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WO1997014962A1 - Systeme de modelisation de posologie - Google Patents

Systeme de modelisation de posologie Download PDF

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Publication number
WO1997014962A1
WO1997014962A1 PCT/US1996/017366 US9617366W WO9714962A1 WO 1997014962 A1 WO1997014962 A1 WO 1997014962A1 US 9617366 W US9617366 W US 9617366W WO 9714962 A1 WO9714962 A1 WO 9714962A1
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WO
WIPO (PCT)
Prior art keywords
therapeutic agent
fluid
bioreactor
loop
diluent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1996/017366
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English (en)
Inventor
Fayez M. Hamzeh
Paul S. Lietman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
Original Assignee
Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Priority to AU75268/96A priority Critical patent/AU7526896A/en
Publication of WO1997014962A1 publication Critical patent/WO1997014962A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the present invention describes a monitoring system, more particularly, a dosage modeling system which delivers therapeutic agents (e.g., drugs) to cultured cells in a bioreactor to simulate human pharmacokinetics and pharmacodynamics.
  • therapeutic agents e.g., drugs
  • an antiviral drug can be tested by culturing or simply placing virus-infected cells into an artificial system which simulates human body characteristics. The cells in the artificial system are exposed to a concentration of the drug throughout the experiment.
  • the artificial system can be used to measure, for example, the drug half-life, i.e., the time required to eliminate half of the quantity of drug that was present in the system relative to the point when the measurement began.
  • the system can also be used to measure other aspects, such as the effectiveness of the drug at various concentrations, and the effect of drug dosing.
  • one problem with the typical in-vitro system is that it continually exposes the pathogen- infected cells to a fixed concentration of the drug.
  • the infected cells and cellular or viral proteins and nucleic acids are then continuously exposed to constant levels of both the drug and its intracellular metabolites.
  • This dosing model is misrepresentative of the drug dosing that would actually occur in any living system.
  • actual drug dosing follows a complicated curve that represents body factors such as abso ⁇ tion rate, the mechanics of how a drug is delivered to a cell, and clearance of the drug.
  • a living system is frequently exposed to two or more drugs, and these drugs may interact in the system.
  • the interaction can be synergistic, whereby doses may be lowered to achieve less side effects from both drugs.
  • Antagonistic drug interactions may be harmful and even fatal.
  • Typical drug dosing models fail to represent the interactions between drugs.
  • conventional modeling systems use a relatively high volume of fluid, e.g. , 100 ml; a relatively low flow rate, e.g., about 1 to 5 ml/min, and the systems require mechanical mixing (magnetic stirrers) and oxygenating coils.
  • the system is relatively large, which can be undesirable, particularly when space is at a premium.
  • the system is subsequently placed in an incubator to control the temperature of the fluid in the system, so the use of a large incubator is required.
  • it can be difficult to find sufficient space for the system, or it may be difficult to find a suitably large incubator.
  • the present invention provides for minimizing or eliminating at least some of the disadvantages of the prior art.
  • the present invention can be used to more accurately evaluate various therapeutic agents to determine an effective dose range and appropriate indications.
  • Embodiments of the invention can be used to provide effective preclinical studies to enhance clinical success.
  • an in vitro cell perfusion system in which both the concentration of one or more therapeutic agents (e.g., drugs and/ or drug candidates), and the rate of elimination of the agent(s) can be manipulated and monitored in order to mimic first order human pharmacokinetics.
  • the model of the present invention can be used to study pharmacokinetics and pharmacodynamics of therapeutic agents such as, but not limited to, antiviral agents, antimicrobial agents (e.g...
  • antibiotics antibiotics
  • antineoplastic agents e.g., antineoplastic agents
  • antiarrhythmic drugs e.g., cardiovascular drugs (e.g., antihypertensive drugs), antiinflammatory agents, immunosuppressive agents, immunostimulatory drugs, drugs used in the test of hyperlipoproteinemias, and asthma, drugs acting on the central nervous system (CNS), hormones, hormone antagonists, vitamins, hematopoietic agents, anticoagulants, thrombolytic and antiplatelet drugs.
  • CNS central nervous system
  • hormones hormone antagonists
  • vitamins, hematopoietic agents e.g., anticoagulants, thrombolytic and antiplatelet drugs.
  • the model can also be used in toxicity studies, including toxicity studies involving metals, metal antagonists, and non-metal toxicants.
  • PK pharmacokinetics
  • PD pharmacodynamics
  • pharmacokinetic terms include C,- ⁇ , the peak (maximum) concentration; AUC, the area under the drug concentration-time curve; T ⁇ , the time of peak drug concentration; Cl (clearance rate), the measure of the body's ability to eliminate the drug (volume per unit time); volume of distribution, the ratio of the amount of drug in the body to the concentration of the drug in the blood: and C m ⁇ n , the concentration before the next dose is administered.
  • Pharmacodynamics is the study of the relationship between drug concentration and intensity of pharmacological effect, i.e., what the drug does to the body. Relevant pharmacodynamic terms include E- ⁇ , the maximum effect, ED 50 , the dose which produces 50% of the maximum effect, and EC 50 , the concentration observed at half the maximal effect.
  • a system can be used to simulate the kinetics of therapeutic agents such as drugs and/or drug candidates.
  • This system allows changing the pharmacokinetic parameters of the drug and/or drug candidate by changing the dosing characteristics.
  • Embodiments of the present invention provide a PK/PD harmacokinetic/pharmacodynamic) system which mimics first order human pharmacokinetics, and defines a dose/response relationship when single or multiple doses are administered.
  • Embodiments of the invention provide an effective PK/PD in vitro perfusion system.
  • Agents of proven efficacy in man can be useful to compare the effectiveness of experimental therapies.
  • antiviral agents such as acyclovir, penciclovir, AZT, and interf erons are useful for comparison studies.
  • the system according to the present invention allows simulation of various dosage techniques, including parenteral dosing, such as subcutaneous, intramuscular, and intravenous (continuous infusion) or oral administrations.
  • parenteral dosing such as subcutaneous, intramuscular, and intravenous (continuous infusion) or oral administrations.
  • Specific parameters involving absorption, distribution, and elimination can be simulated and manipulated for one or more drugs and/or drug candidates according to the present system.
  • Another advantage of the present invention is to simulate the rate with which the drug or drug candidate is being cleared or eliminated from the system.
  • the invention can be used to simulate drug half-life (t., 2 ).
  • the invention can also be used to measure and evaluate other drug dosing parameters such as drug concentration, as well as exposure as measured by the area under the drug concentration-time curve (AUC).
  • the present invention provides for monitoring the relationship of the extracellular concentration of a drug and/or drug candidate to the intracellular concentration of a drug and its metabolites, e.g. , metabolites such as phosphorylated metabolites. This is especially desirable since the intracellular metabolites in humans cannot be measured.
  • the present invention allows one to model the relationship between the concentrations of metabolites of a drug, and the drug's efficiency and toxicity.
  • Embodiments of the present invention provide for monitoring intracellular, extracellular, and/or membrane-associated metabolites of drugs and/or drug candidates.
  • the system allows one to measure, in a time-dependent fashion, the effect of these parameters on pathogen and cellular products, e.g., DNA, RNA (mRNA), and other products.
  • HIV encodes for enzymes such as reverse transcriptase, and integrase.
  • HIV viral genes and their gene products such as env (gpl60, gpl20, gp41); pol (p66, p51, p31); gag (p55, p24, pl7), tat (pl4), and rev (p20), can be measured and monitored.
  • Inhibition of the production of critical genes and viral proteins by new drugs is desired to prevent the formation of, for example, mature core proteins and virions.
  • Other examples include the measurement of antigens such as autoimmune and tumor associated antigens. Autoantigens are associated with diseases (autoimmune thyroiditis, myasthenia gravis, arthritis, lupus).
  • Cell surface tumor associated antigens (TAA) have been associated with RNA and DNA viruses.
  • TAA Cell surface tumor associated antigens
  • Viral antigens have been associated with the following cancer types: nasopharyngeal carcinoma (EBV), cervical carcinoma (human papilloma virus), hepatocellular carcinoma (hepatitis B virus), T cell leukemia and lymphomas (human T-lymphotropic virus). Normal cellular antigens may also be tested.
  • PSA prostate-specific antigen
  • Embodiments of the present invention provide access to the site of action of the therapeutic agent (e.g. , an antiviral drug), while the extracellular concentration is changing in a time-dependent fashion simulating human pharmacokinetics, allowing investigation of the relationship between the extracellular concentration and the intracellular concentration of the drugs and their metabolites, especially the phosphorylated metabolites.
  • the therapeutic agent e.g. , an antiviral drug
  • Intracellular pharmacokinetic and pharmacodynamic parameters of, for example, nucleoside analogues and their phosphates, as a function of changing extracellular drug concentration may provide a better indicator of antiviral activity than extracellular pharmacokinetic parameters.
  • the present invention is especially desirable since intracellular half-life ( ⁇ ) of the active metabolite of different drugs may vary.
  • the present invention provides for studying the relationship between extracellular area under the drug concentration-time curve (AUC) and intra ⁇ ellular AUC by measuring the intracellular triphosphates of several nucleoside analogues.
  • the intracellular AUC where "C" is the intracellular concentration of the triphosphates, can provide a more accurate measure of drug exposure than extracellular AUC.
  • the present invention provides for studying the correlation between intracellular levels of therapeutic agents and their metabolites such as, for example, an antiviral agent such as AZT and its metabolites, and efficacy and/or toxicity.
  • Embodiments of the system according to the present invention can be used to simulate and monitor the interactions between two or more drugs, and to define a dose/response relationship when multiple drugs are administered. Since drugs may interact antagonistically, or synergistically, doses of each drug can be changed to determine an optimum result, and reduce or eliminate drug-related toxicities. Optimization of multiple drug therapies is critical because combination therapy is more commonplace among HIV-infected individuals and cancer patients. Standard antiviral therapy for HTV infection involves three drugs (AZT, 3TC, and a protease inhibitor) or more. Another advantage of the present invention is to minimize the size of the system, e.g., by minimizing the number of parts or components, and/or by reducing their size.
  • embodiments of the invention facilitate mixing and gas exchange while avoiding the need for a stirring element (e.g., a magnetic stirrer) in the central reservoir, and while avoiding the need for oxygenating coils.
  • a stirring element e.g., a magnetic stirrer
  • embodiments of the invention can be carried out without a central reservoir.
  • a dosage simulation system having two or more units, preferably two or more units operating in parallel, can be placed in a relatively small area.
  • the system according to the invention can be a desktop system. Since the therapeutic agent(s) can be expensive and/or available in limited quantities, another advantage of reduced size is that a less of the agent(s) may be needed for testing. Yet another advantage of the present invention is to provide an automated or semi-automated monitoring system.
  • Embodiments of the present invention also provide a connectorized system for a plurality of connected objects. For example, this allows one to avoid confusion between mbes and correctly connect a number of dosing systems in parallel.
  • Figure 1 shows a block diagram of an embodiment of a system according to the present invention, wherein 6 dosing systems are operating in parallel.
  • Figure 2 shows one embodiment of a single dosing system of the present invention.
  • Figure 3 shows a side view of an embodiment of a mixing arrangement according to the invention.
  • Figure 4 shows a side view of a bioreactor cartridge.
  • Figures 5 A and 5B show close-up inside views of the bioreactor.
  • Figure 6 shows a mold used for bundling tubing according to the present invention.
  • Figure 7 illustrates a programmable dosing pump.
  • Figure 8 shows a flow chart of operation to control a programmable dosing pump in accordance with the present invention.
  • Figure 9 shows an embodiment of a single dosing system representing oral absorption.
  • Figure 10 shows the pharmacokinetic curves of a drug infused at different dosing regimens in accordance with the invention. The curves illustrate the extracellular drug concentration.
  • Figure 11 shows the pharmacokinetic curves for three measured metabolites of a drug infused at different dosing regimens in accordance with the invention.
  • the curves illustrate the intracellular drug concentrations of the metabolites.
  • Figure 12 shows an embodiment of a single dosing system providing for dosing with two therapeutic agents.
  • Figure 13 shows an embodiment of a single dosing system including a shunt.
  • Figure 14 shows another embodiment of a single dosing system that can be utilized without a central reservoir.
  • a method of monitoring comprises circulating a simulated body fluid along a first circulation loop in fluid communication with a bioreactor including cells and a dosing element capable of passing at least one therapeutic agent into the first circulation loop; passing the therapeutic agent into the first circulation loop and mixing the therapeutic agent with said simulated body fluid; removing a mixture of therapeutic agent and simulated body fluid from the first circulation loop; and monitoring the effect of the therapeutic agent on the cells in the bioreactor.
  • the method includes passing diluent fluid into the first circulation loop and mixing the diluent fluid with the therapeutic agent and the simulated body fluid; removing a mixture of diluent fluid, therapeutic agent, and simulated body fluid from the first circulation loop; and monitoring the effect of the therapeutic agent on the cells in the bioreactor.
  • the method provides for the dosing of at least one drug.
  • Some embodiments provide for the dosing of at least two drugs.
  • a method of monitoring comprises circulating a simulated body fluid along a first circulation loop in fluid communication with a central reservoir and a bioreactor including cells; mixing at least one therapeutic agent with the simulated body fluid, wherein mixing the therapeutic agent with the simulated body fluid includes passing the therapeutic agent and the body fluid through a mixing arrangement into the central reservoir, the mixing arrangement including a first therapeutic agent port and a simulated body fluid port cooperatively arranged to allow the simulated body fluid to wash the therapeutic agent from the therapeutic agent port; and monitoring the effect of the therapeutic agent on the cells in the bioreactor.
  • the method provides for modeling the dosing of at least one drug, and includes passing a diluent fluid through a diluent port in the mixing arrangement, and washing the diluent fluid and the drug from the diluent fluid and drug ports with the simulated fluid.
  • the method includes measuring the concentration of at least one metabolite of a drug, preferably the intracellular concentration of at least one metabolite of a drug. Some embodiments include measuring the intracellular concentration of at least two metabolites of a drug. Other embodiments provide for measuring the concentration of a plurality of metabolites of a plurality of drugs.
  • the method can also provide for modeling the absorption to simulate oral administration of the therapeutic agent.
  • Embodiments of the invention provide a modelling system comprising a bioreactor includmg cells in fluid communication with a mixing arrangement including at least three ports, the first port being positioned near the second port and the third port.
  • the system also includes a central reservoir.
  • a modelling system comprises a bioreactor loop including a bioreactor having cells contained therein, and a dosing element in fluid communication with the bioreactor loop.
  • Embodiments of the invention provide a device comprising a reservoir; a mixing arrangement that is coupled to the reservoir; the mixing arrangement including a bioreactor loop port, a diluent loop port, and a therapeutic agent port; and the bioreactor loop port being positioned near the diluent loop port and the therapeutic agent port.
  • Embodiments of the invention provide a device comprising a reservoir; a mixing arrangement that is coupled to the reservoir; the mixing arrangement including at least three ports, the first port being positioned near the second port and the third port.
  • a monitoring system comprising a first reservoir; a mixing arrangement that is coupled to the reservoir; the mixing arrangement including a bioreactor loop conduit including a bioreactor loop port, a diluent loop conduit mcluding a diluent loop port, and a therapeutic agent conduit including a therapeutic agent port; the bioreactor loop port being positioned near the diluent loop port and the therapeutic agent port; wherein the diluent loop conduit, the therapeutic agent conduit, and the bioreactor loop conduit each extend into the reservoir.
  • the diluent loop conduit and the therapeutic agent conduit each extend axially into the reservoir.
  • the system provides for modeling the dosing of at least one drug.
  • the system includes additional reservoirs.
  • the system includes an additional reservoir and an additional mixing arrangement, wherein the additional mixing arrangement has at least three ports and is coupled to the additional reservoir.
  • the system includes a central reservoir and a mixing arrangement including a bioreactor loop conduit including a bigreactor loop port, a diluent loop conduit mcluding a diluent loop port, and a therapeutic agent conduit including a therapeutic agent port.
  • the system lacks a central reservoir and/or lacks such a mixing arrangement.
  • An embodiment including a plurality of dosing systems operating in parallel is shown in Figure 1 , and Figure 2 illustrates an embodiment of a single dosing system 1000.
  • like components have like reference numbers.
  • the basic system of the present invention models the human or animal circulatory system, and circulates a fluid that represents the internal fluids of the body, through the bioreactor that simulates a human or animal tissue system.
  • Each dosing system 1000 includes a bioreactor 270 containing the material to be exposed to at least one fluid 232 that, typically, will also contain the therapeutic agent(s) 300.
  • the system circulates the fluid 232, that represents the internal fluids of the body, through the bioreactor 270.
  • the bioreactor which mimics the characteristics of the human or animal tissue system, contains cultured bacterial or mammalian cells.
  • the cultured cells are infected with a pathogen, e.g. , with a virus or bacterium.
  • a pathogen e.g. , with a virus or bacterium.
  • the fluid 232 (sometimes referred to as the simulated body fluid), can be an actual body fluid such as plasma, or a synthetic fluid such as a cell culture medium.
  • a fast pump 260 which operates to circulate fluid through the bioreactor 270, represents a human or animal heart.
  • a dosing system 1000 includes a bioreactor 270, and a first circulation loop (or bioreactor loop) 201 for circulating fluid 232 from a central reservoir 230, through the bioreactor, and back to the reservoir 230.
  • a pump 260 passes fluid 232 through the first circulation loop 201.
  • Bioreactor 270 includes at least
  • sampling port 272 one port, e.g., sampling port 272, and typically includes two or more ports.
  • the system e.g., sampling port 272, and typically includes two or more ports.
  • the dosing system 1000 allows the amount of fluid 232 in the bioreactor loop 201 and the central reservoir 230 r5 to remain essentially constant while adding and removing fluid from central reservoir
  • the dosing system also includes a second circulation (or diluent loop) 203 that allows the amount of fluid added to the central reservoir 230 to be offset by the amount of fluid removed from the reservoir 230.
  • second circulation loop 203 allows diluent fluid 0 200 to be passed from input reservoir 202 into central reservoir 230, and allows fluid from the central reservoir 230 (i.e., the fluid 232 diluted with fluid 200) to be passed into output reservoir 250.
  • a pump 218 passes fluid through the second circulation loop 203.
  • the dosing system 1000 also allows the amount of fluid 232 in the bioreactor loop
  • a plurality of pumps 218 can be used to add and remove fluid.
  • a single pump 218 can be used to add fluid to the loop 201 and 0 remove fluid from the loop.
  • pump 260 (associated with the first circulation loop 201) operates at a higher flow rate than pump 218 (associated with the second circulation loop 203, for example).
  • the system can include additional pumps.
  • the embodiment illustrated in Figure 9 shows two pumps 260, and three pumps 218, and the embodiment illustrated in Figure 14 shows two pumps 218.
  • the system 1000 provides for exposing cells in the bioreactor 270 to the therapeutic agent(s) 300, i.e., by mixing the agent with the fluid 232 to be passed through the bioreactor 270.
  • the system 1000 includes dosing element 224, that, once a pressure differential is created, passes the therapeutic agent 300 (e.g.
  • the system 1000 includes dosing element 224, that, once a pressure differential is created, passes the therapeutic agent 300 (e.g., a drug) into the loop 201.
  • the system can include a plurality of dosing elements.
  • a plurality of dosing elements can be utilized, to pass a plurality of therapeutic agents into the central reservoir 230.
  • Fluid 232 in the reservoir 230 that now typically includes the therapeutic agent
  • fluid 232 is subsequently passed through the bioreactor 270.
  • fluid 232 typically including the therapeutic agent 300
  • the central reservoir 230 has coupled thereto a mixing arrangement 400 that allows fluid passing from the bioreactor 270 to mix with the therapeutic agent 300 and the diluent fluid 200.
  • Fluid 232 which is now a mixture of fluid passing from the bioreactor, diluent fluid, and therapeutic agent, is passed through the bioreactor 270.
  • fluid passing from the bioreactor can be mixed with the therapeutic fluid and diluent fluid without using the mixing arrangement 400.
  • fluid passing from the bioreactor can be mixed with the therapeutic fluid and diluent fluid without using the mixing arrangement 400, and therapeutic agent can be added at any desired location in the system, e.g., via a thin-bore needle.
  • the system may also include a mixing arrangement
  • Embodiments of the invention provide for operation of a plurality of dosing systems, preferably operating in parallel, wherein at least one of the dosing systems is a control. For example, in one embodiment, eight dosing systems are operated in parallel, wherein two of the systems are controls. In some embodiments, a plurality of types of controls are utilized. For example, while at least one control in each embodiment will be free of the therapeutic agent to be tested, embodiments may include additional controls where the additional control is a different therapeutic agent.
  • the additional control can be an FDA approved drug, e.g., AZT, that is used for reference when testing a new drug for treating HIV.
  • the dosing systems allow a comparison between the therapeutic agent to be tested, a therapeutic agent-free control, and an FDA approved drug control.
  • Preferred embodiments provide for parallel operation of at least four, and more preferably, at least six dosing systems 1000 (as illustrated in Figure 1), with each dosing system including a separate bioreactor 270, bioreactor loop 201, and central reservoir 230.
  • each dosing system including a separate bioreactor 270, bioreactor loop 201, and central reservoir 230.
  • the systems can be operated, preferably in parallel, utilizing a common input reservoir 202 and output reservoir 250 if desired.
  • a single input reservoir 202 can contain a sufficient volume of diluent fluid 200 to supply a plurality of dosing systems
  • a single output reservoir 250 can be sufficiently large to contain the fluid removed from the plurality of dosing systems.
  • FIG. 9 illustrates a dosing reservoir 350, a dose diluent input reservoir 302 containing dose diluent fluid 303, a oral dose diluent loop 305, and a mixing arrangement 401 with a plurality of ports, and a dose diluent output reservoir 402.
  • the mixing arrangement 401 coupled to the dosing reservoir 350 allows fluid passing from the oral dose diluent loop 305 to mix with a therapeutic agent 300 and a dose diluent fluid 303.
  • the resultant fluid that is a mixture of therapeutic agent 300, and dose diluent fluid 303, is transferred to the central reservoir 230.
  • Dose diluent fluid 303 lacks therapeutic agent, and has essentially the same composition as diluent fluid 200.
  • dose diluent fluid is an actual and/or synthetic body fluid.
  • a device such as an automated programmable pump can be used to eliminate components such as the dosing reservoir 350, the dose diluent input reservoir 302. the oral dose diluent loop 305, the mixing arrangement 401, and the dose diluent output reservoir 402.
  • the system includes one or more flow control devices 264, preferably unidirectional flow control devices such as check valve(s) to minimize or eliminate the possibility of backflow while the system is operating.
  • flow control device 264 can be a clamp or valve, preferably operated by an automated system.
  • the system can also include one or more vents.
  • input reservoir 202, central reservoir 230, and output reservoir 250 are each associated with a separate pressure balancing tube 212 (212a, 212b, 212c) including an air vent with an air filter 214.
  • Figure 9 illustrates additional reservoirs and associated vents.
  • the system can include a variety of other components such as connectors, tubing, ports, and loops.
  • the system includes a shunt loop 500, communicating with an extra port of bioreactor 270.
  • the bioreactor 270 is suitable for containing the material, e.g., cultured bacterial or mammalian cells, to be exposed to the therapeutic agent(s). If desired, the cells can be bioengineered and exposed to the therapeutic agent(s) in accordance with the invention, e.g. , to monitor gene expression and/or gene therapy.
  • the bioreactor as used herein preferably includes dosage simulating materials, and body simulating materials therein.
  • the dosage simulating materials include, for example, cultured cells, or any other similar material which the therapeutic agent has an effect thereon.
  • the cultured cells are infected with a pathogen, e.g. , a virus, bacterium, fungus (including molds and yeasts), or a protozoan.
  • a pathogen e.g. , a virus, bacterium, fungus (including molds and yeasts), or a protozoan.
  • suitable viruses include DNA viruses, including, but not limited to HSV (he ⁇ es simplex virus), VZV (varicella-zoster virus), HCMV (human cytomegalovirus), and RNA viruses, including, but not limited to, Paramyxoviridae, Reoviridae, Picornaviridae, Rhabdoviridae, Flaviviridae, Retroviridae such as HIV (human immunodeficiency virus), and other viruses.
  • suitable bacteria include, but are not limited to, Cocci (e.g. , Neisseria SPP.. and Streptococcus spp.): Bacilli (e.g., Escherichia coli.
  • Suitable protozoans include, but are not limited to, Ciliates, Flagellates (including Trvpanosoma spp.). and Sporozoa (including Plasmodium spp. and Toxoplasma eondii).
  • Suitable fungi include, but are not limited to, those that cause Candidiasis, Histoplasmosis, and Ringworm.
  • the body simulating materials include structures which simulate, for example, capillaries and extra-cellular spaces, as described herein.
  • bioreactors are suitable for carrying out the invention.
  • the bioreactor provides for physically retaining the cells, e.g. , trapping or immobilizing them in or on the outside of hollow fibers, in ceramic matrixes or between planar membranes; or microencapsulating or immobilizing them in beads.
  • the bioreactor 270 includes a cartridge suitable for culturing the cells.
  • Cartridges for carrying out the invention are commercially available, for example, from Unisyn Technologies, Inc. (Hopkinton, MA).
  • the cartridge which is preferably a fiber cartridge, is typically formed of multiple cellulose or polypropylene capillaries. The capillaries divide the cartridge into two compartments, an intra-capillary compartment and an extra-capillary compartment.
  • Embodiments of the bioreactor 270 are shown in detail in Figures 4, 5 A and 5B.
  • An intra-capillary compartment 404 is used to perfuse the cells.
  • An extra-capillary compartment 402 plates the cells within the extra-cellular space, and, in some embodiments (e.g., as illustrated in Figure 13 including shunt loop 500), is used during supplemental perfusion.
  • the intra-capillary compartment and extra-capillary compartment are connected by micropores that allow selected molecules or materials to move between the two compartments. Molecules which have the proper sizing can move between compartments in this way.
  • intra-capillary compartment 404 includes capillary walls 410 which are perforated 412 to allow the simulated body fluid to enter the extra-capillary compartment 402.
  • shunt loop 500 can provide rapid equilibrium between the intra-capillary and extra-capillary compartments, e.g. , to carry out the dosing protocol more quickly and to increase the transfer of the therapeutic agent.
  • the rate of achieving equilibrium can be varied by, for example, changing the ratio of the lumen of the shunt tubing to the lumen of the inlet of the bioreactor.
  • the drug concentration can be measured both in the intra-capillary space and the extra-capillary space to simulate the effects on different body elements.
  • samples from the intra-capillary compartment 404 and the extra-capillary compartment 402 are taken (e.g., through sampling port 272) for measuring the drug concentration, and for determiriing the viral or bacterial titer.
  • the cells can be harvested from the extra-capillary compartment 402 at specified time points for determination of, for example, metabolites (particularly intracellular metabolites) and/or for the determination of intracellular viral DNA.
  • HPLC high pressure liquid chromatography
  • the intra-capillary compartment is in fluid communication with central reservoir 230.
  • central reservoir 230 contains a quantity of fluid 232 to represent the internal fluids of the body, more preferably the human body.
  • Fluid 232 is a perfusion medium that delivers nutrients, oxygen and one or more therapeutic agent(s) 300 to the cells in the bioreactor 270.
  • the fluid 232 can simulate one or several characteristics of body fluid.
  • the fluid 232 can be an actual body fluid such as, for example, plasma, and/or the fluid can be a synthetic body fluid, e.g., a culture medium such as Dulbecco's
  • the fluid 232 can contain an actual or synthetic component of body fluid.
  • an actual body fluid such as plasma
  • the fluid can be modified, e.g., diluted, before using it according to the invention.
  • the fluid can contain a mixture of actual and synthetic fluids.
  • Fluid 232 can include additional components or ingredients.
  • fluid 232 can also include materials useful for maintaining cultured cells, e.g.. nutrients and/or buffers.
  • at least one therapeutic agent 300 (e.g. , a drug) and diluent fluid 200 will typically also be introduced into central reservoir 230 or first circulation loop 201 and mixed with fluid 232.
  • a first circulation loop (or bioreactor loop) 201 passes the simulated body fluid 232 (that eventually will contain the therapeutic agent 300 and diluent fluid 200) through the bioreactor 270 containing the cultured cells.
  • the fluid in the intracellular compartment continuously exchanges nutrients, oxygen, and carbon dioxide, and therapeutic agent will pass into the extracapillary fluid.
  • Flow allows components of the fluid to pass back and forth between the extra- and intra-capillary compartments and subsequently into the central reservoir through return tube 276.
  • first pump 260 also known as the fast pump
  • first circulation loop 201 The operation of first pump (also known as the fast pump) 260 circulates fluid 232 at a high flow rate through first circulation loop 201 from central reservoir 230, through mbe 262 and bioreactor 270, and back into central reservoir 230.
  • first pump 260 circulates fluid 232 at a high flow rate through first circulation loop 201 bioreactor 270.
  • the flow rate of the first pump 260 is about 15 ml/min or more, and the total amount of fluid in the loop 201 and the central reservoir 230 is about 50 ml or less.
  • the total amount of fluid in the loop 201 and the central reservoir 230 can be greater than 50 ml, particularly if the flow rate of the first pump is about 20 ml/min or more.
  • the total amount of fluid in the loop 201 and the central reservoir 230 is known as the Volume of Distribution (VD).
  • a single fast pump 260 e.g., a peristaltic pump associated with a plurality of tubes
  • a plurality of dosing systems 1000 e.g. , to circulate fluid along a plurality of bioreactor loops.
  • a variety of such pumps are suitable for carrying out the invention and are known in the art.
  • Input reservoir 202 stores diluent fluid 200, that represents new body fluid, i.e. , the internal fluid(s) of the body free of the therapeutic agent(s). Since diluent fluid 200 is new fluid free of the therapeutic agent, it has essentially the same composition as fluid 232 before fluid 232 was exposed to a therapeutic agent. Thus, diluent fluid 200 is an actual and/or synthetic body fluid as described with respect to fluid 232 above, and can contain an actual or synthetic component of body fluid.
  • a second circulation loop 203 passes diluent fluid 200 into central reservoir 230 to simulate the dilution of the therapeutic agent in a body fluid by the washing actions of new fluid(s).
  • the dilution of fluid 232 by the new fluid simulates the half- life of the therapeutic agent(s).
  • the operation of the second pump 218 also known as the dilution pump, or slow pump
  • fluid also known as output fluid
  • dilution pump 218 pumps an amount of the diluent fluid from the input reservoir 202 into the central reservoir 230, and pumps essentially the same amount of ou ⁇ ut fluid at the same rate from the central reservoir 230 into ou ⁇ ut reservoir 250.
  • the amount of dilution of therapeutic agent can be precisely controlled by the dilution pump 218.
  • one or more pumps 218 can be utilized to add diluent fluid to the loop
  • the rates for adding the input fluid and for removing the ou ⁇ ut fluid are substantially equal.
  • the diluent fluid is passed into the central reservoir (and the ou ⁇ ut fluid is passed out of the central reservoir) at a rate of about 2 ml/min or less, e.g., in the range of about .01 to about 1 ml/min, or at a rate of about 0.1 ml/min, or about .2 ml/min.
  • similar rates can also be utilized.
  • a single dilution pump 218 e.g., a peristaltic pump associated with a plurality of tubes
  • a plurality of dosing systems 1000 e.g., to introduce fluid into, and withdraw fluid from, a plurality of central reservoirs and/or bioreactor loops.
  • a single pump e.g., a pump operating different size tubing
  • the single pump can pass fluid along the first circulation loop 201 at a high flow rate, and fluid along the second circulation loop 203 at a lower rate.
  • this single pump can also be utilized with a plurality of dosing systems, as described above.
  • Dosing element 224 introduces the therapeutic agent 300, e.g., a liquid drug, from the agent storage area or chamber 220 into the central reservoir 230 and/or loop 201. This simulates the dosing of the therapeutic agent 300 into the circulator ⁇ ' system.
  • therapeutic agents such as drugs and drug candidates are suitable for carrying out the invention.
  • Illustrative therapeutic agents are drugs such as antiviral drugs, antibiotics, antineoplastic agents, hormones, hormone antagonists, cytokines, anti- depressives, sedatives, hypnotic agents, cardiovascular agents, hematopoietic agents, anticoagulants, anti-inflammatory agents, antimicrobial agents, anti-parasitic agents, agents affecting the nervous system, and agents affecting the immune system.
  • the system can include a plurality of dosing elements 224, and the system can monitor the interaction between a plurality of therapeutic agents 300.
  • the half-lives can be similar for each agent, identical for each agent, or different for each agent.
  • Dosing element 224 is preferably a programmable dosing pump as described below.
  • a programmable dosing pump is especially desirable for delivering very small amounts of the therapeutic agent into the central reservoir 230 (e.g. , Figures 2, 9, 12 and 13) and/or bioreactor loop 201 (e.g. , Figure 14).
  • the therapeutic agent 300, the diluent fluid 200, and the fluid exiting the bioreactor loop 201 are passed into central reservoir 230 through mixing arrangement 400, that has outlet openings or ports leading to the central reservoir that are cooperatively arranged to allow fluid from the bioreactor loop to wash or pull fluid from the therapeutic agent port and from the diluent fluid port.
  • This washing or pulling of fluid from the therapeutic agent po ⁇ and the diluent fluid po ⁇ by the fluid from the bioreactor loop po ⁇ provides efficient mixing, without requiring, for example, a magnetic stirrer in the central reservoir.
  • the mixing arrangement according to the invention provides consistent and continuous flow, rather than stepwise additions of therapeutic agent resulting from large drops.
  • the high flow through the bioreactor 270 obviates these problems, pa ⁇ icularly for those embodiments that also include high flow through mixing arrangement 400.
  • the mixing arrangement 400 has outlet openings or po ⁇ s leading to the central reservoir that are cooperatively arranged to allow fluid from the bioreactor loop po ⁇ to wash or pull liquid from the therapeutic agent po ⁇ and from the diluent fluid po ⁇ .
  • the therapeutic agent and diluent fluid po ⁇ s are spaced across from one another, with the bioreactor loop po ⁇ positioned near the therapeutic agent and diluent fluid ports.
  • the therapeutic agent and diluent fluid po ⁇ s extend axially into the reservoir, more preferably extending further into the central reservoir than the bioreactor loop po ⁇ , so that fluid from the bioreactor loop po ⁇ pours onto at least one of the other po ⁇ s, more preferably onto both the therapeutic agent and the diluent fluid po ⁇ s.
  • the supply tube 204 from the input reservoir 202 leads to po ⁇ or opening 290 (also known as diluent loop po ⁇ 290), and the dosing tube 226 from the dosing element 224 leads to po ⁇ or opening 292 (also known as therapeutic agent po ⁇ 292), and these po ⁇ s 290 and 292 come together at about the same area, separated by space 294.
  • the ou ⁇ ut tube 276 from the bioreactor circulation loop 201 leads to po ⁇ or opening 296 (also known as bioreactor loop po ⁇ 296), and the fluid passing from po ⁇ 296 washes or exhausts into space 294.
  • Space 294 is preferably smaller than the inner diameter of the po ⁇ 296 so that the outflow pours or washes over the ends of tubes 204 and 226 (po ⁇ s 290 and 292).
  • the flow of fluid from the bioreactor loop is washed or poured directly onto the ends of the two tubes 204 and 226.
  • the high flow rate used according to this technique produces an ou ⁇ ut flow which washes out both the therapeutic agent (e.g. , the drug) that remains on the end of tube 226 and the diluent fluid that remains on the end of tube 204 from the input reservoir.
  • the high flow of fluid is continually flowing over these tube ends causing the surface- tension caused drops to continuously be diluted, removed and reformed.
  • the ou ⁇ ut fluid flow splashes into the interior surface of the ports 290 and 292. This operation provides a better continuum and continuous flow of therapeutic agent(s) because it does not require waiting for the drop to fall.
  • the dispersed agent(s) and diluent fluid are essentially immediately washed out of the po ⁇ s 290 and 292.
  • Another advantage of utilizing a mixing arrangement in accordance with the invention is that it provides efficient mixing, without a magnetic stirrer. This is in contrast with conventional systems, that utilize a low flow rate, a larger volume, and a magnetic stirrer.
  • the high flow and low volume also provides for efficient mixing, without a magnetic stirrer.
  • a therapeutic agent can be added at the input reservoir (e.g., to provide for continuous injection), and the high flow provides efficient mixing without a magnetic stirrer.
  • the system provides a mixing area, e.g. , the therapeutic agent can be added to the bioreactor loop 201 via, for example, an injection po ⁇ , and the therapeutic agent mixes with the simulated body fluid.
  • the system includes a plurality of mixing arrangements (400, 401), each arrangement coupled to a separate reservoir, wherein each arrangement includes a plurality' of ports.
  • the po ⁇ s are cooperatively arranged to allow fluid passing from one po ⁇ to wash or pull liquid from the other ports in the arrangement.
  • embodiments of the invention provide efficient gas exchange without oxygenation coils, i.e.. coils with thin walls and high surface areas to oxygenate material. Previous systems of this type used oxygenation coils, which took up a lot of room and required special areas to be dedicated for them. Additionally, or alternatively, efficient gas exchange can be accomplished in accordance with the present invention in a reduced sized system, e.g., by utilizing a CO 2 and/or O 2 injection system, thus avoiding the need for a bulky incubator.
  • the CO 2 injection system provides a CO 2 concentration in the range of from about 2.5 to about 10%, e.g. , about 5%.
  • the present invention is especially suitable for miniaturization.
  • the system can be sufficiently miniaturized to fit on a desktop.
  • the present invention utilizes at least one fast pump 260 that operates at a high flow rate, e.g..
  • the total amount is about 35 ml
  • the flow rate is about 20 ml/min or more, e.g., about 50 ml/min or more, or about 70 ml/min or more. In one embodiment, the flow rate is about 200 ml/min.
  • trickle flow rate e.g., about 4 ml/min or less
  • a larger amount of fluid e.g., about 100 ml.
  • the preferred embodiments of the invention utilize a flow rate that is about 4 times greater, and about 1/3 of the volume, of conventional systems.
  • the high rate of fluid turnover of the present invention assists in providing effective mixing, and thus allows the elimination of magnetic stirrers and/or oxygenating coils, which in turn allows miniaturizing the system.
  • utilizing a high flow rate preferably while, for example, ma taining back pressure in the bioreactor 270, facilitates therapeutic agent (drug) interaction with the contents of the bioreactor. While the mechanism is not well understood, the formation of the back pressure in the bioreactor apparently forces the therapeutic agent to more evenly disperse over the large internal surface area of the cartridge in the bioreactor. Without being held to a pa ⁇ icular theory, it is believed that this back pressure forces the fluid 232 into the simulated intra-capillary spaces in the cartridge. This results in better contact between the drug and the cells, and hence better simulation of their reactions.
  • different diameters are used for the input and the ou ⁇ ut of the bioreactor 270 to provide (in combination with a high flow rate) back pressure in the bioreactor.
  • the diameter of the bioreactor input 278 can be larger than the diameter of the bioreactor ou ⁇ ut 280.
  • input 278 can be a 1 /8-inch diameter tube.
  • Ou ⁇ ut 280 in contrast, can be a tube which has less than half the diameter of the input tube.
  • the ou ⁇ ut tube 280 can be 1/16th to l/64th of an inch.
  • a flow rate controller can be utilized upstream and/or downstream of the ca ⁇ ridge.
  • a flow rate controller can be used to vary the lumen volume of the input of the cartridge as compared to the output.
  • a system according to the invention can be used to model simulate abso ⁇ tion of a drug from the gastrointestinal tract.
  • the dosing reservoir 350 simulates the gastrointestinal tract. Over time, drug in this reservoir will be absorbed. As time passes, less and less drug will be in the gastrointestinal tract.
  • this can be simulated by diluting drug in the dosing reservoir 350 by adding fluid 303 that lacks the drug. As illustrated in Figure 9, this additional fluid 303, that lacks the drug, is passed (using a slow pump) from the dose diluent input reservoir 302.
  • a separate mixing arrangement is coupled to the central reservoir and the dosing reservoir (elements 400 and 401 respectively), and the po ⁇ s are cooperatively arranged as described with respect to Figures 2 and 3.
  • fluid passing from one po ⁇ washes fluid from the other po ⁇ s in each reservoir.
  • the use of a dose diluent ou ⁇ ut reservoir 402 allows the amount of fluid added to dosing reservoir 350 from dose diluent input reservoir 302 to be essentially offset by passing fluid from reservoir 350 into the ou ⁇ ut reservoir 402.
  • the slow pumps 218 operate a different rates and/ or for different periods of time.
  • slow pump 218 associated with second loop 203 can operate continuously at a rate to represent the half-life of the therapeutic agent.
  • Each of the other slow pumps 218, i.e., slow pump 218 inte ⁇ osed between dose diluent reservoir 302 and dosing reservoir 350, and slow pump 218 inte ⁇ osed between dosing reservoir 350 and central reservoir 230, can be operated at different rates for different periods of time to reflect abso ⁇ tion and dosing.
  • the supply of the therapeutic agent 300 is controlled by a dosing element 224 such as a dosing pump, more preferably, a programmable dosing pump.
  • a dosing element 224 such as a dosing pump, more preferably, a programmable dosing pump.
  • These dosing pumps are well known and are commercially available, and are used, for example, for administering drugs to patients.
  • the dosing element provides for adjusting the drug storage position 222.
  • the ou ⁇ ut of dosing pump 224 is a mbe 226 (e.g... a silicon tube) which leads to central reservoir 230, or dosing reservoir 350 ( Figure 9).
  • the ou ⁇ ut of dosing pump 224 leads to loop 201.
  • Another important feature of the present invention is its capability to change the type of therapeutic agent and type of dosage and dosing system that is used.
  • Therapeutic agents such as drugs are absorbed in different ways, depending on the kind of administration. When drugs are dosed intravenously, they are typically immediately adsorbed into the human blood system. In contrast, drugs which are dosed orally often have a lag time until abso ⁇ tion, and are then absorbed slowly. Other abso ⁇ tion characteristics may depend on the drug abso ⁇ tion mechamsm. Different characteristics of the way drugs are absorbed are well known.
  • the dosing pump of the present invention is preferably programmable to simulate these functions and many others. Additionally, the pump should be programmable to provide ascending and/ or descending therapeutic agent concentrations. Specifically, the dosing pump can be programmed to simulate both intravenous and oral administration characteristics.
  • Figure 7 illustrates an exemplar ⁇ ' dosing pump and its controller. As conventional in the an, the dosing element itself is typically a syringe with a pressing element. Chamber 700 includes the therapeutic agent, e.g. , a drug, therein. The chamber 700 is bounded by a moveable plunger 702. Plunger 702 can be moved up and down relative to the holding chamber 700. When plunger 702 is pressed down, it reduces the chamber size and forces some of the drug out deliver ⁇ ' tube 704.
  • Stepper motor 706 produces a controlled and highly precise amount of movement per pulse from the microprocessor. For example, stepper motor might rotate 2° per pulse.
  • the ou ⁇ ut shaft of the stepper motor 706 is connected to a threaded screw 710 that moves downward by a predetermined amount per screw revolution. For example, a screw with 32 threads per mm moves downward 1/32 mm per revolution:
  • each pulse delivers
  • Step 800 obtains an indication of the drug dosage characteristics.
  • the input characteristics can be an intravenous, oral, or any other prestored or user-defined characteristic.
  • the curve envelope shown in step 802 can be loaded for an intravenous (IV) ad ⁇ ninistration.
  • the input characteristic could be oral, in which case the curve envelope shown in step 804 can be loaded.
  • these general curve envelopes are modelled in the form of equations or time-sensitive instructions.
  • the instructions might say for time a do nothing. From time a to time b, gradually increase the slope of the curve by 0.1 per second, time b to time c level off, and from time d on reduce the curve by slope 0.1.
  • a similar characteristic or equation could be used to simulate any desired curve.
  • Step 806 determines the total volume of drug to be administered. This corresponds to the area under either of the curves shown in step 802 or 804. Each of the curves is stored as a unit curve, e.g., intended to be multiplied by a variable X. The total area under the curve determines this multiplicand.
  • the computer translates the two sets of parameters into the amount of time that should elapse between pulses - i.e., how long should there be between deliveries of 1.37 ⁇ l amounts to be dosed at separated time intervals.
  • reservoirs i.e. , the input reservoir 202, the ou ⁇ ut reservoir 250, the central reservoir 230, the dose diluent input reservoir 302, the dose diluent ou ⁇ ut reservoir 402. and the dosing reservoir 350, and are known in the an.
  • the reservoirs 202, 230, 250, 350, 402 and 302 are typically sealed.
  • reservoirs 202, 230, 250, 350, 402 and/or 302 also include a cap such as a cover or stopper that provides an essentially air tight seal for the reservoirs and the conduits such as tubes or pipes entering the reservoirs.
  • caps are suitable for carrying out the invention, and are known in the an.
  • conduits such as mbes or pipes are suitable for providing fluid communication between the elements of the system in accordance with the invention, and are known in the art.
  • suitable tubing can have a circular cross-section or a rectangular inner diameter.
  • Other types of tubing, e.g., including low profile shapes that self-flatten, can also be used.
  • Tubing with rectangular inner diameters might be especially conducive to the tubing connector described herein.
  • input reservoir 202 includes a cap 208; ou ⁇ ut reservoir 250 includes a cap 251, and central reservoir 230 includes a cap 231.
  • System 1000 also includes a supply tube 204 that has an end or port that will remain immersed in diluent fluid 200, e.g., tube 204 can extend to an area 206 close to the bottom of the reservoir into the diluent solution 200.
  • Supply tube 204 provides fluid communication with the mixing arrangement 400 in central reservoir 230.
  • the system also includes an ou ⁇ ut mbe 210 that passes the ou ⁇ ut from central reservoir 230 into the ou ⁇ ut reservoir 250, and additional tubes as noted earlier, e.g. , mbes 226, 262, 276, and 212a-c.
  • the system can also have components such as reservoirs, caps, tubes, and vents, e.g., as illustrated Ln Figures 9, and 12-14.
  • reservoirs 202, 250, 230, 350, 402, and 302 are substantially airtight except for the pressure balancing tubes 212a, 212b, 212c, 212d, 212e, and 212f.
  • Each pressure balancing tube 212a-f preferably includes an air vent with air filter 214.
  • ambient air pressure in the air portion 216 of the input reservoir, the air po ⁇ ion of ou ⁇ ut reservoir 250, and the air po ⁇ ion of the central reservoir are equalized by the vents.
  • air takes its place through a pressure balancing tube 212a
  • As liquid is passed into ou ⁇ ut reservoir 250 air is displaced from the reservoir to the atmosphere through pressure balancing tube 212b.
  • the tubing assembly One disadvantage of conventional systems using cartridges in parallel is the so-called "spaghetti" phenomenon, wherein confusion among the various tubes may lead to failures when attempting to use the cartridges. Since each cartridge or cell might include as many as five to ten tubes, and at least two cartridges should be operated at the same time, i.e. , one as a control, and at least one for testing the parameter of interest, the use of two units in parallel results in at least ten tubes that look similar and which all need to be properly located. Moreover, the mbes are flexible and hence it is very difficult to determine the place where any end is connected. Connecting any mbe incorrectly, e.g., to the wrong pon, could compromise the whole experiment. An advantage of embodiments of the present invention is to obviate this problem by providing a special system, i.e. , tubing assembly, which keeps all of the tubes ordered relative to one another.
  • FIG. 6 One example of a tubing assembly according to the invention is shown in Figure 6.
  • Eight or ten lengths 602, 604 of tubing e.g. , 1/8 inch silicon tubing, are laid side- by-side in a mold 608.
  • a variety of molds are suitable for carrying out the invention, e.g., a formal mold, or sheet(s) of aluminum foil.
  • Color coded beads 610, 612 are preferably placed at the ends of the mbes. Therefore, for example, tube 602 includes beads 610 and 612, both of which are red.
  • Tube 604 includes other colored beads such as blue.
  • the color-coded beads allow identifying each tube of the bundle relative to others.
  • An alternative to the color coding is that each tube of the bundle is identified simply by its position within the bundle.
  • this tubing connector need only identify which mbe is which within the bundle. While the bundle shown herein is a flat bundle which enables distinguishing between mbes by position in the bundle, it could alternately be a tubular shaped bundle, which uses the color codes, by some other marking on the tube, or one which allows determining by some aspect of the position of the mbe within the sphere. Other shapes include, for example, a spiral bundle.
  • a binding agent e.g., an adhesive such as clear silicon
  • a binding agent e.g., an adhesive such as clear silicon
  • a spreader such as a spatula can be used to more evenly coat the top surfaces of the material (e.g. , area 614), and to force the adhesive into the areas 616 between each two adjacent mbes.
  • the ends of the mbing e.g., several inches at each end, are left uncoated to allow some room to connect the tubes.
  • the assembly is removed from the mold, and med over so that the other side can be coated in a similar way.
  • the assembly can be used according to the invention with only one side treated as described above.
  • Figure 1 are preferably tubing assemblies as shown in Figure 6.
  • each input and ou ⁇ ut reservoir may be arranged in a bank, and the bank is connected to elements of these tubing assemblies.
  • Example 1 This Example illustrates simulating different drug half-lives in accordance with the invention.
  • a system is generally arranged as illustrated in Figure 2.
  • the reservoirs are stoppered transparent beakers.
  • the volume of distribution (VD) which is total amount of fluid in the loop 201 and the central reservoir 230 is 50 ml.
  • the fast pump is operated at a flow rate of 35 rrJ/min.
  • the dilution pump 218 is operated to provide a specific clear rate (flow rate) for a specified amount of time, to establish a specific half-life (t m ).
  • the pump setting (% Maximum) is adjusted over time to provide the desired clear rate over time.
  • the pump is initially set at 56.3 to provide a clearance rate of 34.65, and a half-life of 1 hour. The results are provided in Table I. TABLE I
  • Example 2 A system is generally arranged as illustrated in Figures 1 and 2, utilizing 5 dosing systems operating in parallel, wherein one system is a control.
  • the bioreactor includes a cartridge of polypropylene capillaries that is commercially available from Unisyn Technologies, Inc. (Hopkinton, MA).
  • the cells in the bioreactor are a human T-lymphoblastic cell line CEM, infected with HIV.
  • the volume of distribution (VD), which is total amount of fluid in the loop 201 and the central reservoir 230, is 50 ml.
  • the fast and slow pumps are Ismatic Peristaltic Pumps commercially available from Cole-Parmer (Niles, IL).
  • the dosing element is a programmable dosing pump commercially available from Medex, Inc. (Deluth, GA).
  • the fast pump is operated at a flow rate of 35 ml/min.
  • the dilution pump 218 is operated at a flow rate of .5 ml/min.
  • the drug Zidovudine (ZDV) is infused at four different dosing regimens for a dosing period of 24 hours, i.e., 0.1 ⁇ g/ml, continuously at 0.37 ⁇ M; 100 ⁇ g every 8 hours; 150 ⁇ g every 12 hours; and 300 ⁇ g at 24 hours.
  • the control is operated without dosing ZDV.
  • Samples are removed through sampling po ⁇ 272. Extracellular concentrations are determined from the fluid, and intracellular concentrations are determined after harvesting and treating the cells to extract the contents.
  • Drug concentration time curves allow the determination of C ⁇ , C m ⁇ n and AUC.
  • Figure 10 shows the pharmacokinetic curve illustrating the dmg concentration outside the cells (extracellular concentration).
  • Figure 11 shows the intracellular pharmacokinetic profiles for three ZDV metabolites, i.e.. ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate.
  • ZDV-monophosphate i.e.. ZDV-monophosphate
  • ZDV-diphosphate ZDV-diphosphate
  • ZDV-triphosphate The results of this experiment shows that dosing ZDV in these four different ways resulted in different profiles for the intracellular ZDV monophosphate concentration
  • ZDV-monophosphate converts to ZDV-diphosphate and ZDV-diphosphate converts to ZDV-triphosphate inside the cell.
  • ZDV-triphosphate is a metabolite that inhibits HIV reverse transcriptase. which results in the inhibition of HIV replication.
  • the data suggest that the rate limiting step is the conversion of ZDV-monophosphate to ZDV-diphosphate. Therefore, increasing the dose of ZDV results in a relative increase in intracellular ZDV-monophosphate concentration, but that does not result in an increase in the concentration of ZDV-diphosphate or ZDV-triphosphate .
  • Example 2 does not always mean that a higher dose would be beneficial.
  • the PK/PD system of the present invention allows one to optimize dosing of a therapeutic agent, e.g. , as dmg such as ZDV, to humans.

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Abstract

L'invention concerne un système de surveillance tel qu'un système de modélisation de posologie médicamenteuse. Dans les modes de réalisation préférés, le système prévoit un mélange sans agitation mécanique.
PCT/US1996/017366 1995-10-18 1996-10-11 Systeme de modelisation de posologie Ceased WO1997014962A1 (fr)

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WO2010062599A1 (fr) * 2008-10-27 2010-06-03 Caridianbct, Inc. Ensemble de transport de fluide prémonté pour un système d'expansion de cellules et procédé d'utilisation associé à celui-ci
WO2009026938A3 (fr) * 2007-08-26 2010-06-10 Mostafa Adel Mansour Hassan Modèle de technologie d'information dans une application pharmaceutique (mitpa)
US8895291B2 (en) 2010-10-08 2014-11-25 Terumo Bct, Inc. Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions
US8999046B2 (en) 2008-10-27 2015-04-07 Terumo Bct, Inc. Air removal chamber for a cell expansion system and method of use associated therewith

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