A reciprocating machine with a friction bearing crank mechanism having a monitoring device monitoring operating parameters of the friction bearings of the crank mechanism via at least one sensor which is in connection with an evaluation device. At least one sensor is disposed near a part of the crank mechanism which moves in operation relative to the machine""s housing. The connection of the sensor with the evaluation device is guided at least in part via the crankshaft. This provides a simple and effective monitoring option for the friction bearings of the crank mechanism and for other machine-specific parameters.

This application claims the benefit of foreign priority Austrian Patent No. GM 723/99, filed Oct. 18, 1999.
The invention relates to a reciprocating machine with a friction bearing crank mechanism and a monitoring device for monitoring operating parameters of the friction bearings of the crank mechanism via at least one sensor associated with an evaluation device.
The crank mechanism, and in particular a crankshaft with connecting rods and pertaining bearings, is the mechanical hub of a reciprocating machine, such as a combustion engine or a compressor. The forces generated by all cylinders act on the crankshaft and its connecting rods and bearings, adding up to the total power output of the reciprocating machine. Therefore, diagnostic monitoring of the mechanism is particularly important. The invention is based on the premise that the beginning of a malfunction of individually disposed bearings or cylinders can be detected relatively isolated to a particular area.
It is known in the art that bearing defects can be detected by acoustic emission as the defect begins and increases in severity, particularly with high-frequency sound or ultrasound. See, for example Acoustic Emission Testing (Nondestructive Testing Handbook; Vol. 5) by Ronnie K. Miller, Paul McIntire, American Society for Nondestructive Testing, 1987; Diagnostic of sliding pairings by means of sound emission analysis, by A. Sturm, S. Kuhlemann, Mechanical Engineering Berling 34 (1985) 3, pages 129-132; Method and arrangement for detecting the cause of wearing symptoms in friction bearings, by A. Sturm et al., DE 41 23 576 A1 and DE 40 28 559 A1.
Sensors for structure-borne sound having a piezo-electrical measuring element are usually used for analyzing the effect of sound emission on solid body structures. However, resistive, capacitive, inductive, piezo-magnetic or optical sensors for structure-borne sound can also be used. A comparison is made between xe2x80x9cactivexe2x80x9d sensors, which require no auxiliary energy, and xe2x80x9cpassivexe2x80x9d sensors, which typically have to be supplied with electrical current or a stimulating light. Generally, the sensors are designed to be mountable on the structure surfaces and to receive structure-borne sound signals transmitted by the structure that reach the surface of the structure. There are, however, differences in the directional characteristics and in the modal sensitivity of the sensors. For example, a sensor can be designed to detect sound waves arriving radially versus axially with a longitudinal polarization or with a transversal polarization in a certain direction.
Errors in the operation of individual cylinders of an internal combustion engine, such as spark failure, knocking or differences in performance as compared to the performance of other cylinders, can be detected by a standard indication method based on measured gas pressures in the combustion chamber, and can also be used for individual cylinder control and monitoring.
Disadvantages of known methods and devices include the fact that the monitoring sensors usually have to be mounted on the exterior of the static structures, and therefore they are far removed from the hub of the mechanical action, i.e. the crankshaft and bearings. This results in inferior detection of beginning bearing defects and of errors in individual cylinder behavior.
Typical mounting of sound emission sensors on the exterior of components having structure-borne sound contact with the static exterior of bearing-structures causes a significant weakening in the signal and inferior localized sound source differentiation because of long sound paths relative to the wavelength of the high-frequency sounds. Both of the above effects result in an undesirable strength ratio between the wanted signal and the underground or interfering signal, and therefore in an inferior monitoring capability. Furthermore, such apparatus is only able to monitor the main bearings. The apparatus is not able to monitor the connecting rod bearings because the sound signals generated in the connecting rod bearings are weakened on their way to the sensors by the complex structures, joints and lubricating oil films of the crank mechanism and the housing. In some instances, the sound signals are weakened to the point of being virtually unusable.
Using combustion chamber pressure indicators for indicating the behavior of individual cylinders suffers from the extreme stress to which the sensors are subjected. At least the more cost-effective models typically do not provide adequate operational reliability and life-expectancy for monitoring functions, whereby alternative solutions need to be found.
An objective of the present invention is to overcome one or more of the aforementioned disadvantages of apparatus known in the art.
At least one sensor is positioned near the friction bearings to be monitored in the crank mechanism that moves relative to the machine""s housing, and the sensor""s connection with the evaluation device is guided, at least in part, via the crankshaft. The sensor is mounted in close proximity with the event to be monitored whereby it can be reliably located or detected and analyzed. By guiding the sensor signals out via the crankshaft, the structural element, which is mechanically highly stable, can be advantageously utilized and it can also be provided, for example, that individual signal lines are disposed in the lubricant bores, which are usually present in the crankshaft. This provides a fairly simple apparatus for reliably monitoring the friction bearings in addition to various measured variables that affect the crank mechanism.
In another embodiment of the present invention, a non-contact, preferably capacitive, transmission device is provided for feeding the sensor signals in and/or out of the connecting path that runs in or on the crankshaft, and the transmission device preferably operates without any auxiliary energy. In such embodiment, in the proximity of the connecting rod bearings on the connecting rod or the bearings associated therewith, for example, sensors can be provided which, via a capacitive transmission device or other medium transmit respective measuring signals to the crankshaft from where they are led to the outside, for example, via signal lines present in the lubricating oil bores, for example in the area of the toothed rim on the flywheel where, again via a non-contact transmission device, the output to the monitoring device can take place. This allows continuous monitoring of the connecting rod bearing, for example, where the respective measuring signals can be transmitted to the outside quasi continuously.
In another embodiment of the present invention at least one sensor is formed as a sensor for structure-borne sound for high-frequency sound waves which is advantageous for detecting the beginning of bearing damage.
In another embodiment of the invention, at least one sensor is designed for detecting low-frequency mechanical tensions and deformations and it is preferably disposed on the connecting rod bearing, thus allowing the connecting rod stress to be measured so as to provide a measure for the combustion chamber pressure in the pertaining cylinder, and thus allowing an evaluation of the individual cylinders of an internal combustion engine.
In a further embodiment of the invention, at least one sensor is designed for detecting low-frequency hydraulic lubricating oil pressure on at least one friction bearing of the crank mechanism. This allows both the lubricating oil pressure and the bearing play, and thus wear and tear associated therewith, to be monitored.
In another embodiment of the invention, at least one sensor is designed as a combination sensor for detecting various measured variables, both low-frequency and high-frequency, for joint transmission of the combined signal portions to the evaluation device.
It is particularly advantageous to design the monitoring sensor as a combination sensor for sensing ultrasound emission and for sensing lubricating oil pressure present on the bearing. In addition to the sensitivity for the low-frequency lubricating oil pressure and high-frequency sound emission of the adjacent bearings this is particularly advantageous for obtaining an additional high sensitivity for the sound emission of the lubricating oil which is cavitation-related, for example.
The high-frequency signal portion represents the received structure-borne sound and the low-frequency portion represents a further physical measured variable, and the two signal portions are combined and transmitted together. At the time they are evaluated, the two signal portions will be separated according to frequency and further processed. The low-frequency measured variable, for example, can characterize the forces transmitted by the connecting rod via the connecting rod bearing to the crankshaft, so that the measuring signal will indicate cylinder-specifically, among others, spark failure, knocking and any deviations in behavior as compared to the behavior of the remaining cylinders in an internal combustion engine. However, the low-frequency measured variable can also characterize the lubricating oil pressure present on the bearing, so that indications for an inadequate lubricating oil supply and/or lubrication can be detected in the measuring signal.
In another embodiment of the invention at least one sensor can be embedded in the solid body structure of the crank mechanism, preferably in the area of a friction bearing to be monitored, of a crank arm or the connecting rod. As a result of the relatively good sound transmission in the compact structure of the crankshaft, a sensor for structure-borne sound imbedded in the crankshaft will supply dominant signals originating from the closest sound sources. Therefore, one sensor may be sufficient for monitoring the adjacent main bearings and connecting rod bearings. By means of a correlative analysis of the signals of a sensor with the signals of at least a second sensor, such as the next closest sensor, which is mounted in the adjacent crank arm, for example, the sound source can be located. Thus, with this method, it is possible to differentiate which bearing the detected signals originate on, thereby advantageously saving some sensors and thus expenses, provided that the quality of the sound transmission in the crankshaft is adequate.
Accordingly, virtually no additional projecting parts are necessary on the crank mechanism. The sensor is fully encompassed by the measured object and its temperature, and thus it is well protected. Furthermore, the sensor can be optimally adapted to its measuring function. In case of a sensor for structure-borne sound, for example, a high sensitivity and selectivity can be achieved with regard to the longitudinal and transversal oriented sound waves present in the interior of the structure. Also, the style of the sensor and the method of mounting can be designed advantageously for a certain directional characteristic and mode sensitivity, thereby achieving an optimal utilization sensitivity and interfering signal suppression for the selected location where it is mounted and for the locations of interest of ultrasound emission.
A further embodiment is particularly advantageous in which a sensor is designed as a stopper for a boring on the crank arm leading to an exterior for supplying lubricating oil to the friction bearings. This allows a simple design and mounting of the monitoring sensor.
In another embodiment of the invention, at least one sensor is designed as a temperature sensor allowing both low and, when required, temporarily higher defined temperature monitoring of the bearings.
In a particularly advantageous embodiment of the invention electrical or electronic components are provided on the sensor, or at least near the location where the sensor is mounted on the crank mechanism for at least partial signal processing or handling, which components are connected both to the sensor and to the evaluation or monitoring device. Accordingly, at least a portion of signal processing can take place in direct proximity of the sensor with the known related advantages, which makes it considerably easier and less critical to further transmit the signal. For example, an electric filter connection for suppressing interfering frequency components of the sensor signal can also be integrated in the sensor itself, or a high-pass filtering for suppressing low-frequency signal components can take place directly at a sensor for structure-borne sound. On the other hand, with a sensor for a low-frequency measured variable, a low-pass filtering for suppressing high-frequency signal components can occur. In the case of a combination sensor, for example, the high-frequency and the low-frequency signals can be filtered by separate measuring elements, in one case high-pass filtered and in the other case low-pass filtered, before they are combined. Preferably, such electric filter connections contain only passive components (such as resistors, capacitors and inductors, for example) which will not require an additional voltage supply for the components.
In a further preferred embodiment of the invention, a common connection path to the evaluation device can be provided for transmitting the combined signals of multiple sensors during general monitoring wherein a diagnostic association of the signals to the signal-emitting sensor is not required.
In an advantageous embodiment of the invention the sensors themselves can comprise electrically active, preferably piezo-electrical, measuring elements with which the most varied measuring functions can reliably and accurately be carried out because such sensors are also relatively insensitive to high temperatures, vibrations and similar operating conditions.

Reciprocating machine with operating parameter sensor


Methods for promoting immunologic control of human immunodeficiency virus (HIV) in an HIV-infected subject are provided. The methods comprise administering to the subject highly active antiretroviral therapy (HAART) for at least one cycle of an intermittent dosing regimen in combination with administration of a pharmaceutical composition comprising a therapeutically effective amount of interleukin-2 (IL-2) or variant thereof. The combination of daily or intermittent administration of IL-2 (or variant thereof) and intermittent HAART promotes immunologic control of viral replication in the absence of HAART, thereby prolonging the length of time a patient may discontinue HAART before viral rebound necessitates further administration of HAART. Administration of IL-2 therapy in combination with an intermittent HAART dosing regimen provides an effective method for treating a subject infected with HIV.

The present invention is directed to methods of therapy for human immunodeficiency virus (HIV) infection, more particularly to the use of intermittent antiretroviral therapy in combination with interleukin-2 (IL-2) to promote long-lasting immunologic control of HIV.
Infection with the human immunodeficiency virus (HIV) results in progressive deterioration of the immune system in most infected subjects. During disease progression, key cells associated with the immune system become infected with HIV, including, e.g., CD4+ T cells, macrophages/monocytes, and glial cells. Prolonged HIV infection frequently culminates in the development of AIDS. In the late stages of this disease, the immune system is severely compromised due to loss or dysfunction of CD4+ T cells (Shearer et al. (1991) AIDS 5:245-253).
HIV-1-specific CD8+ cytotoxic T lymphocytes appear to be critical in the immunologic control of HIV-1 soon after the acquisition of infection. CTL precursors specific for cells expressing several HIV-1 gene products, including Gag, Pol, and Env antigens, are detectable within three weeks of the primary infection syndrome (Koup et al. (1994) J. Virol. 68:4650-4655). Since CTL activity is antigen driven, the waning in responding T-cell subsets that generally occurs with the passage of time is not unexpected. The clinical significance of this cellular immune response to HIV has been demonstrated in a number of studies, and impairment of this response appears to be associated with more rapid disease progression. Lymphokines elaborated by HIV-specific CD4+ T-cells are critical in supporting the genesis of these mature cytotoxic T lymphocytes directed against HIV-1 (Rosenberg et al. (1997) Science 278:1447-1450), lending credence to the notion that virus-specific T-helper cells are necessary for maintenance of effective immunity to HIV.
Anti-retroviral drugs, such as reverse transcriptase inhibitors, viral protease inhibitors, and viral entry inhibitors, have been used to treat HIV infection (Caliendo et al. (1994) Clin. Infect. Dis. 18:516-524). More recently, treatment with combinations of these agents, known as highly active antiretroviral therapy (HAART), has been used to effectively suppress replication of HIV (Gulick et al. (1997) N. Engl. J. Med. 337:734-9 (see comments); Hammer et al. (1997) N. Engl. J. Med. 337:725-733). However, HAART is primarily efficacious with regard to the prevention of the spread of infection into uninfected cells. This therapy cannot efficiently reduce the residual, latent proviral DNA integrated into the host cellular genome (Wong et al. (1997) Science 278:1291-1295 (see comments); Finzi et al. (1997) Science 278:1295-1300 (see comments); Finzi et al. (1999) Nat. Med. 5:512-517; Zhang et al. (1999) N. Engl. J. Med. 340:1605-1613).
Anecdotal reports of individuals who have discontinued HAART have revealed a rapid relapse of viremia, most often within a few weeks of ceasing anti-viral therapy (Ruiz et al. (2000) AIDS 14:397-403). Consequently, HAART must be administered indefinitely to prevent reactivation of latent virus. Continuous treatment with HAART is problematic, as HAART regimens are expensive, are difficult to comply with, and have many side effects. In addition, prolonged treatment with antiretroviral agents often leads to the emergence of drug resistant viral strains (Larder et al. (1989) Science 246:1155-1158; Kellam et al. (1992) Proc. Natl. Acad. Sci. USA 89:1934-1938; St. Clair et al. (1991) Science 253:1557-1559). The emergence of drug-resistant viral strains is delayed with combination treatment aimed at different points in the HIV replication cycle (D""Aquila (1994) Clin. Lab. Med. 14:393-422). However, a significant portion of patients treated with combination therapy may eventually harbor strains of HIV having multi-drug resistance (Schinazi et al. (1994) Int. Antiviral News 2:72-5).
Recombinant human interleukin-2 (IL-2) has been studied in the setting of HIV disease for over a decade. This immunobiologic agent, when administered intermittently by either the SC or CIV route in conjunction with antiretroviral therapy, produces prominent and sustained increases in the CD4+ T-cell count in the vast majority of HIV-infected patients who have been recently studied (Kovacs et al. (1996) N. Engl. J. Med. 335:1350-1356; Davey et al. Abstract 689, ICAAC (San Francisco, Calif.), September, 1999; Arno et al. (1999) JID 180:56-60; Carr et al. (1998) J. Infect. Dis. 178(4):992-999; Hengge et al. (1998) AIDS 12:F225-F234; Levy et al. (1999) Lancet 353:1923-1929). The increase in the CD4+ T-cell compartment is polyclonal and is characterized by the genesis of CD4+ T-cells that are functional in vitro (Levy et al. (1999) Lancet 353:1923-1929).
Intermittent cycles of IL-2 produce transient rises in plasma viral load in some HIV-infected subjects (Davey et al. (1997) J. Infect. Dis. 175:781-789), however no controlled trials have demonstrated a deleterious long-term effect on viral burden. Indeed, several recent studies indicate IL-2 might have an antiviral effect (Davey et al. (1999) J. Infect. Dis. 179:849-858). The reason an antiviral effect of IL-2 has been observed only in several recent studies might relate to the higher sensitivity of the assays employed and/or the use of HAART.
In vitro data have shown that the IL-2 responsiveness of CD4+ cells of subjects well controlled virologically on HAART is greater than that of cells culled from patients with suboptimal virologic control. However, cessation of HAART in patients receiving cycles of IL-2 therapy in the past frequently leads to virologic rebound (Davey et al. (1999) Abstract 689, ICAAC (San Francisco, Calif.), September, 1999).
Given the problems associated with prolonged treatment with HAART and the tendency for rapid viral rebound following cessation of this combination therapy, even in patients receiving IL-2 therapy in the past, better methods are needed to achieve longterm immunologic control of HIV.
Methods for promoting immunologic control of the human immunodeficiency virus (HIV) in an HIV-infected subject are provided. The methods comprise administering to the HIV-infected subject an antiviral therapy selected from highly active antiretroviral therapy (HAART) or dual protease inhibitor therapy for at least one cycle of an intermittent dosing regimen in combination with administration of a pharmaceutical composition comprising a therapeutically effective amount of interleukin-2 (IL-2) or variant thereof. HAART comprises daily administration of therapeutically effective amounts of at least three antiretroviral agents. An intermittent HAART dosing regimen comprises administering HAART until plasma viral RNA is undetectable in the patient and then discontinuing administration of HAART until plasma viral RNA reaches an acceptable threshold level in the patient. Therapeutically effective doses of IL-2 or variant thereof are administered daily or intermittently during each cycle of an intermittent HAART or other antiviral dosing regimen. The combination of daily or intermittent administration of IL-2 (or variant thereof) and intermittent HAART promotes immunologic control of viral replication, thereby prolonging the length of time a patient may discontinue HAART before viral rebound necessitates further administration of HAART. Administration of IL-2 therapy in combination with an intermittent HAART dosing regimen provides an effective method for treating a subject infected with HIV.
The present invention relates to methods of promoting immunologic control of human immunodeficiency virus (HIV), more particularly HIV-1, in an HIV-infected patient. By xe2x80x9cimmunologic controlxe2x80x9d is intended the ability of an HIV-infected patient to effectively mount a cellular immune defense against actively replicating HIV in the absence of antiretroviral agents. For purposes of the present invention, such immunologic control is manifested in the form of increased anti-viral immune reactivity to actively replicating HIV. By xe2x80x9canti-viral immune reactivityxe2x80x9d is intended proliferative expansion of antigen-selected lymphocytes, more particularly the HIV antigen-specific CD8+ subset of T cells, in response to an increase in viral load, such as that seen with discontinuance of antiretroviral therapy. Proliferative expansion of CD8+ T cells, particularly HIV antigen-specific CD8+ T cells, effectively leads to a subsequent decline in viral load, such that the population of actively replicating virus is maintained below an acceptable threshold level as defined elsewhere herein. The methods of the invention are useful in the treatment of HIV-infected individuals.
Methods of the invention comprise administering to an HIV-infected subject an antiviral therapy for at least one cycle of an intermittent dosing regimen in combination with administration of a pharmaceutical composition comprising a therapeutically effective amount of interleukin-2 (IL-2) or variant thereof. By xe2x80x9cantiviral therapyxe2x80x9d is intended highly active antiretroviral therapy (HAART) or dual protease inhibitor therapy. While the methods of the invention are discussed more fully in terms of HAART, it is recognized that the methods are intended to cover dual protease inhibitor therapy as well. HAART is administered to deplete the population of actively replicating HIV, preferably to an undetectable level, in the HIV-infected subject. IL-2 is administered to maintain a background level of this lymphokine within the HIV-infected patient, with the background level being sufficient to stimulate the immune system, thereby providing for immune reconstitution in the treated patient. An intermittent dosing regimen allows for an HIV-infected subject to have a time period off of HAART, which is beneficial given the expense, complicated regimen, and undesirable long-term side effects associated with continuous HAART. By subjecting an HIV-infected patient to periods on and off HAART, the population of actively replicating HIV virus is allowed to fluctuate between an undetectable level and an acceptable threshold level. During periods of discontinuance of HAART, there is a burst of replicating HIV. However, because the patient is immune reconstituted prior to discontinuance of HAART as a result of IL-2 therapy, an effective immunologic response can be mounted against the replicating virus, thereby returning viremia to within an acceptable threshold level, if not permanently, then for a prolonged period of time. For purposes of the present invention, this ability to return viremia to within an acceptable threshold level is referred to as immunological containment of the virus. Where rebound of actively replicating virus occurs above the acceptable threshold level following a prolonged period of immunological containment, subsequent cycles of intermittent HAART are administered to the patient. The methods of the invention are described in more detail below.
By xe2x80x9cHAARTxe2x80x9d is intended combination therapy with at least three antiretroviral agents, each of which is administered to the subject in a therapeutically effective amount. For purposes of the present invention, antiretroviral agents include any substance that can inhibit, reduce, or eliminate retroviral infection of a cell. A number of these agents are commercially available for administration according to the manufacturer""s recommended dosage. Such antiretroviral agents include, but are not limited to, the two classes known as reverse transcriptase inhibitors and protease inhibitors, as well as agents that are inhibitors of viral entry. Although any combination of three or more of these agents can be used, preferably HAART comprises the administration of therapeutically effective amounts of at least one reverse transcriptase inhibitor and at least one protease inhibitor in combination with at least one additional antiretroviral agent. For example, in one embodiment of the invention, at least two reverse transcriptase inhibitors are administered in combination with at least one protease inhibitor. In another embodiment of the invention, at least two protease inhibitors are administered in combination with at least one reverse transcriptase inhibitor.
A number of reverse transcriptase inhibitors are commercially available for use in administering HAART. Examples include, but are not limited to, nucleoside analogs, which are a class of compounds that are known to inhibit HIV, and non-nucleoside drugs. Nucleoside analogs are exemplified by didanosine (2xe2x80x2,3xe2x80x2-dideoxyinosine or [ddI], available as Videx(copyright) from Bristol Myers-Squibb, Wallingford, Conn.); zidovudine (3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxythymidine or azidothymidine [AZT], available from Glaxo-Wellcome Co., Research Triangle Park, N.C.); zalcitabine (2xe2x80x2,3xe2x80x2-dideoxycytidine [ddC], available as Hivid(copyright) from Hoffman-La Roche, Basel, Switzerland); lamivudine 2xe2x80x2-deoxy-3xe2x80x2-thiacytidine [3TC] (Epivir(copyright), available from Glaxo-Wellcome Co.); stavudine (2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxythimidine [D4T] available as Zerit(copyright)) from Bristol Myers-Squibb); and the combination drug zidovudine plus lamivudine (Combivir(copyright), available from Glaxo Wellcome). These particular drugs belong to the class of compounds known as 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs, which, with some exceptions such as 2xe2x80x2,3xe2x80x2-dideoxyuridine [DDU], are known to inhibit HIV replication, but have not been reported to clear any individual of the virus. Other nucleoside reverse transcriptase inhibitors include abacavir (1592U89, Ziagen(trademark), available from Glaxo-Wellcome Co.). Non-nucleoside reverse transcriptase inhibitors include nevirapine (Viramune(trademark), available from Boehringer Ingelheim Pharmaceuticals, Inc.); delaviridine (Rescriptor(copyright), available from Pharmacia and Upjohn, Kalamazoo, Mich.); and efavirenz (available as Sustiva(copyright), from DuPont Merck).
Examples of protease inhibitors useful in the present invention include, but are not limited to, Indinavir sulfate (available as Crixivan(trademark) capsules from Merck and Co., Inc., West Point, Pa.), saquinavir (Invirase(copyright) and Fortovase(copyright), available from Hoffmnan-La Roche), ritonavir (Norvir(copyright), available from Abbott Laboratories, Abbott Park, Ill.); ABT-378 (new name: lopinavir, available from Abbott Laboratories); Amprenavir (Agenerase(trademark), available from Glaxo Wellcome, Inc.); and Nelfinavir (Viracept(copyright)), and GW141 (available from Glaxo Wellcome/Vertex).
Such examples of reverse transcriptase and protease inhibitors are not intended to be limiting. It is recognized that any known inhibitor, as well as those under development, may be used in the methods of the invention. See, for example, the drugs for HIV infection disclosed in Medical Letter 42(Jan. 10, 2000):1-6, herein incorporated by reference.
Suitable human dosages for these compounds can vary widely. However, such dosages can readily be determined by those of skill in the art. Therapeutically effective amounts of these drugs are administered during HAART. By xe2x80x9ctherapeutically effective amountxe2x80x9d is intended an amount of the antiretroviral agent that is sufficient to decrease the effects of HIV infection, or an amount that is sufficient to favorably influence the pharmacokinetic profile of one or more of the other antiretroviral agents used in the HAART protocol. By xe2x80x9cfavorably influencexe2x80x9d is intended that the antiretroviral agent, when administered in a therapeutically effective amount, affects the metabolism of one or more of the other antiretroviral agents used in HAART, such that the bioavailability of the other agent or other agents is increased. This can allow for decreased dosage frequency of the antiretroviral agent or agents whose bioavailability is increased in this manner. Decrease in dosage frequency can be advantageous for antiretroviral agents having undesirable side effects when administered in the absence of the antiretroviral agent that increases their bioavailability. The therapeutically effective dose of an antiretroviral agent for purposes of having a favorable influence on the phannacokinetics of another antiretroviral agent used in the HAART protocol is typically lower than the amount to be administered to have a direct therapeutic effect on HIV, such as inhibition of HIV replication. When used in this manner, an antiretroviral agent that has undesirable adverse effects at the full dosage required for therapeutic effectiveness against HIV replication can provide a therapeutic benefit a lower doses with fewer adverse side affects.
Thus, in one embodiment, an antiretroviral agent, when administered in a therapeutically effective amount to an HIV-infected subject, decreases the effects of HIV infection by, for example, inhibiting replication of HIV, thereby decreasing viral load in the subject undergoing antiretroviral therapy. In another embodiment, an antiretroviral agent, when administered in a therapeutically effective amount to an HIV-infected subject, favorably influences the pharmacokinetics of one or more of the other antiretroviral agents used in the HAART protocol.
For example, the protease inhibitor ritonavir when administered at full doses is a potent inhibitor of HIV in serum-and lymph nodes. When administered for these purposes, adverse reactions are common, such as gastrointestinal intolerance, hyperglycemia, insulin resistance, new onset or worsening diabetes, increased bleeding in hemophiliacs, circumoral and peripheral paresthesias, altered taste, and nausea and vomiting. Ritonavir can be administered at low doses (for example, 100 to 400 mg bid) with minimal intrinsic antiviral activity to increase the serum concentrations and decrease the dosage frequency of other protease inhibitors (see, Hsu et al. (1998) Clin. Pharmacokinet. 35:275). See, for example, the favorable influence of ritonavir on the protease inhibitor lopinavir (ABT-378) (Eron et al. (1999) ICAAC 39 addendum:18, Abstract LB-20).
Guidance as to dosages for any given antiretroviral agent is available in the art and includes administering commercially available agents at their recommended dosages. See, for example, Medical Letter 42(Jan. 10, 2000):1-6, herein incorporated by reference. Thus, for example, IDV can be administered at a dosage of about 800 mg, three times a day; D4T can be administered at a dosage of about 30-40 mg, twice a day; and Nelfinavir can be administered at a dosage of about 1250 mg, twice a day, or 750 mg three times a day. These agents are generally administered in oral formulations, though any suitable means of administration known in the art may be utilized for their delivery.
For purposes of the present invention, HAART is administered to an HIV-infected subject to effectively reduce the pool of actively replicating virus to an undetectable amount in plasma samples collected from the subject. By xe2x80x9cundetectable amountxe2x80x9d in the plasma is intended the amount of actively replicating HIV is less than about 500 RNA molecules/ml, preferably less than about 400 RNA molecules/ml, more preferably less than about 300 RNA molecules/ml, still more preferably less than about 200 RNA molecules/ml, even more preferably less than about 100 RNA molecules/ml, most preferably less than about 50 RNA molecules/ml. Any method known to those skilled in the art may be utilized to measure viral load in the plasma, including the methods described elsewhere herein. Thus, for example, plasma viral load can be determined using a branched chain DNA assay (bDNA), which has a lower limit of detection (LLD) of 50 HIV RNA molecules/ml (see Jacobson et al. (1996) Proc. Natl. Acad. Sci. USA 93:10405-10410; herein incorporated by reference). When an undetectable amount of replicating virus is present in a plasma sample obtained from an HIV-infected subject, plasma viral RNA is said to be xe2x80x9cundetectablexe2x80x9d in the subject.
In accordance with methods of the present invention, HIV-infected subjects are administered HAART for at least one cycle of an intermittent dosing regimen in combination with IL-2 therapy as defined elsewhere herein. By xe2x80x9cintermittent dosing regimenxe2x80x9d is intended a dosing regimen that comprises administering HAART to an HIV-infected subject for a time period sufficient to decrease plasma viral RNA to an undetectable amount, as previously defined, and then discontinuing administration of HAART until plasma viral RNA reaches an acceptable threshold level in the subject. By xe2x80x9cacceptable threshold levelxe2x80x9d is intended a detectable amount of plasma viral RNA of at least about 200 RNA molecules/ml, preferably at least about 500 RNA molecules/ml, more preferably at least about 1,000 RNA molecules/ml, even more preferably at least about 5,000 RNA molecules/ml, most preferably at least about 10,000 RNA molecules/ml. When plasma viral RNA reaches an acceptable threshold level, a subsequent intermittent HAART dosing regimen is reinstated.
For example, in one embodiment, the HIV-infected subject undergoing IL-2 therapy as noted herein is administered HAART until plasma viral load is decreased to an undetectable level. HAART is then discontinued until plasma viral load reaches at least about 5,000 RNA molecules/ml, preferably at least about 10,000 molecules/ml, at which time HAART is reinstated until plasma viral load is once again decreased to an undetectable level.
The length of time HAART is administered to achieve an undetectable amount of actively replicating virus in the plasma, as well as the length of time to viral rebound following discontinuance of HAART, is a function of several factors, including, but not limited to, the severity of the disease, the combination and dosage of antiretroviral agents administered for purposes of HAART, and a patient""s tolerance of HAART combined with IL-2 therapy, particularly with respect to side effects and adherence to protocol. Generally, in patients receiving cycles of intermittent HAART combined with IL-2 therapy, plasma viral load should be undetectable for at least about 1 week, preferably at least about 2 weeks, more preferably at least about 3 weeks, even more preferably at least about 4 weeks prior to discontinuance of HAART. Similarly, following discontinuance of HAART, an HIV-infected subject undergoing combined treatment with IL-2 therapy in accordance with methods of the present invention should have a plasma viral load that reaches the acceptable threshold level during at least two consecutive determinations, each of which is taken about one week apart, preferably taken about 10 days apart, more preferably taken about two weeks apart, prior to reinstating HAART. Monitoring of plasma viral RNA levels during time periods off of HAART therapy, such as with weekly or biweekly measurements, will ensure that the reconstituted immune system supported by IL-2 therapy has sufficient time to respond to the initial burst of viral replication that follows discontinuation of HAART, thereby preventing premature reinstatement of HAART.
As previously noted, HIV-infected subjects undergoing treatment with at least one cycle of an intermittent dosing regimen of HAART are concurrently undergoing treatment with IL-2 therapy. By xe2x80x9cIL-2 therapyxe2x80x9d is intended administration of IL-2 or variant thereof to a subject, where such administration provides a baseline level of IL-2 or variant thereof within that subject throughout each cycle of an intermittent dosing regimen of HAART. By xe2x80x9cbaseline levelxe2x80x9d of IL-2 or variant thereof is intended an amount of this agent that is sufficient to stimulate the immune system, thereby providing for immune reconstitution in the treated patient in the presence or absence of HAART administration. Immune reconstitution restores immune function and thus immune responsiveness in the patient. Restoration of immune function can be manifested by, for example, an increase in helper/inducer T-cell function, including an increased level of CD4+ cells, restoration of lymphocyte function, and/or an increase in the expression of IL-2 receptors.
IL-2 therapy comprises administering a pharmaceutical composition comprising a therapeutically effective amount or dose of IL-2 or variant thereof to an HIV-infected subject according to a particular dosing regimen throughout each cycle of an intermittent dosing regimen with HAART. Thus, within any given cycle, IL-2 is administered during administration of HAART and following discontinuance of HAART. For purposes of the present invention, a therapeutically effective amount or dose of IL-2 or variant thereof is an amount of IL-2 or variant thereof that, when administered according to a dosing regimen, is sufficient to restore immune function, and thus immune responsiveness, in the HIV-infected subject. The pharmaceutical composition comprising IL-2 or variant thereof can be administered using any acceptable method known in the art. Preferably the pharmaceutical composition comprising IL-2 or variant thereof is administered by way of injection, more preferably intravenous (IV) or subcutaneous (SC) injection, most preferably SC injection. What constitutes a therapeutically effective amount or dose of IL-2 or variant thereof is dependent upon the route of administration and the particular dosing regimen utilized. Additional factors that influence the mode of administration and the respective amount of IL-2 (or variant thereof) administered in combination with intermittent HAART include, but are not limited to, the severity of the HIV infection, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. One of skill in the art could readily determine the amount of IL-2 or variant thereof to be administered in combination with at least one cycle of an intermittent HAART dosing regimen to improve immunologic control of HIV given the description of the present invention.
For example, in one embodiment of the invention, IL-2 therapy is provided by daily administration of low doses of IL-2 or variant thereof. By xe2x80x9clow-dosexe2x80x9d is intended the therapeutically effective amount of IL-2 or variant thereof ranges from about 0.6 mIU/m2 to about 3.5 mIU/m2, preferably from about 0.7 mIU/m2 to about 3.0 mIU/m2, more preferably from about 0.9 mIU/m2 to about 1.5 mIU/m2, still more preferably from about 1.0 mIU/m2 to about 1.3 mIU/m2, most preferably about 1.20 mIU/m2. Although such low doses may be administered with any acceptable route, preferably the IL-2 or variant thereof is administered subcutaneously. Providing IL-2 therapy in this manner minimizes toxicity responses normally associated with IL-2 therapy at higher doses or high-bolus IL-2 administration. Such toxicity responses include, but are not limited to, chronic fatigue, nausea, hypotension, fever, chills, weight gain, pruritis or rash, dyspnea, azotemia, confusion, thrombocytopenia, myocardial infarction, gastrointestinal toxicity, and vascular leak syndrome (see, for example, Allison et al. (1989) J. Clin. Oncol. 7(1):75-80; and Gisselbrecht et al. (1994) Blood 83(8):2081-2085).
In another embodiment of the invention, IL-2 therapy is provided by intermittent administration of intermediate doses of IL-2 or variant thereof. By xe2x80x9cintermediate dosexe2x80x9d of IL-2 is intended the therapeutically effective amount of IL-2 or variant thereof ranges from about 3.0 mIU per day to about 15.0 mIU per day, preferably from about 6.0 mIU per day to about 12.0 mIU per day, more preferably from about 8.0 mIU per day to about 10.0 mIU per day, most preferably is about 9.0 mIU per day. Although such intermediate doses may be administered by any acceptable route, preferably the IL-2 or variant thereof is administered subcutaneously. By xe2x80x9cintermittentxe2x80x9d administration is intended the intermediate doses of IL-2 or variant thereof are administered for a period of time, and then withheld for a period of time. Thus, in one embodiment of the invention, intermittent administration of intermediate doses of IL-2 comprises SC administration of a pharmaceutical composition comprising IL-2 or variant thereof for 5 days, once every 4 weeks, preferably for 5 days, once every 6 weeks, more preferably for 5 days, once every 8 weeks in combination with at least one cycle of an intermittent dosing regimen for HAART.
Thus, methods of the invention comprise administering to an HIV-infected patient at least one cycle of an intermittent dosing regimen of HAART in combination with IL-2 therapy. IL-2 therapy provides a baseline level of this agent within the patient such that an effective immune defense can be mounted against actively replicating HIV in the absence of antiretroviral agents. When HAART is discontinued, these patients, who are immune reconstituted, have increased anti-viral reactivity that is manifested by an increase in CD8+ T cells, more specifically HIV antigen-specific CD8+cells. Proliferative expansion of CD8+ T cells, particularly HIV antigen-specific CD8+ T cells, effectively leads to a subsequent decline in viral load, such that immunological containment of replicating virus is achieved.
Methods of the present invention are beneficial with respect to treatment and/or management of HIV in an HIV-infected subject, particularly subjects with primary infection and those with chronic infection, so long as disease progression has not resulted in permanent loss of HIV-specific immunity. The degree of immunological containment achieved by any given patient is a function of their disease progression, history of the disease, and prior HIV treatment. Generally, HIV-infected subjects receiving at least one A cycle of intermittent HAART and IL-2 therapy in accordance with the methods of the present invention exhibit immunological containment of the virus in the absence of additional antiretroviral agents for a time period that is at least about 10%, preferably at least about 20%, more preferably at least about 25%, 30%, 35%, 40%, 45%, even at least about 50% longer than for a patient receiving intermittent HAART or IL-therapy alone.
Efficacy of the methods of the present invention and any adverse side effects can be monitored throughout the treatment of a subject using any of the methods available in the art, including those described in the examples below. A subject""s vital signs, renal and liver function, glucose levels, etc., can be measured at predetermined time intervals. Activation of T cells can be assessed by IL-2 receptor expression. Blood samples can be analyzed for HIV using any protocol known to those skilled in the art. Polymerase chain reaction (PCR) can be used to determine the presence of HIV nucleic acid in biological samples. PBMCs can be collected from a subject at specific intervals, such as, for example, weekly or biweekly, and tested for infectious HIV by co-culturing with noninfected CD4+ cells. Infection of previously uninfected cells would be indicative of the presence of infectious HIV in the subject. These methods of detection could additionally be used to determine the presence of replicating HIV in lymph node samples obtained from a subject undergoing treatment in accordance with the methods of the invention. For example, the presence of replicating HIV in plasma can be determined using a branched chain DNA assay (bDNA), which has a lower limit of detection (LLD) of 50 HIV RNA molecules/ml (see Jacobson et al. (1996) Proc. Natl. Acad. Sci. USA 93:10405-10410; herein incorporated by reference). The presence of replicating HIV in lymph nodes can be determined using, for example, a co-culture assay (Chun (1999) Nature 401:874-875, herein incorporated by reference).
The term xe2x80x9cIL-2xe2x80x9d as used herein refers to a lymphokine that is produced by normal peripheral blood lymphocytes and is present in the body at low concentrations. IL-2 was first described by Morgan et al. (1976) Science 193:1007-1008 and originally called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. It is a protein with a reported molecular weight in the range of 13,000 to 17,000 (Gillis and Watson (1980) J. Exp. Med. 159:1709) and has an isoelectric point in the range of 6-8.5.
The IL-2 present in the pharmaceutical compositions described herein for use in the methods of the invention may be native or obtained by recombinant techniques, and may be from any source, including mammalian sources such as, e.g., mouse, rat, rabbit, primate, pig, and human. Preferably such polypeptides are derived from a human source, and more preferably are recombinant, human proteins from microbial hosts.
The pharmaceutical compositions useful in the methods of the invention may comprise biologically active variants of IL-2. Such variants should retain the desired biological activity of the native polypeptide such that the pharmaceutical composition comprising the variant polypeptide has the same therapeutic effect as the pharmaceutical composition comprising the native polypeptide when administered to a subject. That is, the variant polypeptide will serve as a therapeutically active component in the pharmaceutical composition in a manner similar to that observed for the native polypeptide. Methods are available in the art for determining whether a variant polypeptide retains the desired biological activity, and hence serves as a therapeutically active component in the pharmaceutical composition. Biological activity can be measured using assays specifically designed for measuring activity of the native polypeptide or protein, including assays described in the present invention. Additionally, antibodies raised against a biologically active native polypeptide can be tested for their ability to bind to the variant polypeptide, where effective binding is indicative of a polypeptide having a confirmation similar to that of the native polypeptide.
Suitable biologically active variants of native or naturally occurring IL-2 can be fragments, analogues, and derivatives of that polypeptide. By xe2x80x9cfragmentxe2x80x9d is intended a polypeptide consisting of only a part of the intact polypeptide sequence and structure, and can be a C-terminal deletion or N-terminal deletion of the native polypeptide. By xe2x80x9canaloguexe2x80x9d is intended an analogue of either the native polypeptide or of a fragment of the native polypeptide, where the analogue comprises a native polypeptide sequence and structure having one or more amino acid substitutions, insertions, or deletions. xe2x80x9cMuteinsxe2x80x9d, such as those described herein, and peptides having one or more peptoids (peptide mimics) are also encompassed by the term analogue (see International Publication No. WO 91/04282). By xe2x80x9cderivativexe2x80x9d is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogues, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, so long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogues, and derivatives are generally available in the art.
For example, amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native polypeptide of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the polypeptide of interest may be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly⇄Ala, Val⇄Ile⇄Leu, Asp⇄Glu, Lys⇄Arg, Asn⇄Gln, and Phe⇄Trp⇄Tyr.
In constructing variants of the IL-2 polypeptide of interest, modifications are made such that variants continue to possess the desired activity. Obviously, any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.
Biologically active variants of IL-2 will generally have at least about 70%, preferably at least about 80%, more preferably at least about 90% to 95% or more, and most preferably at least about 98%, 99% or more amino acid sequence identity to the amino acid sequence of the reference IL-2 polypeptide molecule, such as native human IL-2, which serves as the basis for comparison. A biologically active variant of a native IL-2 polypeptide of interest may differ from the native polypeptide by as few as 1-15 amino acids, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. By xe2x80x9csequence identityxe2x80x9d is intended the same amino acid residues are found within the variant polypeptide and the polypeptide molecule that serves as a reference when a specified, contiguous segment of the amino acid sequence of the variants is aligned and compared to the amino acid sequence of the reference molecule. The percentage sequence identity between two amino acid sequences is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the segment undergoing comparison to the reference molecule, and multiplying the result by 100 to yield the percentage of sequence identity.
For purposes of optimal alignment of the two sequences, the contiguous segment of the amino acid sequence of the variants may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference molecule. The contiguous segment used for comparison to the reference amino acid sequence will comprise at least twenty (20) contiguous amino acid residues, and may be 30, 40, 50, 100, or more residues. Corrections for increased sequence identity associated with inclusion of gaps in the variants"" amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art for both amino acid sequences and for the nucleotide sequences encoding amino acid sequences.
Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. One preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is utilized in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. Another preferred, nonlimiting example of a mathematical algorithm for use in comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding the polypeptide of interest. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the polypeptide of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Also see the ALIGN program (Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.) and programs in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program, where default parameters of the programs are utilized.
When considering percentage of amino acid sequence identity, some amino acid residue positions may differ as a result of conservative amino acid substitutions, which do not affect properties of protein function. In these instances, percent sequence identity may be adjusted upwards to account for the similarity in conservatively substituted amino acids. Such adjustments are well known in the art. See, for example, Myers and Miller (1988) Computer Applic. Biol. Sci. 4:11-17.
For purposes of the present invention, preferably percent sequence identity between the variant IL-2 polypeptide and the reference IL-2 polypeptide, such as native or naturally occurring IL-2, more particularly native human IL-2, is determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math. 2:482-489.
The precise chemical structure of a polypeptide having IL-2 activity depends on a number of factors. As ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. All such preparations that retain their biological activity when placed in suitable environmental conditions are included in the definition of polypeptides having IL-2 activity as used herein. Further, the primary amino acid sequence of the polypeptide may be augmented by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like. It may also be augmented by conjugation with saccharides. Certain aspects of such augmentation are accomplished through post-translational processing systems of the producing host; other such modifications may be introduced in vitro. In any event, such modifications are included in the definition of an IL-2 polypeptide used herein so long as the IL-2 activity of the polypeptide is not destroyed. It is expected that such modifications may quantitatively or qualitatively affect the activity, either by enhancing or diminishing the activity of the polypeptide, in the various assays. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization, and the polypeptide may be cleaved to obtain fragments that retain activity. Such alterations that do not destroy activity do not remove the polypeptide sequence from the definition of IL-2 polypeptides of interest as used herein.
The art provides substantial guidance regarding the preparation and use of polypeptide variants. In preparing the IL-2 variants, one of skill in the art can readily determine which modifications to the native protein nucleotide or amino acid sequence will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition used in the methods of the present invention.
The IL-2 or variants thereof for use in the methods of the present invention may be from any source, but preferably is recombinant IL-2. By xe2x80x9crecombinant IL-2xe2x80x9d is intended interleukin-2 that has comparable biological activity to native-sequence IL-2 and that has been prepared by recombinant DNA techniques as described, for example, by Taniguchi et al. (1983) Nature 302:305-310 and Devos (1983) Nucleic Acids Research 11:4307-4323 or mutationally altered IL-2 as described by Wang et al. (1984) Science 224:1431-1433. In general, the gene coding for IL-2 is cloned and then expressed in transformed organisms, preferably a microorganism, and most preferably E. coli, as described herein. The host organism expresses the foreign gene to produce IL-2 under expression conditions. Synthetic recombinant IL-2 can also be made in eukaryotes, such as yeast or human cells. Processes for growing, harvesting, disrupting, or extracting the IL-2 from cells are substantially described in, for example, U.S. Pat. Nos. 4,604,377; 4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543, herein incorporated by reference in their entireties.
For examples of variant IL-2 proteins, see European Patent Application No. 136,489; European Patent Application No. 83101035.0 filed Feb. 3, 1983 (published Oct. 19, 1983 under Publication No. 91539); European Patent Application No. 82307036.2, filed Dec. 22, 1982 (published Sep. 14, 1983 under No. 88195); the recombinant IL-2 muteins described in European Patent Application No. 83306221.9, filed Oct. 13, 1983 (published May 30, 1984 under No. 109748), which is the equivalent to Belgian Patent No. 893,016, commonly owned U.S. Pat. No. 4,518,584; the muteins described in U.S. Pat. No. 4,752,585 and WO 99/60128; and the IL-2 mutein (des-alanyl-1, serine-125 human interleukin-2) used in the examples herein and described in U.S. Pat. No. 4,931,543, as well as the other IL-2 muteins described in this U.S. patent; all of which are herein incorporated by reference. Additionally, IL-2 can be modified with polyethylene glycol to provide enhanced solubility and an altered pharmokinetic profile (see U.S. Pat. No. 4,766,106, hereby incorporated by reference in its entirety).
Any pharmaceutical composition comprising IL-2 as the therapeutically active component can be used in the methods of the invention. Such pharmaceutical compositions are known in the art and include, but are not limited to, those disclosed in U.S. Pat. Nos. 4,745,180; 4,766,106; 4,816,440; 4,894,226; 4,931,544; and 5,078,997; herein incorporated by reference. Thus liquid, lyophilized, or spray-dried compositions comprising IL-2 or variants thereof that are known in the art may be prepared as an aqueous or nonaqueous solution or suspension for subsequent administration to a subject in accordance with the methods of the invention. Each of these compositions will comprise IL-2 or variants thereof as a therapeutically or prophylactically active component. By xe2x80x9ctherapeutically or prophylactically active componentxe2x80x9d is intended the IL-2 or variants thereof is specifically incorporated into the composition to bring about a desired therapeutic or prophylactic response with regard to treatment or prevention of a disease or condition within a subject when the pharmaceutical composition is administered to that subject. Preferably the pharmaceutical compositions comprise appropriate stabilizing agents, bulking agents, or both to minimize problems associated with loss of protein stability and biological activity during preparation and storage.
In preferred embodiments of the invention, the IL-2 containing pharmaceutical compositions useful in the methods of the invention are compositions comprising stabilized monomeric IL-2 or variants thereof, compositions comprising multimeric IL-2 or variants thereof, and compositions comprising stabilized lyophilized or spray-dried IL-2 or variants thereof.
Pharmaceutical compositions comprising stabilized monomeric IL-2 or variants thereof are disclosed in the copending PCT application entitled xe2x80x9cStabilized Liquid 3Polypeptide-Containing Pharmaceutical Compositions,xe2x80x9d assigned PCT No. PCT/US00/27156, filed Oct. 3, 2000, the disclosure of which is herein incorporated by reference. By xe2x80x9cmonomericxe2x80x9d IL-2 is intended the protein molecules are present substantially in their monomer form, not in an aggregated form, in the pharmaceutical compositions described herein. Hence covalent or hydrophobic oligomers or aggregates of IL-2 are not present. Briefly, the IL-2 or variants thereof in these liquid compositions is formulated with an amount of an amino acid base sufficient to decrease aggregate formation of IL-2 or variants thereof during storage. The amino acid base is an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Preferred amino acids are selected from the group consisting of arginine, lysine, aspartic acid, and glutamic acid. These compositions further comprise a buffering agent to maintain pH of the liquid compositions within an acceptable range for stability of IL-2 or variants thereof, where the buffering agent is an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form. Preferably the acid is selected from the group consisting of succinic acid, citric acid, phosphoric acid, and glutamic acid. Such compositions are referred to herein as stabilized monomeric IL-2 pharmaceutical compositions.
The amino acid base in these compositions serves to stabilize the IL-2 or variants thereof against aggregate formation during storage of the liquid pharmaceutical composition, while use of an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form as the buffering agent results in a liquid composition having an osmolarity that is nearly isotonic. The liquid pharmaceutical composition may additionally incorporate other stabilizing agents, more particularly methionine, a nonionic surfactant such as polysorbate 80, and EDTA, to further increase stability of the polypeptide. Such liquid pharmaceutical compositions are said to be stabilized, as addition of amino acid base in combination with an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form, results in the compositions having increased storage stability relative to liquid pharmaceutical compositions formulated in the absence of the combination of these two components.
These liquid pharmaceutical compositions comprising stabilized monomeric IL-2 or variants thereof may either be used in an aqueous liquid form, or stored for later use in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject in accordance with the methods of present invention. By xe2x80x9cdried formxe2x80x9d is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Examples of pharmaceutical compositions comprising multimeric IL-2 or variants thereof are disclosed in commonly owned U.S. Pat. No. 4,604,377, the disclosure of which is herein incorporated by reference. By xe2x80x9cmultimericxe2x80x9d is intended the protein molecules are present in the pharmaceutical composition in a microaggregated form having an average molecular association of 10-50 molecules. These multimers are present as loosely bound, physically-associated IL-2 molecules. A lyophilized form of these compositions is available commercially under the tradename Proleukin (Chiron Corporation, Emeryville, Calif.). The lyophilized formulations disclosed in this reference comprise selectively oxidized, microbially produced recombinant IL-2 in which the recombinant IL-2 is admixed with a water soluble carrier such as mannitol that provides bulk, and a sufficient amount of sodium dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water. These compositions are suitable for reconstitution in aqueous injections for parenteral administration and are stable and well tolerated in human patients. When reconstituted, the IL-2 or variants thereof retains its multimeric state. Such lyophilized or liquid compositions comprising multimeric IL-2 or variants thereof are encompassed by the methods of the present invention. Such compositions are referred to herein as multimeric IL-2 pharmaceutical compositions.
The methods of the present invention may also use stabilized lyophilized or spraydried pharmaceutical compositions comprising IL-2 or variants thereof, which may be reconstituted into a liquid or other suitable form for administration in accordance with methods of the invention. Such pharmaceutical compositions are disclosed in the copending application entitled xe2x80x9cMethods for Pulmonary Delivery of Interleukin-2,xe2x80x9d U.S. Provisional Application Ser. No. 60/173,922, filed Dec. 30, 1999, herein incorporated by reference. These compositions may further comprise at least one bulking agent, at least one agent in an amount sufficient to stabilize the protein during the drying process, or both. By xe2x80x9cstabilizedxe2x80x9d is intended the IL-2 protein or variants thereof retains its monomeric or multimeric form as well as its other key properties of quality, purity, and potency following lyophilization or spray-drying to obtain the solid or dry powder form of the composition. In these compositions, preferred carrier materials for use as a bulking agent include glycine, mannitol, alanine, valine, or any combination thereof, most preferably glycine. The bulking agent is present in the formulation in the range of 0% to about 10% (w/v), depending upon the agent used. Preferred carrier materials for use as a stabilizing agent include any sugar or sugar alcohol or any amino acid. Preferred sugars include sucrose, trehalose, raffinose, stachyose, sorbitol, glucose, lactose, dextrose or any combination thereof, preferably sucrose. When the stabilizing agent is a sugar, it is present in the range of about 0% to about 9.0% (w/v), preferably about 0.5% to about 5.0%, more preferably about 1.0% to about 3.0%, most preferably about 1.0%. When the stabilizing agent is an amino acid, it is present in the range of about 0% to about 1.0% (w/v), preferably about 0.3% to about 0.7%, most preferably about 0.5%. These stabilized lyophilized or spray-dried compositions may optionally comprise methionine, ethylenediaminetetracetic acid (EDTA) or one of its salts such as disodium EDTA or other chelating agent, which protect the IL-2 or variants thereof against methionine oxidation. Use of these agents in this manner is described in copending U.S. Provisional Application Ser. No. 60/157696, herein incorporated by reference. The stabilized lyophilized or spray-dried compositions may be formulated using a buffering agent, which maintains the pH of the pharmaceutical composition within an acceptable range, preferably between about pH 4.0 to about pH 8.5, when in a liquid phase, such as during the formulation process or following reconstitution of the dried form of the composition. Buffers are chosen such that they are compatible with the drying process and do not affect the quality, purity, potency, and stability of the protein during processing and upon storage.
The previously described stabilized monomeric, multimeric, and stabilized lyophilized or spray-dried IL-2 pharmaceutical compositions represent suitable compositions for use in the methods of the invention. However, any pharmaceutical composition comprising IL-2 or variant thereof as a therapeutically active component is encompassed by the methods of the invention.
The following examples are offered by way of illustration and not by way of limitation.
HIV disease is characterized by an impairment of antigen-induced IL-2 production, particularly in subjects with more advanced disease. Exogenous IL-2, in association with HIV antigen, should enhance expansion of HIV-specific CTLs and augment the effector function of these cells, thus leading to tighter immunologic control of HIV. Prolonged administration of IL-2 might actually promote HIV-specific cellular immunity.
A small uncontrolled trial showed that discontinuation of HAART in 9 subjects with HIV infection receiving low-dose daily subcutaneous IL-2 was followed by a transient surge in viremia commencing a mean of about 19 days after discontinuation of HAART, and peaking roughly two weeks after this (Emert et al. (2000) xe2x80x9cStructured antiviral treatment interruption followed by daily low dose interleukin 2 (IL-2) reveals immune response to HIV,xe2x80x9d Abstract, AIDS 2000 XIII International AIDS Conference, Durban, Jul. 9-14, 2000). The levels of plasma virus subsequently declined in concert with a rise in CD8+ T cells. By 6 weeks after the onset of viremia, the mean virus concentration had decreased to less than 10% of the peak concentration. These data further support a possible role for IL-2 in the immunologic control of HIV infection.
An HIV-specific CTL response is important in subduing HIV infection. There has been difficulty in constructing vaccines that consistently elicit potent and broad HIV-specific CTL responses. A solution might lie in therapeutic xe2x80x9cauto-immunization.xe2x80x9d Through controlled exposure to bursts of HIV during antiviral xe2x80x9cdrug holidays,xe2x80x9d interruptions of HAART may fortify the HIV-specific memory CTL response and, thereby, promote more lasting immunologic control of HIV infection. IL-2 might further strengthen this HIV-specific CTL response and enhance the likelihood of attaining the goal of long-term immunologic containment of infection. Even if permanent immunologic containment is not possible, a decrease in exposure to HAART with antiretroviral xe2x80x9cdrug holidaysxe2x80x9d would be welcomed by patients, for whom HAART pill burdens can be significant and in whom HAART has clearly demonstrated an ability to produce unwanted long-term side effects.
The following controlled study is undertaken to demonstrate the effect of intermittent courses of potent antiviral therapy (HAART), in conjunction with subcutaneous (SC) IL-2, on plasma and tissue viral burden as well as HIV-specific immunity.