Antimicrobial resistance (AMR) and persistence are associated with an elevated risk of treatment failure and relapsing infections. Alhtough AMR has been widely acknowledged as a pressing issue, the incidence of infections and spread of MDR bacteria is still rising. In addition, the increased use of implanted medical devices such as joint prostheses, artifical heart valves, vascular endoprostheses and pacemakers leads to a higher incidence of biofilm assocaited infeciton, which in turn leads to anotehr important phenomenon; antibiotic tolerance. Antibotic resistance frequenlty resutls in dealyed adequate antibiotic treatment, increasing morbidity and mortality. AMR is the inherited ability of microorganisms to grow at high antibiotic concentration. It is usually quantified by measuring the mininum inhibitory concetnraiton (MIC) of a particular antibiotic wherein resistant bacteria are able to multiply and grow at concetnrations of antibiotics, which are fatal to other strains of the same species. In general, Gram-negative bacteria are less permeable than Gram-positive and show an intrinsic resistance to many antibacterial compounds such as the cell was acive glycoppetide vancomycin. These large molecules are unable to cross the outer bacterial membrane of Gram-negative bacteria and thus cannot target the cell wall of Gram-negative bacteria. In contrast, the Gram-positive bacteria Staphylococcus aureus is naturally susceptible to almost every antibiotic that has been developed, but it is well known for quickly acquiring antibiotic resistance by emans of botaining specific genetic modifications. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020).

Essentially any of the accessory genetic elements found in bacteria are capable of acquiring r genes and promoting their transmission; the type of element involved varies with the genus of the pathogen. In the streptococcie, miningococci and related genera, the exchange of both virulence and pathogenicity genes is highly promiscuous; the principal mechanisms for DNA traffic appears to be transformation. (Davies, “origins and Evolution of Antibiotic Resistance” Microbiology and Molecualr Biology Reviews, 2010, p. 417-433).

Antibiotic presistence or heterotolerance is defined as the ability of a bacterial subpopulation to surive high bactericidal drug concentrations to which the bacteria are fully susceptible. One major chacteristic of antibiotic peristence is a biphasic killing, with a rapid killing of the susceptible populations and a slower killing of the persister subpopulation. Persistent infections are ongoing infections, which are not cleared by the host, whereas antibiotic persistence is used to describe a bacterial population that suvives antibiotic exposure wihtout being resistant. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020)

To combat damage to themselves by antibiotics, bacteria have evolved stratgies that include both intrinsic and acquired resistance. Intrinsic resistance is ahceived by invactivaiton of antibiotics by hydrolytic or chemically modified enzymes, modification of antibiotic targets, cell membrane permeability barriers and antibiotic efflux pumps. In contrast, acquired antibiotic resistance is acieved through HGT, including transformation (bacteria absorb naked DNA for the eternal environment and insert it inot the genome), transduciton (chromosomal and extrachromosomal DNA is transferred between donor and recipitn bacteria via a bacteriophage intermediate), conjugation (mobile genetic elements such as plasmids and integratie and conjugative elements (ICEs) are transferred form one bacterium to another) adn membrane fesicle transport (vesicle-mediated transfer of DNA). These stregies ahve greatly helped bacteria to acquire resistance to antibiotics adn certian extensively ARB such as carbapenemase-producing colistin-resistant Klebsiella pneumonaie are now resistant to almost all antibotics.

Horizontal Gene Transfer:

Acquisition of foreign DNA material through horizontal gene transfer (HGT) is one of the most important drivers of bacterial evolution and it is frequently responsible for the development of antimicrobial resistance. Most antimicrobial agents used in clinical practice are (or derive from) products naturally found in the environmment (mostly soil). Bacteria sharing the environment with these molecules harbor intrinsic genetic determinants of resistance and there is robust evidence suggesting that such environmental resitome is a prolific source for the acquisition of antibiotic resistance genes in clinically relevant bacteria. Classically, bacteria acquire external genetic material through three main strategies (i) transformation (incorporation of naked DNA), ii) transduction (phage mediated) and, (iii) conjugation (bacterial “sex”). (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

The ecological role of naturally produced antibiotics should be to inhibit competitors. Given this role, resistance genes should have evolved to counteract antimicrobial action, so taht the function of each of these determinants is to avoid the activity of one specific antibotic (or a family of antibiotics with similar structures). Work with genes acquired by horizonatal gene transfer (HGT) confirm this idea. Plasmid-encoded beta-lactamases serve to resist beta-lactam antibiotics and are not active agaisnt other kinds of drugs. The same applies for mutation-drived resistance; quinolone reistance is the consequence of mutations in topoisoerases genes, which do not alter bacterial susceptibility to other antibotics. The only impact that hte acquisition of resistance is usually supposed to have on bacterial physiology is to produce a general metabolic burden. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Mutations/modifications of an antibiotic target molecule:

Spontaneous mutations or post translational modifications of an antibiotic target molecule can elad to conformational changes that result in ineffective target binding and attenuation of antibiotic activity.

–Mutations in ribosomal protein genes: 

Ability to pump antibiotics out of cells: The production of complex bacterial machineries capable to extruTetracycline resistance is one of thede a toxic compound out of the cell can result in antimicrobial resistance.  classic examples of efflux-mediated resistance, where the Tet efflux pumps extrdue tetracyclines using protein exhcange as the source of energy. Currenlty, more than 20 different tet genes have been described, most of which are harbored in MGEs. (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

Decreased permeability of the bacterial cell membrane: Intracellular concentrations of antibiotics can be kept low via decreased permeability of the bacterial cell membrane, such as by reducing or mutating proins that would allow entry of antibiotic into the cell. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020)

Modifying antibiotics: Antibiotics can be inactivated by the transfer of chemical groups to vulnerable sites thus inhibiting target binding and function or directly destroyed by hydrolysis. Inactivation of antibiotics by addition of a chemical group, such as acyl, nucleotidyl, phosphate or ribitoyl groups, resulting in steric hindrance can casue antibiotic resistance.

Bacteria can also become resistant to antibiotics by the production of enzymes that digest/metabolize the antibiotic. (Popl, “ARDB-Antibiotic Resistance Genes Database” Nucleic Acids Research 2009, 37)

The main mechanisms fo beta-lactam resistance relies on the destruction of these compound by the action of beta-lactamases. These enzmes destroy the amide bond of the beta-lactam ring, rendering the antimicrobial ineffective. Beta-lactamases were first described in the early 1940s one year before penicillin was introduced to the market; however, there is evidence of their existence for millions of years. (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

Phenotypic Resistance:

Phenotypic resistance refers to those transient situations in which a bacterial population, otherwise susceptible to a given antibiotic, is refractory to its action. This transient resistance does not require a genetic change and thus it is not inheritable. The growth rate is the first parameter that impacts the phenotype of susceptibility to antibiotics of bacterial populations. The relevance of growth rate on the activity of penicillin was already described in 1944, in a study that showed that the activity o this antibotic was impared when cells grew slowly. However, this effect is not restricted to just beta-lactam antibiotics. It has been shown that resting cells are fully resistant to ampicillin or to tetracylcine, whereas streptomycin or ciprofloxacin is still active agaisnt cells in the stationary phase, although their activity is lwoer than that obeserved for exponentially growing bacteria. This situation has been called “drug indifference” and can be relevant for the persistence of bacterial infections even in patients under antibiotic treatment, when bacterai are in a host location that restricts grwoth or when the microoriganisms have ocnsumed the host resources and their growth is impaired. This can be particulalry important in the case of long lasting infections, becasue it was demonstrated that the concentraiton of antibiotics required to cure an experimental infection increases with the duration of the infection. The existance of resting or slow-growing cells which are refractory to treatment, is supposed to be the reason for the need to use prolonged regimes for treating infections by organisms such as Mycobacterium tuberculosis where hypoxia triggers dormancy and TB the organisms undergoes significant metabolic reprograming, with upregulation of stress-related gens and down-regulation of many central metabolism pathways. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Metabolic Shifts:

Two other situations that might confer phenotypic resistance are persistence and growth in biofilms. Whereas it was first propsoed that this situation might be due to the existence of a fraction of nongrowing cells into any bacterial population that is phenotypically resitant to antibiotics, more recent work indicates that there are several difference mechanisms that can lead to prersistence. Metabolic enzymes, global regulationrs and toxin-antitoxin systems are among those elements that are relevant for devleoping persiter pehnotype. Among genes coding for metabolic enzymes, it was ofund that the lnockout of ygfA, which codes for a putative t-formyl-THF cycloligase involved in folate biosynthesis or of yigB, which may clock metabolism by depleting the pool fo flavin monoculcoetide, dereases persistence. Folate deficiency impaired the biosynthesis of purines, thymidilate and methionine. Overepxression of YgfA increased tolerance to ofloxacin. These reuslts support the existence of a linkdage between some specific metabolic pathways and bacterail persistence. Detailed anlsyes of P. aeruginosa biofims showed that the response to antibiotics was highly dependent on the metabolic state of each population. For instance, aerobically growing bacteria were sensitive to ciprofloxacin, but resistant to polymyxin, whereas the opposite was found for cells grwoting in the deepest part of the biofilm, which present an anaerobic metabolism. While some metabolic shifts can make bacteria phenotypically resistant to a particular antibotic, the opposite might occur as well where some specific grwoing conditions can make bacterai more susceptible during infection than when growing in vitro. For example, Listeria monocytogenes is an intracellular pathogen that grows inside mammal cells using hexose phosphates present in the host cytosol. For frwoing into host cells, Listeria upregulates a set of virulence detemrinatns, whose expression is under control of the transcriptional regulator PrfA. One of the PrfA0regulated determiants required for intracellular growth is the sugar phosphate transporter Hpt. This transporter is highly expressed during Listeria intracellular growth, but its expression is very low when bacteria grwo in vitro in the media regulalry used for suspectiblity tests. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Given that antibiotic susceptbility can change as a consequence of laterations in the bacterail metabolism, it is conceivable that bacterail colonization of novel habits might select antibiotic resistance even int he absence of antibiotic selective pressure. One of the best known chronic infectious diseases is cystic fibrosis, which is the most prevalent inherited disease in the Caucasion population. CF pateitns suffer chronic infections in their lungs by different bacterail species such as P. aeruginosa. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Introduction:

Type II diabetes or non-insulin-dependnet diabets mellitus accounts for over 90% of the diagnosed cases of diabetes and affects more than 16 million people in the US and some 200 million people around the world. Type II diabetes is a metabolic disease characterized by hyperglycemia and is a worldwide major public health care problem, affecting some 194 million worldwide.

Pathology:

Decreased Insulin Receptors/Insulin Resistance: Contrasting with type I diabetes, type II diabetic individuals have normal or even greatly elevated insulin levels. Their symptoms arise from an apparent paucity of insulin receptors on normally insulin responsive cells. It has been hypothesized that the increased insulin production resulting from overeating, consequent obesity, eventually, suppresses the synthesis of insulin receptor.  In Type II diabetic people, either the body does not produce enough insulin or the cells ignore insulin. Thus, instead of being absorbed by the cells, the glucose remains in blood leading to hyperglycemia.

Studies of genetically predisposed individuals have indicated that insulin resistance in the muscle is the primary or initating defect leading to the ultimate development of T2D. Thus, skeletal muscle is a mian target to fight against development of insulin-resistance in prediabetic state or during early stage of T2D. (Magnan, “Regnerating islet-derived protein 3 alpha: a promising therapy for diabetes. Preliminary data in rodents and in humans” Heliyon 8 (2022).

Inflammation: Type 2 diabetes is associated with a high cardiovascular risk, which is even increased if renal damage is superimposed. Peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokines are key factors linking type 2 diabetes and atherosclerosis. Navarro (Nephrol Dial Transplant (2008) 23: 919-926) discloses disclsoses isolating PBMCs from diabetic patients with normal renal function and different stages of diabetic nephropathy and showing a relationship between inflammatory activation of PBMCs by enhanced mRNA expression of TNF-alpha and IL-6.

Causes of Diabetes

Obesity is the most important nutritional disorder in the western world, with the estimates of its prevalence ranging from 30-50% within the middle aged population. It is usually defined as a body weight more than 20% in excess of the ideal body weight. Obesity is associated with an increased risk for cardiovascular diseases, diabetes, stroke, muscular dystruphy and infertility. In particular, obesity can evolve to type II diabetes in successvie phases.

Obesity is defined by the WHO as an abnormal or excessive fat accumulation that may impair health. It is defined by body mass index (BMI), weight in kilograms divided by the square of height in meters, in adults over 30 kg/m2. (Chavda, Molecules 2022, 27, 4315).

The genetic basis for obesity and diabetes has gradually progressed. Zhang cloned the mouse obesity (ob) gene and its human homologue in 1994 (Zhang, Nature, 372 (2994) 425-432). Mutation in ob leads to symptoms of obesity. Several other single gene mutations resulting in obesity in mice have been identified. For example, the yellow mutation at the agouti locus has been found to cause a pleiotropic syndrome which causes moderate adult onset obesity, a yellow coat color, and a high incidence of tumor formation (Herberg and Coleman (1977), Metabolism 26: 59) and an abnormal anatomic districution of body fat (Coleman (1973) Diabetologia 14: 141-148).

The most widely used method to gauge obesity is the body mass index BMI which is equal to weight-height2in kg-m2.  A BMI of 30 is most comonly used as a thereshold for obesity. A BMI between 25’30 should be viewed as medically significant.

Complications/Symptoms of Diabetes:

see WebMD (discusses diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy).

Prevention/Treatment Strategies:

Treatment of T2D includes lifestyle changes such as diet, exercise, and mutraceuticals, as well as administration of medications such as metformin. Conventionally, two sorts of therapeutic agents have been used for the treatment of T2D. (Chavda, Molecules 2022, 27, 4315).

The obesity market is currently dominated by Novo Nordisk and Eli Lilly. Several companies including Pfizer, Roche, Viking Therapeutics and Rivus Pharmaceuticals are also developing obesity therapeutics. Some leverage the GLP-1 pathway, while others commit to different mechanisms, including amylin analogs and mitochondrial uncoupling. Other players in the space include Veru Pharmaceuticals, Regneron, Altimmune, Amgen and Amylyx.

Currenlty, various pharmacological approaches are avialbe for treating hyperglycemia and subsequently, T2DM. These may be groups into the following classes:

Insulin secretogogues: including sulphony-ureas (e.g., glipizide, glimepiride, glyburide), meglitinides (e.g., nateglidine, repaglinide), dipeptidyl peptidase IV 9DDP-IV inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin, saxogliptin), and glucagon-like peptide-1 receptor (GLP-1R) agonists (e.g., liragludie, albiglutide, exenatide, lixisenatide, dulaglutide, semaglutide) which enahce secretion of insulin by acting on the pancreatic beta-cells. Suponyl-ureas adn meglitinides have limited effiacy and tolerability, cause weight gain and often induce hypoglycemia. DPP-IV inhibitors have limited efficacy. Marekted GLP-1R agonists are peptides adminsitered by subcutaneous injection. Liraglutide is additionally prroved for teh treatment of obesity.

Biguanides (e.g., metformin) are thought to act primarily by decreasing heaptic glucose production. Biguanides often casue gastrointestinal disturbances and lactin acidsosis, further limited their use.

Inhibitors of alpha-glucosiase (e.g., acarbose) decreases intestinal glucose absorption. These agents often cause gastrointestinal disturbances.

Thiazolidinediones (e.g. pioglitazone, rosiglitazone) act on a specific receptor (peroxisome proliferator-activated receptor-hamma) in the liver, musle and fat tiessues. They regualted lipid metabolism subsequently enhancing the response of these tissues to the actions of insulin. Frequent use of these drugs may lead to weight gain and may induce edmea and anemia.

Insulin is used in more severe cases, either alone or in combination with the above agents, and frequent use may also lead to weight gain and carries a risk of hyoplycemia.

Sodium-glucose linked transporter cotransporter 2 (SGLT2) inhibitors (e.g., dapagliflozin, empagliflozin, canaglifloxin, ertugliflozin) inhbit reabsorption of glucose in the kidneys adn thereby lwoer glucose levesl int he blood. This emerging class of drugs may be assocaited with ketoacidosis and urinary tract infections.

Lectins:

–Regenerating islet-derived protein 3alpha (Reg3alpha, Reg3A) (hepatocarcinoma-intestinal-pancreas/pancreatitis-associated protein (HIP/PAP)): is a 16 kDA type C lectin protein with a single carbohydrate binding domain. Reg3alpha is also secreted and carries out its biological activites in an autocrine and paracine manner. It could also act in an endocrine manner since it is also detected in the bloodstream. This protein has been shown to drive tissue repair and regeneration as well as protection against oxidative sttress and cell death in the liver, pacreas and intestine. It also promotes motor neuron axonal survival and guidance and Schwann cell proliferation. It shows anti-inflammatory activites and yeilds antibacterial activiteis in the digestive tract. The prtoein shows an intrinsic ROS cavenger actiity targeted on the extra-cellular matrix in the context of inflammation. Administration of Reg3alpha has been shown to control glucose homeostasis and could potentially be a new target of interest in the treatment of type 2 daibetes. In this respect, recombinant human Reg3alpha prtoein was adminsitered to insulin resistant mice fed a high fat diet. An increase in insulin sensitivity during an oral glucose tolerance test and decrease in teh pro-inflammtory cytokine C-X-C Motif Chemokine Ligand 5 (CXCL5) was evidenced. There was also an increase in glucose uptake in the skelatal musle. (Magnan, “Regnerating islet-derived protein 3 alpha: a promising therapy for diabetes. Preliminary data in rodents and in humans” Heliyon 8 (2022).

Biomarkers Related to Diabetes:

Lower Your A1C levels:

When you have diabetes you should check your blood sugar regularly. Also recommended is to check A1C levels, which measures the amount of sugar which attaches to the protein hemaglobin. In general, A1C levels should be below 7%. When you take steps ot lower your A1C, you can reduce complications such as nerve damage, eye problems and heart disease.

Incretins (“Incretin homones):

Incretins stimulate beta cells to release insulin and were discovered in the early 1972. These products are secreted in the intestine, affecting the functioning of beta cells. The most commonly known incretins include glucagon-like peptide-1 (GLP-1) and glucose-dependent inulinotropic polypeptide (GIP). GLP-1 and GIP are peptide hormones that are secreted with the benefit of utilizing cells from intestines called enteroendocrine cells in reaction to the consumption of nutrients, and they have an important function in posprandial metabolism. With the identification of glucose equilibrium, their most favorable condition, the incretin effect, begins to improve the glucose-stimulated release of insulin from the pancreas. GIP is considered to be the essential incretin hormone accounting for this effect.( Chavda, Molecules 2022, 27, 4315)

GLP-1 is a 30 amino acid long incretin hormone secreted by the L-cells in the intestine in response to ingestion of food. GLP-1 has been shown to simulate insulin secretyion in a physiological adn glucose-dependent manner, decrease glucagon secretion, inhibit gastric emptying, decrease appetite, and stimulate proliferation of beta-cells. In non-clinal experiments GLP-1 promotes continued beta-cell competence by stimulating transcription of genes important for glucose-dependent insuline secretion and by promoting beta-cell neogensis. In a healthy individual, GLP-1 plays an important role regualting post-prandial blood glucose leves by sitmulating glucose-dependet insulin secretion by the pancrease resutling in increased glucose absorption in the periphery. GLP-1 also suppresses glucoan secretion leading to reduced heaptic glucose output. In addition, GLP-1 delays gastric emptying and slows small bowel motility delaying food absorption. In people with T2DM, the normal post-prandial rise in GLP-1 is absent or reduced. (WO 2018/109607)

In 2019, the FDA approved Novo’s Rybelsus for the first oral GLP-1 for type 2 diabetes. Considered by many to be the next frontier in obesity treatment, oral medicines are under development by a number of companies, including Novo, whose amycretin is in phase I trials  and Lilly, which has a Phase III trial underway for orforglipron. Brazilian owned multinational company Eurofarma also has a Phase III study under way for its oral candidate sibutramine/topiramate XR. Phizer recently accouned that it would move forward with a modified release formulation of its candidate danuglipron and in December 2023, San Francisco-based Structure Therapeutics accouned postive resutls form a Phase IIa trail of its weight-loss pill GSBR-1290. (clinicaspace, “Obesity and Diabetes Markets Explode”, 2024).

Glucagon-like peptide 1 (GLP-1) Agonists:

GLP-1 receptor agonists make up a class of medications used for the treatment of type 2 diabetes and obesity. The first GLP-1 receptor agonist to reive FDA approval was exenatide in 2005 and the FDA subsequently approved several other drugs with the same mechanisms of action. More than 15 years latter, these products remain costly with monthly net prices rising to more than $600 in 2017. The mediuan annual out of pocket cost in Medicare Part D exceeded $15000 in 2019. Manufacturers earned mroe than 10 billion on GLP-1 agonists in the US alone in 2021. (JAMA, “Patents and Regulatory Exlusivities on GLP-1 Receptor Agonists”, 2023, 330(7)).

Other FDA approved GLP-1 receptor agonstis include Victoza (Liaglutide), Bydureon (Exenatide), Saenda (Liraglutide), Adlyxin (Lixisensatide), Xultophy (liraglutide), Soliqua (lixisenatide)and Ozempic, Rybelsus (Semaglutide).

Most marketed GLP-1 receptor agonists are drug-device combinations with active ingredients sold together with their subcutaneous inector pens. Device patents expiring later than other patents may force generic firms to either wait until these patents expire before market entry or undertake lenghty and costly patent challenges. (JAMA, “Patents and Regulatory Exlusivities on GLP-1 Receptor Agonists”, 2023, 330(7))

GLP-1 is a hormone that is screted by the small intestine, generally promotes the biosynthesis and secretion of insultin, inhibits the secretion of glucagon. (Song, WO 2008/082274)

Song, WO 2008/082274 discloses an insulinotropic peptide for promoting the syntehsis and expression of insulin such as GLP-1 conjugate haivng improved in vivo duration and stability which includes an insulinotropic peptide, a non-peptide polymer and an immunoglobulin Fc region which are covalently linked to each other. If the insulinotropic peptide to be coupled at a site other than the N-temrinus, a reactive thiolgroup can be introduced to the site of amino acid resiude to be modified in the native amino acid sequence to form a covalent bond using a maleimide linker at hte non-peptidyl polymer. Alternatively, a reactive amine group can be introduced to the site of amino acid resiude to form a covalent bond using an aldehyde linker at the non-eptidyl polymer.

–Exenatide (Gyetta, AstraZeneca):  is a GLP-1 receptor agonist released from the gut and acts to increase glucose-dependent insulin secretion from pancreatic beta cells, suppress glucagon secretion, delay gastric emptying, and reduce food intake. The binding of the drug to pancreatic GLP-1 receptors mediates these actions. Administration of exenatide has shown restoration in the insulin response that is usually defective in type 2 diabetic patients. Researchers also observed the prolonged release of insulin in response to elevated glucose levels was also observed in patients treated with exenatide. Treatment with exenatide leads to the decreased release of glucagon during hyperglycemic periods, which reduces hepatic glucose output as well as decreases insulin demand. Delayed gastric emptying decreases the rate at which glucose arrives in the bloodstream. Exenatide is available as an immediate-release solution and extended-release suspension for subcutaneous administration. The administration of these injections should be in the thigh, abdomen, or upper arm. Additionally, patients should receive instruction to use a different injection site for each dose to prevent infection.

Exenatide is made by Amylin Pharmaceuticals and commercialized by AstraZeneca. Exenatide was approved by the FDA in April 2005, for people whose diabetes is not well controlled on other oral medications.

–Semaglutide (Ozempic, Novo Nordisk): is a GLP-1 RA and widely used. An oral form of Simaglutide to be adminsitered once a wekk is also available. (Chavda, Molecules 2022, 27, 4315)

–Tirzepaptide (Mounjaro™): is comparable to GLP-1 agonists, as it is a dual GIP/GLP-1 agonist. The FDA has approved tirzepatide under the brand name Mounjaro, which has become a revolutionary agent for the management and treatment of T2D and achieving weight loss. The most common side effects assocaited with tirzepatide are related to gastrointestinal tract like nausea, vomiting and diarrhea. Tirzepatide is a single molecule that combines dual agonism of glucose-dependent insulain tropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. Tirzepatide binds to GIP and GLP-1 receptors with high affinity that is similar to that of native GIP and about 5 fold weaker than that of native GLP-1. Native GIP and GLP-1 are incretin hormones that stimulate insulin secretion and decrease glocagon secretion. GIP also plays a role in nutrient and energy metabolism, while GLP-1 also delays gastric emptying, suppresses appetide and improved satiety. Eli Lilly is developing irzepatide for the treatment of type 2 diabetes mellitus (T2DM), obesity, cardiovscular disorders in T2DM, hert failure, non-alcoholic steatohepatitis, obstructive sleep apnoea and for reducing mortaility/morbidity in obsity. (Syed Druges (2022) 82: 121-1220).

Tirzepatide is a synthetic linear peptide molecule containing 39 amino acids. Residues derive form GLP-1, GIP and semaglutide and few residues are unique. The strucure is based on the native GIP sequence and includes C20 fatty diacid moeity (eicosanedionic acid) linke via hydrophilic linkesconnecte to lysine reisudes at C20 position. The peptide sequence contains two non-coded amino acid resiudes at position 2 and 13 which are responsible for its long half-life and high affinity to albumin. The main critical improvements are modificaiton of residues in the peptide backbone to obtain GIP receptor activating activity. Tirzepatide is the first agent that functions as a dual agonist for the two main human GLP-1 and GIP incretins. It has impressive glycemic efficacy. Moreover, it is the first effective drug to have demonstrated notable body weight loss in a phase 3 study in patients with T2D. Trizepatide has significanlty better therapetuic efficacy than current drugs. It is also superior to semaglutide and insulin degludec. Phase 1 clinicl trails which lasted for 4 weeks, followed by 4 weeks of safety investigation, for tirzepatide showed a remarkable statistical decrease in HbA1c, and postprandial glucose levels were also found to be diminished. A 26 week long phase 2 clinical trials, which also included a dulaglutide group, showed better efficacy over dulaglutide. It also contributed to reduction of weight and decreased appetide in the subjects. In the SURPASS-1 phase 3 clinical trails, which included six countries worldwide, patietns who had ongoing treatment with SLGT2 inhibitors were included. Soytel with or without metformin, a dose-dependent impact on HbA1c and weight loss was observed, which was larger than that of the slective GLP-1RA dulaglutide. Meanwhile, in a 12-week phase 2 trial with moderate dose escalation regimens, gastrointestinal tolerability improved when starting with a lower dose and slowly increaseing to the highest effective doses. Other additional effect of tirzepatide were noted to lowering the concentrations of very low-density lipoproteins and triglycerides, in addition to a reduction in blood pressure, as well as an elevation of high density lipoprotein concentraiton. The adverse events most commonly reported were nausea, vomiting and diarrhea, which were mild to moderate and occurred mostly during the dose-escalation period.   (Chavda, Molecules 2022, 27, 4315) Besoe most common adverse events were gastrointestinal events, including nausea, vomiting and diarrhea. Most of these events were mild to moderate. n (US 2020/0023040) disclsoes administration of tirzepatide from a lower starting escalation dose to a higher maintenance dose.

Metformin: is an FDA approved drug to treat Type 2 diabetes. It is an oral medication approved for people as young as 10. While Mounjaro provides better blood glucose control and more potential weight loss than metformin, metformin is often more cost effective for many people. Metformin has been a mainstay when it comes to treating type 2 diabetes for decades. Metformin remains the go to choice for many providers when prescribing a Type 2 diabetes medication because it works and is cost effective. Metformin effectively lowers hemoglobin A1C (HbA1C or A1C). Your A1C represents your average blood glucose over 3 months.

Metformin is a biguanide. Similarly to Mounjaro, it lowers the amount of glucose the liver makes. But Metformin does not increase pancreas release of insulin. Instead, it makes the body more sensitive to the insulin one makes naturally. It also lowers the amount of glucose one absorbed from the food. Because Mounjaro and metformin work in two different ways, they can be combined for additional blood glucose improvement.

–Metformin and Herbal Formula:

Metformin and a Chinese herbal formula may ameliorate type 2 diabetes with hyperlipidemia via enriching beneficial bacteria, such as Blautia and Faecalibacterium. The herbs used were Rhizona Anemarrhenae, Momordica charantia, Coptis chinensis, Ale vera and red yeast rice (Zhao, “Structural alteration of gut microbiota during the amelioration of human type 2 diabetes with hyperlipidemia by Metformin and a traditional chinese herbal formula: a multicenter, randomized, open label clinical trial” mBio, May/June 2018, volume 9).

Naltrexone and Bupropion (Takeda, Contrave): is indicated for weight loss. BUPROPION; NALTREXONE is a combination of two drugs that helps one lose weight. The product is used with a reduced calrie diet and exericse. The product can also help in maintaining weight loss. Pateints should watch out for new or worsening depression or thoughts of suicide.

liraglutide (Novo Nordisk, Victoza): is approved in 2010 for type 2 diabetes.

Insulin: insulin or its analogs, such as insulin lispro, insulin aspart, and oral agents like glipizide, glimepride, metformin, acarbose, pioglitzone and saagliptin. In addition to these agents, a short acting insulin that can be inhaled just before nutrient intake –Exubera® –haary 2006. (Chavda, Molecules 2022, 27, 4315)s been approved by the FDA in Junuary 2006. (Chavda, Molecules 2022, 27, 4315)

–NovoLog is a modified type of medical insulin used to treat type 1 and type 2 diabetes. It is generally used by injection under the skin but may also be used by injection intoa vein. It works like human insulin by increasing the amount of glucose that itssues take in and decreasing the amount of glucose made by the liver. It is a manufactured from of human inslun where a single amino acid hs been changed (protein with aspartic acid at the B28 potion). It was approved by the FDA in 2000. In 2022, it was the 76th most commonly prescribed medication in the US. Manufacturing includes eyast, which have had the gene for insulin aspart put into their genome.

Inhbitors of sodium-glucose cotransporter 2 (SGL2):

–Empaglifloxin (Jardiance): is an antidiabetic mediciton used to improve glocuse control in people with type 2 diabetes. It is taken orally. The use of empaglifloxin has been shown to improve outcomes in epople with established CVD and it can help to slow the rate of kidney funciton decline. It is an inhibitor of the sodium glucose co-transporter-2 (SGLT02) and works by increasing sugar loss in the urine. It was developed by Boehringer Ingelheim and is co-marketed by Eli Lilly. It is on the EHO Model List of Essential Medicines

–Farxiga (Dapagliflozin): is used to treat type 2 diabetes. It is also used to treat adults with heart failure and chronic kidney disease. It reversibly inhibits sodium-glucose co-transporter 2 (SGL2-) in the renal proximal convoluted tubule to reduce glucose reabsorption and increase urinary glucose excretion. It was developed by Bristol-Myers Squibb and AstraZeneca. It is on the WHO Model List of Essential Medicines

Organ transplantes: In the late 1990s, ressearchers first showed that pancreatic islet cells recovered form deease donors could help patients with T1D live an insulin-free existence. However, only a limited number of donor pancreases are available each year and those organs are often of variable quality. (Eli Dolgen Nature Biotechnology, 40, March 2022, pp. 291-295)

Stem Cell Protocols:

Stem cel differentiation proteocls (initially for naked administraiton together with immunosuppression)geard toward producing insulin-producing beta cells has been of intense focus given the limited availability and quality of deceased donor pancreatic islet cells. In parallel, the field has endeavored to obviate the need for immunosuppression through the use of immune privileged devices or protective materials; an approach called “islet encapsulation”. ViaCyte for example is focused on adminsitering its cells inside ouches deisgned to keep out immune cells. This encapsulation device named “Encaptra” is a pouch made of polytetrafluoethylene membranes with 0.45 um pores that is inserted into a skin flap The device relies on a well-characterized human enbryonic stem cell (hESC) line known as CyT49, The cmpany then oconverts those cells into pancreatic progenitors through a stepwise differentiation protocl. Over the course of 12 days in the laboratory, the cells progress sequentially through various stages of develoment –mesendoderm, anterio definitive endoderm, primitve gut tube, posterio foregul, before forming the pancreatic endoderm cells which go into the transplantable packets. (Eli Dolgen Nature Biotechnology, 40, March 2022, pp. 291-295)

Transmissible spongiform encephalopahties (TSEs) are an unusual group of diseases characterized by the presence of numerous small cavities in the brains of affected individuals. These cavisties are casued by the loss of neurons, and can take decades to form following the initial infection by the causative agent.

TSEs include scapie in sheep, bovine spongiform encepholopathy (BSE( or “mad cow” disease, chronic wasting disease in deer, and Creutzfeld-Jakob disease (CJD) in humans.

TSEs can be transmitted experimentally by ijecting infected brain tissue into a recipient animal’s brain. TSEs can also spead via tissue transplants and apparently by consumption of tainted food. In the late 20th century, the diease kuru was discovered among member of the Fore people of Papua New Guidea. These members engaged in ritualistic consumption of their dead which lead to the spread of a commonly fatal TSE through the tribes.

Mad cod disease spread widely among cattle herds of England in the 1990s becaseu cows were fed bone meal prepared form sheep and cattle carcasses to increase the protein content of their diet. Like the Fore, teh cattle were eating the tissues of animals that had died of the disease.

TSEs are casued by prions (proteinaceous infectious particles).

American Parkinson Disease Association

Introduction:

Parksinson’s diease (PD) is a progressive neurological disorder known for its characteristic motor symptoms, which include tremor, regidity, and slowness of movement. Among these, rest or resting tremor (RT) – a shaking that occurs when muslces are relaxed -is one of the most recognizable yet least understood. (“Parkinson’s symptomk resting remor, relates to dopamine in unexpected way” Noember 18, 2024.)

Pathology: 

Alpah-synuclein (alpha-Syn) and other misfolded proteins: 

Parkinson’s Disease (PD) is a neurodegenerative disease casued by progressive accumulation of abnormal intracellular aggregates of alpha synuclein (alpha-Syn) protein existing as Lewy bodies, the pathological hallmark of the disease. Lewy bodies first appear in the olfactory bulb and medulla and gradually spread to midbrain, at which time, the first motor sign of PD appear. Concomitantly, inflammatory responses form resident microglia result in T-cell recruitment, setting off an exacerbating inflammatory cascade. ogether, these events lead to the progressive demise of nigrostriatal dopaminergi nuerons, resulting in the classical clinical signs of bradkykinesis, rest tremor and regidity. Symptomatic relief is provided by dopamine replacement, but the underlying disease process continues unabated. Cao (US Patent Application No: 2017/0196948, published as US 10,653,759)

Structurally, human alpha-Syn is an intrinsically disordered 140 amino acid long protein consisting of three distinct regions: an N-temrinal region (residues 1-60) which forms a helical structure and interats with the cellular membrane, a central highy aggregation-prone non-Abeta component region (residues 61-95) and a C terminal region (residues 96-140) that is highly enriched in acidic residues and prolines. Cao (US Patent Application No: 2017/0196948, published as US 10,653,759)

Preclinical evidence has suggested that other misfolded proteins including hyperphosphorylated tau, prior proteins, huntington, TAR DNA binding protein 43, and mutant superoxide dismutase 1 (SOD1) can also be targeted for immunotherapeutic strategies Cao (US Patent Application No: 15/313,810, published as US 10,653,759)

Alpha-Synuclein has been identified as the major component of such inclusions and it is found in the brains of PD patients and patients wtih other degenerative disorders such as the LB variant of Alzheimer’s disease.

Dodel (US 2013/0052200) discloses naturally occurring autoantibodies that bind to alpha-Synuclien from human sera and commercial IgG preparations (IVIG).

Alpha-synuclein is a protein which builds up in PD pateints. (Morreale, “Cancer Immunotherapy applied to Parkinson’s Disease” June 20, 2024). Aggregates of the brain protein alpha-synuclein (alpha-Syn) are generally considered to have a major role in the pathological development and progression of PD. Cao (US 10,653,759)

–Associated Proteins with Alpha-Synuclein:

Dawson and others have discovered that a cell surface marker, Aplp1, binds to Lag3 and drives PD. Interestingly, Aplp1 has been associated with the spread of alpha-synuclein throughout the brain once binding with Lag3. Aplp1 bound to Lag3 allows brain cells to accmulate clumps of alph-asynuclein, which resutls in cell death promoting PD symptoms. The binding of aplp1 and Lag3 suggests that by targeting this interaction, PD symptoms could be significanlty delayed. This discovery is exciting becasue Lag3 checkpoint inhibiotrs is alreay FEDA approved and could reasibly apply to PD pateints in cominbation with other treamtents. Researchers discvoered that mice lacking Aplp1 and Lag3 had 90% reduction of alpha-synuclein adsorption. Anti-Lag3 also prevented the adsorption of alpha-synuclein and significantly slowed PD symptoms. (Morreale, “Cancer Immunotherapy applied to Parkinson’s Disease” Immunology, June 20, 2024)

Dopamine levels: 

Parkinson’s Disease (PD) is a degenerative neurological condition linked to reduced dopamine levels in the brain, caused by degeneration and death of “dopaminergic” neurons. PD is the second most common neurodegenerative disorder globally as it affects about 1% of the population over 65 years old worldwide. It is clinically characterized by resting tremor, slowness of movement, muscular rigidity and impairment of postural reflexes. The progressive loss of dopaminergic neurons in the substantin migran and formation of fibrillar cytoplasmic inclusions term “Lewy bodies” and “Lewy neurites” are the nueropathological hallmarks of PD.

However, a new study from the Champalimaud Foundation, led by scientifists at the Nueral Circuits Dysfunction Lab in collaboration with the Neuropsychiatry and Nuclear Medicine Labs, indicated the preserved dopamine in certain brain regions may actually contribute to remor symptoms, challenging common beliefs. Paradoxically, they discovered that patietns who exhibit tremor have ore dopamine preserved in the caudate nucleus, a part of the brain important for movement planning and cognition. This challenges traditional udnerstanding of how dopamine loss related to PD symptoms and highlights the importance of looking beyond general classificaitons in PD and underscores the need for mroe nuanced approches informed by underlying biology, which culd ultimately hep to inform on therapeutic strategies. Dopamine loss in brain regions including the putamen, assocaited with movement regulation, is a well established hallmark of PD. Dopamine loss is also necessary for rest tremor to be present. However, while some patients experience significant tremor relief with dopamine replacement therpaies, such as L-DOPA, toehrs see little to no improvement, or even a worsening of symptoms. This suggests that the link between dopamine depletion and RT is more complex than a simple DA dose dependency model, the term noted. Resting remor (RT) is a Parkinson’s disease symptom with an unclear relationship to the dopaminergic system. By combining imaging data with measurements from these sensors, they observed a clear link between dopamine function in the caudate nucleus and global severity of resting tremor. The analysis suggested that the more dopamine activity preserved in the caudate, the stronger the tremor. An in more intriguing finding was that the more dopamine was preserved in the caudate on one side of the brain (each hemisphere has its own caudate), the more tremor there was on teh same side of the body. (usually, each side of the brain controls movement on the oppsite side of the body) The study also noted that not all dopamine cells are alike. They have different genetic maekups, connections and functions. This means that which cells a patient loses or keeps could affect theri symptoms. For example remor might be tied to the loss or preservation of specific dopamine populations that connect to certain brain areas. This variation in cell type loss could further expalin the wide range of symptoms among PD patietns. “Parkinson’s symptomk resting remor, relates to dopamine in unexpected way” Noember 18, 2024.)

Leucine-rich repeat kinase 2 (LRRK2): is a protein implicated in the pathogenesis of Parkinson’s disease and progressive supranuclear palsy (PSP). Increased activity and expression of LRRK2 are linked to the development of these neurological diseases.

Genetic Variants:

Although therapeutic modificaiton of several genetic targets has reached the clinical trail stage, a major obstacle in conducting these trials is that PD pateints are largely unaware of their genetic satus and, therefore, cannot be recruited. Expanding the number of investigated PD related genes and genes related to disorders with overlapping clinical features in large, well phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene targeted clinical trails. (Hanseen, “Relevance of genetic testing in the gene-targeted trails era: the Rostock Parkinson’s disease study” Brain 2024: 147; 2652-2667).

The genetic landscape of PD and related phenotypes is multifaceted. Even when considering only monogenic causes of classical PD, pathogenic variants in seven genes (LRRK2, PRKN, PINK1, SNCA, PARK7, VPS35 and CHCHD2) are implicated. Furthemore, heterozgous changes in GBA1 are a strong risk factor for PD. In addition, >30 other more complex monogenic movement disrders may present with atypical parkinsonism or may have parkinsonism as a prominent or even predominant clinical feature in at elast a subset of patients. (Hanseen, “Relevance of genetic testing in the gene-targeted trails era: the Rostock Parkinson’s disease study” Brain 2024: 147; 2652-2667)

The ROPAD study is an observational clinical study which assesssed the frequency and type of pathogenic variants in known PD related genes adn genes realted to other movement disorders or dementia in a multicentry, international setting. The study is registed at clinicaltrials.gov (NCT03866603) and is a collaboration between CENTOGENE GmbH (Rostock, Germany), the University of Lubeck and Denali Therapeutics (San Francisco, CA).

Treatment: 

Treatments to manage Parkinson’s disease (PD) symptoms include medications that target depression, tremors, pain, and pysical movement. Most of the medications increase the levels of dopamine in the brain, which is a chemical that helps relay information from your brain to the rest of your body. However, new therapeies are being investiated to better control and slow down the progression of PD (Morreale, “Cancer Immunotherapy applied to Parkinson’s Disease” June 20, 2024).

There are about 20 therapies on the market that address the syptoms of Parkinson’s diease. However, currently there are no dieasese modifying treatments becasue the underlying cause of the disease is still unclear. (McKenzi, “7 Alzheimer’s and Parkinson’s Programs Discarded in 2024” BioSpace, November 4, 2024).

Dopamine Receptor Stimulators:

–Rotigotin: is a dopamine receptor stimulator that has been used to treat Parkinson’s since the 1990s. 

A rotigotine transdermal path is sold by UCB Manufacturing.

–Tavapadon (Cerevel Therapeutics aquired by Abbvie)is a partial agonist that activates dopamine D1/D5 receptors, which regulate motor activity. It is taken once daily and can be used as a monotherapy or in combination with other drugs.

–Vyalev (Abbvie) combines two drugs that help boost dopamine levels in the brain and keep symptoms under control for a full 24 hours. Instead of taking pills throughout the day, this treatment is delivered through a small pump that continuously injects the medication under the skin. This new treatment offers an alternative to oral medications, which can become less effective as Parkinson’s progresses. The most common side effects include skin reactions where the injection is given, involuntary movements (dyskinesia), and hallucinations (seeing or hearing things that aren’t there).

Alpha-Synuclein Inhibitors:

–Anti-alpha-synuclein:

Cao (US Patent Application No: 15/313,810, published as US 10,653,759) discloses methods of protecting against dopaminergic neuron cell death which includes administering an anti-alpha-synuclein peptide to a subject in need thereof, where dopaminergic neurons are protected from cell death due to alpha-synuclein mediated neurodegeneration. In preferred dmeobdiments, the anti-alpha-synuclein peptide anitibody is specific for the N-temrinal region of the alpa-synuclein protein.

It has also been reported that anti-alpha-Syn mAbs directed against the C terminal of alpha-Syn enhanced the clearing of intracellular alpha-Syn aggregates. Cao (US Patent Application No: 15/313,810, published as US 10,653,759)

Leucine-rich Repeat Kinase 2 (LRRK2) Inhibitors:

–Proteolysis-Targeting Chimeras (PROTACs): are an emerging therapeutic approach for neurodegenerative diseases like Parkinson’s, by targeting degrading disease causing proteins such as LRRK2. PRTOACs are molecuels designed to induce the degradation of specific proteins by recruiting the body’s natural protein degradation machinery, the proteasome.

Arvinas ARV-102 (a novel orgal PROTAC designed to garget LRRK2 and currently in phase 1 clinical studies. It is designed to cross the blood-brain barrier and target LRRK2.

Vaccines:

—-DC Vaccines:

Dendritic cell (DC) vaccination is a cell based therapy that elicits an immune response by using antigen-loaded DCs as the vehicle for immunization. DCs loaded with peptide directly interact with the immune system without eliciting generalized inflammation that typically occurs in adjuvant-containing vaccines. Moreover, peptide sensitized DC vaccines trigger a longer laster antigen-specific T cell response unlike the shorter respones to traditional vaccines. Cao (US Patent Application No: 15/313,810, published as US 10,653,759)

—-Against Alpha-Syn:

Antigen sensitved DCs have been used as vaccines for treating PD. For example, Cao (US Patent Application No: 15/313,810, published as US 10,653,759) discloses a DC vaccine against alpha-synuclein. In some embodiments, more than one antigen sensitized DC is utilized in the mixture of compositions. In certain embodiments DCs sensitived with full lengh rh-alpha-Syn or with peptide fragments from h-alpha-Sun are effective in triggering the generation of anti-alpha-Syn antibodies in a Tg alpha-Sun mouse model of synucleinopathy.

The term thromobitic microangiopathy (TMA) encompasses a group of conditions that are defined by, or result from, a similar histopathological lesion. Hemolytic uremic syndrome HUS), thrombotic thrombocytopenic purpura (TTP) and several other conditions are assocaited with TMA. (Kplan, Pedian ephtrol (2008) 23: 1761-1767). Other notable causes for TMA iclude infection (e.g., bloody diarrhea associated with E. coli infection), medications (e.g., quinine, bevacizumab), connective tissue dsieases (e.g., systemic lupus erythematosus, antiphospholipid anitobdy syndrome, scleroderma), cancer, vasculitis, pregnancy, malignant hypertension, organ transplant and metabolic disorders. uraaemic syndrom (HUS) and thrombotic thrombocytopenic purpra (TT) are the clincial entitles comprising TMA, with predominantly renal manifestrations in the former, while the latter more often present with systemic and neurological findings. There are no standard laboratory values that define TMA, but the clinical traid of renal failure, thrmobocytopenia and microangiopathic haemolytic anaemia is considered the hallmark of TMA syndromes. Nephrol Dial Transplant (2006) 21: 3038-3045). 

Thrombotic microangiopathy (TMA) is a microvascular occlusive disorder characteried by preedominantly platelt thrombi in the renal and/or systemic circulations. Haemolytic 

Hemolytic Uremic Syndrome (HUS): See Kidney Diseases

Another major cause for TMA is atypical hemolytic uremic syndrome (aHUS), a discorder caused by dysregulation of a prt of the complement system. About 50% of aHUS pateints are found to have ither a genetic mutation in the complement system or an aut-antibody that inteeres with the reulation of complement. 

Hemolytic uremic syndrome (HUS) is characterized by the triad of thrombocytopenis, Coomb’s test negative microaniopathic hemolytic anemia, and acute renal failure. HUS is classified as eitehr D+ when it is associated with a preceding diarrhoeal illness, which in most people is caused by infection with E. coli 0157, or less commonly nondiarrhoeal associated (D_) (also called “atypical”). D- HUS may be sporadic or familial. Mutations have been reproted in the complement regulatory protein factor H in both sporadic and familial HUS. (Goodship, PNAS, 100(22), October 28, 2003). 

 Immune Thrombocytopenic Purpura (ITP)/ Thrombotic Thrombocytopenic Purpura (TTP): 

ITP refers to a rare disease characterized by microangiopathic hemolytic anemia, causing blood clots to form in small blood vessels throughout the body. These clots can cause serious problems if they block blood vessels and limit blood flow to the brain, kidneys or heart. Blood clots form when blood cells called platelets clump together. ITP is an autoimmune disease characterized by platelet clearance mediated by pathogenic platelet-specific antibodies. Autoantibody-coated platelets induce Fc receptor mediated phagocytosis by macrophages, primarily but not exclusively in the spleen. Thus the spleen is the key organ in the pathophysiology of ITP not only because platelet autoantibodies are formed in the white pulp but also because macrophages in the red pup destroy immunoglobulin coated platelets. If bone marrow megakaryocytes cannot increase production and matain a normal number of circulating platelets, thrombocytopenia and purpura develop. In the US, the annual incidence of chronic ITP is stimated to be about 6.6 cases per 100k persons. In adults, women are affectied about 3 times more frequently than men. Adults may be affected at any age, but most cases are diagnosed in women aged 30-40 years. Onset ina patient older than 60 years is uncommon, and a search for other causes of thrombocytopenia is warranted. The disease is characterized by reduced blood platelets, which cause visible skin blemishes from bleeding or brusing. Symptoms can include bleeding, red dots on the skin, red dots on the mouth membranes, purplish mouth membrane areas, bleeding nose, bleeding gum, digestive bleeding, urinary bleeding and brain bleeding. To establish a diagnosis of ITP, other causes of thrombocytopenia are excluded, such as leukemia.

Thrombotic thrombocytopenic purpura (TTP) is due to low activity of a protein called ADAMTS13. Some individuals are born with a mutation in the gene for ADAMTS13, although most affected pateitns have an acquired auto-antibody that blocks the activity of ADAMTS13. 

Causes/Pathophysiology:

Role of complement system:

(Hughes Am J Physiol Renal Physiol, 278: F747-F757, 2000) discloses that in an immune-meidated thromobtic microangiopathy model both CVF treatment and C6 deficiency reulsted in 10-25 fold reudction in the number of apoptotic cells. The results indicated that complement is the principal inducer of endothelial cell apoptosis in antibody-mediated glomerulenphritis and that this effect is mediated primarily by sublytic C5b-9. The data also strongly suggests that the C5b-9 membrane attack complext may play an important role in the induction of endothelial cell apoptosis in vivo, particularly in diseases associated with antiendothelial antibodies such as systemic lupus erhematosis, scleroderma, hemolytic uremic syndrome and the systemic vasculitides. 

Mutations have been reproted in the complement regulatory protein factor H in both sporadic and familial HUS with mutations identified in 10-20% of cases studied. (Goodship, PNAS, 100(22), October 28, 2003)

Tumour Immunology Strategies

• cancer vaccines refers to therapeutic immunization in patients with cancer, with the main aim of breaking tolerance toward self-tumor-associated antigens. One strategy is to pulse antigen presenting cells such as  with tumor antigens in the hope that that such DCs will induce host protective and therapeutic antitumor immunity. 

The identification of the optimal antign to target for a pparticular tumour type has long been a priority for cancer immunotherpay. Tumour antigens can generally be categorized as being tumor associated which are overexpressed such as the HER2 receptor or tumour specific such as HPV which is associated with cervical cancer. (Hu, “Toward personalized, tumour-speciic, therapeutic vaccines for cancer” 2018). 

• Introduce genes encoding MHC Ags,  agonists, costimulatory molecules or into tumor cells. The goal here again is to improve the immunogenicity and the Ag-presenting capability of tumor cells. For example, DCs can be raised from bone-marrow stem cells upon culture with cytokine combinations. During the differentiation culture they can be transfected with recombinant viral vectors encoding cytokines like IL-12, IL-7, or IL-2. If such cells are then injected intratumorally they capture antigen, migrate to draining lymph nodes and orchestrate a potent CTL response. Artificial expression of the transgene greatly enhances the antitumor immune response. 

Interestingly, most of the pulsing has been done with peptides rather then proteins (which would require DCs to process the antigen). This is because when take DCs and put them into plastic culture dishes, giving them cytokines, etc., this matures them. So most of the DCs become mature. One way to counteracti this is to do this in viov. For example, FLT3L is a way to increase DCs in vivo to increase DCs prior to taking out the blood to grow the DCs in culture. Morese et al, JCO, 2001 found that if they systemically gaive Flt3 ligand to mice, DC population increased. Then use IL-14, GM-CSF and TNFa for growing the DCs (CD34+ HPC) in vitro.

Patients given subcutaneous GM-CSF administration increase mobilization of CD80+ DCs. Studies have shown that just giving GM-CSF is therapeutic. Newer treatments include taking tumor cells from patients, growing them, transfecting them with plasmid encoding with GMCF, then irradiating them to use them as a vacine. Trials have shown this stimulated DCs leading to increased CD8+ cells at tumor sites.

Another approach is to use TLR agonists to activate local immune response. For example, the TLR7 agonist, imidazoquinolines and TLR 9 CpG are being used to activate the innate immune system.

• Direct Tumor Transfection with Co-stimulatory Molecules: Another strategy is to take the tumor and turn it into an APC. Take tumor and put in rv-B7.1 to induce T cell activation and induce tumor recognition. Researchers have found that by doing this they were able to generate gp100- and MARt-1-specific T cells found in most A2+ patients.

• Adoptively transferring T Helper Cells to initiate CTL Immunity: Infusion of CD4+ T cell clones specific for P815AB reported shown tumor destruction through a CD8 mediated destruction. Other reports have shown that such transferring induces a broad immune response.

• Cancer Specific Antibodies: About 70 humanized monoclonal antibodies (mAbs) against tumor antigens are currently in human clinical trails, and 3 mAbs, Rituximab, herceptin and Alemtuzumab have been approved by the FDA. Although their mechanisms of action have been controversial, a major antitumor mechanisms of mAbs is thought to be by antibody-dependent cellular cytotoxicity (ADCC). mABs specific for tumor cell surface antigens bind to the tumor cell through their antigen binding domains and, through the Fc region of the mAb, bind to cells such as NK cells and neutrophils that express Fc receptors. This binding event is throught to activate the FcR-expressing cell, which results in ADCC.

• T Cell Activation with Treg Depletion: Knutson et al. 2005. Treg cells are known to express CTLA-4. Prudhomme G, J Luekocyte Biol, 2004 have given mice anti-CTLA4 and vacine against gp100. However, side effect get large autoimmunity.

• CAR T-cell Immunotherapy:  See outline

• Induce apoptosis. In models utilizing animals engrafted with human tumors, treatment with nduces significant tumor-specific apoptosis, tumor regression, and improved survival with no identifiable toxicity. TNF, LT-alpha1Beta2, FasL, and TRAIL are all expressed and used to kill cancer cells by professioanl cytotoxic cells, such as CD8+ CTLs and NK cells. In addition, Fasl, TNF, and TRAIL are expressed by activated CD4+ T cells, B cells and macrophages. Human immature DCs also express these TNF family ligands on their cell membrane. Cell surface expression and secretion of these cytotoxic ligands appears to be tightly regulated and differentiation stage-dpependent with the dendritic cell lineage. The possible regualtory mechanism might include activiteis of cytokines and/or metalloproteases.

• Induce phagocytosis. Cells undergoing apoptosis express specific signals, including lipids like phosphatidylserine (PS) that facilitate recognition and ingestion by macrophages. The possibility exists that one might be able to stimulate phagocytosis of tumor cells in the absence of a death signal to thereby delete cancer cells without even having any associated bystander effects observed during conventional chemotherapeutic treatment which is based on the induction of apoptosis.

• Attracting DCs to tumors with Chemokine: For example, injection of the chemokine CCL20 attracts DCs into the tumor, leading to induction of anti-tumor response. Intratumoral CpG can also activate tumor DC, leading to induciton of systemic anti-tumor immunity.
• Combination therapies: Strategies such as taregting multiple neopeitopes adncomining a personalized vaccines with complementary therapies will be important to prevent immune espacpe. For example, a mouse study showed that the comibnation of four components, a tumour antigen-targeting antibody, IL-2 aanti-PD1 therapy and a T cell vaccine) could liminate large tumour burdens. (Hu, “Toward personalized, tumour-speciic, therapeutic vaccines for cancer” 2018).

MedlinePlus

Most, if not all, chemotherapeutic agents kill cancer cells through the induction fo apoptosis.

Specific Chemotherapeutic agents

Colchicine: has been investigated as an anti-cancer agent and has proven to be effective in treating cancerous cells. However, colchicine brutally damages a cell’s inner layers by blocking the action of a protein called tubulin. Tubulin plays an important role as cells divide into two. Thus, colchicine effectively halts cell division. Stopping cell division is a vital part of potential cancer drugs. Unfortunately, colchicineattacks not only cancer cells, but healthy ones as well, classifying it as a poison.  One strategy to deal with this has been proposed by Laurence Patterson, Professor of Drug Discovery and Institute Director of the Institute of Cancer Therapeutics at the University of Bradford. Patterson uses a drug delivery system that targets colchicine directly to the target. Many cancers produce a lot of enzymes called matrix metalloproteases (MMPs) which are useful to the cancer because they can disolved extracellular matrix around cells and give the cancer cells access to new blood vessels. Patterson attach long molecular tails to colchicine molecules which target the colchicine directly to the cancer cells. The colchicine is nontoxic until it comes into contact with MMP1, a metalloprotease protein released by tumors. 

Side Effects oc Chemotherapeutic agents

Chemotherapy suffers from problems like drug toxicity and drug resistance (due to increased drug metabolism, increased DNA repair mechanisms and decreased drug import into cells). Although chemotherapeutics and radiation exposure can effectively kill rapidly proliferating tumor cells, the rapidly dividing stem cells of the host’s mucosal epithelium, including the stem cells of the interstinal crypts and epidermis, are also damaged or killed, leading to the clinical condition termed “mucositis”. Oral and gastrointestinal mucositis can be sufficiently painful or debilitating to deter patients from continuing their course of treatment. Cytotoxic chemotherpay is known to cause mucosal injury (MI) both in the oral cavity and to mitotically active intestinal crypt cells. The manifestations of oral mucositis include erythema, ulcer formation, bleeding and exudates. Most of patietns treated for head and neck cancer and almost half of the patients receiving chemotherpay for non-head and neck cancer develop oral complications. The effect of radiation therpay on oral cavity primarily results form local tissue changes. These changes are initiated by a reduction in the prolfieration of basal epithelial cells, causing atrophy. Compromise of the mucosal barrier can also contribue to local invasaion by colonizing microorganisms and, subsequently, to systemic infection (Khan, “Infection and Mucosal Injury in Cancer Treatment, 2001).

Recombinant human keratinocyte growth factor (rhKGF, sold as Kepivance by Amgen, Inc, used as pretreatment, has been shown to cause an increase in measures of mucosal thickness and a 3.5 fold improvement in crypt surfival in the small intestine.

Drugs which Counteract Side-Effects

Granulocyte colony stimulating factor: is used to stimulate neutrophil production in patients udnergoing chemotherapy. Amgen, for example, sells Neupogen® (filgrastim) which is a recombinantly products version of G-CSF and FDA approved for cancer patients receiving myelosuppressive chemotherapy, actue myeloid leukemia patients receiving induction or consolidation chemotherapy, patients udneroing bone marrow transplantation, autologous periopheral blood progenitor cell collection and therapy and patients with severe chronic neutropenia. Sandoz has recentrly received approval for a biosimilar under the provisions of the Biologics Prcie Competition and Innovation Act (BPCIA). 

Treatment of invasive cervical cancer is affected by the stage of the disease, which is based on clincal evaluation.

Chemotherapy: 

Single-agent chemotherpay can be divided into those who received plantinum-based therapy and those who received nonplatinum-based therapy. Cisplatin is regarded as the most active agent in carcinoma of the cervix. Platinum analogs such as Carboplatinhave been investigated in an effort to reduce toxicity.

Combination chemotherapy: 

Combination chemotherapy incldues drugs that have single agent-activity, nonoverlapping toxicity, and additive or synergistic activity with no incrase in toxicity in order to improve response rates and survival. For example, cisplatin has been combined with 5-fluoruracil, bleomycin, ifosfamide, gemcitabine, vinorelbine, paclitaxel, and topotecan in phase II-III trails. These trials showed advantage in response rates when compared with single-agent cisplatin.

Chemoradiotherapy 

Chemoradiotherapy is the treatment of choice for patients with stage IB2 disease (tumour >4cm), invasive carcinoma, confined to cervix). An RCT showed that adding weekly cisplatin to pelvic radiotheraphy before hysterectomy reduced the risk of recurrence of disease and death compared with radiotheraphy and hysterectomy alone.

Three RCTs have shown that improvements in progression free survival and overall survival are greater for chemoradiotheraphy than for radiation alone in patients with locally advanced stage IIB-IVa disease. Platinuum based chemoradiotheraphy is now the standard of care for these patients.

Patients with early stage disease have a high risk of recurrence postoperatively if they have one of the following risk factors: postive nodes, parametrial invasion, or positive surgical margins. Such patients should receive adjuvant cisplatin based chemoradiotheraphy after hysterectomy, as shown by an RCT.

There is a lot of conflicting published work regarding the treatment of builky stage Ib-IIa cervical cancer. While some centers are performing primary surgery as for Ib1 disease followed by tailored postoperative radiation with or without chemotheraphy, the others are in favor of primary chemo-radiation therapy. Neoadjuvant chemotheraphy followed by radical surery has emerged as a possible alternative which may imporve a surfival in patients with stage Ib2 disease.

Concomitant chemoradiation is becoming a new standard in treatment of advanced disease, because it has been clearly shown to improve disease-free, progression-free and overall survival.

The management of recurrent cervix carcinoma has not improved significantly with modern chemotherapy due to several factors. First, carcinoma of the cervix has limited sensitivity to cytotoxic agents, especially when it recurs in the irradiated pelvis. Second, the median response duration is usually 3-70 months. Third, there is a difficulty in showing a meaninful incrase in survival of the patient population as a whole. Fourth, there is refractoriness among patients who have failed any prior chemotherapy.

EGFR Inhibitors:

Activation of EGFR triggers a cascade of events leading to cell reproduction. 

Tarceva (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazo-linamine): has been shown to be useful for treatment of tumors caused by HPV. (see US Patent NO: 6,900,221). 

Radiotheraphy: 

Patients with early stage disease have an intermediate risk of recurrence postoperatively if they have two of the following factors: large tumour size, deep stromal invasion, or involvement of the lymphovascular space. An RCT evaluating 277 women with stage IB disease versus “no further treatment” and at least two risk factors showed that adjuvant radiotheraphy decreased the rate of recurrence and imporved disease free survival.

–Radical hysterectomy:

Radical hysterectomy is the treatment of choice for young healthy patients because it preserves ovarian function. It is thought to be equally effective for patients with early stage disease.

Most often, stage Ib1 cervical cancer is treated by radical hysterectomy with pelvic lymphadenectaomy. Laparoscopically assisted radical vaginal hysterectomy has shown similar efficacy and recurrence rates.

Natural Medicines

Artemisinin: US Patent Publication No. US2007/0142459 discloses methods of treating proliferative cervical disorders by administering a therapeutically effective amount of artemisinin-related compounds. In some embodimetnes, artemisinin or an artemisinin derivative, combined with other anti-viral or anti-cancer therapies such as radiation theraphy are used. Other agents known for their use in the inhibition of cervical cancer include interleukin-2,5′-fluorouracil, nedaplatin, methotrexate, vinblastine, doxorubicin, carboplatin, paclitaxel (Taxol), cisplatin, 13-cis re-moic acid, pyrazoloacridine, and vinorelbine.

Artemisinin (Oinghasosu) is a naturally occuring substance, obtainec by purificatoin from sweet wormwood, Artemisia annua. Artemisinin and its analogs are sesquiterpene lactones with a peroxide bridge. The very low toxicity of these compoudns to humans is a majro benefit.

Formulations of the artemisinin-related compounds suitable for oral administration may be in the form of capsules, pills, pwoders, etc. The topical formulations may include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples include 2-pymolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcholo, dimethyl sulfoxide and azone.

N,N-dimethylglycine: US Patent Publication Number 2006/0183801A1(Assignee, FoodScience, Corporation, Essix Junction, VT) discloses a method of treating, inhibiting the metasis of, or preventing cervical cancer by administering to a patient an effective amount of N,N-dimethylglicine or a pharmaceutically acceptable salt thereof.

See also therapeutic applications of antibodies   See also Antibodies as toxic drug carriers in drug delivery. 

Companies:  Immunogen 

 Often membrane-associated polypeptides are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such polypeptides are more abundantly expressed on the surface of cancer cells. The identification of such tumor associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody based therapies. About 70 humanized monoclonal antibodies (mAbs) against tumor antigens are currently in human clinical trails, and 3 mAbs, Rituximab, herceptin and Alemtuzumab have been approved by the FDA. Such antibodies may also be conjugated to a toxin which can kill the tumor cell. 

Such tumor antigens include receptors of the erbB class such as EGFR, HER2-4, insulin growth factor recetpor I (IGF-RI), c-met, receptors of the tumor necrosis factor family such as DR4-5, DcR1, DCR2, IL-17 recetpor, IL-23 receptor, IL-12 receptor and interferon-alpha receptor (EP2546268).

The mere binding of antibodies to cells does not always lead to their destruction. Thus, attempts have been made to render antibodies cytotoxic by attaching a drug, toxin or radiolabelled isotope to them, so called “magic bullets”. (Segal US 4,676,980) See Drug Delivery

Mechanism of Action

A major antitumor mechanisms of mAbs is thought to be by antibody-dependent cellular cytotoxicity (ADCC). mABs specific for tumor cell surface antigens bind to the tumor cell through their antigen binding domains and, through the Fc region of the mAb, bind to cells such as NK cells and neutrophils that express Fc receptors. This binding event is thought to activate the FcR-expressing cell, which results in ADCC. Most antibodies that mediate ADCC can also cause CDC. However, CDC does not play a big role in the mAb mediated destruction of tumor cells, as these cells tend to overexpress complement regulatoyr proteins (e.g., CD46, CD55, CD59). Interference with the function of these proteins may promote CDC. Therapetic anti-cancer mAbs may also induce apoptosis of cancer cells by blocking binding of growth factors to their receptors and/or sensitive cancer cells to chemotherapy and radiotherapy as well as act as anti-angiogenic agents by blocking the binding of VEGF to its receptor. Therapeutic may also block intracellular signaling pathways. Ionnello (Cancer and Metastusis Reviews 24: 487-499, 2005). For example, bevacizumab binds to the soluble VEGF and thus inhibits angiogenesis, which is important for maintenance of tumor vaculature and tumor health. Goswami (Antibodies 2031, 2, 452-500)

Tumor Antigens and Antibodies which Target These Antigens

B7H4: Terret (WO2009/073533) discloses B7H4 antibodies which are useful for treating cancer. B6-H4 exerts its physiologic function by binding to a receptor on T cells, which in turn induces cell cycle arrest and inhibits the secretion of cytokines, the development of cytotoxicity and cytokine production of CD4+ and CD8+ T cells. Chen (US2008/026235) also discloses administering an effective amount of a B7-H4 antagonist such as an antibody which can be used to enhance an immune response to treat cancer. 

CALLA: The monoclonal antibody J5 is specific for Common Acute Lymphoblastic Leukemia Antigen (CALLA) and can be used to target cells that express CALLA (e.g., acute lymphoblastic leukemia cells).

c-Met: is the cell surface receptor for hepatocyte growth factor (HGF), also known as scatter factor. The c-Met receptor is a disulfide linked heterodimer consisting of extracellular alpha and beta chains. The alpha chain, heterodimerized to the amino terminal portion of the beta chain, forms the major ligand binding stie in the extra cellular domain. HGF binding induces c-Met receptor homodimerization and phosphorylation of two tyrosine residues within the catalytic site, regulating kinase activity. HGF mediated activation of c-Met results in a complex genetic program referred to as invasive growth, consisting of a series of physiological processes, including profliferation, invasion, and angiogenesis that occur under normal physiological conditions during embryonic developmetn and pathologically during oncogenesis. In tumour cells, c-Met activation causes the triggering of a diverse series of signalling cascades resulting in cell growth, proliferation, invasion and protection from apoptosis.

A variety of c-Met pathway antagonists such as monoclonal antibodies with potential clinical applications are currently under clinical investigation. Examples include the anti-c-Met 5D5 antibody generated by Genentech (WO96/38557). WO2009/007427 also describes mosue monoclonal antibodies to c-Met and chimeric variants in which the antigen binding domains of the mosue monoclonal antibody are coupled to the constant region of human IgG1.

Hultberg 9US2012/0156206) discloses a combination of antibodies binding to the human c-Met protein and more specifically combinations of two or more c-Met antibodies which bind to distinct, non-overlapping epitopes. Such antibodies have advantageous properties in that the combinations can produce potent inhibition of HGF independent activaiton of the human c-Met receptor.

CD4: An anti-CD4 Mab that is useful for T cell depletion therapy is IDEC-151 (US 6,136,310). 

CD20: In 1997, the FDA approved RITUXAN® (IDEC Pharmaceuticals Corp. and Genetechn, Inc.,) (also referred to as rituximab), a chimeric anti-CD20 antibody for the treatment of non-Hodgkin’s lymphoma.

Rituximab is a mAb consisting of a human kappa constant region, a human IgG Fc portion and a murine variable region. The exact antitumor mechanisms of rituximab remains unclear, but it is assumed that it exerts its effects by various mechanisms including activaiton of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and signaling of apoptosis by binding to the CD20 antigen specifically expressed on B cells. Although most patients with B cell non-Hodgkin lymphoma respond to initial rituximab treatment, a significant number become unresponsive. It has been shown that complement regulatory proteins such as CD55 and CD59 are associated with acquisition of resistance. (K. Takei, Leukemia Research 30 (2006) 625-631. 

CD22: is a 140 kDa member of the Siglec family of cell surface proteins that is expressed by most mature B cell lineages. . It is also a member of the sialadhesin family of adhesion molecules that includes sialoadhesin and myelin-associated glycoprotein. Sialoadhesin and CD22 mediate cellular interactions by recognizing sepcific cell surface sialylated glycoconjugates. Remarkably, mAbs which blocked CD22 ligand binding were shown to accelerate mature B cell turnover and inhibit the survival of adoptively transferred normal and malignant B cells in vivo. In addition, mAbs that boudn CD22 ligan binding domains induced significant CD22 internalization, depleted marginal zone B cells and reducd mature recirculating B cell numbers by 75-85% in the mouse (Haas, J Immunol. 2006, 177(5): 3063-73). 

CD22 is an attractive molecular target because of its restricted expression. It is not exposed on embvyonic stem cells but is highly expressed on B cells in NHL. A murine anti-CD22 mAb has been developed for imaging and treatment of NHL. Anti-CD22 antiboides also have potential as carriers of cytotoxic agents since they rapidly internalise and human CD22 has been trageted using mAb conjugates linked to toxins such as derivatives of Pseudomonas exotoxin. A particularly exciting possibility is using CD22 targetted RNAses. This has the advantage of overcoming limitations experienced in using toxins. RNAses are not immunogenic in animal studies nor does a naked RNAse cause immunological problems in humans (newton, Expert Opin. biol. Ther: (2001) 1(6): 995-1003).

Jones (US 8,389,688) discloses humanized versions of anti-CD22 mouse mAbs which can be used in the treatment of B cell malignancies.

Epratuzumab is a humanized antihuman CD22 IgG1 antibody which has shown promising clinical activity both as a single agent and in combination with rituximab in patients with non-Hodkin’s lymphomas (NHL) Wang, Clin Cancer Res. 2003, 9 (10 Pt2): 3982S-90S

CD33: The monoclona antibody MY9 is a murine IgG1 antibody that binds specifically to the CD33 antigen and be used to target cells that express CD33 (e.g., acute myelogenous leukemia (AML) cells. 

CD88: IDEC-114 is an anti-CD80 MAb for treating autoimmune diseases and preventing organ transplant rejection (US 6,113,898). 

CD200: Bowdish (WO2007/084321) discloses anti CD200 anitobides for treatment of cancer. 

EGFR: Aberrant expression of epidermal growth factor receptor (EGFR) has been observed in a variety of solid tumors, and often correlates with a poor clinical outcome for patients. These features make EGFR an important target for cancer therapeutis, linke mAbs and tyrosine kinase inhibitors (TKI). Anti-EGFR mAbs paneitumumab (Vectibix) and cetuximab (Erbitux) are established agents in the treatment of colorectal cancer. Yang, “Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency” mAbs (2015). 

IGF-IR (type 1 insuline-like growth factor receptor): has been implicated in promoting oncogenic transformation, growth and survival of cancer cells. WO20007/110339 discloses antibodies that bind to the extracellular domains of IGF-IR and inhibit receptor activation. 

VEGF (Human vascular endothelial growth factor): VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors as well as intracoular disorders. VEGF is a homodimeric glycoprotein that has been isolated from several sources. It shows highly specific mitogenic activity for endothelial cells. It ahs important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life. Anti-VEGF nuetralizing antibodies suppress the growth of a variety of human tumor cell lines in mice. WO 94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 refers to antibodies against VEGF.

Humanized monoclonal antibody bevacizumab (sold under the trade name Avastin) is an anti-VEGF antibody used in tumor therpay (WO 98/45331).

Ranibizumab (trade name Lucentis) is a monoclonal antibody fragment derived form the same parent murine antiboy as bevacizumab. It is much smaller than the parent and has been affinity matured to provide stronger binding to VEGF-A (WO 98/45331). It is an anti-angiogenic that has been approved to treat the “wet” type of age-related macular degneration, a common form of age related vision loss. 

Another anti-VEGF antiboy HuMab G6-31 is described in US 2007/0141065. 

ANG-1/ANG-2 (Human angiopoietin-2): ANG-1 and ANG-2 were discovered as ligands for the Iites, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. Corneal angioenesis assay have shown that both ANG-1 and ANG-2 had similar effects, acting syntergistically with VEGF to promote growth of new blood vessels. 

WO2011/117329 discloses a bispecific, bivalent antibody that includes a first antigen bidning site for human VEGF and a second antigen binding ste for human ANG-2. 

Histones: It has been shown that extracellular histones released in response to inflammatory challenge are mediators contributing to endothelial dysfunction, organ failure and death during spesis. Esmon (US2009/0117099) discloses that inhibitors of histones such as antiboides that bind to H1, H2A, H2B, H3 and H4 can also be used to alleviate toxicity in cancer cuased by chemotherapy, radiation and cytokines.  Svanborg (US2006/0233807) also disclsoes using a moeity which specifically binds to hisone such as an antibody such that the chromatin assembly or remodeling activity of the cell is inhibited. The method is disclosed as being useful for the treatment of cancer. 

Antigens which are Overesxpressed in Certain Types of Cancers and Monoclonal Antibodies which Target these antigens

Claudin-18.2: is overexpressed in up to 80% of gastrointestinal adenocarcinomas and 60% of pancreatic tumors, in addition to other solide cancers. Astellas has a monoclonal candicate IMAB362 that targets this tight junction protein.

Antibodies against Immune check point proteins

Immuen checkpoint proteins are a set of proteins that act as either stimulators or inhibitors of the immune system. By dampening or attenuating the “off-switch” an immune response is enhanced. The most well known checkpoint targets are the following:

cytotoxic T lymphocyte-assocaited antigen 4 (CTLA-4):  ipilimumab is an anti-CTLA-4 soled as Yervoy by Bristol-Myers Squibb. (WO2016063263) discloses that the bacterium Bacteroides fragilis improved the antitumor efficacy of an anti-CTLA-4 antibody. 

programmed cell death 1 (PD-1):  Pembrolizumab is an anti-PD-1 sold as Keytruda by Merck

programmed death ligand 1 (PD-L1). nivolumab is sold as Opdivo by Bristol-Myers Squibb.  (US20150352206) discloses that Bifodobacterium improved the antitumor efficacy of an anti-PD-L1 antibody in a mouse model of melanoma. 

T cell receptor therapeutics:

TCR arm – antibody domain; TCR like antibodies:

As part of normal immune surveillance, intracellular.proteins are degraded by the proteasome into short peptide fragments of about 8-10 amino acids which are presented on the cell surface bound to MHC molecules, known as human leukocyte antigen (HLA) molecules in humans. Circulating T cells bind specific peptide MHC complexes with their TCRs, continuously monitoring for intracellular ref flags that are processed and displayed on the cell surface. Immunocor’s tebentafusp is approved by the FDA for uveal melanoma. The bispecific biologic molecules combines a TCR arm that recognizes tumour cells with an antibody domain to bind and activate T cells. For tebentafusp, Immunocore uses a TCR domain to recognize an epitope of gp100, a transmembrane glycoprotein that is located on the ER and epxressed in melanocytes and melanoma –as presented on HLA-A*02:01. The T cell engaing arom fo the bispecifiv binds CD3on T cells. For this therapy, one just needs ti know gp100 (a tumor specific antigen) and the patien’ts HLA. (Nature Reviews Drug Discovery, 21, May 2022, 321). 

Roche has made it into the clinic with a TCR mimicking bespecific called RG6007. One antibody domain recognizes a fragment of the cancer associated transcription factor WTI presented on HLA-A&*02 and the other binds CD3 on T cells. (Nature Reviews Drug Discovery, 21, May 2022, 321)

HLA restriction poses one challenge for TCR based approaches. There are multiple HLA genes, and each has numerous variants. But first generation TCR therapies can only bind theri targets in patients harbouring a certain HLA allele. Some alleles are more common in particular populations, providing starting points for frug developers. Tebentafusp, for example, takes aim at the HLA-A*02 subtype, which is present in about 40% of the global population and over 95% of white populations. But patients with other subtypes will need different therapeutics. A greater challenge for teh field is choosing the best TCR target. Finding a protein expressed exclusively in tumour cells is just the begninng, as MHC presentation of the peptidome is a complex and dynamic process. (Nature Reviews Drug Discovery, 21, May 2022, 321)

T cell redirecting antibody (TRAB):

Antibodies which bind a tumor cell specific antigen on one arm and CD3 on a T cell by the other arm thereby inducing cytotoxicity against the tumor cell by bridging the tumor cell and the T cell have been developed. (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012). 

MedLinePlus

Frederick National Laboratory

Unpredictability in Cancer Treatment

Non-correlation between In Vitro and In Vivo treatment: Cancer treatment is highly unpredictable. For example, even though the EGFR was identified in some cancers as a drug target, the in vitro effectiveness of a drug in inhibiting the EGFR turned out to be a poor proxy for how effective that drug actually was in treating cancer in vivo. Numerous EGFT inhibitors that showed promising in vitro activity failed for a variety of reasons. These included poor pharmacokinetics due to poor adsorption or rapid metabolism (or both), undersirable drug-drug ineractions, drug toxicity due to drug binding onto healthy cells, sdrug toxicity due to binding onto other receptors and metabolite toxicity. OSI Pharmaceuticals v. Apotex (USPTO, No: IPR2106-01284)

Challenges with Experimental Models: Despite some successes, many cancer still have a high mortality rate and no effective treatment. For many cancers, survival rates have not changed in decades – pancreatic cancer remains almost 100% lethal, and the oeverall survival rate for lung cancer has improved only from 13 to 16 %. The key difficulty is that cancer is a complex and heterogenous disease: many genes are amplified, deleted, mutated, and up- or down-regualted. Many pathways are activated or suppressed. These chances vary substantially in different cancers, in different patients with the same cancer, and even in different tumor samples from the same patient (Fortney, 2011, p. 465, last ¶). Although mice are crucial for cancer research, cancer is a complex, three-dimensional disease that changes, evolves and spreads through the body. Mice, easy to breed and genetically manipulate, and with a completed genome sequence, are the obvious choice for scientists who wanted to pick apart these processes and test new drugs. Bust despite having broad similarities, mice have significant differences. For one, most mouse trmours originate in different types of tissues from humans and unlike humans, their lethalaly cells can maintain the ends of their chromosomes, a key factor influencing which mutations tumour cells develop. One of the key mouse models, involves grafting human cancer cells into mice and seeing how the resulting tumours respond to treatment. But human cells are likely to behave differently in a mouse than in a human body, making results hard to interpret (Carina Dennis, Nature, (2006), pp. 739-741)

Combination Therapy and Dosing

It is recognized in the art that a benefit of using chemotherpaeutic agents in conjunction with other therpaies, e.g., an antibody antagonist, can be useful for allowing adminsitraiton of lower dooses of chemotherpaeutic agetns, thereby potentially resulting in a reduction in toxic side effects. It is also known in the art that therpaeutically effective dosages of chemotherpaeutic agents can vary when these drugs are used in treatment combiantions. Methods for experimetnally determining therapeutically effective dosages of chemotherapeutic drugs and other agents for use in combination treatment regimends are descried in the literature. For example, the use of metronomic cheotherapy dosing, i.e., providing more frequent lower doses in order to minimize toxic side effects, has been described extensively in the literature. A combiantion treatment regimen encompases treatment regimens in which administration of a chemothreapetuic agent is initiated prior to, during, or after treatment with the second agent, e.g., an antibody, and continues until any time duringtreatment with the other agent or after termination of treatment with the other agent. It It also includes treatment in which the agents being used in combiantnion are adminsitered simultaenoeusly or at differnt times and/or at decreasing or increasing intervals during the treatment period. (Van Epps (US 2007/0048325).

Cancer Treatment Strategies

Surgical debulking: If surgical removal is possible, this is a favored treatment strategy. However, surgery may miss micrometastases.

Chemotherapy: Most, if not all, chemotherapeutic agents kill cancer cells through the induction fo apoptosis. Cisplatin, for example, is extensively used for the treatment of a broad spectrum of turmors. Chemotherapy suffers from problems like drug toxicity and drug resistance (due to increased drug metabolism, increased DNA repair mechanisms and decreased drug import into cells). It also has devastating side effects such as gut-tering vomitus and diarrhea, alopecia, and fatal vulnerability to infections. Cisplatin, for example, has preogressive, irreversible side effects including nephrotoxicity and ototoxicity. Evidence indicates that cisplatin ototoxicity is closely related to the increased production of reactive oxygen species (ROS).

Tumor Immunology: Tumor cell destruction should be possible by the immune system. Strategies along this line of thought include the following:

(i) Cancer Vaccines refers to therapeutic immunization in patients with cancer, with the main aim of breaking tolerance toward self-tumor-associated antigens. One strategy is to pulse antigen presenting cells such as dendritic cells (CDs) with tumor antigens in the hope that that such DCs will induce host protective and therapeutic antitumor immunity. These approaches are currently limited for clinical application because few human tumor antigens on a tumor cell that can activate the immune system have been found. In the best studied human melanoma where a class of tumor associated proteins have been identified, it is unclear which of the identified tumor associated Ags is the best choice to induce effective tumor rejection in vivo or how effective they are.

The advent of massively parallel sequences has now made it possible to sequence the entire gehome of exome (coding regions) of tumor and matched normal cells to identify all of the muations that have occurred. The subset of those mutations that alters protein coding sequences creates personal, novel antigen “neoantigen” which may provide the “foreign” signal needed for cancer immunotherpaty This is important because the use of self antigens which are selectively expressed or overexpressed in tumors requires overcoing both central tolerance (whereby autoreactive T cells are deleted in the thymus during development) and periopheral tolerance (whereby mature T cells are suppressed by regulatory mechnisms). (Hacohen, “Getting Persoanl with Neoantigen-based therpaeutic cancer vaccines” DOI: 10 (2013).

Another interesting therapeutic approach is the use of peptides as vaccines. Phage-display libraries are panned against therapwutic antibodies. Peptides identified that resemble the original antigen to which the antibody binds have been called “mimotopes”. The resulting mimotpe may be used to elicit humoral and cellular respones. (Brissette “Identificaiton of cancer targets and therapeutics using phage display” Current Opinion in Drug Discovery and Development 2006, 9(3) 363-369).

(ii) Introduce genes encoding MHC Ags, toll-like receptor agonists, costimulatory molecules or cytokines into tumor cells. The goal here again is to improve the immunogenicity and the Ag-presenting capability of tumor cells. For example, DCs can be raised from bone-marrow stem cells upon culture with cytokine combinations. During the differentiation culture they can be transfected with recombinant viral vectors encoding cytokines like IL-12, IL-7, or IL-2. If such cells are then injected intratumorally they capture antigen, migrate to draining lymph nodes and orchestrate a potent CTL response. Artificial expression of the transgene greatly enhances the antitumor immune response.

Interestingly, most of the pulsing has been done with peptides rather then proteins (which would require DCs to process the antigen). This is because when take DCs and put them into plastic culture dishes, giving them cytokines, etc., this matures them. So most of the DCs become mature. One way to counteract this is to do this in vivo. For example, FLT3L is a way to increase DCs in vivo to increase DCs prior to taking out the blood to grow the DCs in culture. Morese et al, JCO, 2001 found that if they systemically gaive Flt3 ligand to mice, DC population increased. Then use IL-14, GM-CSF and TNFa for growing the DCs (CD34+ HPC) in vitro.

Patients given subcutaneous GM-CSF administration increase mobilization of CD80+ DCs. Studies have shown that just giving GM-CSF is therapeutic. Newer treatments include taking tumor cells from patients, growing them, transfecting them with plasmid encoding with GMCF, then irradiating them to use them as a vacine. Trials have shown this stimulated DCs leading to increased CD8+ cells at tumor sites.

Another approach is to use TLR agonists to activate local immune response. For example, the TLR7 agonist, imidazoquinolines and TLR 9 CpG are being used to activate the innate immune system.

(iii) Direct Tumor Transfection with Co-stimulatory Molecules: Another strategy is to take the tumor and turn it into an APC. Take tumor and put in rv-B7.1 to induce T cell activation and induce tumor recognition. Researchers have found that by doing this they were able to generate gp100- and MARt-1-specific T cells found in most A2+ patients.

(iv) Adoptive immunotherapy: (CD4+ or CD8+ cells): Adoptive immunotherpay of cancer refers to a therapeutic approach in which immune cells with an anti-tumor reactivity are adminstered to a tumor bearing host, with the aim that the cells mediate, either directly or indirectly, the regression of an established tumor. Transfusion of lymphocytes, particularly T lymphocytes, falls into this category and investigators at the National Cancer Institute (NCI) have used autologous reinfusion of peripheral blood lymphocytes or tumor infiltrating lymphocytes (TIL) T cell cultures from biopsies of subcutaenous lymph nodules, to treat several human cancers (US 4,690,914). For example, T cells that have a natural or genetically engineered reactivity to a patients’ cancer are expanded in vitro using a variety of means and then adoptively transferred into the cancer patient.One approach with respect to T helper cells is to adoptively transfer T helper cells to initiate CTL immunity. Infusion of CD4+ T cell clones specific for P815AB has shown tumor destruction through a CD8 mediated destruction. Other reports have shown that such transferring induces a broad immune response. For example, TIL expanded in vitro in the presence of IL-2 have been adoptively transferred to cancer patients, resulting in tumor regression in select patients with metastatic meloma. Melanoma TIL grown in IL2 have been identified as activated T lymphocytes CD3+ HLA DR+, which are predominantly CD8+ cells with unique in vitro antitumor properties. Many long term melanoma TIL cultures lyse autologous tumors in a specific MHC class 1 and T cell antigen receptor dependent manner.

It has been deomonstrated that CD8+ cytotoxic T lymphocytes (CRLs) recognize epitope peptides derived from tumor-associated antigens (TAAs) presented on MHC class I molecules and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological apporaches. Some of the discovered TAAs are now in clinical development as targets of immunotherapy. TAAs discovered so far include MAGE, gp100, SART, and NY-ESO-1. For a listing of human tumor antigens recognized by T cells see (Cancer Immunol Immunother (2001) 50:3-15).

Chimeric antigen receptor T-cell (CAR-T) therapies:

CAR T cells combine the specificity of a mAb with the cytolytic capacity of a CAR T cell. This is achieved by fusing the scFv of a mAb (or another antigen recognition domain) to a transmembrane domain and intravellular signaling domains capable of elicity a T cell response. CARs that contain only the CD3-epsilon endodomain are known as first generation CARS. those that contan one costimulatry domain (such as CD28 or 41BB) are known as second-generation CARS; and those that contain two or more costimulatory domains are known as third generation CARS. To date, most clinical experience and sucess has been amassed with CAR T cells targeting CD19, a surface protein involved in B cell signlaing that is expressed on B cell malignancies. Given that its expression is restricted to the B cell lineage, and patients can live without healthy B cells, CD19 has emerged as a promising target for CAR T cell immunotherapy. Other targets that have demonstrated clinical sucess in B cell malignancies include CD22 fo B-ALL and BCMA for multiple myeloma. T date, most CAR T cell trials have used autologous T cells for transduction. A cancer patient’s T cells are collected, activated with antibodies or antibody coated beads, and then transduced, most commonly with a lentivirus or retrovirus, to express the CAR molecule. CAR T cells are then expanded in vitro to sufficient numbers to infuse back into the pateint. Patiens often recieve lympho depleting chemotherapy before T cell infusion. Majzner (“clinical lessons learned from teh first leg of the CAR T cell journey, Nature Medicine, 25, pp. 1341-1355, 2019)

Numerous factors can affect the potency and quality of a CART-T therpay including the production process (e.g., CD4/CD8 T cell ratios, T cell phenotype, levels of non-transduced cells, duration of activation, transgene construct (high or low epression, insolators, etc), vector choic (retroviral, adenoviral or transposon), CAR design (e.g., scFv affinity, stability and immunogenicity, spacer lenght and signaling domains) and input donar blood cells (e.g., starting cell number and exposure to different treatments and conditioning regimens). (Nature Biotechnology, 35(10), October 2017.

–Kymriah: which is developed by Novartis was approved by the FDA for treating relapsed B cell acute lymphoblastic leukemia in 25 and under.

–Yescarta, made by Kite Pharma, a Gilead Science company for example has been approved for treatment of certain types of non-Hodgkin lymphomoa in which patient’s white blood cells are extracted, modified, and then injected back into the patient. It was shown to cure 36% of patients completely and to reduce tumors in 82% of patients. It costs about $373,000.

Cancer Specific Antibodies: See outline

T Cell Activation with Treg Depletion: Knutson et al. 2005. Treg cells are known to express CTLA-4. Prudhomme G, J Luekocyte Biol, 2004 have given mice anti-CTLA4 and vacine against gp100. However, side effect get large autoimmunity.

Induce apoptosis.

   –TNF, FasL and TRAIL:

In models utilizing animals engrafted with human tumors, treatment with TRAIL/Apo2L induces significant tumor-specific apoptosis, tumor regression, and improved survival with no identifiable toxicity. TNF, LT-alpha1Beta2, FasL, and TRAIL are all expressed and used to kill cancer cells by professioanl cytotoxic cells, such as CD8+ CTLs and NK cells. In addition, Fasl, TNF, and TRAIL are expressed by activated CD4+ T cells, B cells and macrophages. Human immature DCs also express these TNF family ligands on their cell membrane. Cell surface expression and secretion of these cytotoxic ligands appears to be tightly regulated and differentiation stage-dependent with the dendritic cell lineage. The possible regualtory mechanism might include activiteis of cytokines and/or metalloproteases.

Complement factor or receptor antagonists: Goldenberg (US2006/0140936) discloses multispecific antagonists that react specifically with at least one complement factor or complement regulatory protein in the thearapy of various inflammatory diseases including cancer. In preferred embodiments, the complement factor includes C3, C5, C3a, C3b and C5a.

Medof (US12/920293) disclose that administration of a C3aR antagonist and a C5aR antagonist such as an an antibody to C3aR and an antibody to C5aR induced apoptosis in a cancer cell expressing a c3a recetpor (C3aR) and a C5a receptor (C5aR). Lambris (US2011/0044983A1) also discloses treating cancer by admistering complement inhibitors such as inhibiotrs of C3aR and C3aR.

Corticosteroids: are used in the treatment of patients with blood disorders such as multiple myeloma. corticosteroids may work by causing programmed cell death of certain cells. Corticosteroids are also used in the treatment of cancer because they decrease inflammation. This cn decrease swelling around tumors in the spine and brain.

Induce phagocytosis. Cells undergoing apoptosis express specific signals, including lipids like phosphatidylserine (PS) that facilitate recognition and ingestion by macrophages. The possibility exists that one might be able to stimulate phagocytosis of tumor cells in the absence of a death signal to thereby delete cancer cells without even having any associated bystander effects observed during conventional chemotherapeutic treatment which is based on the induction of apoptosis.

Attract DCs to tumors with Chemokine: For example, injection of the chemokine CCL20 attracts DCs into the tumor, leading to induction of anti-tumor response. Intratumoral CpG can also activate tumor DC, leading to induciton of systemic anti-tumor immunity.

Counteracting Angioenesis: Counteracting angioenesis hampers tumor growth and spread. Angiogenesis involved the action of endogenous growth hormones such as vascular endotherlial growth factor (VEGF-A/B, VEGF-C and/or VEGF-D). Thus, counteracting the action of such growth factor indirectly counteracts tumor growth because angiogenesis is at least in part prevented. Placental growth factor (PIGF) is also involved in angiogenesis. Whereas PIGF is primarily involved in angiogenesis in tumour tissue, VEGF plays an important role in angiogenesis in toehr (non-tumour) tissue as well.

Tumour laser thermotherapy: has been shown to be superior to surgical exicsion, partly due to the laser induced immunological effect such as increased number of tumour infiltrating macrophages and CD8 lymphocytes (Ivarsson, British J. of Cancer, 2005, 93, 435-440).

–Photodynamic therapy (PDT): is a systemic administration of tumor localizing photosensitzers and subsequent irradiation with light of the appropriate wavelenght. The combination of drug uptake in maliganant tissues and selective delivery of laser generated light provides effective therapy, with efficient tumor cytotoxicity and minimal normal tissue damage. Activated photosensitivers interact with molecular oxygen to produce singlet oxygen that destroys neoplastic cells with minimal normal tissue dage. PDT utilizing the hematoporphyrin derviative (Hpd) has been used clincially for palliation of obstructive lesions of the esophagus, and the tracheobronchial tree, for treatment of bladder tumors and for loca control of various tumors on the skin surface (Canti, Anti-Cancer Drugs 1994, 5, 443-447). Photodynamic therapy is a chemotherapeutic approach that utilizes a bifunctional reagent that localizes to the target tissue relative to the surrounding tissue and is toxic to the arget tissue when epxosed to light.

Inhibition of Detrimental Cytokines: It is now accepted that inflammation is a driving force behind cancer and reflects that the inflammation is a protective attempt to remove the injury. However, disease progressio in cancer is dependent on the coplex interaction beween the tumor and the host inflammatory response. There is substantial evidence in advanced cancer that host factors such as weight loss, poor performance status and the host systemic inflammatory response are linked. For example, elevated level of C-reactive protein is now included in the definition of “cachexia” as a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. The inflammatory cytokines that have been involed in wasting diseases are IL-6, TNF-alpha, IL-1beta and interferon-gamma (Argiles, European J Pharmacology 668, 2011, S81-S86. IL-6 psotive staining in carcinoma of the esophagus and also been assocaited with shorter survival (Chen, Mol Cancer, 2013, 12:26).

Immunotoxins: In this treatment an anti-tumor cell antibody is used to delvier a toxin to the tumor cells. However, in common with chemotherpaeutic approaches, immunotoxin therapy also suffers from drawbacks when applied to solid tumors. Tumor mass is generally impermeable in macromolecular agents such as antibodeis and immunotoxins. (Thorpe (US 2006/0228299)

Vasculature targetting: A promosing treatment strategy is to target the vasculature of solid tumors. Targeting the blood vessels of the tumors, rather than the tumor cells themselves, has certain advantages in that it is not likely to lead to the development of resistant tumor cells, and the targeted cells are readily accessible. Thorpe (US 2006/0228299)

Thorpe (US 2006/0228299) discloses that phosphatidylerine (PS) is a specific marker of tumor vasculature which has led to the development of new anti-PS immunoconjugates for delivering anti-cellular agents, toxins and coagulation factors to tumor blood vessels. Other examplary phosphatidylservine binding prtoeins includes annexins such as annexin V.

Precision Cancer Treatment:

Researchers can now sequence a person’s entire genome in mere days. Doing such sequencing can often point treatment i the right direction becasue they can reveal genetic mutations in genes that may be important in the type of cancer. Knowing the specific type of cancer can lead to personalized treatment for this type of cancer.

Treatment of Cancer Side Effects:

Pegfilgrastim-apgf injection is used to treat neutropenia (low white blood cells) that is caused by cancer medicines. It is a synthetic (man-made) form of a substance that is naturally produced in your body called a colony stimulating factor. Pegfilgrastim-apgf helps the bone marrow to make new white blood cells. This medication is usually given at least 24 hours after chemotherapy to stimulate the growth of new, healthy, white blood cells (WBC). Pegfilgrastim is a longer acting form of filgrastim and the manufacturer recommends that it should not be given within 14 days prior to chemotherapy.