Growth environment assembly and method of use thereof

Toxicokinetics (TK) is generation of kinetic data for systemic exposure and toxicity assessment of the drug. These studies help us to estimate the observed toxicity to that dose. TK evaluation is very important in drug development phase in both regulatory and scientific perspective. There are several guidelines to conduct TK study in animals recommended by regulatory bodies (OECD). TK evaluation is useful in selection of dose, dosing form, alternative dosing route, evaluation of toxicological mechanism, and also used for the setting safe dose level in clinical phases. This TK studies also used to reduces the animal number (replacement, reduction and refinement). On the other hand, TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection. Email:marketing@medicilon.com.cn web:www.medicilon.com

A method and assembly for treating biological environments with varying doses of fluids in vitro. The assembly comprises a vessel, a closure and a tubular membrane suspended in the vessel, so as to provide rapid and uniform equilibration of fluids into the vessel. The assembly provides means for carrying out pharmacokinetic and toxicokinetic studies.

1. Field of the Invention

This invention relates to a method and assembly for treating living and non-living biological environments with varying doses of fluids, solutes, drug molecules or diffusible agents in vitro, and more particularly to a vessel and closure assembly having means for releasing and/or delivering fluids, solutes, drug molecules or diffusible agents in environments that may include cell lines and tissue cultures.

2. Description of Related Art

The use of in vitro cell lines and tissue cultures in carrying out pharmacokinetic and toxicokinetic studies related to safety evaluations of new drugs is a growing area of scientific development. By using cell lines and tissue cultures, the need for large animal studies is reduced.

Pharmacokinetic and toxicokinetic studies depend upon the control rate of drug delivery and dose of drug to a cell line for multiple drugs simultaneously. Repeated drug administration can influence the toxicity of the drug by changing its metabolism and stimulating the cell's synthesis of certain proteins that can effect the drug's activity on the cell line.

Current technologies for studying in vitro pharmacokinetics and toxicokinetics use either a closed monolayer culture system (MCS) or a dynamic perfused cell system (DPCS). The MCS employs an immediate total dose application of the test drug. Its limitation is that it operates in essentially a single dose range which can be toxic due to over dose.

On the other hand, the DPCS acts as an in vivo circulating model. The in vitro routes of bolus administration does not mimic the in vivo method of drug administration since their are no tissue masses to act as the sustain release matrix.

A need exists for a delivery system in order to carry out in vitro pharmacokinetic metabolic pathway studies of drugs, proteins, growth factors and other such biologicals. The need arises because it is experimentally undesirable to deliver fluids, such as drugs to cell lines and tissue cultures in bolus concentrations. Bolus concentrations cause the pharmacological dose to quickly and uncontrollably extend into the toxicological range.

Therefore, a special need exists for a drug delivery and/or releasing system which permits the control of drug delivery and/or release and provides an assessment of drug concentration on cell lines and tissue culture environments so as to establish a correlation of drug action with adverse reactions.

Such a need for a new type of drug delivery and/or releasing system for treating biological environments has not been suggested or taught in the literature.

SUMMARY OF THE INVENTION

The present invention is a method and assembly for delivering and/or releasing fluids, solutes, drug molecules or diffusible agents to environments, such as living and non-living biological environments that may include such as cell lines and tissue cultures.

Most preferably, the present invention is an assembly comprising a vessel, a closure and means for delivering fluids, such as drugs, proteins, growth factors and other such biologicals into the vessel. Most preferably, the means for delivering fluids into the vessel is at least one tubular membrane extending through and suspended into the vessel.

Preferably, the vessel is a flask, roller bottle, tube, spinner flask, stirred bioreactor or any vessel that will facilitate cell culture viability. Most preferably, the vessel is a flask or roller bottle. Vessels that are applicable to this invention include those described in U.S. Patent Nos. 5,272,084; 4,770,854; 5,139,952; 4,334,028; 4,289,248; 4,387,822; and 5,047,347, which are incorporated by reference.

Desirably, the closure is a cap, push cap, threaded cap, screw cap or stopper.

Preferably, the tubular membrane extends from an entry port located on the vessel and ends at an evacuation port preferably located on the vessel opposite the entry port. The tubular membrane comprises a first end and a second end, wherein the first end is connected to the entry port and the second end is connected to the evacuation port. The tubular membrane comprises a hollow cavity containing the means to distribute fluid substances.

The evacuation port provides the means for the drainage and/or withdrawal of excess fluid that is not distributed by the membrane into the vessel. Therefore, stagnation of fluid in the membrane is minimized and efficiency of fluid flow through the membrane is enhanced.

Most notably, the assembly of the present invention provides a continuous controlled release of fluids which diffuse from the membrane and into the vessel with minimal stagnation of fluid in the membrane. The present invention eliminates the delivery of bolus concentrations of fluids which causes the pharmacological dose to quickly and uncontrollably extend into the toxicological range.

The tubular membrane is an imperfect barrier separating two fluids and hinders free diffusion of a substance in an isotropic medium. Diffusion is the tendency of molecules to migrate from a region of high concentration (potential and/or activity) to a region of lower concentration (potential and/or activity).

The use of the assembly of the present invention permits the control of fluid delivery and concentration to cell lines and tissue cultures and permits the development of steady state drug diffusion for studying possible mechanisms for the passage of nutrients and drugs and for studying in vitro pharmacokinetic studies on living cells.

The present invention provides important advances over methods used to perform pharmacokinetic and toxicokinetic studies. The assembly of the present invention provides the ability to maintain an in vitro culture system wherein the cells are subjected to an environment which permits them to function in a manner closely simulating that encountered in vivo. The invention also permits the efficient removal of desired products from the cells being cultured and provides important advantages in the efficiency, economy and flexibility of operation.

An alternate embodiment of the assembly of present invention comprises multiple tubular membranes extending through the vessel so that more than one drug solute may be injected into the tubular membrane at one time or at varying time intervals.

A further advantage of the present invention include its use in drug therapy by providing quantitative evaluations of cell populations responding to drug therapy. Whereby, kinetic rate constants, activation energies, entropies and other basic pharmacological parameters can be evaluated. Another feature of the present invention is that metabolic substances such as uremic poisons can be delivered to cells for the purpose of investigating the effects of metabolites on in vitro cultured cells.