Chapter 4 — Quiz

Twenty-two questions.

Multiple choice

1. The enzyme that cleaves native GLP-1 within minutes is: a) pepsin b) DPP-4 c) trypsin d) neprilysin

2. Native GLP-1's circulating half-life is approximately: a) 1–2 minutes b) 1–2 hours c) 1–2 days d) 1 week

3. Semaglutide's half-life is approximately: a) 13 hours b) 3 days c) 1 week d) 1 month

4. The kidney filters molecules freely below approximately: a) 500 Da b) 5,000 Da c) 50,000 Da d) 500,000 Da

5. Semaglutide's position-8 modification (Ala→Aib) primarily: a) increases receptor affinity b) blocks DPP-4 cleavage c) enables albumin binding d) improves solubility

6. Semaglutide's position-34 modification (Lys→Arg) exists primarily to: a) extend half-life b) increase potency c) leave a single fatty-acid attachment site for manufacturing control d) reduce immunogenicity

7. The C18 fatty acid on semaglutide works by: a) increasing membrane permeability b) binding albumin, defeating renal filtration c) blocking proteases directly d) improving oral absorption

8. Oral semaglutide's bioavailability is approximately: a) 1% b) 10% c) 40% d) 80%

9. The default route of administration for peptide drugs is: a) oral b) intravenous c) subcutaneous d) intranasal

10. Steady state on regular dosing is reached after approximately how many half-lives? a) 1 b) 2 c) 4–5 d) 10

11. First-pass metabolism refers to: a) the first dose of a drug b) clearance by the liver of drug absorbed from the gut before it reaches general circulation c) initial receptor binding d) renal filtration

12. Which route is designed to have essentially zero systemic bioavailability? a) subcutaneous b) intranasal c) oral, locally acting (e.g. linaclotide) d) depot injection

13. The most serious consequence of anti-drug antibodies is: a) injection site reactions b) reduced efficacy c) neutralization of the drug d) cross-reaction with the patient's own endogenous peptide

14. GLP-1 agonist doses are titrated upward mainly because: a) low doses are ineffective b) gastrointestinal side effects are much worse if started at target dose c) the receptor needs time to upregulate d) insurance requires it

15. During the GLP-1 shortage, the binding manufacturing constraint was primarily: a) peptide synthesis b) aseptic fill-finish and injector pen assembly c) raw amino acids d) regulatory review

16. Aib is: a) one of the twenty proteinogenic amino acids b) a non-natural amino acid that must be incorporated chemically c) a fatty acid d) a permeation enhancer

Short answer

17. Name the five barriers of the oral gauntlet and state which one cannot be solved by protecting the peptide from enzymes.

18. Explain why defeating only one of the two elimination routes is insufficient for a long half-life.

19. Explain why the oral semaglutide label has unusual administration instructions and the injection label does not.

20. Why does aggregation of a peptide in a vial matter beyond simple loss of drug?

Applying the rating discipline

21. Chapter 4 issues no evidence ratings. Explain why delivery information, despite being decisive in many cases, is not itself a rating.

22. A claim fails the delivery filter — the route is one the molecule cannot survive and no bioavailability has ever been measured. Chapter 2 said mechanism is "strong evidence against, weak evidence for." Explain how this case illustrates that, and state precisely what you are and are not entitled to conclude.

Answer key **1.** b — DPP-4 (dipeptidyl peptidase-4), which cleaves two residues from the N-terminal end. **2.** a — one to two minutes. **3.** c — approximately one week, enabling once-weekly dosing. **4.** b — roughly 5,000 Da, with the cutoff softening up to tens of thousands depending on shape and charge. Most peptides fall below it. **5.** b — it abolishes the DPP-4 cut site. Receptor activity is preserved. **6.** c — manufacturing control. GLP-1 has two lysines; the fatty acid attaches to a lysine, so with two available you would get a mixture. Removing one produces a single defined product. **This modification has no pharmacological benefit to the patient.** **7.** b — binding albumin (~66,000 Da), producing a complex far too large to filter, and shielding the peptide from proteases. **8.** a — roughly 1%, which is why the tablet contains far more drug than the injection. **9.** c — subcutaneous. **10.** c — roughly four to five. **11.** b. **12.** c — a locally acting oral peptide such as linaclotide is designed not to be absorbed; its target is in the gut. **13.** d — cross-reaction with the endogenous peptide, which can produce a deficiency worse than the original condition. Rare, and the reason immunogenicity is taken so seriously. **14.** b — tolerability, not efficacy. **15.** b — aseptic fill-finish capacity and injector pen assembly, the final steps, which require large highly regulated facilities that are slow to build. **16.** b — 2-aminoisobutyric acid, non-proteinogenic, not encoded by any gene and not incorporable by a ribosome. Its use is one reason semaglutide is made by chemical synthesis rather than grown in bacteria. **17.** (1) stomach acid and pepsin; (2) pancreatic proteases in the small intestine; (3) brush-border peptidases; (4) crossing the intestinal epithelium; (5) first-pass hepatic metabolism. **Barrier 4 is the one that protection from enzymes does not address** — an entirely intact peptide still has to cross a cell layer it is too large and too polar to traverse, and the transporters that exist handle only di- and tripeptides. **18.** Because the two exits are independent. A protease-resistant peptide that is still under the renal filtration cutoff will be removed intact by the kidney; a peptide bound to albumin but still carrying an intact protease cut site will be clipped while bound. Closing one leaves the other fully open, which is why semaglutide required both a substitution *and* a fatty acid. **19.** Because the tablet's absorption depends on a fragile, local, transient environment created by a permeation enhancer in a small region of the stomach lining. Food, extra fluid, and other oral medications all disrupt it, and the absorption window is brief. The injection has almost no instructions because subcutaneous delivery bypasses all five oral barriers and has essentially no delivery problem. **The tablet's instructions are the delivery problem, written down.** **20.** Because aggregated peptide is substantially more **immunogenic** than properly formulated peptide. Aggregates present repeating structures that the immune system is particularly good at recognizing, raising the risk of anti-drug antibodies — including, rarely, antibodies that cross-react with the patient's own endogenous molecule. So improper storage or handling changes not just how much active drug is present but the immunological risk profile, without any change to the sequence. Regulated manufacturing controls for this explicitly; an unregulated supply chain does not. **21.** Because a rating attaches to a **claim about a clinical outcome in a population**, and delivery information addresses a prior question — whether the molecule can physically reach its target in useful quantity. Failing the delivery filter tells you a claim is implausible; it does not tell you what a trial found, because in these cases there usually is no trial. Delivery narrows the space of plausible claims. Ratings evaluate the evidence for what remains. **22.** This is the asymmetry at its sharpest. **Evidence against is strong:** a molecule that cannot reach its target in useful quantity cannot produce the claimed effect, and that conclusion does not require a trial. It is a physical constraint, and physical constraints do not have exceptions for compounds you like. **You are entitled to conclude:** the specific claim, by this route, at this dose, is not supported and is probably not physically available. **You are not entitled to conclude:** that the molecule does nothing by *any* route, that the underlying biology is wrong, or that a properly delivered version would fail. Those are different claims requiring different evidence — and several compounds have gone from "cannot be delivered" to "approved drug" when someone solved the delivery problem. Oral semaglutide is one of them.