HIV Pre-Exposure Prophylaxis: New and Upcoming Drugs to Address the HIV Epidemic.

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) provides a critical intervention toward ending the HIV epidemic and protecting people with reasons to utilize PrEP. PrEP options continue to expand as new administration modalities offer the potential to tailor PrEP use for individual success. We have provided the evidence for new and emerging antiretroviral agents for PrEP (cabotegravir, lenacapavir, dapivirine, and broadly neutralizing antibodies), divided into pharmacology, animal model, and human data, accompanied by a summary and suggested place in therapy. Cabotegravir is a US Food and Drug Administration (FDA)-approved intramuscular injection given every 2 months with a strong body of evidence demonstrating efficacy for HIV PrEP, lenacapavir administered subcutaneously every 6 months is currently under investigation for HIV PrEP, dapivirine vaginal ring is an available PrEP option for women in certain areas of Africa, and broadly neutralizing monoclonal antibodies have been challenged in demonstrating efficacy in phase 1-2 study for HIV PrEP to date. Clinical literature for individual agents is discussed with data from major studies summarized in tables. This review provides a detailed overview of recently available and premier candidate PrEP drugs.

Early Experience Implementing Long-Acting Injectable Cabotegravir/Rilpivirine for Human Immunodeficiency Virus-1 Treatment at a Ryan White-Funded Clinic in the US South.

Background: Long-acting injectable (LAI) antiretroviral therapy (ART) has the potential to improve medication adherence, reduce human immunodeficiency virus (HIV) stigma, and promote equity in care outcomes among people with HIV (PWH). We describe our early experience implementing LAI-cabotegravir/rilpivirine (CAB/RPV) for maintenance HIV-1 treatment. Methods: We launched a pilot LAI-ART program at a large Ryan White-funded clinic in the Southeast, which accept provider-initiated referrals from April 14, 2021 to December 1, 2021. Our interdisciplinary program team (Clinician-Pharmacy-Nursing) verified clinical eligibility and pursued medication access for eligible patients. We describe (1) demographic and clinical variables of PWH referred and enrolled and (2) early outcomes among those accessing LAI-CAB/RPV. Results: Among 58 referrals, characteristics were median age 39 (Q1-Q3, 30.25-50) years, 74% male, and 81% Black, and payor source distribution was 26% Private, 21% Medicare, 19% Medicaid, and 34% AIDS Drugs Assistance Program. Forty-five patients (78%) met clinical eligibility for LAI-CAB/RPV; ineligibility concerns included evidence of confirmed or possible RPV resistance (n = 8), HIV nonsuppression (n = 3), possible RPV hypersensitivity (n = 1), and pregnancy (n = 1). Among 45 eligible PWH, 39 (87%) enrolled and 15 (38%) initiated LAI-CAB/RPV after a median of 47 (Q1-Q3, 31-95) days since enrollment. Conclusions: Implementing LAI-ART at a Southern US Ryan White-funded clinic has been challenged by the following: substantial human resource capital to attain drug, administer injections, and support enrolled patients; delayed therapy initiation due to insurance denials; patient ineligibility primarily due to possible RPV resistance; and inability to provide drug regardless of payor source. These barriers may perpetuate disparities in ART access and outcomes among PWH and should be urgently addressed so that LAI-ART can be offered equitably.

Cabotegravir: a novel HIV integrase inhibitor combined with rilpivirine as the first long-acting injectable program for the treatment of HIV infection.

Cabotegravir, a novel HIV integrase inhibitor, shares structural similarity with dolutegravir and bictegravir. Its oral half-life is 32 hours, but cabotegravir nanosuspension for intramuscular injection yields half-lives ranging from 25 to 54 days, enabling extended interval dosing. Drug interactions are minimal, although oral doses require spacing from polyvalent cations, and potent uridine glucuronosyltransferase induction (e.g., rifampin, carbamazepine) requires avoidance due to anticipated subtherapeutic cabotegravir exposure through extended intervals. Randomized clinical trials combined cabotegravir treatment with rilpivirine to demonstrate treatment efficacy in patients living with HIV who had attained virologic suppression, lacked known/suspected mutations to either component, and had not experienced prior HIV treatment failure. Together, oral cabotegravir and rilpivirine maintained viral suppression in the LATTE study while the combination, given intramuscularly, performed comparably to conventional oral therapy in LATTE-2. FLAIR and ATLAS, respectively, demonstrated HIV suppression maintenance for monthly injections in treatment-naive participants and treatment-experienced patients, with ATLAS-2M supporting the efficacy of injections given every 2 months. Investigations to date show an excellent safety profile. Injectable cabotegravir causes short-lived, mild injection site reactions (primarily administration site pain/soreness) that decrease in frequency over time, produce attributable discontinuation rates of at least 2%, and generate satisfaction scores that favor injectable therapy over oral therapy. Virologic failure with resistance development is rare, primarily occurs in the first year of therapy, and is associated with baseline proviral DNA mutations to coadministered rilpivirine. A key component of the first U.S. Food and Drug Administration (FDA)-approved injectable maintenance treatment program for HIV, injectable cabotegravir heralds a new era in HIV treatment innovation. Here we provide a detailed review of the clinical pharmacology, administration and available formulations of the novel HIV integrase inhibitor cabotegravir with in-depth analysis of the clinical trial data, safety, satisfaction and viral resistance development when combined with rilpivirine as the first long-acting injectable program for the treatment of HIV infection.

Assessment of hospital emergency medication kit use at a large academic medical center with automated dispensing machine technology.

PURPOSE: Hospital emergency medication kits (HEMKs) are used to provide certain critical medications in emergent situations, despite many technological advancements for patient safety and medication distribution. We sought to evaluate HEMK usage and analyze associated costs to identify and recommend process improvements. METHODS: Mayo Clinic in Rochester, MN, is a large multisite academic medical center with 2 hospital campuses and many ambulatory clinics. All documentation of the approximately 250 HEMKs in circulation was analyzed from January to November 2017. The primary outcome was HEMK use. Secondary outcomes included individual medication usage and associated costs. These data were then used to recommend process improvements. RESULTS: Of 880 HEMKs evaluated, 675 (76.7%) were used, resulting in expiration 23.3% of the time. A total of 1,024 emergency medications were used, most commonly for hypoglycemia. Many of these medications are also available in automated dispensing machines for patient care use. Cost analysis revealed an average annual cost of nearly $200,000 associated with HEMKs. The results of our analysis indicated little added benefit of HEMKs in the setting of automated dispensing machine optimization. Steps for HEMK retirement are described. CONCLUSION: HEMKs offered little added benefit considering technological advancements that have been made in patient safety and medication distribution since their inception. Retirement of HEMKs is anticipated to increase pharmacy operational efficiency by using automated dispensing machine technology and appropriate emergency response protocols to ensure optimal patient care.

Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer.

PURPOSE: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. EXPERIMENTAL DESIGN: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. RESULTS: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing. CONCLUSIONS: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.

Evaluation of CML model cell lines and imatinib mesylate response: determinants of signaling profiles.

Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.

Antibody-based profiling of the phosphoinositide 3-kinase pathway in clinical prostate cancer.

PURPOSE: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited. EXPERIMENTAL DESIGN: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples. RESULTS AND CONCLUSIONS: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity.

BACKGROUND: Stat5(1) (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. METHODS: Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4',6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies. RESULTS: Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was approximately 70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity. CONCLUSION: We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines.

Analysis of the phosphatidylinositol 3'-kinase signaling pathway in glioblastoma patients in vivo.

Deregulated signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is common in many types of cancer, including glioblastoma. Dissecting the molecular events associated with activation of this pathway in glioblastoma patients in vivo presents an important challenge that has implications for the development and clinical testing of PI3K pathway inhibitors. Using an immunohistochemical analysis applied to a tissue microarray, we performed hierarchical clustering and multidimensional scaling, as well as univariate and multivariate analyses, to dissect the PI3K pathway in vivo. We demonstrate that loss of the tumor suppressor protein PTEN, which antagonizes PI3K pathway activation, is highly correlated with activation of the main PI3K effector Akt in vivo. We also show that Akt activation is significantly correlated with phosphorylation of mammalian target of rapamycin (mTOR), the family of forkhead transcription factors (FOXO1, FOXO3a, and FOXO4), and S6, which are thought to promote its effects. Expression of the mutant epidermal growth factor receptor vIII is also tightly correlated with phosphorylation of these effectors, demonstrating an additional route to PI3K pathway activation in glioblastomas in vivo. These results provide the first dissection of the PI3K pathway in glioblastoma in vivo and suggest an approach to stratifying patients for targeted kinase inhibitor therapy.

Tissue microarray analysis of signal transducers and activators of transcription 3 (Stat3) and phospho-Stat3 (Tyr705) in node-negative breast cancer shows nuclear localization is associated with a better prognosis.

PURPOSE: Although a high frequency of tumors contain constitutively activated signal transducers and activators of transcription 3 (Stat3), its relationship to breast cancer and patient survival has not been determined in a large retrospective study of node-negative tumors. To further elucidate the role of Stat3 in breast cancers, the expression patterns of Stat3 and Phospho-tyrosine residue 705 (Tyr705) Stat3 were correlated with survival outcome and clinicopathological parameters in a large cohort of node-negative breast cancer tumors. EXPERIMENTAL DESIGN: Immunohistochemical analysis of Stat3 and Phospho-Stat3 was performed on a breast cancer tissue microarray of 346 node-negative breast cancer specimens. These results were correlated with overall survival and other clinicopathological data. RESULTS: Positive Stat3 cytoplasmic expression was seen in 69.2% of tumors, and positive Phospho-Stat3 (Tyr705) cytoplasmic expression was seen in 19.6% of tumors. Neither cytoplasmic expression showed significant association with survival or other clinical parameters. However, 23.1% of tumors had positive Stat3 nuclear expression, and those patients had a significantly improved short-term survival (P = 0.0332) at 5 years of follow-up. Upon analysis of positive Phospho-Stat3 (Tyr705) nuclear expression, seen in 43.5% of tumors, positive tumors had a significantly improved survival at both short-term 5-year survival (P = 0.0054) and long-term 20-year (P = 0.0376) survival analysis. Additionally, positive Phospho-Stat3 (Tyr705) nuclear expression is an independent prognostic marker of better overall survival node-negative breast cancer by multivariate analyses that included the variables of nuclear grade, Ki-67, estrogen receptor staining, progesterone receptor staining, Her2 staining, age, and tumor size. CONCLUSIONS: These findings support a role for Stat3 and Phospho-Stat3 (Tyr705) overexpression in node-negative breast cancer and provide initial evidence that Phospho-Stat3 (Tyr705) may be a marker for improved overall survival independent of other prognostic markers.

Tissue microarray-based analysis shows phospho-beta-catenin expression in malignant melanoma is associated with poor outcome.

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the beta-catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of beta-catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated beta-catenin, as detected by a phospho-specific antibody, may be useful to predict outcome in melanoma. We use immuno-histochemical analysis of beta-catenin and phospho-beta-catenin, first to verify the specificity of the phospho-beta-catenin antibody and then to assay expression in a tissue microarray-based study. The subcellular localization of beta-catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho-beta-catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho-beta-catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho-beta-catenin was more common in metastatic lesions. High levels of nuclear phospho-beta-catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho-specific antibodies to beta-catenin define a unique subset of cases and that monitoring of phospho-beta-catenin expression may be useful for assessing prognosis in malignant melanoma.