Tag Archives: Pylarify

Prostate Cancer, Round 2

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Note: What follows are my observations and information from my oncologists and what is scraped off the interwebs. I try to seek information from either primary research literature, medical textbooks or from credible secondary sources. For treatment, I stick to a university medical institution and medical school faculty managing my treatment. I tend not to believe in dietary or nutraceutical approaches. It has been my observation that the origins of cancer are biochemically different from curative or preventative biochemistry. In other words, preventative measures by diet or supplements are mechanistically distinct from the treatment of cancer cells. Divine intervention is not testable, driven by faithful wishing and is supported only by anecdote. I believe that if something truly happens in the universe, it will have an observable mechanism and therefore be measurable.

Oh yes, if you’re squeamish with talking about your prostate because it’s part of your reproductive apparatus, get over it. Part of successfully living with cancer is being able to talk about and learning from it. I’d rather die at least knowing about it.

Because of modern medicine, my experience with both throat and prostate cancer has not been a rocket sled ride to the hereafter. It’s been said that some cancers can be thought of as a treatable, chronic condition and for me that has been true thus far. As luck would have it, my throat cancer was viral in origin and consequently highly treatable by IMRT irradiation and cisplatin. Since 2013 I have had yearly checkups that have all indicated no visible return of the cancer. Since I go to a university medical center, I have had medical students and various head and neck residents also peering down my throat from a camera threaded through my nose picturing my gullet in all of its pink glistening majesty.

The prostate story is a bit different. Before diagnosis the cells had already left the prostate (stage 4) and were judged to be Gleason 9 by histopathology. This was unfortunate. Outside of the prostate capsule they began to wander around through the lymphatic system, lodging in the lymph nodes. Since there was no unified target for surgery or concentrated radiation, The cutters were not called in. Elvis had left the building. After IMRT radiation of the prostate, seminal vesicles and suspected nearby lymph nodes along with 2 years of hormone ablation, my PSA returned to 0.01 ng/mL. Things had taken a turn for the better.

But, the other shoe had to drop eventually. After 9 years, my stage 4 prostate cancer has begun to ramp up steeply. The PSA curve over time (below) is looking more and more like a hockey stick. The borderline PSA value for treatment is 4.00 ng/mL. When it pops up over that value the oncologists begin to take notice. Whether this is based on some statistical mortality data or because of what insurance companies will likely cover is unclear to me. Importantly, PSA may also indicate non-cancerous conditions like prostatitis and benign prostatic hyperplasia. PSA is only an indicator and alone is not definitive. Biopsy is needed to verify and grade the tissue. Of this whole adventure, the biopsy was the worst of it for me. During the procedure, the urologist asked questions about my hobbies -his was carpentry- but I was too distracted to talk about airplanes.

Stage 4 is indicated by histology and backed up by the PET scan revealing radioactive (avid) spots outside of the prostate. Thankfully, this time around nothing was found in the head & neck, chest, prostate or bones. That was good news.

However, the PET/CT scan did show the presence of 5 or so avid lymph glands along the aorta from below the chest to above the prostate.

A proper prostate cancer diagnosis requires more than just a PSA value. An abnormal prostate is detected by digital examination by a urologist and the presence of cancer cells is confirmed by biopsy by a histologist.

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My PSA curve.

But, wait a minute. Exactly what is PSA and what does it do? According to Wikipedia, Prostate Specific Antigen (PSA) is a peptidase enzyme (a protein) secreted by the epithelial cells of the prostate gland. It’s immediate job is to liquify the semen in the seminal coagulum, allowing sperm to swim freely. It is also thought to be involved in dissolving cervical mucus, allowing sperm to enter the uterus. Amounts of PSA above a certain threshold are not normally found in the blood. Elevated PSA is associated with prostate cancer. It’s just a marker.

The glycoprotein PSA, prostate specific antigen. It is an enzyme of the serine protease variety. A protease will cleave peptide bonds in the amino acid backbone of a protein.

Serine protease enzymes like PSA have a serine amino acid in the active site of the enzyme which is capable of connecting temporarily with a carbonyl carbon of a (C=O) peptide bond. Since proteins are long chains of peptide bonds, cleaving a peptide bond snips the protein into smaller pieces.

Chemists are all about the mechanisms of chemical transformations and the following has been proposed for a serine protease.

Source: Wikipedia. Graphics by Gaussling.

All this said, it turns out that when castration resistance sets in, things begin take a turn for the worse. Prostate cancer cells begin to accumulate in the bone marrow, they begin to interact and develop into tumors that are essentially beyond the reach of treatment. The spine is a common place for them to go, but they can spread to other organs as well.

Of particular interest is the spread of prostate cancer to bone. Prostate cancer cells have an affinity for bone marrow tissue. In my case, the PET/CT scans gave no indication of being present in the head & neck, the chest or bones. That’s good news. In my first round of treatment, I was given 18F-Glucose diagnostic for the PET scan. This time I was given the more receptor-selective 18F PSMA diagnostic called Pylarify. While it is selective for a particular receptor on the cancer cell, it also shows up elsewhere in the body in the PET scan as a result of circulation and transport out of the system. Receptor-specific drugs will bind to the intended receptor, but only after they wander around and stumble into it. This is made less than random due to active transport or solubility partitioning. The effectiveness also benefits by resistance to metabolism and excretion.

Pylarify is a kind of pseudo-peptide containing two modified amino acids, lysine and glutamic acid, joined at the nitrogen atoms as a urea linkage. The key step is the nucleophilic aromatic substitution of trimethylammonium by 18F on the pyridine ring. The presence of abundant heteroatoms (nitrogen and oxygen) groups is not uncommon for pharmaceuticals and is absolutely ordinary for proteins. Heteroatoms serve as hydrogen bond donors and acceptors which is critical in biochemical transformations. A hydrogen bond donor can reversibly bind to a hydrogen bond acceptor and keep the molecules in close proximity long enough for a transformation as well as participate in it.

Sarah Piron, Jeroen Verhoeven, Christian Vanhove, Filip De Vos, Recent advancements in 18F-labeled PSMA targeting PET radiopharmaceuticals, Nuclear Medicine and Biology, Volumes 106–107, 2022,
Pages 29-51, ISSN 0969-8051, https://doi.org/10.1016/j.nucmedbio.2021.12.005.

The interaction of prostate cancer cells in the bone marrow environment is fairly complex and is well described by Zhang X. Interactions between cancer cells and bone microenvironment promote bone metastasis in prostate cancer. Cancer Commun (Lond). 2019 Nov 21;39(1):76. doi: 10.1186/s40880-019-0425-1. PMID: 31753020; PMCID: PMC6873445.

The interaction of prostate cancer cells with bone marrow cells is a topic for another day.

18F-Glucose

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The story of PET, Positron Emission Tomography, has evolved over decades of advancement. To begin, tomography, detectors and computers had to be invented. Separately, positron emission as a medically viable radiation source had to be identified and validated. Positron decay occurs when a neutron deficient nucleus emits a positron and a neutrino to convert a proton to a neutron. This brings the p/n ratio to a more stable state.

A substance for delivering a dose of isotope must be found. In the case of 18Fluorine, it is prepared as an inorganic salt like K18F or elaborated as an organic molecule like 2-deoxy-2-[18F]fluoro-D-glucose.

How did it come about that the 18Fluorine in the position where it is? I’ve not found mention of this in the literature so far. Looking through Chem Abstracts I have noticed there are numerous synthetic pathways leading to fluorine at that position. Could it have been placed there because research found that it was most biochemically similar to glucose? Or was it the more mundane reason that fluorination at position 2 gave the best yields and purity or was the cheapest and easiest to make?

18F has replaced the oxygen (OH) group at position 2 of glucose, thus the prefix “2-deoxy-2-[18F]fluoro-“

There are recent radioligand compounds that are used as PET (Positron Emission Tomography) diagnostic agents which selectively bind to the prostate specific membrane antigen receptor where they can undergo positron emission revealing the site of prostate cancer cells. 18F-glucose was first synthesized in 1967 in Czechoslovakia at Charles University by Dr. Josef Pacák and was first tried as a radiotracer by Abass Alavi in 1976 at the University of Pennsylvania on volunteers. PET scanning came along later. Cancer cells consume glucose a bit faster than normal cells so the 18F-glucose will tend to accumulate to a slightly greater extent and reveal their position by positron annihilation. This yields two 511 keV x-rays 180o apart and is identified by a ring coincidence detector. A single detection event is discarded.

Today, 18F-glucose is being superseded by many 18F PET preparations that are designed to interact with specific receptors. This interaction is called “conjugation”. In the case of Prostate Cancer there is PSMA, Prostate Specific Membrane Antigen, targeted by Pylarify (piflufolastat F 18) which is designed to bind with fatty acid binding protein 3 (FABP3). I just received a 6 millicurie (222,000 Becquerel) intravenous dose of this positron emitter just today for a PET/CT scan.

Synthetic Strategies Affording 18F-glucose

First, I have to say that the name 18F-glucose is a bit of a misnomer in that it is not glucose nor did it ever even start out as glucose. It is a 2-deoxy-2-18fluoro analog of D-glucose. It originates from D-Mannose whose OH groups were specially protected from side reaction by capping 4 of them with acetyl (Ac) groups and carrying away the hydrogens. The OH at position 2 of the D-Mannose precursor is converted to a triflate (OTf).

In chemical synthesis there is usually more than one possible strategy for getting to a target molecule. In the case of 18F-glucose, whatever pathway we choose must be rapid and efficient owing to the very short half-life of the 18F. The preparation must be done in as few half-lives as possible.

When it comes to a great many sugar derivatives, synthesizing them from scratch is just crazy. They are structurally and stereochemically complex. They have numerous hydroxyl groups in chemically different locations on the molecule and selective modification of one and not another can be quite involved. The world is awash in sugars (e.g., sucrose, starch and cellulose) from natural sources and many varieties are commercially available for developmental use. Better to adapt available sugars for modification than starting from earth, air, fire and water.

Source: Gaussling.

Getting 18F attached to a sugar can go along on one of two basic strategies- electrophilic addition of fluorine or nucleophilic addition. The first is called “electrophilic” addition where electrophile means “electron loving”. In electrophilic addition, the 18F reagent must be electron deficient requiring that the intended carbon skeleton is relatively electron rich. Electron rich means that there are oxygen or nitrogen atoms present with their lone-pair electrons, or pi-bonds present with their off-axis pi-electrons. Equations (a) and (b) below show two examples of electrophilic addition of 18F to a sugar analog.

The fluorinating reagents are (a) 18F enriched F2 and (b) acetyl hypofluorite, [18F]AcOF. Both fluorinating reagents feature fluorine atoms that are electron deficient and therefore electrophilic. Atomic and molecular fluorine are by nature quite electrophilic, but negatively charged fluoride is nucleophilic.

Source: Cole EL, Stewart MN, Littich R, Hoareau R, Scott PJ. Radiosyntheses using fluorine-18: the art and science of late stage fluorination. Curr Top Med Chem. 2014;14(7):875-900. doi: 10.2174/1568026614666140202205035. PMID: 24484425; PMCID: PMC4140448.

Nucleophilic addition of 18Fluoride is shown in reaction (c) wherein the OTf group is installed specifically to be displaced from the back side by 18F anion. A “nucleophile” is an attacking species that is able to bond directly with a carbon nucleus by virtue of having a lone pair of electrons available for bond making. A nucleophile is frequently negatively charged but can also be neutral in some cases.

The general strategy for the nucleophilic substitution synthesis of 18F-glucose is this: Protect all of the hydroxyl groups of D-Mannopyranose as an acetate except for one which serves as a “leaving group“. This leaving group is called a trifluoromethanesulfonate, or just “triflate“. This triflate is then displaced by 18Fluoride anion by an SN2 substitution. In plain English, 18Fluoride anion forms a C-F bond as the triflate anion is breaking its C-O bond in a process called nucleophilic substitution.

Oh, one more thing. The 18fluoride anion(-) must be made more reactive by keeping the inhibiting potassium cation (K+) in a “cage” so it can lose some of its electrostatic attraction to the negatively charged 18fluoride. Strong electrostatic attraction of K+ to 18F– will impede fluoride’s aptitude for triflate displacement. See below for Kryptofix 222. K+ wrapped in neutral Kryptofix 222 is called a “weakly coordinating ion”.

Ok, so there are some funny things you ought to know about this substitution business on a 6-member ring. Hydrogen atoms are not drawn because it is a pain. First, carbon always wants to have 4 bonds to it and oxygen just two bonds. Second, a 6-carbon ring with all single bonds can be twisted into several shapes or conformations. One of them is favored by virtue of having the least “strain” in it. That would be the “chair” conformation. It looks vaguely like a lawn chair.

Source: Gaussling. Shapes that cyclic, 6-carbon rings can take. In reality, the rings flip back and forth across the different conformations, but they tend to spend the most time in the lowest ring strain shape which is the chair.

Selective chemical synthesis happens only because some reaction pathways are fast while others are slow. Some possible reaction pathways are so slow that effectively they do not happen.

Making 18F

The 18F isotope does not exist in nature due to its 1.83 hour half-life. It decays by positron and neutrino emission to stable 18O. 18F must be prepared by slamming a suitable precursor nucleus with a nuclear particle like a proton or a deuteron with a cyclotron or linear accelerator. Yes, commercial cyclotrons are available for purchase.

Some Sugar Facts

What helps when thinking about sugars is to detach them from the matter of sweetness. Sugars are far too diverse and important to get hung up on sweetness.

Look at the blue O-H groups on the α-D-Mannopyranose and compare it to the  α-D-Glucopyranose shown above. See how they are hanging on the ring? One is directed up and the other is pointing outward and down a bit. This simple inversion in orientation produces the chemical difference between the two sugars making them distinctly different chemical substances.

Source: Gaussling. How the 18-Fluorine gets attached to a sugar. D-Mannose is first derivatized by capping off 4 of the hydroxyl groups as acetates, OAc, and one as a triflate, OTf. 18-Fluoride backside attack will displace the triflate, –OTf. Of the –OTf and the –OAc, the –OTf is displaced much faster. The faster pathway dominates. The Ac groups are removed from their oxygens by base hydrolysis leaving OH groups on the ring behind. This results in the 18Fluorinated glucose.

In the reaction scheme above the 18F– is shown displacing the –OTf group from below, establishing a C-18F bond and causing the C-H to flip to the upper side like an inverting umbrella. The scheme is only partially correct. What isn’t shown is the positive counterion to the 18F– anion. The fluoride must be charge balanced by a positive ion which could be just a theoretical bare-naked ion or solvated potassium ion, K+.

In solution, ions or dipolar molecules interact with solvent molecules by Van der Waals forces or stronger dipolar influence. Going down the Group 1 elements on the periodic table from Lithium to Francium, all form 1+ cations, but also the radius of the ion increases. If you think of the ionic radius as being the distance from the nucleus to the distance that a solvent molecule can bump into, the Van der Waals radius, then as we drop down Group 1, a square picometer of “surface” of the ion carries less and less of the cationic charge at any given moment. This means that attractive or repulsive forces with that square picometer diminish as we go down the group, thus lowering the attractive forces. Very often potassium cation is acceptable, but it can be helped along.

While much of the time K+ is sufficiently non-interfering, but as happens occasionally the fluoride anion tends to bind to the potassium cation a bit too tightly. This can substantially slow the rate of transfer of 18F anion to the carbon of the sugar ring. To get around this, either the potassium must be replaced with another more charge diffuse cation like tetrabutylammonium+ or cesium+, the K+ can be “wrapped” in a protective organic “jacket or shield” that will prevent the K+ and the 18F– ions from getting too close to one another and bound too tightly. We would call the protected K+ a non-interfering or charge diffuse cation.

The cyclic amino polyether “ligand” that is used in this case in Kryptofix [2.2.2]. The single positive charge of the K+ is somewhat spread over the surface of the much larger Kryptofix [2.2.2]-potassium complex and diffuses the positive charge. This has the effect of “separating or loosening” an otherwise tight ion pair (K+F–) in solution. Once detached from potassium, the 18F– ion is able to react much faster to form the 18F-Glucose.

18F-Glucose must be synthesized in a radiopharmacy, also called a nuclear pharmacy, nearby the point of administration to the patient given its very short half-life. The 18F is produced in a commercially available cyclotron or linear accelerator either by proton bombardment of stable but scarce 18O enriched water or by deuteron bombardment of the stable isotope 20Neon.

18F-glucose is a sugar and undergoes metabolic trapping by phosphorylation with hexokinase inside the cell, giving it a phosphate group with a negative charge, inhibiting its transport to outside the cell. This allows the phosphorylated 18F-glucose to accumulate inside the cell, concentrating 18F to release more positron decays from the cell.