Drug formulation Flashcards
What do we mean by tumour-associated hypoxia and what is the signalling pathway
involved?
Tumor-associated hypoxia refers to the condition of low oxygen levels within the tumor microenvironment. It occurs when the supply of oxygen to tumor cells is insufficient to meet their metabolic demands. Hypoxia is a common characteristic of solid tumors due to the rapid proliferation of cancer cells, abnormal blood vessel formation, and inadequate oxygen delivery.
hypoxia-inducible factors (HIFs), prolyl hydroxylases (PHDs), and von Hippel-Lindau tumor suppressor protein (VHL).
Under normoxic (normal oxygen) conditions, PHDs hydroxylate specific proline residues on HIF-α subunits, marking them for degradation via the proteasomal pathway. This occurs with HIF-α subunits, typically HIF-1α and HIF-2α, which are constitutively produced in cells. The VHL protein recognizes hydroxylated HIF-α subunits and binds to them, leading to their degradation.
However, under hypoxic conditions, PHDs are inhibited due to low oxygen levels, preventing hydroxylation of HIF-α subunits. This allows the HIF-α subunits to accumulate and translocate to the cell nucleus. In the nucleus, HIF-α forms a heterodimer with the constitutively expressed HIF-β subunit to create the active HIF transcription factor. The HIF transcription factor binds to specific DNA sequences called hypoxia response elements (HREs) in the promoter regions of target genes.
The binding of HIF to HREs initiates the transcription of various genes involved in adaptive responses to hypoxia. These genes include those regulating angiogenesis (formation of new blood vessels), erythropoiesis (production of red blood cells), glucose metabolism, cell survival, and many other processes. The upregulation of these genes helps tumor cells adapt to the hypoxic environment and promotes their survival and progression.
Which factors in the tumour microenvironment can affect the uptake/effectiveness of drugs
(and how)?
Hypoxia: Tumor-associated hypoxia can reduce drug efficacy. Low oxygen levels can hinder the penetration of drugs into tumor tissues, limiting their access to cancer cells. Additionally, hypoxia activates various cellular responses, such as the upregulation of drug efflux pumps and DNA repair mechanisms, which can confer resistance to chemotherapy.
Tumor vasculature: The abnormal and disorganized blood vessels within tumors can affect drug delivery. Tumor vasculature is characterized by leaky vessels, irregular blood flow, and high interstitial fluid pressure.
Extracellular matrix (ECM): The ECM in the tumor microenvironment consists of proteins, proteoglycans, and other components. It can create physical barriers that hinder drug diffusion into tumor tissues. The dense and stiff ECM can limit drug penetration and distribution within the tumor, reducing its effectiveness.
Acidic pH: Tumors often have an acidic microenvironment due to increased glycolysis and poor perfusion. Acidic pH can affect drug stability and alter its pharmacokinetics. Some drugs may become less soluble or undergo chemical degradation in an acidic environment, reducing their efficacy.
Immune cells: Immune cells, such as tumor-associated macrophages and regulatory T cells, can influence drug response. They can contribute to drug resistance by promoting immunosuppression, creating a protective barrier around cancer cells, or inducing changes in tumor signaling pathways that affect drug sensitivity.
Stromal cells: The presence of stromal cells, including fibroblasts and myofibroblasts, can impact drug efficacy. These cells can produce factors that promote tumor growth and drug resistance. They can also contribute to the formation of a physical barrier around tumor cells, preventing efficient drug delivery.
Tumor heterogeneity: Tumors often exhibit heterogeneity, with distinct subpopulations of cancer cells possessing different genetic and phenotypic characteristics. This heterogeneity can result in variations in drug response among different tumor regions or cell populations. Some cells may be more resistant to the drug, leading to incomplete eradication of the tumor.
What is a bioreductive drug and can you provide examples ?
A bioreductive drug, also known as a hypoxia-activated prodrug (HAP), is a type of medication that is designed to be selectively activated in the hypoxic (low oxygen) regions of tumors.
AQ4N: AQ4N is a prodrug that is selectively converted into a cytotoxic agent in hypoxic conditions. It undergoes enzymatic reduction to generate a DNA-interacting metabolite that can induce cell death. AQ4N has been studied in preclinical and clinical settings as a potential therapeutic agent. The drug is low cytotoxity and no DNA interactions in its prodrug forum however through reduction this flips
What is the function of carbonic anhydrases and which once are linked to cancer?
Carbonic anhydrases (CAs) are a family of enzymes that catalyze the reversible conversion of carbon dioxide (CO2) and water (H2O) to bicarbonate ions (HCO3-) and protons (H+). They play a crucial role in maintaining acid-base balance and pH regulation in various tissues and organs throughout the body.
In the context of cancer, certain carbonic anhydrases have been found to be overexpressed or dysregulated, and their aberrant expression is associated with tumor progression and metastasis.
Carbonic Anhydrase IX
CAIX is induced by hypoxia through the action of hypoxia-inducible factor (HIF) transcription factors. Its expression helps cancer cells adapt to the hypoxic microenvironment by regulating pH homeostasis and promoting survival and angiogenesis.
HIFs have been shown to directly regulate the expression of carbonic anhydrase IX (CAIX) and carbonic anhydrase XII (CAXII). Hypoxia-induced HIFs promote the transcription and subsequent overexpression of CAIX and CAXII in cancer cells.
Define liposomes and how they can be generated from thin lipid film and MLV.
Generally, liposomes are definite as
spherical vesicles with particle sizes
ranging from 30 nm to several
micrometres. They consist of one or
more lipid bilayers surrounding
aqueous units, where the polar head
groups are oriented in the pathway of
the interior and exterior aqueous
phases.
S4 liposome formation by hydration method consists of adding phospholipid + cholesterol and the drug into the solid surface, such as a round-bottom flask or a glass vial.
The liquid is evaporated leaving a thin lipid layer which is then rehydrated using a buffer which forms multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) or Small unilamellar vesicles (SUV).
Extrusion is then performed a technique were the liposome suspension is passed through a membrane filter of defined pore size
Finally filtration chromatography removes any unbound drug leaving only liposomes.
MLVs: The film is gently agitated or vortexed to induce the formation of multilamellar vesicles (lipid bilayers stacked on top of each other), which can have a range of sizes.
LUVs: The hydrated film is subjected to sonication, high-pressure extrusion, or other methods to break down the multilamellar vesicles and form smaller, unilamellar vesicles. These LUVs typically have a more homogeneous size distribution.
Differentiate between passive and active loading of lipsome drugs
Passive loading: drug encapsulation occur during the vesicle formation process.
Hydrophilic drugs in hydrating buffer
Lipophilic drugs in mixture to generate thin dry film
Free, unbound removed
Active loading: drug is entrapped after the formation of vesicles. Weakly acidic or
alkaline drugs
Explain the stability of liposomes.
They are subject to hydrolysis of their ester bonds on their phospholipids additionally the unsaturated fatty acids are subject to peroxidation which can reduce stability.
Fatty acid side-chains - reduce compactness, reducing stability
➢ Polar head chemistry - Neutral, positive, negative charged
➢ Chain length - Lipids with long acyl chain length are most commonly used because high phase
transition temperature.
➢ Degree of unsaturation - Lipids with unsaturated acyl chains are subject to oxidative
degradation, which then causes changes in phase transition temperature, consequently affecting
the stability of the liposome.
➢ Phospholipid:cholesterol - A higher cholesterol to phospholipid ration stabilises liposomes.
➢ PEGylated - coating with polyethylene glycol, a hydrophilic polymer that can prevent the
liposomes from recognition and subsequent clearance by Reticuloendothelial System (RES)
What are the advantages of liposomes
Liposomes increases efficacy and therapeutic index of drug.
2. It increases stability via encapsulation.
3. It reduces the toxicity of encapsulating agent.
4. They help to reduce the exposure of sensitive tissue to toxic drugs.
5. They have flexibility to couple with site specific ligands to achieve
active targeting.
6. They are non-toxic, flexible, completely biodegradable, biocompatible
and non immunogenic for systemic and non systemic administration.
7. Ability to trap both hydrophilic and lipophilic drugs
What are the disadvantages of liposomes ?
Liposomes having low solubility.
2. Short half life.
3. Sometimes phospholipids undergoes oxidation and hydrolysis-like
reaction.
4. Leakage and fusion of encapsulated drugs/molecules.
5. Production cost is high.
6. Fewer stable.
What does cholesterol to phospholipid ratio have t to do with drug release?
More cholesterol is more stable
liposomes however there needs to be a balance.
Explain the steps of active drug loading?
This is usually used for weakly acidic or alkaline drugs.
- the liposome is in a citrate buffer
- Na2Co3 is added making a gradient of protons within the liposome
- the drug is then added which diffuses across into the liposome
What are Factors contributing to the passive uptake of nanoparticles?
Tumour vascular permeability alows liposmes to be taken up easily due to leaky vasulator additionally
Lymphatic drainage is low so the liposmes are retained well.
The level of angiogenesis is poor which does not help drug uptake.
A high Interstitial fluid pressure (Intratumoural pressure) Elevated IFP in tumors leads to increased hydraulic pressure within the tumor interstitium. This increased pressure can impede the convective transport of liposomes within the tumor.
Explain active Active - functionalising the nanoparticles
These liposomes contain area which bind specifically to areas within tumours to increase specificity e.g The adhesion/homing molecule CD44, implicated in cell-cell and cell-matrix adhesion, major
surface receptor for hyaluronic acid (HA) functionalised liposomes.
CD44 is overexpressed in tumour cells and tumour-associated macrophages
Paradox that higher CD44 expression (and potentially the best binders) showed the slower/more
difficult internalization of HA carrier structure in macrophages
or
Doxorubicin encapsulated in liposomes, allows for a greater lifetime cumulative dose of doxorubicin to be
administered (dose-dependent cardiac myopathy).
