PHT 540: Drug Delivery Systems King Saud University College of Pharmacy Department of Pharmaceutics PHT 540: Drug Delivery Systems Parenteral Drug Delivery Systems Principles and Applications

Outlines Introduction Molecular Biotechnology Experimental conditions Experimental Design Novel Parenteral Formulations Formulation Considerations and Potential Ingredients

Why? Why choose a parenteral system? This system ensures that the drug will reach specific target areas of the body via blood and lymphatic systems. It allows the resarcher to have control over pharmacological parameters, serum levels, tissue concentrations, elimination of the drug from the body.

Introduction The parenteral administration of proteins and peptides today offers assured levels of bioavailability and the ability of the product to reach marketplace first. It is safe to assume that over 95% of the protein therapeutics approved by FDA today are injectable products since parenteral administration avoids physical and enzymatic degradation. The most applicable route of parenteral administration are: Intramuscular, Intravenous, Subcutaneous, Epidural and Intrathecal.

Introduction…(cont.)

Introduction…(cont.)

Introduction …(cont.) Applied formulation scientists today face formidable challenges in their quest to formulate stable recombinant protein therapeutics. We are dealing with High MW, highly purified, heat-unstable molecules with high tendency to aggregates. As this happens, chemical and physical changes occur, leading to a great deal of instability. Before prefomulation begin, it is crucial for the formulation scientists to do extensive research on the physicochemical properties of the active protein, peptide, or monoclonal antibodies.

Molecular Biotechnology Complementary DNA: In genetics, a double-stranded DNA or RNA strand consists of two complementary strands of base pairs, which are non-covalently connected via two or three hydrogen bonds. Since there is only one complementary base for any of the bases found in DNA/RNA, one can reconstruct a complementary strand for any single strand. This is essential for DNA replication. For example, the complementary strand of the DNA sequence: A G T C A T G is T C A G T A C A= adenine; G= Guanine; T= thynine; C= cytosine; U= uracil 8

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Recombinant DNA Technology Investigators tried to manipulate the genetic properties of all kind of cells. To achieve this, they simply added foreign DNA to microbial cells. All these attempts failed. Reasons: - Only a limited number of bacteria species is able to take up DNA spontaneously. - Foreign DNA, if taken up at all, is in general not maintained in the receptor cell. DNA brought into a cell from outside will only be maintained if it is able to replicate autonomously, or if it is integrated in the recipient genome. In all other cases foreign DNA will not be propagated and will eventually be degraded through the activity of cellular nucleases. 12

Recombinant DNA Technology …cont. Recombinant DNA technology enables the fusion of any DNA fragment to DNA molecules able to maintain themselves by autonomous replication (such molecules are called replicons). Replicons used as carrier for foreign DNA fragments are termed vectors. The vectors exploited in the DNA technology include plasmids from bacteria or yeast, or DNA from bacteriophages, animals viruses or plant viruses. Recombinant DNA technology or DNA cloning technology: fusion of foreign DNA to the isolated plasmid in order to create a recombinant DNA molecule. 13

Recombinant DNA Technology …cont. Plasmid with only one recognition site for restriction enzyme. The double stranded DNA is then cut. The isolated foreign DNA is also cut. Principle of cloning a foreign DNA fragment. 14

Recombinant DNA Technology …cont. DNA fragments are brought together, the various single stranded ends may recombine due to the presence of a complementary bases. The enzyme DNA ligase, able to catalyze the formation of phosphodiester bonds, is used to create a closed circular recombinant DNA molecule. Principle of cloning a foreign DNA fragment. 15

Recombinant DNA Technology …cont. Some bacterial cells are able to take up DAN under physiological conditions. This process is described as natural transformation. Other method involves the transfer of bacterial cells to package DNA in a bacteriophae capsid and then mimic the normal bacteriophae infection procedures. Phage as a mediator for transfer of recombinant DNA. 16

Recombinant DNA Technology …cont.. DNA Sources: Any DNA can be used to construct recombinant DNA molecules. In protein production based on recombinant DNA technology very distinct pieces of DNA are required. This may be accomplished by using synthetic DNA. This approach allows the choice for a certain amino acids. 17

Recombinant DNA Technology …cont. Production by Recombinant DNA Technology: Human Insulin Human Growth Hormone (hGH) 18

Experimental conditions Experimental conditions for initial preformulation studies: pH Range Buffers Chelating Agents Antioxidants Preservatives Glass Vial Selection Rubber Stoppers Membrane Filter Selections Degradation Mechanisms Formulation Bulk Active Drug

Experimental conditions…(cont.) Chelating Agents The use of chelating agent is a requirement, and recommended dosages may range from 0.01 to 0.05% to bind or chelate the metal ions present in the solution. Antioxidants Since oxidation is one of the major factors in protein degradation, the use of antioxidants may be required. Ascorbic acid and monothio-glycerol have been used for this purpose.

Experimental conditions…(cont.) Glass vial selection The vial should be of type I glass, as stated by USP. Glass is not an inert material. Glass surfaces must be taken into consideration to study the adsorptive properties of the repective protein. Rubber stopper selection The variety of composition of rubber stoppers in parenteral formulations of biopharmaceuticals requires studies on compatibility with proteins, involving chemical extractants from the rubber composition into the protein solution over periods of stability at varying temperatures.

Experimental conditions…(cont.) Membrane Filter Since all protein formulations are aseptically filled for final sterilization of the product, selection of the membrane filter and its media composition is very important. The potential toxicity of the filter and the product’s compatibility with the membrane must be determined. Of all the filter tested, polycarbonate and polysulfone were found to be the most compatible with several proteins, with minimal amounts of protein binding and deactivation.

Novel Parenteral Formulations A pharmaceutical company’s objectives are based upon: Clinical application. Physicochemical properties of the drug. Bioavailability Efficacy Simple formulation Patients acceptability

Novel Parenteral Formulations Vaccine Delivery Nano-Emulsion Technology

PYROGENS AND PYROGEN TESTING

PYROGEN A pyrogen is a material which when injected into a patient will cause a rise in body temperature (pyrexia). The lipopolysacchride that comprise a a major part of the cell wall of gram-negative bacteria are called endotoxins, and it is these that are the most commonly encountered pyrogens. Bacterial cells may be pyrogenic even when they are dead and when they are fragmented, and so a solution or material that passes a test for sterility will not necessarily pass a pyrogen test.

PYROGEN… cont. Two main procedures are used for the detection of pyrogens. The traditional method: It requires the administration of the sample to laboratory rabbits whose body temperature is monitored for a period of time thereafter.

PYROGEN… cont. Steps: 1- Render the syringes, needles, and glassware free form pyrogens by heating at 250 °C for not less than 30 minutes. 2- Warm the product to be tested to 37 °C ± 2 °C 3- Inject into an ear vein of each of three rabbits 10 ml of the product per kg of body weight. 4- Record the temperature at 30-minute intervals between 1 and 3 hours subsequent to the injection

PYROGEN… cont. Steps: 5- If no rabbit shows an individual rise in temperature of 0.5 °C or more above its respective control temperature, the product meets the requirements for the absence of pyrogens. 6- If any rabbit shows an individual temperature rise of 0.5 °C or more, continue the test using five other rabbits.

PYROGEN… cont. Steps: 7- If no more than three of the eight rabbits show individual rise in temperature of 0.5 °C or more and if the sum of the eight individual maximum temperatures rises does not exceed 3.3°, the material under examination meets the requirements for the absence of pyrogens.

PYROGEN… cont. Limulus Amoebicyte Lyste Test (LAL) In recent years, it has been shown that an extract from the blood cells of the horseshoe crab (Limulus polyphemus) contains an enzyme and protein system that coagulates in the presence of low levels of lipopolysaccharides. The discovery has led to the development of the Limulus Amoebicyte Lyste Test (LAL) for the presence of bacterial endotoxins.

PYROGEN… cont. Limulus Amoebicyte Lyste Test (LAL) … cont. In the test procedure, the lysate is mixed with equal volume of the test solution in a depyrogenated container, such as a glass tube. The tube is then incubated undisturbed at 37 °C for a period of about 60 minutes. The test is a pass or fail test. The end point is identified by gently inverting the glass tube. A positive result is indicated by the formation of a solid clot. The clot doesn’t disintegrate when the tube is inverted. A negative result is indicated if no gel clot has been formed.

GEL PYROGEN… cont. Limulus Amoebicyte Lyste Test (LAL) … cont. Proclotting enzyme Endotoxin Activated clotting enzyme GEL Coagulation Clottable protein The Lysate Clotting Mechanism

Formulation Considerations and Potential Ingredients Due to stringent requirements of the parenteral products, only few excipients are acceptable for parenteral delivery. The excipient selected for the parenteral systems should be biocompatible, sterilizable, available as non-pyrogenic grade, non-irritant to nerves and non-hemolytic. Very few excipients comply with all these requirements. For example, the sugar surfactants are biocompatible and have fairly good solubilization potential but they have been found to be hemolytic.