptide modulators according to the present invention are traditionally administered intravenously, for example, by means of a 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 to a subject, each unit containing a defined amount of active substance selected to achieve a desired therapeutic effect while being in the necessary diluent; i.e. media or environment.

The compositions are administered in a manner that is compatible with the medicinal composition and in a therapeutically effective amount as described herein. Suitable modes of administration are variable, but generally represent the first administration followed by repeated doses at intervals of one or more hours as a subsequent injection or by another route of administration. As an alternative, continuous intravenous infusion is considered, which is sufficient to maintain the blood concentration in the ranges defined for ip mmo treatment methods.

Within the scope of this description, the terms "pharmaceutically acceptable", "physiologically tolerable" and their grammatical variations, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials can be administered without significant or debilitating toxic side effects.

Pharmaceutically useful compositions containing the modulator of the present invention can be formulated according to known methods, such as by admixture with a pharmaceutically acceptable carrier. Examples of such media and methods of composition can be found in Ketipdiopye5 Rpagtasetschiisa! Zsiepse5. 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

Methods of measuring coagulation activity

Standard coagulation tests include plasma-based prothrombin time (PT) and activated partial thromboplastin time (APT) assays, both in plasma and whole blood, and whole blood-based activated clotting time (AEC) assays. While the activators used to trigger clotting in each of these assays are different, they all share a common characteristic - clot formation is the end point of the assay. It is important that in the indicated ip migo analyzes for the formation of a sufficient amount of fibrin necessary to reach the end point, sufficiently low levels of thrombin, "10-30 nm. The indicated thrombin level represents the conversion of only 3-595 prothrombin to thrombin, and is comparable to the amount of thrombin produced during the initiation phase of the coagulation reaction (Vshepavkh et al., 2003; Mapp et al., 2003). Thus, these analyzes report largely on the initiating phase of the coagulation reaction, and do not fully reflect the value of deficiency or inhibition of coagulation factors primarily involved in the propagation phase of coagulation.

The manner in which standard coagulation-based assays reflect CYH/IXa activity is illustrated by their ability to detect or fail to detect abnormal coagulation parameters in a patient with severe hemophilia A (EMII deficiency) or B (GICH deficiency). A sign of hemophilia is an isolated lengthening of AChT, since a patient with hemophilia has pathological AChTCh, but normal IF (Vokop-Madoz and Ravi,

2003). A cellular model of coagulation explains the paradox of why patients with EMI! or SIN determine normal RTs. The IF assay begins with supraphysiological levels of tissue factor sufficient to induce clot formation after 11 to 15 seconds. Therefore, high levels of the complex tissue factor-

The EMIIa used to initiate the reaction rapidly lead to the formation of EXa in amounts sufficient to generate enough thrombin to achieve clot formation as an end point, even in the absence of EMIIIA or RIC. Thus, even a significant inhibition of the activity of ХХ/ХХ would not affect the value of the PU assay, since the role of ХХ in initiating coagulation in the specified assay is masked or bypassed.

Thus, pharmacological inhibitors of RiXa, such as the anti-riXa aptamer KVO06, are not expected to increase PU values.

Both plasma or whole blood APTC assays are triggered by a charged participant such as celite or kaolin, a phospholipid surface, and calcium in amounts sufficient to form a clot after “28-35 seconds. In patients with hemophilia B (and A), pathological values of AChT are registered; however, the magnitude of the increase in aPTT in these patients is finite (i.e., of 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 its value

APT, because APT depends on other coagulation factors in addition to SIN. Therefore, the predominant criterion for interpretation is how pharmacological inhibition of RiKh will affect the analysis

АХТЧ, there is an analysis of SIN plasma. Plasma GRIH analysis is a variation of the standard APTT method in which, prior to APTT, the tested plasma is diluted in a buffer and mixed with PHI-deficient plasma in such a way that the level of GRIH in the tested plasma is the primary determinant of clot formation time. This test is usually used to determine the severity of hemophilia B (that is, to determine the levels of GHI) or to diagnose acquired GHI inhibitors. The results of the SIN analysis are interpreted by comparing the time of clot formation in the tested sample with a standard curve of the PX-level, obtained by serial dilution of normal plasma in a buffer before mixing with PX-deficient plasma. Table 1 shows a typical standard curve for the level of PIX obtained for normal human plasma. (Please note: The absolute times of APTC in the indicated analysis depend on the reagents). As can be seen from Table 1, at RIC levels that are 2595 from normal (i.e., a decrease of 7595), the times of AChT clot formation exceed the norm by 1.4 times. At RIC levels that are -395 from normal (i.e., a decrease of 9795), APTC clot formation times exceed 2 times the norm, and at RIC levels that are "195 from normal (i.e., a decrease of "99905), APTC clot formation times exceed 2.5 times the norm. In carriers of hemophilia B (ie levels

ХХ -5095 from the norm) determine the normal values of АХТХ (Voyop-Madoz and Ravi, 2003), which is consistent with the data of the standard curve of the ХХ level. Taken together, the obtained observations indicate that a significant percentage of the activity of РІХ must be inhibited before the AChT will be prolonged.

Table 1

Standard curve of the analysis of SIN activity in human plasma to the level Time of formation Coefficient

EX | sgortkaza AUTOU | clot formation time exceeded "10095 SIN level represents a 1:5 dilution of normal pooled human plasma in buffer

Because ACE assays are primarily used in operating rooms and catheterization laboratories to monitor anticoagulation during interventions, there is limited data on the effect of ACE reduction on RIC/RICH activity, as hemophiliacs are typically treated with factor replacement therapy (or similar therapy) before undergoing such procedures. However, since

CHAS is an assay whose end point is the formation of a blood clot triggered by charged particles, so the effect of pharmacological inhibition of RiX in the CHAS assay is likely a reflection of that observed in the AChT assay. Thus, it is expected that a prolongation of the CAZ will not be observed until a significant degree of RiX inhibition has been achieved (25095). Therefore, by analogy with the APTT assay, the magnitude of the prolongation of the ACE is likely to be modest compared to the prolongation observed with unfractionated heparin.

Finally, in response to inhibition of RiXa, the assay is likely to reach saturation. Such a similarity in the reactions of AChT and

CAZ was demonstrated in monkeys treated with various doses of KVO0O6 in nonclinical toxicity studies.

The effects of the KESI1 anti-saline system on salinity indicators

Preliminary data show that anti-biIXa aptamers do not prolong PF both ip and after intravenous administration to the animal (Kizsopi et al., 2004, My Biol. 22 (11):1423-8; Kizsopi et al., 2002,

Maishte 419 (6902):90-4; Buke, 2006, Sigsshayop.114 (23):2490-7). As shown in fig. 3, KVO0b reveals a dose-dependent increase in AChT in pooled normal human plasma and migo. The obtained data indicate that for KVO0b АХТЧУХ the dose-response curve is most sensitive from 0 to 30-50 μg/ml, and then reaches a plateau.

These features, including the rise phase and the plateau phase of the APTC dose-response curve, are consistent in plasmas from all species in which "VO0O06b or previous anti-PIH aptamers show cross-reactivity, including human, pig, mouse, and monkey (Kizsopi et al., 2004 , Mai Viotesppoi. 22 (11):1423-8). The maximum value of APTC achieved in response to treatment of plasma with an anti-RIIX aptamer depends on the APTC reagent and species used. It is important, however, that the indicated maximum AChT is consistent with complete or almost complete inhibition of RIX activity. This is supported by the fact that the maximum APTT value in response to the anti-PIX aptamer is equivalent to the APTT in human plasma containing PIK 195 of normal (but normal with respect to the levels of all other coagulation factors), and the APTT in the plasma of PIK-knockouts. mice Kizsopi et al., 2004, Mai Viotesppoi. 22 (11):1423-8). Thus, the plateau of AChT in response to

KVO06 probably reflects the saturation of inhibition of ХХ/ХХ by the aptamer.

In addition, a comparison of the data in Fig. With the standard curve of the SIN analysis of plasma in Table 1, an understanding of the activity of "B006. АХТЧУХ increases by -1.4-fold in response to EVO06 at a concentration of FVO06 of -5 μg/ml, indicating that this concentration of VO06 is sufficient to inhibit 7595 activity of plasma РХ. In addition, based on the analysis of РІХ of plasma, almost 9595 inhibition of ІРХ plasma (a 2.0-fold increase in АХТЧУЧ) is achieved at a concentration of КВООб from 10 to 15 μg/ml.

In order to assess the individual variability of the effect of the anticoagulant EVO0b, i.p. migo studies were carried out by measuring the concentration-dependent prolongation of AChT in patients' plasma. A comparison of the ip migo EVO06 APTC dose-response curve in pooled normal human plasma versus patient plasma is presented in Fig. 4.

As shown in fig. 4, the concentration-dependent increase in EB006 AChT is very similar for the plasma of each subject. In addition, the concentration-dependent increase in VO06 AChT in patient plasma is very similar to that in pooled normal human plasma (20 donors per pool). EVO06 also increases blood clotting time as measured in the CAZ assay (Kizsopi et al., 2004, May Viostesppoi. 22 (11):1423-8). However, interpretation changes

CHAS as a function of concentration "KVO006 is currently limited due to the difficulty of performing dose-response studies with CHAS, as this analysis requires fresh whole blood and is time sensitive.

The neutralization of the anti-inflammatory activity of KVO0b6 by the antidote KVO07 was measured using an assay

AChTU As shown in fig. 5, as the concentration of KV007 is increased relative to the fixed concentration of KVOO6 in the pooled human plasma, the change in the value of AChT returns to the initial levels, indicating the complete neutralization of the antiplatelet activity by means of KV007. The minimum molar excess of КВО07, which is necessary for complete neutralization of КВО06 and migo in human plasma, is approximately 3- to 4-fold (ie, the molar ratio of the antidote to the oligonucleotide part of the aptamer). This is consistent with the measured thermodynamic stability of the duplex KV006-АВОО?7 (Тпл.-907С).

The data presented in fig. 5, also serve as a basis for choosing the ratio of the dose of the antidote KVO07 to the dose of the medicinal product KVO06 used in non-clinical pharmacological safety studies and toxicity studies and clinical trials. The minimum molar excess of KVO07 relative to EVOO06, which is necessary to achieve complete neutralization of KVO0O6 and migo in human plasma, is 3- to 4-fold. Given the difference in molecular weight of KVO07 (5269 Da, sodium salt) and KVO006 (-50964 Da, sodium salt), this translates into an antidote:drug weight ratio of 0.5:1. Because this is a result of ip mimo and therefore does not predict how the pharmacokinetics of each component will affect the neutralization of the ip mimo drug, an antidote:drug weight ratio of 0.5:1 reflects the minimum ratio of antidote that would be expected to effectively neutralize drug. Therefore, as an initial dosage in non-clinical and clinical studies, a small multiple of the minimum ratio of the effective dose of ip miigo, a weight ratio of antdot:drug of 2:1, was chosen.

Summarizing the above, the anti-RIXa aptamer KVOO06 is a potent inhibitor of the coagulation factor RiXa, capable of complete or almost complete inhibition of the activity of RiXa and migo. The anti-erosive activity of EVO06, as well as the neutralization of aptamer activity with the help of KVO07, can be effectively monitored using the АХТЧУ and ЧАЗ assays. From ip migo studies, the relationship between the percentage of inhibition of РІХ and changes in

APTCs were well defined for KVO06. An appropriate molar ratio of antidote to aptamer sufficient to achieve complete inhibition of aptamer activity was also determined in i.p. migo studies, from which the 2:1 mg/kg antidote:aptamer dose ratio selected for the RECI1 anti-inflammatory system was derived.

Non-clinical pharmacology, drug distribution and toxicity

The pharmacological activity of the anti-inflammatory system РКЕС1И and its individual components, the drug and the antidote (or the less active prototypes of the drug and the antidote, named "ВО002 and КВО04, respectively) have been demonstrated in vitro and in clinically relevant animal models.

The anti-erosive activity of the anti-RIX aptamer was evaluated in systemic anticoagulation studies in pigs (Kizsopi et al., 2004, Mai Vioitesppoi. 22 (11):1423-8), in models of extrapulmonary circulation in sheep, and in a pharmacological safety study in cynomolgus monkeys. The antithrombotic activity of the anti-RIX aptamer was also demonstrated in a model of arterial injury in mice (Kizsopi et al., 2004, May

Vioyesnppoi. 22 (11):1423-8). The neutralizing activity of the antidote in relation to the medicinal product has been demonstrated i.p. in human plasma (Kizsopi et al., 2002, Maishge 419 (6902):90-4), in systemic anticoagulation models in pigs, in models of surgical trauma in mice (i.e., tail crossing in animals on the background of pronounced anticoagulation) (Kib5sopita et al. 2004, May Biosciences 22 (11)1423-8), in models of extrapulmonary circulation in sheep and in a pharmacological safety study in Javan macaques.

In addition, in studies of systemic anticoagulation in pigs, the ability of the drug to be re-administered shortly after the antidote of the previous dose of the drug has been demonstrated.

The characterization of the pharmacokinetics of the KES1 anti-inflammatory system required a bioanalytical strategy based on a new methodology for quantifying aptamer, antidote, and aptamer/antidote complex levels in plasma samples. These methods were applied to samples collected after IP mmo toxicity studies, which allowed determination of the pharmacokinetics of all three molecular components under conditions of single and repeated dosing in monkeys and mice.

A comprehensive safety study of the KES1 anti-seize system was conducted. Primary toxicity studies were performed in monkeys and mice under dosing conditions simulating the intended use of the agent in the initial clinical trials (ie, sequential administration of the aptamer followed by an antidote three hours later). 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 monkey studies, multiple treatment groups were formed to receive single doses of aptamer, antidote, or both test sites in a schedule that mimicked the intended administration in the original clinical trials. In addition, the 14-day mouse study and the single- and multiple-dose toxicity studies in monkeys included two-week repeated-dose groups (14 daily doses for mice and 7 doses administered every other day for two weeks for monkeys ). Specialized endpoints were included in toxicity studies to assess pharmacodynamic responses, exposure to KES1 components, and effects of oligonucleotides as a class of compounds.

Basic toxicity studies were supplemented by a pharmacologic safety assessment in monkeys (using radiotelemetry), a list of genetic toxicity tests, and a blood compatibility study.

Study of anti-inflammatory and neutralizing activity of the drug in pigs

The ability to re-introduce the KVO0Ob aptamer after neutralization of the initial dose of the aptamer with the KVO07 antidote was evaluated in a model of systemic anticoagulation in pigs. In these studies, the second dose of the drug was administered 30 minutes after the administration of the antidote. A 30-minute window between the administration of the antidote and the re-administration of the aptamer was chosen in order to obtain a clear experimental demonstration of neutralization of the antiplatelet 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 actually the same for the initial dose of the aptamer, which indicates that re-introduction of the aptamer after neutralization of the first dose of the aptamer with an antidote is feasible. The obtained data are consistent with the observed pharmacokinetics of KVO07 in both mice and monkeys, indicating that KVO07 has a very short plasma half-life (ie, several minutes) and does not accumulate in plasma to detectable concentrations even at significantly higher doses than those used in this study. Given the half-life of the antidote, it is likely that the aptamer can be effectively re-administered at a shorter time interval than minutes after the administration of the antidote.

Effectiveness of KES1 antithrombotic system in coronary artery bypass grafting (CABG) with extrapulmonary circulation in sheep

KESI can be used as a reversible anticoagulant antidote for coronary revascularization interventions (coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI)) as a reversible anticoagulant antidote for use in patients, including people, with acute coronary syndromes. and as an anticoagulant in other indications where it would be beneficial to use a reversible antidote for antithrombotic or anticoagulant therapy. The studies described in this application are intended to determine the range of doses of the antiplatelet component of KES1, KVOO06, necessary to maintain an extrapulmonary shunt (EPS) in an open state in an animal undergoing surgery

CABG with SRV, and determination of the appropriate dose of the antidote component of KESI, КВО07, necessary for neutralization of КВОО6 in this model.

KVO06 (an anti-asthenic agent) was administered intravenously to 10 sheep at the beginning of an aorto-coronary bypass surgery. At the end of the operation, КВО07 (an agent that neutralizes ЕВО06) was administered intravenously to completely neutralize the effects of КВОО6. After 28:23 days, all animals were euthanized.

Representative samples of the right and left kidney, liver, lung, and whole brain were collected. Hearts were washed with a jet of Ringer's solution with lactate or physiological saline until blood was removed, and fixed by perfusion under pressure at -100 mm Hg. 1095 neutral buffered formalin (NBU) for at least 6 hours. After completion of fixation, hearts were placed in 1095 MVE.

Representative tissue samples collected during autopsy were fixed by immersion at 1095 MVE.

Hearts were transversally dissected approximately every 1 cm (like a loaf of bread) and examined for pathological changes. Ten sections of each heart were collected and embedded in paraffin. Three of the ten sections included: anastomosis of the ІС, aortic anastomosis, and the middle of the graft. The other seven sections included: right atrial wall, left atrial wall, atrial septum, right ventricular wall proper, left ventricular wall proper, ventricular septum, and apex. All paraffin blocks containing myocardial tissue were cut into two parts, one for staining with hematoxylin and eosin (HEE) and the other for staining with Mazzop'5 Methotrexate (MME). Kidney, liver, lung, and brain samples were embedded in paraffin and cut as follows: one section of each kidney, one section of liver, one section of lung, and one section of each of the four brain tissue samples, for a total of eight sections. All obtained micropreparations were stained

NaIE.

Correlations between macroscopic and histological observations in this study indicate that most of the pathological changes are related to either the surgical procedure (eg, adhesions) or euthanasia (eg, foam in the trachea and bronchi). Adhesions are a common side effect of this type of procedure and were not considered excessive in this study.

In one animal, a small, minimally attached thrombus was found in the aortic anastomosis.

The thrombus did not appear to impede blood flow in the graft. There were no characteristic microscopic signs indicating this. Microscopy data at the anastomotic site were similar in type and amplitude to those of other experimental animals in both groups. With one exception, there was no macroscopic sign or thrombosis of occlusion within any part of the coronary artery shunt in any experimental animal. Incidental thrombus formation is very common in this model; therefore, the connection with the introduction of EVO0b is considered doubtful.

Pharmacodynamic activity of the KESI1 anticonvulsant system in Javan macaques

The antiplatelet activity of KVO0Ob in the plasma of Javan macaques was reflected as a concentration-dependent prolongation of clot formation time in the AChT analysis. As can be seen in fig. 7, "B006 APTC 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. The dose-response curves for monkeys and humans are similar except that the response range is greater in humans. In human plasma, there is a concentration-dependent increase in AChT up to about 200 μg/ml, whereas in monkey plasma, the concentration-response curve reaches a plateau at about 50 μg/ml. The plateau of the curve for human plasma is reached at a value of АХТЧУХ, equivalent to that determined for human plasma containing 190 of the activity of РХХ in plasma, and probably occurs due to saturation of the target, РХХ. Plasma SIN analyzes were performed to facilitate the interpretation of KV006 АХТЧУ dose-response curve for monkey plasma. As shown in Table 2, AChT in monkey plasma is sensitive to the level of RIC. However, the magnitude of the response to the decrease in the level of РІХ is small. 75905-not a decrease in the level of RICH leads to a 1.4-fold increase in AChT, and x»9595-not a decrease in the level

RICH leads to a doubling of AChT, and a reduction of 99.995 of CGH in plasma leads to a 2.5-fold increase in AChT.

Table 2

Standard curve of the analysis of SIN activity in the plasma of Javan macaques 96 level of AChT time Coefficient

10095 PIX clotting time increase 10095 PIX level represents a 1:5 dilution of normal pooled cynomolgus macaque plasma in buffer. PIX-deficient human plasma (Seogde Kipd Vioteaisai) was used as a source of PIX-deficient plasma.

Comparing the data in fig. 7 with the data presented in Table 2 indicates that "6 μg/ml of KVO006 is required to inhibit approximately 9095 of plasma GRIH activity in monkeys (i.e., a 1.6-fold increase in AChT occurs at this concentration) and that 29595 inhibition of plasma GRIH activity occurs at concentrations

ABOO6 10-12 μg/ml. Ip migo EB006 APTT dose-response curve of monkey plasma plateaus at approximately 2.5-fold increase from baseline (initial value “24 seconds, peak APTT value -60 seconds), which is consistent with the amplitude of the APTT increase observed in the assay Plasma SIN at a SIN level of "0.195 of the normal SIN level (see Table 2). Therefore, the plateau for КВО0б6 АХТЧ of the dose-response curve probably represents the saturation of the target in the plasma of the monkey (ie, complete inhibition of the activity of ХХ). In conclusion, the 9o inhibition of CYC versus CVO06 concentration in the plasma of monkey ip migo is generally similar to that observed in human plasma ip migo, with the main differences being that the range of concentrations of CVO06 from initial to maximal AChT is wider in humans, and the dose-response curve is more flattened for human plasma than for monkey plasma.

IP mimo activity of KVO06 and KVO007 in Javan macaques

The relationship between the anti-inflammatory properties of KVO0b and the KVO06/KVO07 complex and the levels of these compounds in plasma was evaluated in a pharmacological study of the safety of KES1-TOHO0O1 in monkeys. Briefly, 12 monkeys were divided into three treatment groups. Group 1 received the anti-BIIXa aptamer KVO0Ob, group 2 received the antidote KVO06, KVO07, and group C received the anti-inflammatory system RESI1, i.e. KVO06, after which - KVO07 (after three hours). Doses were doubled, with the first dose administered on study day 4 and the second dose administered on day 13. To better understand the dose-response relationship for KVO0b, four monkeys enrolled in group 1 (KVO06, aptamer only) were divided into two groups on day 13, where two animals received a low dose (group Ta, 5 mg/kg KWOOb) and two animals received a high dose (group 16, 30 mg/kg KWOO0b).

As shown in fig. 8, introduction of KVOOb in doses ranging from 5 to 30 mg/kg led to pronounced anticoagulation in monkeys. The mean value of АХТХХХ for each dose level exceeded 60 seconds for 0.25 to 24 hours after the administration of КВООб, which is equivalent to a level of SIN plasma in monkeys "0.190 of normal. In response to the introduction of KVO06, there is a dose-dependent increase in AChT.

However, the dose-response relationship is not immediately obvious due to the fact that up to the b-hour period after the introduction of KVO0b, the level of KVO0Ob in the plasma exceeds the concentration at which the dose-response curve reaches a plateau (40-50 μg/ml; see . table C and Fig. 7). At time points that are 6 hours after the introduction of KVO0b, when the concentration of KVOOb decreases below the specified level, the dose-response relationship is more obvious. AChT in monkeys receiving doses of "VO06 5 and 15 mg/kg" was determined until it returned to the initial value. The mean APTC value returned to baseline 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 both the i.p.migo APTC dose-response curve (Fig. 7) and the half-life , which is observed, which is approximately 12 hours for KVO0Ob in monkeys (see Table 3). Whole blood activated coagulation time (ACT) data mirrored that of AChT (data not shown).

Toxicokinetics data were collected at several time points during the first 24 hours after administration

KVO06b, using EGISA analysis with hybridization of double oligomers. As shown in Table 3, the concentration of KVO0Ob increased as a function of the administered dose, and the half-life of KVO0b was in the 12-hour range. Consistent with the data presented in fig. 8, a comparison of the levels of KVOOb in plasma (Table 3) and migo with the dose-response curve presented in Fig. 7, indicates that the animals were in a state of deep anticoagulation during the first 24 hours after administration of KVO0b at all dose levels. The indicated dose levels significantly exceed the proposed clinical range. There is an excellent correspondence between the average value of the concentration of KVO06 24 hours after administration in animals in group 1a and the average value of AChT in these animals. The mean concentration of KVOb in animals receiving 5 mg/kg KVO006b after 24 hours was 15.9 μg/ml, and the mean APTC value was 61.1 seconds. This is in very good agreement with the expected result based on the ip migo response-dose-response curve of KVOO06 in monkeys (see Fig. 7). Therefore, this study confirms the practical value of APTQ for monitoring the level of anticoagulation in monkeys receiving KVOOb, and the obtained data support the use of APTQ to monitor anticoagulation status in humans receiving KVOO06 in initial clinical studies.

Table C

Levels of KVO06 in plasma in animals of group 1, KES1-TOKHOO1 (μg/ml)

Time after animal/dose level go n-2 n-2

Before 3 | 666 | 14562325 | 328.9 7.6 | Yagi | 01534134 | 2753 "On the 13th day of the experiment, the animals were divided into groups 1a (5 mg/kg) and 15 (30 mg/kg). For the indicated dose levels, the average level in the plasma of two animals per dose level is presented.

KVOO06 in animals of groups 1 and 16 at the time before administration is residual from the dose of 15 mg/kg on day 4. The GO of this analysis is "0.04 μg/ml.

In animals of group 2, which were administered only the antidote KVO07, the average values of АХТЧУХ and ЧАЗ did not change in response to the administration of КВОО07 at all tested dose levels (30 and 60 mg/kg). Toxicokinetics data were recorded at different time points during the first 24 hours after the introduction of KVO07, using EGISA analysis with double oligomer hybridization. As shown in Table 4, low but detectable levels of antidote were present in the plasma of animals receiving KVO07 0.25 hours after injection of 30 mg/kg on day 4 or 60 mg/kg on day 13. The levels obtained were very variable, but 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 when only the antidote is administered, it has a very short half-life in plasma, and is largely eliminated from the circulation 15 minutes after injection.

Table 4

Levels of KVOO07 in plasma in animals of group 2, KES1-TOHOO1 (μg/ml)

777.76 | wow | "002 "1 animal at "(OO 0.01 was included in the calculation of I 3 animals at "OO 0.01 were included in the calculation of poor average! 00

The data of AChTCH in animals that were injected with KVOOb three hours after which KVOO7 was administered (group 3) are shown in fig. 9. In agreement with the data on animals that were injected with KVO0b alone, the administration of KVOOb at the indicated dose levels leads to a state of pronounced anticoagulation, with an average value of AChT of 0.25 and 3 hours after administration, which is consistent with, as a matter of fact, complete inhibition of RICH at both levels dose Next input

KVO07 rapidly and completely neutralized the anti-obesity effects of KVO06b in monkeys, with the mean APTC returning to baseline within 15 min of KVO07 administration (first time point) at both KVO06/KVO07 dose levels tested. In animal groups 3, which were injected with 30/60 mg/kg, KV006/28007,

AHTCHU was determined within 5 days after the introduction of KVOO06. AChTU data collected over this time period indicate that the anticoagulation effects of KVO0b were neutralized for a long time without signs of resumption of anticoagulation for more than 120 hours, or approximately 10 half-lives of EVO0b in the monkey (Fig. 9).

The duration of neutralization of the anticoagulant activity of КВООб by the antidote КВО07 is fully consistent with the thermodynamic stability of this observed complex, the drug-antidote.

Toxicokinetics data were collected within 24 hours after the administration of KVO06 in animals of group C (table 5). For animals of group C, plasma concentrations of both free EVO0006 (ie EVO06, not bound to КВОО7) and complex with "8006 (ie КВОО06 bound to КВО007) were measured. In agreement with the data of АХТЧУЧ, presented in fig. 9, the average values of "BO06 concentration in plasma at 0.25 and 3 hours after administration were very high. Within minutes after the introduction of KVO07, the average concentration of free KVO0b decreased 5,000-10,000 times to levels below the lower limit of measurement (LOM) of the assay used. Accompanying the decrease in the levels of free EVO0b6, the average plasma concentration of KVO0b in the complex increased from less than 00 analysis to from "125 to 220 μg/ml at the dose levels of 15/30 and 30/60 mg/kg, respectively, indicating that the rapid the decrease in the concentration of free КВОО06 was due to binding of КВО07 with КВО06. Free concentration

KVO06 remained below the GO analysis for at least 3 hours after the introduction of KVO07, which is consistent with the results of AChTU. At 21 hours after administration of KWOO7 (24 hours after administration of KWOOb), very low levels of KWOO6 were determined in several animals (mean value only 0.17 μg/ml or less). However, the levels of VO06 obtained were too low to show a measurable antiplatelet effect, consistent with the lack of APPT prolongation at 24 hours or longer in animals administered the KESI1 antiplatelet system.

Table 5

Group Z KES1-TOHOOT: plasma levels of free and complex KVOOb (mg/ml)

Time from the moment of injection of the complex of the complex about Pre-introduction | "004... | nv | 0053001 | (nv ( 77771.7025 | 28024643 | nv. | 4676367. | - nv 77717176... | «004 | 9875205 | «004 | 18483289 Sh ( "1 animal at GO "0.04 μg/ml was included in the calculation "3 animals at GO "0.04 μg/ml were included in the calculation

KVO07 was administered at 1-3 hours immediately after the 3-hour blood sampling. (n.v.) not defined.

The essence of non-clinical pharmacological studies on monkeys

The presented studies demonstrate that KVO0Ob is a powerful anticonvulsant agent in monkeys, which is able to achieve virtually complete inhibition of the activity of SIN for 24 hours or longer after a single intravenous injection of the drug in doses exceeding clinical doses. A comparison of ip migo studies of the antispasmodic activity of KWOOb in monkeys with the data of APTC and toxicokinetics as a result of a pharmacological safety study demonstrates a good correspondence between the expected and prolonged APTC observed, depending on the concentration of ЕVO0Ob in plasma. Therefore, APTT analysis will serve as a useful tool for monitoring anticoagulation caused by KVO06 administration. Similarities between ip migo EVO06-

APTC dose-response curves for humans and monkeys suggest that the data obtained in this study in monkeys (KES1-TOHOOT1), as well as the large general toxicity study conducted in monkeys (AES 1-TOHO03), will serve as useful guidance in predicting a person's response to the introduction of KVOO06.

Finally, АХТЧУ and the toxicokinetics data of КЕС1-ТОХО001 demonstrate that КВО07 is a very effective antidote

KVO006. Within 15 minutes after intravenous bolus administration of KVO07 to the animal to which it was administered

КВО06, the average values of AChT returned to the levels before the introduction of КВООb and remained at this initial level during the entire monitoring period (up to 120 hours). The observed neutralization of the anti-inflammatory activity of КВОО6 by means of КВО07 was fully confirmed by toxicokinetic data and is consistent with the measured thermodynamic stability of the КВО006-К8007 complex. Toxicokinetics studies demonstrated that the levels of free КВО0Об decreased to values below the GH ОО analysis within 15 minutes after the introduction of КВО07, accompanying a significant increase in the concentration of КВООb in the complex, and without a significant increase in the levels of free "ВО0О06 during the toxicokinetic analysis (24 hours after administration

VVOO06). Therefore, data obtained in monkey studies demonstrated that the KES1 anticoagulation system performs as predicted in achieving stable, long-lasting, and monitorable anticoagulation after a single intravenous injection of aptamer, followed by rapid, complete, and long-lasting neutralization of aptamer activity. with intravenous bolus injection of the antidote. These characteristics of the KES1 anticoagulation system were achieved at low to high multiples of the intended clinical dose range (ie, doses suitable for toxicity studies), but without adverse effects in animals.

Toxicokinetics of KES1

Bioanalytical methods were developed and approved for the quantitative analysis of concentrations of free aptamer (KVOO06b), free antidote (KVOO7) and aptamer/antidote complex (Ж80О06/К8007) in the plasma of monkeys and mice. The specified methods were applied to the analysis of samples obtained during a pharmacological safety study in monkeys (study Me KES1-TOX01), a 14-day study in mice (study Mo KES1-TOX002) and a study of single/multiple administration in monkeys (study

Mo KES1-TOHO03). For all three studies, separate groups of animals were formed, which were either given only the aptamer, or only the antidote, or the aptamer, which was followed by the antidote three hours later. Multiple dose levels for each administration condition were tested in all studies, and two of these studies (a 14-day study in mice and a single/multiple-dose study in monkeys) also included repeated administration of the test agents. Aptamer dose levels 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. The doses of the tested antidote were two times higher than those for the aptamer (ie, up to 90 mg/kg in monkeys and 45 mg/kg in mice). This ratio is similar to that expected to be used in clinical trials.

For all three studies, the toxicokinetics results were similar in describing the following properties of the KES1 anti-inflammatory system: - Aptamer plasma concentrations after intravenous injection were dose-proportional over a wide dose range, with a modest degree of inter-animal variation. No gender differences were evident in either monkeys or mice. - Aptamer clearance from plasma was relatively slow (ie, the estimated half-life was at least 12 hours in monkeys and 8 hours in mice). Such slow clearance was expected based on the structure of the PEGylated aptamer and is consistent with literature reports on the pharmacokinetics of other PEGylated oligonucleotides. The minimal clearance of the aptamer, in combination with its high degree of inhibition of factor IX, is presented for a relatively stable degree of anticoagulation during 6 hours, based on the measurement of pharmacodynamic markers, ie activated partial thromboplastin time and active clotting time. This profile is a desirable property of the aptamer component of the KESI1 anti-adhesion system. - Intravenous injection of only the antidote (without prior administration of the aptamer) resulted in very low plasma levels, even at the first time of sampling after the injection (10-15 minutes). Antidote levels measured at the indicated early time points were orders of magnitude lower than those for aptamer (ie, compared to aptamer levels in aptamer alone groups), even though antidote dose levels were twice as high. Taken together, the antidote data indicate that it has a very short plasma half-life when administered alone. Application at 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 plasma. - For groups that received aptamer and three hours later received antidote (i.e., the complete KES1 antiplatelet system), the concentration of free aptamer decreased dramatically within minutes of antidote administration to a level below or slightly above the limits of quantification (using a highly sensitive assay based on hybridization), which indicates complete binding of the circulating aptamer by the antidote. As noted when treated with only one antidote, the levels of free antidote were very low under these conditions. Antidote aptamer binding was associated with virtually complete neutralization of aptamer activity (i.e., normalization of coagulation parameters), which is consistent with the expected action of the KESI1 anticoagulant system. - In parallel with the elimination of free aptamer, the aptamer/antidote complex was detected in plasma at levels comparable to the complete binding of the aptamer by the antidote. The complex was eliminated from plasma at a rate slightly faster than that of the free aptamer (i.e., comparable to the aptamer clearance rate in the aptamer-only groups), but at a much lower rate than the free antidote, as expected due to the presence of the polyethylene glycol group in composition of the complex (derived from the aptamer). Large elimination of the aptamer/antidote complex from plasma was observed within 21 hours after administration of the antidote. When the aptamer and antidote (KES1 coagulation system) were repeatedly administered to monkeys daily for two weeks, there was no accumulation of the complex or free aptamer in the blood, no change in the pharmacokinetics of the aptamer (i.e., during the time period before antidote administration), and no evidence of cumulative anticoagulation by the aptamer . - The only difference between the pharmacokinetics in mice and monkeys was the slightly longer half-life of the aptamer in monkeys (at least 12 hours, compared to 8 hours in mice).

Clinical application of KESI1 in humans

When choosing a method of anticoagulation for use in an individual patient or patient population, clinicians take into account the characteristics of different pharmacological strategies. Given that the major adverse effect of anticoagulation is bleeding (i.e., abnormally increased pharmacological activity), for intensive care indications the ideal anticoagulant would be 1) delivered intravenously or subcutaneously, 2) immediately therapeutic, 3) easily dosed so as not to require frequent control, and most importantly, 4) immediately and predictably reversible. The KES1 anti-laxative system was developed in response to an unmet medical need for an effective, safe and rapidly reversible anti-laxative agent.

KES1 can be used in many clinical situations to treat humans and other animals in need of such treatment. For example, KESI can be used in coronary and peripheral vascular revascularization procedures associated with arterial disease and occlusions as a neutralized antidote to an anticoagulant. In particular, KES1 can be used as an antidote-neutralized anticoagulant in coronary revascularization procedures (coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI)), as an antidote-neutralized anticoagulant for use in patients with acute coronary syndromes, and as anticoagulant for other indications in which it would be advantageous to use an antidote-neutralized agent for antithrombotic or anticoagulant therapy.

Diseases and procedures for which the methods of the present invention may be used include, but are not limited to, peripheral vascular transplant procedures, including those related to iliac, carotid, brachial, aortic, renal, mesenteric, femoral, popliteal, tibial, and peritoneal vessels; prevention of deep vein thrombosis; prevention of pulmonary embolism after orthopedic surgery or in cancer patients; prevention of atrial fibrillation; prevention of thrombotic stroke; and for indications requiring extracorporeal blood circulation, 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 develop blood clots in the heart or with peripheral embolization; and patients with other hypercoagulable conditions.

The methods of the present invention may also be useful for the prevention of OMT and pulmonary embolism in immobilized patients and for maintaining the function of indwelling intravenous catheters and systems for intra-arterial or intravenous administration.

The range of doses of the anticoagulation component of KES1, EVO0b, will depend on the indication. For example, dose

KVO06b in humans can range from about 0.1 mg/kg to about 10 mg/kg. For specified indications, dosage ranges 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, from 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. With the specified indications, the medicinal component will be used in the dose necessary to maintain the opened state during the procedure. With certain indications, only EVO0b will be administered, without the subsequent administration of a neutralizing antidote.

The appropriate dose of the antidote component of KESI, KVO07, required for neutralization or partial neutralization of KVO0b, depends on the amount of KVO0Ob injected. The antidote dose may vary in the antidote:drug mass ratio (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 from about 7.5:1, from about 2.5:1 to about 6:1, from about 3:1 to about 5.

The most important property of the RECI1 antidote system, which contributes to the development of confidence in its safe clinical use, is the well-established ability of the antidote to neutralize the pharmacological activity of the aptamer in a dose-dependent manner.

Evaluation of the KES1 anti-inflammatory system in humans

This study was the first to evaluate the KES1 anti-inflammatory system in humans. Studies of single intravenous increasing doses (IM) of the KESII anti-inflammatory system were conducted on healthy volunteers. Subjects in this study were randomized to receive either test agent or placebo in one of three regimens at one of four (4) different dose levels. In each regimen at each dose level, subjects were randomized 7:11, treatment versus placebo, to receive KES1 or placebo. For all placebo injections, injections of 0.995 sodium chloride OBR were used. Subjects were randomized to receive RECI1 or placebo at each dose level.

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

Scheme 1: the active antidote component KVOO07 emerged after the placebo drug, or the placebo antidote emerged after the placebo drug.

Scheme 2: after the active medicinal product KV00b, the active component of KV07 emerged, or after the placebo of the medicinal product, the placebo of the antidote emerged.

Scheme 3: after the active drug EVO0b followed the placebo of the antidote, or after the placebo of the drug followed the placebo of the antidote.

In Scheme 1, the antidote component of the KES1 anti-sedation system (VO007) was evaluated. Each subject according to the specified scheme received a placebo injection at time 0 (ie, the time when the first bolus injection was given). Three (3) hours later, subjects received an intravenous injection of the active antidote component (EVO07), while one (1) subject received a placebo.

Scheme 2 evaluated the combination of the active medicinal component of the anti-inflammatory system KES1 (KVOOb), followed by the active antidote component of the anti-inflammatory system РЕС1 (8007). Subjects in this regimen received an injection of the active drug component (KVOO06) at time 0, and one (1) received a placebo. Three (3) hours later, subjects receiving the active drug component received an injection of the active antidote component (KVO07), while one (1) subject receiving a placebo instead of the drug component received a placebo instead of the antidote .

In scheme C, the active medicinal component of the KES1 anti-inflammatory system (КВО006) was evaluated. Subjects in this regimen received an injection of the active drug (EVO06) at time 0, and one (1) received a placebo instead of the antidote. Three (3) hours later, all subjects received a placebo instead of the antidote.

The active medicinal component under investigation (ACD) was administered in four (4) dose levels: (1) low dose (15 mg of ACD); (2) intermediate low dose (30 mg of KVO0b); (3) intermediate high dose (60 mg КВООб); and (4) high dose (90 mg EVO06). The starting dose and subsequent escalating doses were chosen to achieve peak plasma concentrations that determine three (3) key aspects of the ip migo AChT dose-response curve for CVOBb in pooled normal human plasma: a low dose to achieve peak plasma concentrations, at at which AChT begins to increase in KVOO0b and migo of the dose-response curve (4 μg/ml); two (2) intermediate doses to achieve concentrations in plasma that limit ISvo in ip migo КВ0О06 АХТЧУ dose-response curve (8-16 μg/ml); and a high dose to achieve the plasma concentration at which the dose-response curve begins to reach a plateau (25 μg/ml).

The study active antidote component (KVO07) was administered at four (4) respective dose levels equivalent to twice the drug dose level (ЕВО0О06) on a mg/kg basis: (1) low dose (30 mg

KVOO7); (2) intermediate low dose (60 mg KVOO7); (3) intermediate high dose (120 mg KVOO7); and (4) high dose (180 mg KWOO07).

Table 6 summarizes the doses for each group in Phase 1A of this study.

The investigational drug component (EVO06), the investigational antidote component (KVO007) and their corresponding placebo were injected over one (1) minute. The study drug component KESI1 or placebo was administered at time 0 and the antidote component or placebo was administered at three (3) hours.

Table 6

Planned doses for the three phase 1a administration regimens

Scheme 1: placebo « (Scheme 2: drug (EVOOb), Scheme 3: medicinal

Antidote group (KVOO07), mg mg same antidote (KVOO7), mg agent (KVOOV), mg ' ' placebo

Dose Level 2: Intermediate Venada 10000061

Dose level 3: intermediate defect 00016 (Dose level 4: high dose | 180 ...-:-/ | 90 2 2 shЮ(| 180 | .-:.Г 90 2 D /

EESI1 was evaluated in healthy volunteers to determine the safety profile and describe the RK and RO response of the anti-inflammatory system of KES1. This study was the first in which an antiplatelet system using an aptamer and an oligonucleotide antidote to the aptamer was used in humans. The results indicate that a dose-response response was detected after the intravenous bolus injection of the antidote drug, with a rapid and long-lasting return to the initial level of the antidote after the intravenous bolus injection of the antidote. A similar pattern was observed for CHAZ, as well as for АХТЧ. IF remained unchanged compared to baseline.

Subjects were administered EVO06 or 0.995 normal saline as an intravenous bolus injection at time zero, and the antiplatelet effect was assessed over time by measuring plasma AChT (Fig. 10). APT values for each treatment group are presented in the form of mean value x s.p.s. specific AChT. Specific APTFE is the value of APTFE for an individual subject at this moment in time, divided by the initial value of APTFE before the introduction of KVO06 for this subject. A value of 1 indicates no response to CVOB, and a value of 21 indicates an anticonvulsant effect. A pronounced response to the dose in terms of the value of the specific AChT is observed when increasing the dose of КВООб from 15 mg to 60 mg. The half-life as a pharmacodynamic activity of KWOOb as determined by the AChT assay appears to be at least 12 to 18 hours, since this is the time at which the average specific AChT of subjects administered 60 mg of EVO06 reaches the maximum specific AChT determined in subjects who were injected with 30 mg of KVO06.

Subjects were administered KVO0b or 0.995 saline (placebo) by intravenous bolus injection at time zero, and either then KVO007 or placebo by intravenous bolus injection 3 hours after administration of KVO0b. The anti-erosive effect after the introduction of KVO0b was detected for a long time by measuring the plasma APTCH (Fig. 11). The values of AHTCHUCH for each treatment group are presented in the form of the average value of the s.p.s. specific AChT. A characteristic of АХТЧУХ is the value

APTFE for an individual subject at this point in time, divided by the initial value of APTFE before the introduction

EVO06b for this subject. A pronounced response to the dose in terms of the value of the specific AChT is observed when the dose of KVOO06 is increased from 15 mg to 90 mg. The introduction of KVO07 led to a complete, rapid (within 5 minutes) and long-lasting neutralization of the pharmacological activity of KVO0O06, which is confirmed by the return of the specific AChT to initial values after the introduction of KVO07.

Treatment schemes were carried out as described in the above figs. 10 and 11. A comparison of the pharmacodynamic response in subjects who were administered 60 mg of EVO0b, which was followed three hours later by the administration of KVO07 or placebo, demonstrates the rapid and long-lasting neutralizing activity of VO007 (Fig. 12). The introduction of EVO07 effectively eliminates the state of subsequent anticoagulation in subjects, as shown by comparing the area under the curve of the APTC response with C to 24 hours in the presence or absence of EVO07 administration.

The possibility of administration of the antiplatelet system KESI as an intravenous bolus injection that does not result in complement activation in primates is unexpected, given the association of complement activation and, therefore, toxicity seen with administration previously seen with similar bolus injections of other types of oligonucleotide molecules. See, for example, Sairgaip et al. (1994). and I emip, A. A., Mopiey, 0. K.,

G eedv", 9. M., MiscKiip, R. G., Seagu, K. 5., Vsheg, M., Tetriip, M. M., and Nepgu, 5. R. (1998). Tohisyu oi oiidopisieoyide

Ipegaresiis adepiv, Ip Naparsok oi Ehregitepia!| Rnagtasoiodu, S MV is. and. That's it. (Vepip: Zrgipdeg-Mepad), 169-215.

Strategic analysis of dosage parameters

In fig. 13 shows a more detailed analysis of the relative increase in AChT in comparison with the original ambassador 0-

From hours for all subjects receiving KVO06b. In agreement with the data of tests on monkeys, the level

AHTCH reaches a maximum and reaches a plateau within several hours. The data were analyzed by determining the area under the curve of the specific АХТХХУЧ in comparison with the initial value, measured during the first three hours after administration. In fig. 19 shows how the response to KVOOb correlates with 90 SIN inhibition. The obtained data demonstrate that it is possible to inhibit 29995 SIN activity in a stepwise manner using an anti-inflammatory agent.

In fig. 14 presents AOSo-z for each subject, given in the order of the level of the dose of KVOO6b (15, 30, 60 or 90 mg). Since the relative effect is measured over 3 hours, a value of "3" indicates no response, a value of 6 indicates an average 2-fold increase over baseline, etc.

In fig. 15 shows the weight-based dose of KVO06 as a function of EVO06 dose level. In fig. 16 shows the relationship between the pharmacodynamic effect of ABOO6 (АЧОсо-з) and the "weight-standardized" dose of KVO06. The weight-adjusted dose is in the range of 0.2 mg/kg to 1.6 mg/kg, with an AsCo-z range of about 3 to 10 units.

The graph demonstrates that there is a clear relationship between response and weight-normalized anticonvulsant dose, with fairly low intersubject variability.

As shown in fig. 20 and 21, there is a clear relationship between the body mass index (BMI) of the registered subjects and the level of the KVOO0b dose. A BMI of 19-25 is normal, 25-30 is overweight, and 230 is obese.

The subjects in this study ranged in BMI from about 16 to a BMI greater than 35. Body mass index (BMI) is a measure calculated based on 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 of measuring body fat, such as underwater weighing and dual-energy X-ray absorptiometry (DXA). BMI can be considered an alternative to direct methods of measuring body fat.

IIMT is calculated equally for adults and children. The calculation is based on the following formulas:

Formula: weight (ku)/height (m)|g

Calculation method:

Kilograms and meters (or (weight (kgurist (mu/height (M)! . centimeters) in the metric system, the formula for calculating BMI is weight in kilograms divided by the square of height in meters. Since height is often measured in centimeters, to express height in meters, it is necessary to divide the height in centimeters by 100.

Formula: weight (IU)Lrist (ip))"x703

Calculation method:

Pounds and inches Iwaga (IBurist (ipu/height (ip)|)x703

To calculate BMI, you need to divide the weight in pounds (IB) by the square of the height in inches (in) and multiply by the conversion factor 703.

In fig. 17 shows BMI-normalized dosing for subjects receiving KVO06b as a function of KVO0Ob dose level. In fig. 18 shows the relationship between AOSoz for KVO0Ob with the dose normalized by BMI.

Dosages ranged from 0.5 mg/MTt to approximately 4.5 mg/MTtT. The range of AsSo-z was from approximately 3 to 10 units. As can be seen in the diagram, between the pharmacodynamic parameters and the dosage normalized to

BMI, there is a clear connection. This relationship is even more pronounced than the relationship with weight-adjusted dose, with less variability. The association of BMI with specific IsSo-z indicates that the drug is likely to be distributed primarily in the central part of the body, rather than in the periphery or in the relevant tissues.

This distribution provides additional support for the use of the KES1 system as an anticonvulsant for parenteral administration.

Evaluation of the KES1 system in patients with stable angina pectoris

The study was conducted on 50 patients with stable angina who were taking aspirin and/or clopidogrel. Patients were randomly divided into three groups (only EVO0Ob, VOO06, followed by

ЕВО07, or only placebo) with 4 dosage levels "ВО06 and 28007.

Baseline characteristics included a median age of 61 years (interquartile range (IQR) 56-68), 2090 women, 8095 with a history of percutaneous coronary intervention and 3495 with a history of coronary artery bypass grafting. The median AHTCHU 10 minutes after a single intravenous (IM) bolus injection of low, intermediate low, intermediate high and high doses of KVO0Ob was 29.2 seconds (OK 28.1-29.8), 34.6 seconds

(OA 30.9-40.0), 46.9 seconds (OK 40.3-51.1) and 52.2 seconds (OK 46.3-58.6), p«e0.0001, (normal range

Achtchuch 27-40 seconds). KVO07 returned AChT to "1095 above the upper limit of normal with a median of 1 minute (OK 1-2) (fig. 1) without reversible increase up to 7 days. Despite the use of dual antiplatelet therapy, 3,895 subjects had no significant bleeding or other serious adverse events.

In fig. 20 presents the results of the comparison of the AChTUCH reaction at four aptamer/antidote dosages in comparison with placebo. Group 1 "low dose" was administered 15 mg of KVO006 at time 0 and 30 mg of antidote KVO07 at time 3 hours by intravenous bolus injection. Group 2 "intermediate low dose" was administered 30 mg of KVO0b at time 0 and 60 mg of antidote KVO07 at time 3 hours as an intravenous bolus injection. Group C "intermediate high dose" was administered 50 mg of "VO006 at time 0 and 100 mg of the antidote

KVO07 at the time of the hour From the hour in the form of an intravenous bolus injection. Group 4 "high dose" was administered 75 mg of KVO0b at time 0 and 150 mg of the antidote KVO07 at time 3 hours as an intravenous bolus injection. Both at 50 and at 75 mg/kg ЕВ00б6, a pronounced increase in AChT was determined, which was completely neutralized by the introduction of KVO07 at a 2-fold concentration of the aptamer.

Re-dosing of the KES1 system

The study was conducted on 38 patients with general good health. Three treatment groups were formed: group 1, in which subjects received a single dose of the aptamer (0.75 mg/kg "VOOb" on days 1, 3, and 5, followed by a fixed dose of the antidote (1.5 mg/kg E8B007 ) in an hour; and groups 2 and 3, in which subjects received a single dose of the KWOOb aptamer (0.75 mg/kg) on days 1, 3, and 5, followed by single different doses

ABO07, administered after one hour. Dose titration of KVOO07 in subjects in groups 2 and 3 is presented in Table A below.

Table A

Ratio of doses of antidote (KVOO7) and medicinal product (KVOOb) for groups 2 and З

Relation

She - vvO07 day| antidote (mg/cu):k 8006 (mg/kg) 1 The antidote:study drug ratio on day 5 was between 0.11 1171 and was based on the AChT results on days 1 and 3. 2 The antidote dose was from 0.075 mg/kg to 0.75 mg/kg.

The dose of VO06 (0.75 mg/kg) was selected based on the weight-normalized response to VO06. On average, the indicated weight-normalized dose of KVO0b increased the AChT of subjects by 2 times. Aptamer KVOO06, antidote and their respective placebos were injected over one (1) minute. In fig. 21 shows the time-weighted AHTCHU after administration of KVOOb (0.75 mg/kg) on days 1, 3 and 5 at different dosages of the antidote.

In fig. 22 shows the percentage of the effect on AChT from the introduction of KVOO06 in the respective groups. An approximately 27,090 increase in APTT was noted after administration of 0.75 mg/kg aptamer in all three groups and was not significantly different over the three days of treatment.

In fig. 23 shows the average AChT in groups that were injected with KVO0b (0.75 mg/kg) and KVO07 in different ratios to KVO0b. KV0b was administered at time 0, and KV007 in the listed ratios was administered after one hour. As can be seen from the diagram, KVO07 varied the anticoagulant dose of the antidote to the aptamer. In addition, as can be seen from fig. 23, the inversion effect of EEVO07 for each of the ratios tested was relatively stable over time with a gradual decrease in the pharmacodynamic activity of KVO06 over time, as expected for this compound.

In fig. 24 shows the percentage of recovery of the time-weighted AChT in groups that were injected with KVO06 (0.75 mg/kg) and KVO07 in different ratios with KVO06. KVO0b was administered at time 0, and KVO07 in the listed ratios was administered one hour later. At the lowest ratio tested, 0.125:1, KVO07 neutralized the effect of KVO0O06 by about 4095. At 0.2:1, EVO0O07 neutralized the effect of KVO06 by about 5095. At 0.31 EVO007, it neutralized the effect of KVO0O06 by about 7595. At 0.511, KVO007 neutralized the effect

KVO0O at approximately 8595. | at higher ratios, 1:11 or 2:11, KEVO07 effectively completely neutralized the effect of KVOO06.

x And at Na g that a

Initiation of the cell, point of the tissue KK mimne -- "tor - -

Shi U Pa uh Hia M o M Ma go Usha ul stromsott Du)

ХИХ / 1:00.

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Activated platelet

Their Distribution about Fig. 1 voob sqO07 Complex apartment

Sazvgu a and, . hits 5:'s-« ve o E KI sh-s v s-e s tya se

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Active Inactive

Fig. 2

"THAT TS 1 GO pod t Y pnshny ni ni ni VIS y Y i yi Her Ni I and her NN ri ! I 400 -n -- ss ni nyny a y SH YN GI, shya vo pen: shin v ni sope ftettyutttvnsya - I'm not TT e------5ШШЧ. Ts SS

With MISHINNYNO now tires are not tires

Where did they drink today?

OO won shu Matt --y p a A OZ ren: o INNNNNINNN NIM ENN NI NI SHANY 1 10 1300 to

KBO06 μg/ml , ! Fig. With 150 and United normal human plasma 1494 pcs" M279, M/55 i! 130-s M343, M/23 sn 6 120 ee MZ320, P/49 E o

And mine about Mm38b, K/21 di about sh 2 100 a - 8, 80 dovo; sh 70 p 60 whatever

And long live the ski slopes at 10 20 30 40 5О0 70 85 100 115 130 145

ИКВООб| µg/ml

Fig. 4

50 I VVO0b-88007 -

At 40 5, sh

with zo'

X-y t kozh - 10 : y o i ik || in and in I 0 2 4 6 8 10 12 14 16 18 20

Molar ratio of antidote drug

Fig. 5

A INJECTION v Injection of antidote antidote Re-INJECTION o Re-INJECTION 15o drug 1 drug 7 "25 A Y be vo i y

E V soyu r» o - 75 Z, zo

YOU Fo 50. yu : 25. io o o yo 0 10 15 20 25 00 95 15 15 29 25

Time after INJECTION (HOURS) Time after INJECTION (HOURS)

Fig. 6

' 109-,. Ф Plasma of Javan macaques al 90 Jewish plasma 5 a vo schi p p o to p . sh! 8, 60 | h e e -

And in 594; "40 and Eh; for 20 rnntnntnnTTTNtNTi at 10 20 from 40 BO 150 200 250 300 350 200

IQVOObI μg/ml : Fig. 7

АХТЧгрупы 1 КБЕС1-ТОХО001 120 ОЗ АХТВРВ5 mo sho АХТВ at 5 mg/kg ЕВ006 100 о АХТВ at 15 mg/kg ЕВО06 le ee АХТВ at 30 mg/kg КВО06 in 90

He evo v o , to t kovo y - 5 50 F 5 40 . h z o g 5 i

OO 12 24 35 48 B6BO 72 84 96 198 120 132 144 156 168 150 192

Time after injection (h)

Fig. 8

ACHICH group 3 KES1-TOHO01 2 АХТВ ев 119 o АХТВ ВЕС 15/30 me/kg 1Х F АХТВ КЕ 30/60 mg/kg 1 - 803, - 1 ' -8, 704U other sh bo - vo 0 zo or drink as au l. DN call OV 0020 vali s 0 12 24 36 48 bo 72 84 96 108 120

Щ Time after injection of KVOOb (h)

Injection of KVOO7

Fig. 9

In 2.6 I and

With? | About 50 mg of medicinal product/placebo antidote (item 5)

Z "Zh F 30 mg of medicinal product/placebo antidote (p-7) and

At 22 | schu D 15 mg of medicinal product/placebo antidote (p-5) iv s x - 8 2.94 9 A A Placebo/placebo (p-5) 5, in s - in 8 and 2 5 she 5 s 146 e i sh se - that 1.45 in she: with And Her 8 KK: - D-YOU got used to the SAME

A 1. ' cheh s v EI cho Payer "V cha Prove at 08 t

B, o Z b 9 12 15 18 21 24

Time after drug administration (hours)

Fig. 10 l-- 3.8 3. o

Injection of an antidote that per sg dose I

Е 3.0 И), SH 90 mg of medicinal product/180 mg of antidote (p-3) 5 - ze and O 60 mg of medicinal product/120 mg of antidote (p-7) 5 : 24 and Ф 30 mg of medicinal product/60 mg of antidote (p--6) 522 g of AD I15 mg of medicinal product/30 mg of antidote (p-6) in B 250 Ki 5 2 448 and A Placebo/placebo (p-5) ozh o 23 -3- I) t 4.6 o 8 14 -

V 12 No A «F- - -.3 s - Usyu v oda fs Ann o pre Koba - 08 : 0 1 2 Z a 5 b 7 8 9 10 Di Y 12

Time after drug administration (hours)

Fig. 11 tt 28 t Injection of antidote yes 5 2

B 2. IF 50 mg of drug/placebo antidote (p-5) and 2.2 O 60 mg of drug/120 mg of antidote (p-7)

Z 5 2. A Placebo/placebo (pi-5) "vi

B" vo o A 4. vro a I ee

Shi andB--in-

B desvlrtnnnia sno

From 1. "Kk po es nen and "0. 5 o'clock! g! From 145 18.21.24 c

Time after drug administration (hours)

Fig. 12 sh 90 mg KBO0b o 60 mg KBOoyb t ee 30 mg KVO0b i 40 A 15 mg KBOOb

Ex 3,084 A Placebo t 3.

Go r 3. - 8 3.2 for 9 3.0 - 5 2.8 o 5 2. i BA n - 22 - - 1.8 pinni

WHAT 1.

In 1.4, it is not necessary to write in 1.2.

I 08 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Time after drug administration (hours)

Fig. 13 - " o di - 5 o is 8. o

In (F) gi 7 5) - 6 Z va ko

In i-th 4 in B (With 2 in 5 of them 2: 1 0 15 30 45 6о 75 90

Dose of KWOO6 (mg)

Fig. 14 o o o o

Yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes, yes.

Dose of KVOO06 (mg/kg)

Fig. 15 11 - in o 7 ode) o i-th x 6 in o

At 5 o'clock Ff n

I vo -e o b 3 Fe o

From 2 « 1 o 00 02 04 06 08 10 12 14 16

Dose of KWOO6 (mg/kg)

Fig. 16 o o o o o - 2.7 is ke and o ФооШшо

Beats ist

WITH

- that's it

From ОХ and 1 о о 00 05 10 15 20 25 30 35 40 45

Dose of KVOO06 (mg/BMI)

Fig. 17 11 t o x 5 o o (F) 5 1 o o o

In o

At 5 a.m

I am talking about it

F o o 2 3 own

From 2 « 1. 00 05 10 45 20 25 30 35 40 45

KVOO06 dose (mg/IMT)

Fig. 18

8 38 t

With 90 mg of EVO06 t Z.: 1 o. e 3.0 F r. with 2 Fa. 60 mg of EVO0b6 t - - B. And in 22 t ZO mg of BVO0Ob

In Shch 18 m

OBO" e 15 mg KVO0Ob that "s, i " Sha 1.

A it 01 1 10 100 (9; .

B, about the activity of ЕХХ

Fig. 19 60 4 Intravenous bolus Intravenous bolus administration KVOO06 at the time of 7 VEVO07 at the time of time time 0 hours From hour 55 da dey - BO And in sh in i; 45 She o V y - 40

E- oo zv 2

With | rah and ; sh ts ) and puti fr net vla i V NIVY itsk 7.

F 25

CI) 1 2 of 4 5 6 7 of 9 10 11 12

Time (hours)

Low dose Intermediate high dose (50 2-4 Placebo

I (15 mgkv00b/3Omgkv007) /02-- mg BVO06/100 mg KVO007) (0.995 saline solution at both time points) «--- Intermediate low dose (30 mg daily High dose (75 mg

КВО06/60 mg КВО07) КВО06/150 mg КВО007)

Fig. 20

100 Z

And in

And eat

І во и и» re | | wat orogogam no. Kn Kiya to o Ushk KOM and a ooo

K "s So 2 o sh. 90000 oyu 2 ! 41: it: PN nia Maky ves. vtovooo o NE kmotsei KK 0, 1891 xx, that M Y cows Koosoyuo orni KK pet Kooooouya Ko

Chi KY se And beans Koko boi still SZHK royu i NOV Ky n- we Oros 24 93 oyu I soyu moe

SEN pos ANN Shi Shnny since 17 ? many RO eevrnnnv,

44 no.

TE promised ШИ Ko poch 7 And that's it: ШИ SSO ooo,

Ny -o that of my ooo korogvy ven Go) . o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o

And both Fo, ooo this language is tota. au ooo, moe ia mogo s 99, 9.0, Fo, oh MO SHK you

Hm! - with. OO oyu with lacebo f-t: shk bo SHI with Placebo 1 with with Placebo kasa and acebo (1 2 with ---t- ptn tn н з Group - ; 2 Х;:| «5 52 5-- 5 en

Fig. 21 oo 290 -- Placebo -schk-- - - Group I o st -- Group2 -3-GroupZ o (pi) (fr) 260 ni 50 (pazu (eteHlntu 40

From zo (tov, "(psho) t-e 220 ! - 210

There are 200 o'clock in 190

F is yours

Y g 170 i 160 vka 150 140 1530 120 ' 10 (pshkhy) Number of subjects

The upper limit of 95705 confidence 00 Zav) became the interval

Ivo g3 7 )) 5 Average value in Lower limit of 9595 confidence interval of 1 g in

Day

Fig. 22

The ratio of the antidote to the Placebo //------- 21 drugs is 1:11, and 0.51! b 05:10 - 56 BTe- 0.2:1 in pe 1. ti eoo 0.125: ! I. AND

Yes, she." I. TOI FIGHTED

HER sun - 1 MI t v i skh

With ! t 503 | No pi

R, ? ; i pa v ONY v i khi Shamo - | ; NOT tan - in

She is on the 1st. -2--44.21121-11.. ! m: YOU OPP oo V ! ---- In o 1 2 c

Fig. 23 120 o 100 20 . r r

I am 8 o'clock

B хх я ХА в щ во 9 в 50 Iii o u s : call; i | horse d

For o oyu ie 20 pe and OO and you i xx To mine

OS

- Kk

I 9,000,000 o - shi XX i zba 21 1 i VI 0.31 0.21 0.123511

Matching the antidote to the medicinal product

Fig. 24

Claims (20)

1. The method of introducing the aptamer, which includes: a) measuring the body mass index (BMI) of the subject; b) determination of the desired pharmacodynamic response; 1 c) administering to the subject a dose of the aptamer to achieve the desired pharmacodynamic response based on the comparison of the dose to BMI with the pharmacodynamic response, in which the aptamer contains 5EO IU MO I, 1 where the desired pharmacodynamic response is the maximum level of anticoagulation. 2. The method according to item I, which additionally includes administering to the subject a dose of the aptamer antidote, where the antidote dose is based on a known dose of aptamer administered earlier, 1 antidote:.aptamer ratio is based on the desired decrease in aptamer activity. 3. The method according to claim 2, in which the aptamer is administered at a dose of 4 mg/ BMI or more. 4. The method according to item I, in which the desired pharmacodynamic response is an anticoagulation level of approximately 75 9o from the maximum, and in which the aptamer is administered at a dose of approximately 3.0-4.0 mg/ BMI. 5. The method according to item I, in which the desired pharmacodynamic response is an anticoagulation level of about 50 9o from the maximum, and in which the aptamer is administered at a dose of about 20-30 mg/ BMI. 6. The method according to claim 1, in which the dose of the anticoagulant is from 0.1 to 10 mg/ BMI. 7. The method according to item I, in which the dose of the antiplatelet agent is approximately 5 mg/ BMI. 8. The method of introducing the aptamer, which includes: a) measuring the subject's weight in kilograms; b) determination of the desired pharmacodynamic response; c) administering to the subject a dose of the aptamer to achieve the desired pharmacodynamic response based on a comparison of the dose per kilogram with the pharmacodynamic response; and, 4) administration of an antidote dose of the aptamer to the subject, where the dose of the antidote is presented based only on the ratio of the dose of the aptamer, in which the aptamer contains 5EO IU MOI, and the dose of the antidote is approximately 1.4 mg/kg or more, and the desired the pharmacodynamic response is the maximum level of anticoagulation, while the method includes additional administration to the subject of an antidote dose of aptamer, where the dose of antidote is based on a known dose of aptamer administered earlier, 1 antidote:aptamer ratio is based on the desired decrease in aptamer activity. 9. The method of claim 856, wherein the desired pharmacodynamic response is an anticoagulation level of about 75 90 of the maximum, 1 wherein the dose of the anticoagulant is about 1.0 mg/kg. 10. The method according to claim 8, in which the desired pharmacodynamic response is an anticoagulation level of about 50 90 of the maximum, 1 in which the dose of the anticoagulant is about 0.6-0.8 mg/kg. 11. The method according to claim 8, in which the dose of the anticonvulsant is from 5 to 10 mg/kg. 12. The method according to claim 1 or 8, in which the antidote is an oligonucleotide antidote. 13. The method according to claim 1 or 8, in which the pharmacodynamic response is measured in the coagulant activity assay. 14. The method according to claim 1 or 8, in which the aptamer is administered by intravenous bolus administration. 15. The method according to item I or 8, in which the aptamer is administered by subcutaneous injection. 16. The method according to claim 2 or 8, in which the aptamer and the antidote are administered in a ratio of 1.1. 17. The method according to claim 2 or 8, in which the aptamer and the antidote are administered in a ratio of at least 2: 1. 18. The method according to claim 2 or 8, in which the aptamer and antidote are administered in a ratio of 0.5: 1 or less. 19. The method according to claim 2 or 8, in which the activity of the aptamer is reduced by less than 90 Fo. 20. The method according to claim 2 or 8, in which the activity of the aptamer is reduced by about 50 95.